WO2023041978A1 - SYNERGIC COMPOSITIONS OF COMPOUNDS AGAINST THE HETERODIMERIC COMPLEX K-RAS4B/PDE6δ FOR THE TREATMENT OF PANCREATIC CANCER - Google Patents

SYNERGIC COMPOSITIONS OF COMPOUNDS AGAINST THE HETERODIMERIC COMPLEX K-RAS4B/PDE6δ FOR THE TREATMENT OF PANCREATIC CANCER Download PDF

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WO2023041978A1
WO2023041978A1 PCT/IB2021/062509 IB2021062509W WO2023041978A1 WO 2023041978 A1 WO2023041978 A1 WO 2023041978A1 IB 2021062509 W IB2021062509 W IB 2021062509W WO 2023041978 A1 WO2023041978 A1 WO 2023041978A1
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compounds
pancreatic cancer
compound
ras4b
treatment
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PCT/IB2021/062509
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Spanish (es)
French (fr)
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Paola BRISEÑO DÍAZ
Miguel Angel VARGAS MEJÍA
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Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • compositions with pharmaceutical activity that are useful for the treatment of diseases, particularly with pharmaceutical compositions for the treatment of pancreatic cancer, more particularly with pharmaceutical compositions that exhibit synergistic effects to reduce tumor growth, without generating adverse effects. and that comprise compounds against the K-Ras4B/PDE6 ⁇ heterodimeric complex, decreasing the activation of K-Ras4B.
  • PDAC Pancreatic ductal adenocarcinoma
  • K-Ras4B mutations induce aberrant activation of K-Ras4B, resulting in continued activation of K-Ras4B-dependent signaling pathways such as AKT and ERK [5, 7, 8].
  • DNA synthesis inhibitors such as Gemcitabine, 5-FU and Oxaliplatin, which generate side effects in patients, such as elevated liver enzymes, leukopenia, neutropenia, venous collapse, pain and loss of bone mass [9].
  • Deltarasin which interacts with PDE6 ⁇ with a K d of 38 nM, and prevents the recognition of the post-translational modification present in K-Ras4B, which would concentrate K-Ras4B in the cytosol, thus preventing its activation and tumor progression; this compound was named as the first generation of PDE6 ⁇ inhibitors [20].
  • this compound was evaluated in non-cancerous cell lines of the pancreatic duct and a high cytotoxicity was observed, considerably affecting cell viability at low concentrations [21-26].
  • Deltazinone presenting a dissociation constant of Kd 38 nM to Kd 4 nM, proving to be a compound with better interaction energy. than the first generation.
  • Deltazinone showed cytotoxic effects on pancreatic cancer cell lines at a concentration of 24 ⁇ M, however it took around 8 h to have an anti-proliferative effect on pancreatic cancer cell lines, while Deltarasin at a concentration of 5 ⁇ M in one hour showed the same effect as its analogue, so considering these data, the first generation of PDE6 ⁇ inhibitors have a greater effect than the second generation [27, 28].
  • Deltasonamides analogue to Deltarasin
  • Transport of the K-Ras4B protein is mediated by the PDE6 ⁇ protein from the endoplasmic reticulum to the plasma membrane for subsequent activation, thus forming the K-Ras4B/PDE6 ⁇ heterodimeric complex in the cytoplasm. [23, 32, 33].
  • K-Ras4B/PDE6 ⁇ was thought to be transported as a dimer and is now known to form a cluster of 6-12 proteins or 3-6 dimers [32]. Due to this, our working group searched for a model of the heterodimer using the crystal of the heterodimeric complex in a group of 6 proteins, obtaining a representative dimer of the K-Ras4B/PDE6 ⁇ multiprotein heterocomplex, finding two compounds called D14 and C22 that bind and stabilize the K-Ras4B/PDE6 ⁇ heterodimeric complex [23].
  • the analog called P8 presents higher interaction energy on the mutated complexes in silico, thus presenting greater cytotoxic effects in cell lines and primary cultures of pancreatic cancer with mutated K-Ras without damaging the cell line and non-cancerous primary cultures.
  • both compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways, however the compound P8 presents an IC504 times lower than the compound C14, being up to now the best compound found in our research group.
  • compositions that comprise the combination of the C14/P8 compounds present a synergistic effect, inducing cytotoxicity in cell lines and primary cultures of pancreatic cancer that are greater than those obtained by the compounds evaluated individually.
  • compositions of the invention that comprise the combination of compounds C14 and P8 decrease tumor growth from 90 to 95% in subcutaneous and orthotopic xenograft models as the dose increases, do not induce adverse effects or genotoxicity as presented by first-line chemotherapy with Gemcitabine, decrease K-Ras4B activation and decrease malignancy markers in remnant tumors.
  • the compositions of the invention that comprise the combination of compounds C14 and P8 decrease up to 99% of tumor growth in PDx models of pancreatic cancer.
  • the compositions of the invention showed better antineoplastic effects in the Subcutaneous Xenograft, Orthotopic and PDx models, decreasing tumor growth by up to 97% without inducing side effects such as those presented by Gemcitabine.
  • C14 stabilizes the K-Ras4B/PDE ⁇ complex and inhibits the growth of human pancreatic cancer cell lines.
  • D Representative bright field images of the cell lines ARPE-19, hTERT-HPNE, PANC-1 and MIA PaCa-2 treated with C14100 ⁇ M, DMSO as vehicle and untreated control cells.
  • A-B Evaluation of cell viability after treatment with P1, P2, P3, P4 and P5 analogues of C14 at 90.18 ⁇ M in MIA PaCa-2 (A) and hTERT-HPNE (B) cells are observed;
  • C-D evaluation of cell viability after treatment with P6, P7, P8, P9 and P10 analogues of C14 at 90.18 ⁇ M in MIA PaCa-2 (C) and hTERT-HPNE (D) cells;
  • E-F evaluation of cell viability after treatment with P11, P12, P13, P14 and P15 analogues of C14 at 90.18 ⁇ M in MIA PaCa-2 (E) and hTERT-HPNE (F);
  • G-H Evaluation of cell viability after treatment with P16, P17, P18, P19 and P20 analogues of C14 at 90.18 ⁇ M in MIA PaCa-2 (G) and hTERT-HPNE (H).
  • P8 stabilizes the K-Ras4B/PDE ⁇ complex and inhibits the growth of PDAC cell lines better than C14.
  • D interaction of P8 with the K-Ras4BG12V/PBDE6 ⁇ complex;
  • E-H) relative cell viability of PDAC PANC-1, MIA PaCa-2 and Capan-1 cells and the normal pancreatic cell line hTERT-HPNE treated with different concentrations of P8 for 72 h (n 5);
  • FIG. 4 P8 and C14 decrease K-Ras activation and AKT and ERK phosphorylation in PDAC cell lines with K-Ras mutation.
  • A-D Western blot images representative of cell lines A) hTERT-HPNE, B) PANC-1, C) MIA PaCa-2 and D) Capan-1 treated with the IC50 of P8, C14, Gemcitabine and Deltarasin for 3 p.m.
  • Total protein extracts were precipitated using RAF-RBD beads.
  • Total RAS (Ras-T) and GAPDH are shown as loading controls.
  • K-Ras GTP pixel intensities were normalized to total RAS and GAPDH;
  • E-G Western blot representative of cell lines
  • Figure 5 Characterization of PDAC tissues and primary cells.
  • A–E Effects of P8 and C14 at various concentrations (5, 10, 30, 50, 100, 150, and 200 ⁇ M) are observed for 72 h in non-cancerous primary cultures PBD033 and JGC028, and PDAC primary cultures MGKRAS003, MGKRAS004, and MGKRAS005;
  • F-H clonogenic assays of the primary cultures of PDAC, MGKRAS003, MGKRAS004 and MGKRAS005 treated with the IC fifty from P8, C14, Gemcitabine and Deltarasin;
  • I-J Cell death analyzes of PBD033, JGC028 MGKRAS003, MGKRAS004, and MGKRAS005 were determined by flow cytometry after staining with annexin-V, 7-AAD, and cytocalcein violet; J) quantification of the percentages shown in I).
  • A-E Western blot images representative of JGCD28 (A), PBDD33 (B), MGKRAS004 (C), MGKRAS003 (D) and MGKRAS005 (E) cells treated with the IC are shown.
  • Total protein extracts were precipitated using RAF-RBD beads.
  • Total RAS (Ras-T) and GAPDH are shown as loading controls.
  • FIG. 12 The combination of P8 and C14 reduces tumor growth in subcutaneous and orthotopic xenograft models.
  • A) The effects of P8, C14 and C14/P8 at different concentrations (5, 10, 30 and 60 mg/kg, and a combination of 30 mg/kg C14 + 30 mg/kg P8) are observed in a model subcutaneous xenograft using MGKRAS004 cells; B) final effect after treatment with P8, C14 and C14/P8 at different concentrations; C) body weight was measured daily during treatment with P8, C14 and C14/P8; D) representative images of MGKRAS004 tumors obtained from each group; E) Effects of P8, C14, and C14/P8 at different concentrations (5, 10, 30, and 60 mg/kg, and a combination of 30 mg/kg C14 + 30 mg/kg P8) in a subcutaneous xenograft model (n 6) using MGKRAS005 cells; F) final effect of treatment with P8, C14 and C14/P8 at different concentrations for x d; G) body weight was measured daily during treatment with P8, C14 and C14/P8;
  • the present invention provides synergistic compositions that comprise the combination of the C14/P8 compounds, inducing cytotoxicity in cell lines and primary cultures of pancreatic cancer greater than those obtained by the compounds evaluated individually, which makes them a viable, effective and useful alternative. for the treatment of pancreatic cancer.
  • Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis of all human cancers, as it is highly resistant to chemotherapy. This leads to the search for new pharmacological alternatives to improve the quality of life of patients with pancreatic cancer.
  • Compounds have been designed that can inhibit the signaling and transport pathway of the K-Ras4B oncoprotein.
  • compositions of the present invention that comprise the combination of the C14/P8 compounds have a synergistic effect, inducing cytotoxicity in cell lines and primary cultures of pancreatic cancer that are greater than those obtained by the compounds evaluated individually.
  • the compositions described here decrease tumor growth from 90 to 95% in Subcutaneous and Orthotopic xenograft models as the dose increases, likewise they do not induce adverse effects or genotoxicity as presented by first-line chemotherapy with Gemcitabine, they decrease activation of K-Ras4B and decrease malignancy markers in remnant tumors.
  • compositions of the invention decrease up to 99% of tumor growth in PDx models of pancreatic cancer, showing better antineoplastic effects in Subcutaneous Xenograft, Orthotopic and PDx models, decreasing tumor growth by up to 97% without inducing side effects.
  • the compositions described herein are configured as new and efficient chemotherapeutic solutions with better properties than conventional chemotherapy.
  • compositions described herein comprise: a) Compound C14 (formula I) or its pharmaceutically active salts in a concentration of 90.18 ⁇ M to 154.24 ⁇ M with respect to compound P8, b) Compound P8 (formula II) (analogous to compound C14 which has amino groups, benzenes, pyridines and non-aromatic heterocycles) or their pharmaceutically active salts in a concentration of 18 ⁇ M to 150 ⁇ M, and c) A pharmaceutically
  • a modality of the invention is the adaptation of the active principles to be used in pharmaceutical compositions for enteral, parenteral administration and topical use, including inhalation.
  • the effective doses for the patient of the active ingredient will also be adjusted in accordance with preclinical and clinical studies, but based on the findings of the present invention.
  • Examples of pharmaceutically acceptable excipients accompanying the active principle of the invention are, for example, for oral administration as tablets or tablets, agents comprising, for example, diluents, binders, stabilizers, bulking agents, thickening agents, such as povidone, cellulose microcrystalline, lactose, etc., disintegrating agents such as cross-linked carboxymethylcellulose, surfactants such as sodium lauryl sulphate, lubricating or slipping agents such as magnesium stearate, colloidal silicon dioxide, etc., where said excipients can be formulated for preferably slow or prolonged release for a systemic effect.
  • agents comprising, for example, diluents, binders, stabilizers, bulking agents, thickening agents, such as povidone, cellulose microcrystalline, lactose, etc., disintegrating agents such as cross-linked carboxymethylcellulose, surfactants such as sodium lauryl sulphate, lubricating or slipping agents such as magnesium stearate, colloidal silicon dioxide
  • Solutions for intravenous or intraperitoneal administration of the active ingredient can be prepared first dissolved in an organic solvent such as DMSO, ethanol, or dimethylformamide and subsequently in aqueous buffers, such as PBS.
  • an organic solvent such as DMSO, ethanol, or dimethylformamide
  • aqueous buffers such as PBS.
  • compositions described herein can be obtained by combining the compounds C14 and P8 with pharmaceutically compatible vehicles known in the art, in the amounts and/or concentrations that correspond to what is described herein, and may include known compounds in the art for obtaining said compositions.
  • administration of such compositions can be done depending on the conditions of the patient, which will determine the doses and frequency of administration necessary to achieve an effective treatment of the condition in each particular case.
  • compositions described manage to reduce the viability of cancer cells without affecting the viability of healthy cells, and were tested for their ability to prevent the appearance of tumors or to reduce tumor size, for which said compositions are an excellent alternative for treatment. of neoplasms in pancreatic tissue.
  • compositions described herein can be used as antineoplastic or anticancer to treat mammals, including humans; Said compositions are effective and safe at the appropriate doses, which can be calculated by specialists in the field of the invention and for each individual requirement, as well as the routes of administration and the appropriate formulations that allow reaching the target organ and tissue, based on the principles provided by the present invention.
  • One of the modalities of the invention refers to a method for treating pancreatic cancer using the compositions that comprise the combination of compounds C14 and P8, including the elimination/reduction of tumors caused by said condition.
  • Another modality of the invention refers to the method for treating pancreatic cancer in a specific way using compositions that comprise the combination of compounds C14 and P8, without said compositions causing adverse reactions by only affecting cancerous tissue and not affecting healthy tissue.
  • compositions that comprise the combination of compounds C14 and P8, without said compositions causing adverse reactions by only affecting cancerous tissue and not affecting healthy tissue.
  • analogues to compound C14 were identified.
  • crystallographic KRas4B/PDE6 ⁇ complex and performing Molecular Dynamics tests we identified the interaction energy of the compounds.
  • Cell viability and type of death were evaluated using flow cytometry, as well as clonogenic assay.
  • RAS-GTP Pulldown and Western blot assays were performed to measure K-Ras activation and its signaling pathways.
  • MIA PaCa-2 cells were implanted in subcutaneous and orthotopic xenograft models in Nu/Nu mice and the PDX model was made using primary cultures of pancreatic cancer to be treated with compounds C14, P8 and the combination C14/P8.
  • the analog called P8 presented higher interaction energy on the in silico mutated complexes, thus presenting greater cytotoxic effects in cell lines and primary cultures of pancreatic cancer with mutated K-Ras without damaging the cell line and non-cancerous primary cultures.
  • both compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways, however the compound P8 presents an IC504 times lower than the compound C14, being up to now the best compound found in our research group. .
  • the evaluation of the compound C14 and its analogue P8 using preclinical models of pancreatic cancer approved by the FDA is reported.
  • the results showed that the Compound C14 and P8 have a greater specific cytotoxic activity than compounds D14 and C22 previously reported by our work group.
  • the combination of the compounds C14 and P8 included in the compositions of the present invention has a synergistic effect, both in cell lines and in primary cultures and in murine models.
  • the compounds C14 and P8 do not present side effects like Gemcitabine, therefore they can be considered as new chemotherapeutic agents.
  • Compound C14 presents higher interaction energy and greater decrease in cell viability in pancreatic cancer cell lines with mutated K-Ras4B.
  • C14 is a small organic molecule with a molecular weight of 344.8 g/ mole (Table 1). Table 1. Interactions of compound C14 and P8 on the K-Ras4B/PDE6 ⁇ complex. Results obtained from the virtual selection analysis in the crystallographic heterodimeric complex. To analyze the interactions and coupling energies of the C14 compound on the heterocomplex, we made modifications to obtain the K-Ras4B complexes. w.t.
  • Cell viability was determined by measuring the ATP concentrations in the cell after three days of treatment with C14 at different concentrations (5, 10, 30, 50, 100, 150 and 200 ⁇ M), where the cell lines MIA PaCa-2 and PANC -1 presented sensitivity to dose-dependent treatment, obtaining an IC50 of 90.18 ⁇ M for MIA PaCa-2, 103.5 ⁇ M for PANC-1 and 171.4 ⁇ M in the normal hTERT-HPNE cell line. ( Figure 1E–F). These results suggest that C14 has strong specific activity in cell lines with K-Ras4B mutations. The determination of the type of cell death produced by the C14 compound is very important for its subsequent approval for cancer treatment.
  • the identification of analogues of the C14 compound yields a compound with higher chemical properties.
  • the identification and selection of organic molecules analogous to the leader compound C14 was carried out using a database from the ENAMINE chemical library, where 335 analogues of compound C14 were analyzed.
  • MOE Molecular Operating Environment
  • the 20 analogues to the leader compound retain the chromene group and the acetamide group, where the modifications made with respect to the structure of the leader compound were with the addition of amino groups, benzenes, pyridines and non-aromatic heterocycles, with the purpose of increasing the interactions. with the molecular complex K-Ras4B/PDE6 ⁇ .
  • Table 3 Analogs to the leader compound C14 selected by means of bioinformatics programs. For the selection of the analogues of the C14 leader compound with the greatest cytotoxic effect on the MIA PaCa-2 cell line, the C14 analogues were evaluated using the IC50 of the C14 compound as a reference, which is 90.18 ⁇ M at 24, 48 and 72 hours later.
  • MIA PaCa-2 and hTERT-HPNE cell lines were cultured in 96-well plates, which were treated with the 20 analogues at a concentration of 90.18 ⁇ M for 24, 48 and 72 hours to select for the compound(s). which has(have) a greater cytotoxic effect on the cell line MIA PaCa-2 and hTERT-HPNE used as control ( Figure 2).
  • the results obtained ( Figure 2) with the first five analogues of compound C14 show a decrease in the viability of the hTERT-HPNE control cell line, but not in the Mia-PaCa 2 cell line, where the same result is observed in compounds P11 to P20 ( Figure 3).
  • compound P8 ( Figure 1G-H) has a greater effect on cell viability in MIA PaCa-2.
  • a significant decrease in cell viability was obtained at a concentration of 90.18 ⁇ M of compound P8 ( Figure 1G-H), in addition to showing a decrease in viability with the presence of the compound.
  • leader C14 despite the fact that these compounds show 80% similarity with the pharmacophore, being that 95% of the analogues do not retain the cytotoxic effect on the cell line
  • the P8 analogue presents higher interaction energy and greater cytotoxic effect on cell lines with mutated K-Ras4B, four times greater than the leader compound C14.
  • Cell viability was determined by measuring the ATP concentrations in the cell lines after three days of treatment with the leader compound and its analogue at different concentrations (5, 10, 30, 50, 100, 150 and 200 ⁇ M), where the analogue Called P8, it presented a greater cytotoxic effect than its leading compound, presenting an IC50 of 51.18 ⁇ M in PANC-1, 24.18 ⁇ M in MIA PaCa-2 and 28.96 ⁇ M in Capan-1, 2 to 4 times lower than compound C14, being that these IC50 concentrations do not affect the viability of the normal hTERT-HPNE cell line with an IC50 of 103.45 ⁇ M.
  • the determination of the type of cell death produced by the compounds C14 and P8 was determined by flow cytometry, observing that the compound P8 promotes cell death by apoptosis in cell lines MIA PaCa-2, PANC-1 and Capan-1 with a greater sample percentage of cells than compound C14 at lower IC50 concentrations without causing damage to hTER-HPNE normal pancreatic duct cells.
  • Compound P8 has been shown to be a better compound than its parent compound, inducing cytotoxicity, clonogenic decline, and inducing apoptosis with concentrations 2 to 4 times lower than the parent compound.
  • Compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways in PDAC cell lines depending on their oncogenic addiction.
  • pancreatic cancer cell lines with different oncogenic addiction Obtaining and characterization of primary culture. To obtain pancreatic cancer samples from patients, the Department of Genomic Medicine and the Department of oncology and general surgery of the Hospital 1o. October ISSSTE in Mexico City, following the provisions of the national project 002.2015. Pancreatic cancer samples were collected in the operating room and transported to the Laboratory for processing.
  • pancreatic cancer Nineteen samples of pancreatic cancer were obtained from patients between 40 and 100 years of age, with a higher frequency of 40-59 years and with a higher incidence in women, where said information contradicts what was previously described in the sense that the higher incidence of this type of cancer is reported in men aged 60 to 80 years.
  • 2 epithelial tissue samples were obtained from healthy patients PBDD33 and JGCD28 as a control for our study. Once the samples were obtained, they were processed and obtained 3 primary cultures of pancreatic cancer MGKRAS003, MGKRAS004 and MGKRAS005, and 2 primary cultures derived from epithelial tissue, performing several passages until obtaining passages 5.
  • markers of pancreatic origin Ck7 and Ck19 markers of pancreatic origin Ck7 and Ck19 and the Malignancy markers most used in several countries such as CEA, MUC1, MUC4, MUC16, EFGR, VIMENTIN, cytoplasmic B-Catenin and E-Caterin and Ki-67 (Table 4).
  • pancreatic cancer 8 ( Figure 7C), obtaining as a result MGKRAS003 WT, MGKRAS004 G12V and MGKRAS005 G12C, where the two mutations found are the second. and the third most frequently worldwide;
  • the G12V mutation represents the mutation with the highest chemoresistance that has been reported in pancreatic ductal adenocarcinoma.
  • Primary cultures of pancreatic cancer show greater sensitivity to compounds C14 and P8 than to conventional therapy.
  • the determination of the type of cell death produced by the compounds C14 and P8 on the primary cultures was determined by flow cytometry, where this determination showed that the compound P8 promotes cell death by apoptosis in cell lines MGKRAS003, MGKRAS004 and MGKRAS005, with a higher percentage cell sampling than compound C14 at lower IC50 concentrations without causing damage to non-cancerous primary cultures PBDD33 and JGCD28.
  • Compound P8 has been shown to be a better compound than its lead compound C14 inducing cytotoxicity, clonogenic downregulation and inducing apoptosis with concentrations 2-fold lower than the lead compound on primary cultures of pancreatic cancer.
  • compound P8 decreased ERK activation from 50 to 80% in MGKRAS003, MGKRAS004, and MGKRAS005, indicating that compounds C14 and P8 are directly affecting the K-Ras signaling pathway in primary pancreatic cancer cultures. .
  • Compounds C14 and P8 exhibit synergistic effects in pancreatic cancer cell lines and primary cultures.
  • mice were used, to which 30.60 mg/kg of C14 and P8 were administered intraperitoneally, the combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg each, 1:1 w/w ratio), Gemcitabine 40mg/kg, vehicles (0.05% carboxymethyl cellulose in PBS with 0.5% DMSO) and na ⁇ ve mice as controls, being administered one dose for 24h (figure 11 D-G) and once a day for 15 days to complete the treatment scheme (figure 11 H-K).
  • mice After 24 hours of treatment, the mice were sacrificed and the urine was collected to evaluate protein, pH, bilirubin and glucose, obtaining an increase of 300 mg/dl of protein, 70 mg/dl of bilirubin and 250 mg/dl of glucose in urine.
  • gemcitabine-treated mice none of the variations of the above parameters were detected in the mice treated with the compounds C14, P8 and with the combination C14/P8, indicating that our compounds did not present side effects after the first 24 hours of treatment.
  • the treatment was extended for 15 days, observing during the treatment several side effects in the BALB/c mice treated with Gemcitabine (Table 5), such as diarrhea, rectal prolapse, intestinal torsion syndrome, decreased muscle mass.
  • mice treated with Gemcitabine had to be sacrificed, while the mice treated with C14, P8 and the C14/P8 combination did not present any of the aforementioned symptoms. Table 5. Side effects obtained in BALB/c mice treated with Gemcitabine, C14, P8 and C14/P8. N.Normal; N.D.
  • FIG. 13 shows the representative images of the different concentrations of C14, P8, Gemcitabine and Vehicle, observing in brown the positive immunoreaction in different markers, where the immunoreaction of Ck-19, CA-125 and Ki-67 decreases as the concentrations of compounds C14 and P8 increase; The decrease in these markers is more evident when using 30 mg/kg of C14 and P8, where more than 70% of the proliferating ductal neoplastic cells are decreased, however, with 60 mg/kg of P8, more than 90% of the cells are decreased.
  • neoplastic ducts (figure 13 B-D).
  • the combination of compounds C14 and P8 decreases tumor growth in PDX models.
  • the primary cultures MGKRAS004 and MGKRAS005 were used since they present the G12V and G12C mutations, which represent the third and second most frequent in pancreatic cancer. with greater chemoresistance ( Figure 14).
  • mice were measured, obtaining a decrease of more than 20% of the weight in the mice treated with Gemcitabine.
  • MGKRAS004 we decided to evaluate the antineoplastic effect in the PDX model using the primary culture MGKRAS005 which presents the G12C mutation.
  • Deltarasin interacts with PDE6 ⁇ , it is impossible for it to recognize the farnesyl present at the carboxyl end of K-Ras4B, thus preventing its transport to the plasma membrane, where it performs its function by allowing the activation of the different cell signaling pathways. related to oncogenic processes.
  • one of the disadvantages of Deltarasin is that it can inhibit a wide variety of cell signaling pathways since Kras4B is not its only target for transport by PDE6 ⁇ [20].
  • the increase in the affinity of the P8 compound is given by the presence of a piperazine, which has two amino groups, increasing the interaction sites with the heterodimeric complex, making it more stable when interacting with the WT and mutated K-Ras4B/PDE6 complexes. ⁇ .
  • the presence of piperazine in compound P8 provides greater solubility, presenting a partition coefficient of 3.99 and a solubility constant of -4.4, on the other hand, compound C14, which does not present piperazine in its structure, has a coefficient partition of 3.63 and a solubility coefficient of -4.4.
  • the increase in the partition coefficient of P8 with respect to C14 makes it even more soluble and permeable upon contact with the plasma membrane.
  • One of the pending tests to be carried out in the present invention is obtaining the affinity values of the compound C14 and P8 by means of biochemical methods such as BIACOR or by means of microcalorimetric techniques, which will allow us to confirm and obtain real quantitative data, by same as mass spectrometry to identify all possible targets of compounds C14 and P8.
  • the cytotoxic effects obtained by compounds C14 and P8 on pancreatic cancer cell lines suggest that depending on the mutation present in K-Ras4B, the cells present different cell permeability and therefore present different IC50, allowing us to obtain greater cytotoxic effects in cell lines. with the G12C and G12V mutations which present greater chemoresistance in pancreatic cancer, this without affecting the non-cancerous cell line.
  • pancreatic ductal adenocarcinoma is characterized by the presence of activating mutations in K-Ras4B;
  • the compounds C14 and P8 and their combinations are capable of decreasing the activation of the oncoprotein in pancreatic cancer cell lines without affecting the non-cancerous cell line and therefore decrease the signaling pathways depending on the oncogenic addiction. that each cell line presents towards K-Ras4B.
  • Deltarasin decreased the activation of K-Ras4B in the normal cell line, implying the reduction of its signaling pathways, making clear the non-specificity that this compound presents.
  • One of the trials considered to verify the antineoplastic effects of compound P8 and compound C14 is the performance of tumorigenesis trials in in vivo models, where one of the results encouraged us to perform these trials in reducing the clonogenic capacity of the 80% using compound C14 and 96% with compound P8 in cell lines and primary cultures of pancreatic cancer, this being more evident with the combination of C14 and P8 where the clonogenic capacity decreased by more than 99% in both cases.
  • the FDA-approved preclinical models for the evaluation of drugs with possible chemotherapeutic effects are subcutaneous xenograft, orthotopic xenograft, and patient-derived xenograft, with which the influence of the niche and cellular heterogeneity present in each of the models can be observed [ 38].
  • the antineoplastic activity of compounds C14 and P8 decreased tumor growth in Subcutaneous and Orthotopic xenograft models as the dose increased, without inducing adverse effects or genotoxicity (as if presented by first-line chemotherapy with Gemcitabine), they decrease the K-Ras4B activation and malignancy markers decrease in remnant tumors.
  • compounds C14 and P8 decrease tumor growth in PDx models of pancreatic cancer, while the combination of C14/P8 showed better antineoplastic effects in Subcutaneous Xenograft, Orthotopic and PDx models, further decreasing tumor growth without inducing it. side effects such as those presented by Gemcitabine.
  • the top 30 London dG score results were further refined using energy minimization with the MMFF94x force field and re-scored using the Affinity dG score.
  • Example 2. Simulation of molecular dynamics. MD simulation of the protein-ligand complex was performed using the AMBER 16 package [21] and the ff14SB forcefield [22]. Ligand charges for unparameterized residues in proteins were determined using the AM1-BCC level and the Amber General Force Field (GAFF) [23] for the protein-ligand complex, a 15 ⁇ rectangular-shaped box of the ligand model.
  • GFF Amber General Force Field
  • TIP3P water [24] was applied to solvate the complex; and the Cl- and Na ions + for the protein-ligand system were placed in the model to neutralize any positive or negative charges around the complex at pH 7.
  • the system Prior to MD simulation, the system was minimized by 3000 steepest descent minimization steps followed by 3000 minimization steps. of the conjugate gradient. Then, the system was heated from 0 to 310 K for 500 picoseconds (ps) of MD with position constraints under an NVT ensemble, successively an isothermal isobaric ensemble (NPT) of MD was carried out for 500 ps to adjust the density of the system. solvent followed by 600 ps constant pressure equilibrium at 310K using the SHAKE algorithm [25] on hydrogen atoms and Langevin dynamics for temperature control.
  • the equilibrium run was followed by a 100 ns long MD simulation without position constraints under periodic boundary conditions using a 310K NPT assembly.
  • the particle mesh Ewald method was used to describe the electrostatic term [26], and a 10 ⁇ limit was used for van der Waals interactions.
  • Temperature and pressure were conserved using the weak coupling algorithm [27] with coupling constants ⁇ T and ⁇ P of 1.0 and 0.2 ps, respectively (310 K, 1 atm).
  • the MD simulation time was set to 2.0 femtoseconds and the SHAKE algorithm [25] was used to constrain the bond lengths to their equilibrium values. Coordinates were saved for analysis every 50 ps.
  • AmberTools14 was used to examine the time dependence of root mean square deviation (RMSD), radius of gyration (RG), and clustering analysis to identify the most populous conformations during equilibrated simulation time.
  • RMSD root mean square deviation
  • RG radius of gyration
  • Example 3 Calculation of free bond energies. The calculation of the binding free energies was carried out using the MMGBSA approach [28-30] provided in the AMber16 suite [21]. 500 snapshots at 100 ps time intervals were chosen from the last 50 ns of MD simulation using a 0.1 M concentration and the Generalized Born (GB) Implicit Solvent Model [31].
  • the binding free energy of the protein-ligand system was determined as follows: where ⁇ EMM represents the total energy of the molecular mechanical force field that includes the electrostatic ( ⁇ Eele) and van der Waals ( ⁇ Evdw) interaction energies.
  • ⁇ G solvation is the free energy rate of desolvation upon complex formation estimated from the implicit GB model and solvent accessible surface area (SASA) calculations yielding ⁇ Gele.sol and ⁇ Gnpol.sol.
  • T ⁇ S is the solute entropy that arises from the structural changes that occur in the degrees of freedom of free solutes in forming the protein-ligand complex.
  • Example 5 Cell culture.
  • Example 6. Cell viability.
  • Pancreatic cancer cell lines were seeded in 6-well plates at a density of 300 cells per well and cultured overnight.
  • Cell lines and primary cultures PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004 and MGKRAS005 were treated with final concentrations of 0.496 ⁇ M gemcitabine (PiSA Laboratories, Mexico), the IC50 concentration of C14 and P8 for 72 h. , and Deltarasin 5 ⁇ M. Subsequently, the medium was replaced with fresh medium supplemented every third day for a total of 10 days. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min and washed with distilled water.
  • PFA paraformaldehyde
  • Example 8 Apoptosis assay. Approximately 5 x 10 were planted 5 cells in 6-well plates for 24 h. Cells were then treated with an IC50 concentration of C14 and P8 and vehicle for 24 h. Cells were harvested with 0.25% trypsin, washed with phosphate buffered saline (PBS) and collected together by centrifugation.
  • PBS phosphate buffered saline
  • Apoptosis was determined using the Apoptosis/Necrosis Detection Kit (Abcam, catalog number ab176749, Cambridge, England) according to the manufacturer's instructions and analyzed by flow cytometer on a FACSCalibur instrument (BD Biosciences) followed by a data analysis using FlowJo software (Tree Star Inc). All experiments were performed in triplicate.
  • hTERT-HPNE, PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004, and MGKRAS005 cells were cultured to have 3x10 6 cells, and lysed in ice cold lysis buffer (400 ⁇ L) supplemented with cOmpleteTM Ultra Protease Inhibitor Cocktail without EDTA and 1xPhosSTOPTM (Sigma-Aldrich). The lysates were centrifuged and the protein (300 ⁇ g) was collected. Lysates were incubated by end-to-end rotation with 100 ⁇ g Raf-RBD-conjugated beads for 1 h.
  • Example 10 Western blot.
  • Cell lines were serum starved for 16 hours and pretreated with an IC50 concentration of C14, P8, gemcitabine and deltarasin for 3 hours after pretreatment. Cells were stimulated with epidermal growth factor at 100 ng/ml for 10 min.
  • Protein extracts were forced 10 times through a 22-gauge needle and centrifuged for 10 min at 14,000 rpm at 4°C, and protein concentration was determined using the PierceTM BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Tissue samples were weighed, quick frozen, and ground in liquid nitrogen in a mortar and pestle. Samples were transferred to a microfuge tube and lysed using ProteoJETTM Mammalian Cell Lysis Reagent, followed by centrifugation at 2,000 x g for 15 min and protein quantification. SDS-PAGE was carried out using 30 ⁇ g of protein from each sample. Proteins were transferred to PVDF membranes (Merck Millipore) and blocked for 1 h.
  • Nu/Nu immunodeficient male nude mice were maintained at 6 weeks of age (CINVESTAV, Mexico) under pathogen-free conditions on irradiated chow. Animals were injected subcutaneously in the torso with 5x10 6 MIA PaCa-2 cells per tumor in 0.2 ml of high glucose DMEM matrigel medium.
  • mice were sacrificed in a CO 2 chamber and the xenograft tumors were excised, fixed in 4% buffered formalin and embedded in paraffin. The tumors were cut with a microtome obtaining 2 ⁇ m sections.
  • hematoxylin and eosin (H&E) staining tissues were deparaffinized in xylene, hydrated in dry alcohol starting from absolute ethanol to distilled water, stained for 2 min with Harris hematoxylin, destained with 0.5% acid alcohol and were fixed for color in lithium carbonate for 1 min, washed in distilled water, in 96% ethanol and stained with Sigma Eosin, washed and dehydrated in gradual changes of alcohol until reaching absolute alcohol, allowed to dry at room temperature, mounted and observed, to identify the site of the lesion.
  • H&E hematoxylin and eosin
  • the tissues were deparaffinized in xylene, hydrated in depleted alcohols starting from absolute ethanol to distilled water, epitopes were unmasked with 10 mM Citrate Buffer pH 6.03 in the Tender Cocker for subsequent washing with PBS pH 7.4; endogenous peroxidase was blocked with H 2 EITHER 2 0.9% for 15 min, blocked with 3% BSA for 1 h, while Ki-67 (BIOCARE MEDICAL API 3156 AA), CK 19 (GENETEX GTX110414) and CA125 (BIOCARE MEDICAL CM 101 CK) antibodies were diluted with 1% PBS and 1% BSA, where the primary antibody was incubated at room temperature for 40 min, washed with PBS for 3 min, incubated with the biotinylated secondary antibody for 20 min at room temperature, washed with PBS for 3 min, incubated with streptavidin for 15 min, and washed with PBS for 3 min.
  • Example 13 Primary cultures of pancreatic cancer from patients with PDAC.
  • the pancreatic cancer tissues were provided by the 1st Regional Hospital. of October of the Institute of Security and Social Services for State Workers (ISSSTE) in the framework of project 002.2015 in Mexico City. Tissues were collected in the operating room of said hospital, placed in transport medium (DMEM base medium without fetal bovine serum and 5% antibiotic), always keeping the medium at 4°C.
  • transport medium DMEM base medium without fetal bovine serum and 5% antibiotic
  • Tissue was sectioned until 3mm3 fragments were obtained, which were placed in 6-well plates with DMEM medium high in 20% glucose, 80% fetal bovine serum, and 3% antibiotic, this until tumor cells adhered to the plate. . The percentage of serum was decreased until the cells could survive with 10% serum and 1% antibiotic.
  • Example 14 Patient-derived subcutaneous xenograft model. Nu/Nu immunodeficient male nude mice were maintained at 6 weeks of age (CINVESTAV, Mexico) under pathogen-free conditions on irradiated chow. Animals were injected subcutaneously in the torso with 5x10 6 primary culture MGKRAS004 and MGKRAS005 cells per tumor in 0.2 ml of high glucose DMEM matrigel medium.
  • Example 15 Model of orthotopic xenograft in Nu/Nu mice.
  • Nu/Nu immunodeficient male nude mice were maintained at 6 weeks of age (CINVESTAV, Mexico) under pathogen-free conditions on irradiated chow. Mice were anesthetized and sedated with xylazine and ketamine. The mouse spleen was located on the left side, subsequently a 0.5 cm incision was made in the skin and peritoneum, the spleen was removed, allowing visualization of MIA PaCa-2 cells after 1 million cells were inoculated. in 50 ⁇ l of serum-free minimal essential medium without phenol red directly into the pancreas. Organs were relocated within the mouse and peritoneum, and the skin was sutured with self-absorbing suture.
  • Example 16 Cellular immunofluorescence. Cells were grown on coverslips in 24-well plates to desired confluence, fixed with paraformaldehyde for 20 min at 37°C, then washed with 1X PBS and permeabilized with 1:1 methanol/acetone or 0.2 X100 triton.
  • Tissues were deparaffinized in xylene, hydrated in degraded alcohols starting from absolute ethanol to distilled water, epitopes unmasked with 10 mM Citrate Buffer pH 6.03 in Tender Cocker, washed with PBS pH 7.4, endogenous peroxidase blocked with 0.9% H2O2 (decreases erythrocyte autofluorescence) for 5 min, autofluorescence was reduced with 0.05M NH4Cl for 30 min at 37°C and washed with PBST three times; the primary antibody was diluted with 1% PBS and 1% BSA, in this way the non-specific binding site was blocked, while the primary antibody (Sup M 1) was incubated at room temperature for 60 min, to subsequently perform washes with PBS for 3 min, incubate with fluorocorm-labeled secondary antibody for 40 min at room temperature, and wash with PBS for 3 min.
  • Genomic DNA from human samples diagnosed with pancreatic cancer (MGKRAS-003 to MGKRAS-005) was extracted from frozen tissue with the GenElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich G1N70).
  • GenElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich G1N70).
  • PCR and sequencing PCR was performed with approximately 60 ng of hybridized DNA using the following sense and antisense primers at a concentration of 10 pmol: Sense: RASO15'-AAGGCCTGCTGAAAATGAC-3', Antisense: RASA25'-TGGTCCTGCACCAGTAATATG-3.
  • PCR was performed in a TC-512 TECHNE thermal cycler with 20 cycles of endpoint PCR (65°C initial run temperature, decreasing 0.5°C per cycle) and 15 cycles at 55°C run temperature. PCR products were purified using the QIAGEN QIAprep Miniprep Kit. Purified PCR products were sequenced in the reverse direction. Example 20. Genotoxicity test. For the evaluation of the genotoxic effect of C14 and P8, the micronucleus assay with bone marrow cells was carried out according to the method described above.
  • Test compounds were administered intraperitoneally once, as a solution (at a concentration of 40 mg/kg gemcitabine (n: 5), 60 mg/kg C14 and P8 (n: 5) and a combination of 30 mg /kg of C14 + 30 mg/kg of P8 (n: 5) and vehicle (n: 5)) and using naive mice as control (n: 5).
  • Bone marrow cells were obtained 24 h and 15 days after treatment and stained with Giemsa-Wright (Diff-Quick; Harleco; Gibbstown, NJ).
  • HE Two thousand polychromatic erythrocytes per animal were counted using a light microscope at 100x magnification to determine the number of micronucleated polychromatic erythrocytes.
  • Example 21 Example 21.

Abstract

The present invention describes synergic compositions useful for the treatment of pancreatic cancer comprising combination of the compounds: Formula I, Formula II or pharmaceutically active salts thereof, which induce cytotoxicity in primary pancreatic cancer cell lines and cultures greater than that obtained for the compounds evaluated individually, thus making same a viable, effective and useful alternative for the treatment of pancreatic cancer. The antineoplastic evaluation of the compositions described showed the specificity thereof for primary pancreatic cancer cell lines and cultures, without affecting primary non-cancerous cell lines and cultures, decreasing the activation of K-Ras4B and the signalling pathways for same, whereby said antineoplastic activities improved with the synergic antineoplastic effect of the combinations of compounds C14 and P8, thereby almost completely erradicating the presence of pancreatic cancer tumours in preclinical models without exhibiting adverse effects or genotoxicity. The synergic compositions of the invention are novel pharmacological alternatives against pancreatic cancer with better properties than conventional chemotherapy.

Description

Composiciones sinérgicas de compuestos contra el complejo heterodimérico K-Ras4B/ PDE6δ para el tratamiento de cáncer de páncreas Campo de la invención. La presente invención se relaciona con composiciones con actividad farmacéutica que resultan útiles para el tratamiento de enfermedades, particularmente con composiciones farmacéuticas para el tratamiento de cáncer de páncreas, más particularmente con composiciones farmacéuticas que exhiben efectos sinérgicos para disminuir el crecimiento tumoral, sin generar efectos adversos y que comprenden compuestos contra el complejo heterodimérico K-Ras4B/PDE6δ, disminuyendo la activación de K-Ras4B. Antecedentes de la invención. El adenocarcinoma ductal de páncreas (PDAC) tiene el peor pronóstico entre todos los cánceres humanos, debido a su alta tasa de diseminación y resistencia a la quimioterapia. La supervivencia de los paciente con PDAC es de aproximadamente 3 a 6 meses en más del 95% de los casos [1-3]. Aproximadamente el 99% de los casos de PDAC presentan mutaciones activadoras en el oncogén K-RAS, donde estas mutaciones correlacionan con la adicción oncogénica que presenta PDAC para su supervivencia, antiapoptosis y quimiorresistencia, es por esto que dichas mutaciones activadores se consideran como el factor más importante para la progresión y mantenimiento del adenocarcinoma ductal pancreático [4-6]. Las mutaciones activadoras en K-Ras4B consisten principalmente de sustituciones en los residuos de aminoácidos G12, G13 y Q61 de dicha proteína, las cuales alteran la hidrólisis intrínseca de GTP mediada por proteínas GAP. Estas mutaciones inducen la activación aberrante de K-Ras4B, lo que da como resultado una activación continua de las vías de señalización dependientes de K-Ras4B como AKT y ERK [5, 7, 8]. Actualmente en México el tratamiento de primera línea para PDAC son los inhibidores de la síntesis de DNA como Gemcitabina, 5-FU y Oxaliplatino los cuales generan efectos secundarios en los pacientes, tales como elevación de enzimas hepáticas, leucopenia, neutropenia, colapso venoso, dolor y pérdida de masa ósea [9]. Después de los hallazgos sobre la presencia de las mutaciones en K-Ras4B y su importancia en la formación, mantenimiento y progresión de las neoplasias más mortales como lo es el PDAC [10, 11], se han realizado estudios para descubrir y desarrollar inhibidores farmacológicos contra la activación de K-Ras4B oncogénico [10, 11]. La búsqueda de nuevas alternativas farmacológicas dirigidas contra K-Ras4B para disminuir su activación y así mejorar la calidad de vida de los pacientes con PDAC ha llevado unos 40 años, donde se han probado anticuerpos monoclonales como Ramucirumab [12], Matuzumab [13], Trastuzumab [14] y Figitumumab [15] los cuales están dirigidos a los receptores tirosina-cinasa transmembranales para disminuir la activación de K-Ras4B, presentando del 9 al 40% de inhibición en la activación de K- Ras4B en el adenocarcinoma ductal pancreático [12]. Con el fin de encontrar un compuesto orgánico que fuese capaz de inhibir la activación de K-Ras4B, se realizaron estudios para dirigir una molécula específica hacia el sitio de localización de la mutación de K-Ras4BG12C, la cual es la más frecuente en cáncer de pulmón [16]. Uno de los compuestos estudiados fue el denominado SCH-54292 [17], el cual es capaz de unirse a las hélices α2 y α3 de K-Ras4B, donde este compuesto presenta actividad solo en las líneas celulares que presentan K-Ras4BG12C; con este hallazgo, los investigadores proponen identificar y estudiar los análogos de SCH-54292 que presenten un efecto citotóxico mayor en las líneas celulares cancerosas [17]. Otro grupo de investigadores, creó un análogo de GDP denominado SML-8-73-1, el cual podía unirse covalentemente a la cisteína de K-Ras4BG12C, esto sin tener en cuenta la afinidad de GDP con su sitio de unión en K-Ras4B [16]; en el 2020 se logró obtener un compuesto dirigido al sitio de mutación de K-Ras4B él cual fue denominado como Sotorasib, presentando más del 70% en la inactivación de K-Ras4B en líneas celulares y en modelos murinos, con estos resultados el Sotorasib se encuentra en fase clínica-I [18, 19]. Se han identificado compuestos capaces de bloquear el transporte de K-Ras4B a la membrana plasmática como Deltarasin, el cual interactúa con PDE6δ con una Kd de 38 nM, e impide el reconocimiento de la modificación pos-traduccional presente en K-Ras4B, la cual concentraría a K-Ras4B en el citosol, evitando así su activación y la progresión tumoral; este compuesto fue denominado como la primera generación de inhibidores de PDE6δ [20]. Sin embargo este compuesto fue evaluado en líneas celulares no cancerosas de ducto pancreático y se observó una alta citotoxicidad, afectando considerablemente la viabilidad celular a bajas concentraciones [21-26]. En el año 2016 se reportó el análogo del compuesto Deltarasin el cual fue llamado Deltazinona, presentando una constante de disociación de Kd 38 nM a Kd 4 nM, mostrando ser un compuesto con mejor energía de interacción que la primera generación. Deltazinona mostró efectos citotóxicos sobre las líneas celulares de cáncer pancrático a una concentración de 24 µM, sin embargo tardó alrededor de 8 h para tener un afecto anti-proliferativo en las líneas celulares de cáncer pancreático, en cambio Deltarasin a una concentración de 5 µM en una hora mostraba el mismo efecto que su análogo, por lo que considerando estos datos, la primera generación de inhibidores de PDE6δ presentan mayor efecto que la segunda generación [27, 28]. En el 2017 se reportaron otros análogos al Deltarasin denominados como Deltasonamidas, los cuales presentan mayor energía de interacción que la primera generación, mayores efectos citotóxicos en líneas celulares de cáncer pancreático a concentraciones de 1 a 12 µM [29, 30]. A principios del 2020 surgen nuevos análogos denominados como Deltaflexin, sin embargo a pesar de los esfuerzos en buscar análogos con mayores propiedades que Deltarasin, estos no han presentado los mismos efectos citotóxicos que la primera generación de inhibidores de PDE6δ [31]. El transporte de la proteína K-Ras4B está mediado por la proteína PDE6δ desde el retículo endoplásmico a la membrana plasmática para su posterior activación, formando así el complejo heterodimérico K-Ras4B/PDE6δ en el citoplasma. [23, 32, 33]. Este mecanismo de transporte representa una gran oportunidad para dirigir compuestos que pueden estabilizar el complejo heterodimérico en el citoplasma y así prevenir la activación de K-Ras4B en la membrana plasmática [23, 32, 33]. Se creía que K-Ras4B/PDE6δ se transportaba como un dímero y ahora se sabe qué forma un grupo de 6-12 proteínas o 3-6 dímeros [32]. Debido a esto, nuestro grupo de trabajo buscó un modelo del heterodímero utilizando el cristal del complejo heterodimérico en un grupo de 6 proteínas, obteniendo un dímero representativo del heterocomplejo multiproteico de K-Ras4B/PDE6δ, encontrando dos compuestos denominados D14 y C22 que se unen y estabilizan al complejo heterodimérico K-Ras4B/PDE6δ [23]. La evaluación in vitro e in vivo de los compuestos D14 y C22 demostró un efecto citotóxico específico en líneas celulares de cáncer pancreático con mutaciones en K-RAS, disminuyendo la viabilidad celular, induciendo muerte por apoptosis y disminuyendo la activación de K-Ras y sus vías de señalización como AKT y ERK en un 50% [21]. Por otro lado, estos compuestos redujeron el crecimiento tumoral en un 50% en comparación con el vehículo, conforme las dosis aumentaban de D14 y C22 [21]. Analizando nuestros resultados anteriores decidimos evaluar a un compuesto de los 38 compuestos evaluados en nuestro primer estudio de estabilización del complejo heterodimérico K-Ras4B/PDE6δ [21]. El compuesto C14 presentó mayor actividad citotóxica a 200 µM que los compuestos D14 y C22, se ha demostrado que al realizar modificaciones sutiles en la estructura primaria de los compuestos líder se pueden mejorar las propiedades químicas y su actividad biológica, para lo cual la búsqueda de análogo de C14 podrían presentar mayor actividad que su compuesto principal. Por lo anterior y en vista de las soluciones mencionadas no resultan del todo suficientes para un tratamiento que resulte efectivo para el cáncer de páncreas, sigue existiendo la necesidad de contar con mejores soluciones y que al mismo tiempo no generen efectos adversos en el paciente bajo tratamiento. Breve descripción de la invención. El adenocarcinoma ductal de páncreas (PDAC) tiene el pronóstico más desalentador entre todos los cánceres humanos, ya que es muy resistente a la quimioterapia. Esto conlleva a la búsqueda de nuevas alternativas farmacológicas para mejorar la calidad de vida de los pacientes con cáncer de páncreas. Se han diseñado compuestos que puedan inhibir la vía de señalización y transporte de la oncoproteína K-Ras4B. Teniendo en cuenta que la interacción de KRas4B con PDE6δ es fundamental para su transporte y posterior activación en la membrana plasmática, en la presente invención se identificó y evaluó en modelos preclínicos al compuesto C14 y su análogo P8, los cuales podrían estabilizar al complejo heterodimérico KRas4B/PDE6δ. El análogo denominado como P8 presenta mayor energía de interacción sobre los complejos mutados in silico, de este modo presenta mayores efectos citotóxicos en las líneas celulares y cultivos primarios de cáncer pancreático con K-Ras mutado sin dañar a la línea celular y cultivos primarios no cancerosos. De este mismo modo ambos compuestos C14 y P8 disminuyen la activación de K-Ras y sus vías de señalización, sin embargo el compuesto P8 presenta un IC504 veces menor que el compuesto C14, siendo hasta el momento el mejor compuesto encontrado en nuestro grupo de investigación. Por otro lado, composiciones que comprenden la combinación de los compuestos C14/P8 presentan efecto sinérgico, induciendo citotoxicidad en líneas celulares y cultivos primarios de cáncer pancreático mayores que los obtenidos por los compuestos evaluados individualmente. Las composiciones de la invención que comprenden la combinación de los compuestos C14 y P8 disminuyen del 90 al 95% el crecimiento tumoral en modelos de xenoinjerto Subcutáneo y Ortotópico a medida que la dosis se incrementa, no inducen efectos adversos ni genotoxicidad como lo presenta la quimioterapia de primera línea con Gemcitabina, disminuyen la activación de K-Ras4B y disminuyen los marcadores de malignidad en tumores remanentes. De este mismo modo las composiciones de la invención que comprenden la combinación de los compuestos C14 y P8 disminuyen hasta el 99% del crecimiento tumoral en modelos PDx de cáncer pancreático. Las composiciones de la invención mostraron mejores efectos antineoplásicos en los modelos de Xenoinjerto Subcutáneo, Ortotópico y PDx disminuyen hasta un 97% el crecimiento tumoral sin inducir efectos secundarios como los presentados por Gemcitabina. Los compuestos C14 y P8 son nuevos quimioterapéuticos con mejores propiedades que la quimioterapia convencional. Breve descripción de las figuras. Figura 1. C14 estabiliza el complejo K-Ras4B/PDEδ e inhibe el crecimiento de líneas celulares de cáncer de páncreas humano. Se observa A) interacción del compuesto C14 (rojo) con el complejo K-Ras4Bwt (violeta) PDE6δ (azul); B) interacción del compuesto C14 con el complejo K- Ras4BG12D/PBDE6δ; C) interacción del compuesto C14 con el complejo K-Ras4BG12C/PBDE6δ; D) imágenes representativas de campo claro de las líneas celulares ARPE-19, hTERT-HPNE, PANC- 1 y MIA PaCa-2 tratadas con C14100 µM, DMSO como vehículo y células de control sin tratar. Barra=20 µm; E) viabilidad celular relativa después del tratamiento de las células PDAC MIA PaCa- 2, PanC-1 y la línea celular pancreática normal hTERT-HPNE con diferentes concentraciones de C14 durante 72 h.; F) porcentaje de viabilidad de líneas celulares PDAC a las 72 h.; G-H) viabilidad celular relativa después del tratamiento de la línea celular PDAC MIA PaCa-2 (G) y la línea celular pancreática normal hTERT-HPNE (H) con diferentes compuestos que se sabe, se dirigen al complejo K-Ras4B/PBDE6δ (n=5). Figura 2. Evaluación e identificación de análogos del compuesto C14. Se observa A-B) evaluación de la viabilidad celular después del tratamiento con análogos P1, P2, P3, P4 y P5 de C14 a 90,18 µM en células MIA PaCa-2 (A) y hTERT-HPNE (B); C-D) evaluación de la viabilidad celular después del tratamiento con análogos P6, P7, P8, P9 y P10 de C14 a 90,18 µM en células MIA PaCa- 2 (C) y hTERT-HPNE (D); E-F) evaluación de la viabilidad celular después del tratamiento con análogos P11, P12, P13, P14 y P15 de C14 a 90,18 µM en MIA PaCa-2 (E) y hTERT-HPNE (F); G- H) evaluación de la viabilidad celular después del tratamiento con análogos P16, P17, P18, P19 y P20 de C14 a 90,18 µM en MIA PaCa-2 (G) y hTERT-HPNE (H). Figura 3. P8 estabiliza el complejo K-Ras4B/PDEδ e inhibe el crecimiento de líneas celulares PDAC mejor que C14. Se observa A) interacción del compuesto P8 (azul oscuro) con el complejo K- Ras4Bwt (rosa)/PDE6δ (azul); B) interacción de P8 con el complejo K-Ras4BG12D/PBDE6δ; C) interacción de P8 con el complejo K-Ras4BG12C/PBDE6δ; D) interacción de P8 con el complejo K- Ras4BG12V/PBDE6δ; E-H) viabilidad celular relativa de las células PDAC PANC-1, MIA PaCa-2 y Capan-1 y la línea celular pancreática normal hTERT-HPNE tratada con diferentes concentraciones de P8 durante 72 h (n=5); I-K) ensayos clonogénicos de las líneas celulares PDAC PANC-1, MIA PaCa-2 y Capan-1 tratadas con el IC50 de P8, C14, gemcitabina y deltarasina (n=5); L-M) análisis de muerte celular por citometría de flujo en células hTERT-HPNE, PANC-1, MIA PaCa-2 y Capan-1 tratadas con la IC50 de P8 y C14, DMSO como vehículo o medio solo después de la tinción con Anexina-V, 7- AAD y violeta de citocalceína; M) Cuantificación de las parcelas mostradas en L) n=5. Figura 4. P8 y C14 disminuyen la activación de K-Ras y la fosforilación de AKT y ERK en líneas celulares PDAC con mutación K-Ras. Se muestran A-D) imágenes de Western blot representativas de las líneas celulares A) hTERT-HPNE, B) PANC-1, C) MIA PaCa-2 y D) Capan-1 tratadas con la IC50 de P8, C14, Gemcitabina y Deltarasin para 3 h. Los extractos de proteínas totales se precipitaron usando perlas RAF-RBD. El RAS total (Ras-T) y GAPDH se muestran como controles de carga. Las intensidades de píxeles de K-Ras GTP se normalizaron a RAS y GAPDH totales; E-G) Western blot representativo de líneas celulares E) PANC-1, F) MIA PaCa-2 y G) Capan-1 tratadas con la IC50 de P8, C14, Gemcitabina y Deltarasin o vehículo, para AKT y ERK totales y fosforilados utilizando GAPDH como control de carga. La cuantificación de las intensidades de píxeles de pERK y pAKT en relación con los niveles totales de ERK y AKT respectivamente, se muestran en los gráficos de la derecha. Los datos se muestran como SDM; n=5, *** p<0.001. Figura 5. Caracterización de tejidos PDAC y células primarias. Se observa A) expresión de CK19 en tejidos PDAC MGKRAS003, MGKRAS004 y MGKRAS005 y células primarias mediante microscopía de inmunofluorescencia confocal (Leica SP8, Barcelona, España). Barra=50 µm; n=3; B) expresión de MUC1 en tejidos PDAC MGKRAS003, MGKRAS004 y MGKRAS005 y células primarias mediante microscopía de inmunofluorescencia confocal. Barra=50 µm; n=3; C) secuencias de nucleótidos e histogramas de secuenciación del exón 2 que contiene KRAS en tejidos y células primarias MGKRAS003, MGKRAS004 y MGKRAS005. Figura 6. Caracterización de cultivos de células primarias derivadas de la piel. Se observa A) expresión de IB-10 en cultivos primarios derivados de piel PBD033 y JGC028 mediante microscopía de inmunofluorescencia confocal (Leica SP8, Barcelona, España). Barra=50 µm; n=3; B-C) expresión de CD90, CD105, CD73, CD34, CD45 y HLADR en cultivos primarios derivados de piel PBD033 y JGC028 analizados por citometría de flujo después de la corrección de autofluorescencia. Figura 7. P8 y C14 inhiben el crecimiento e inducen la apoptosis en cultivos de células primarias PDAC. Se observa A-E) efectos de P8 y C14 a diversas concentraciones (5, 10, 30, 50, 100, 150 y 200 µM) durante 72 h en cultivos primarios no cancerosos PBD033 y JGC028, y los cultivos primarios de PDAC MGKRAS003, MGKRAS004 y MGKRAS005; F-H) ensayos clonogénicos de los cultivos primarios de PDAC, MGKRAS003, MGKRAS004 y MGKRAS005 tratados con el IC50 de P8, C14, Gemcitabina y Deltarasin; I-J) los análisis de muerte celular de PBD033, JGC028 MGKRAS003, MGKRAS004 y MGKRAS005 se determinaron mediante citometría de flujo después de la tinción con anexina-V, 7-AAD y violeta de citocalceína; J) cuantificación de los porcentajes mostrados en I). Los datos se muestran como SDM; n=5, p<0.001. Figura 8. Evaluación de la viabilidad de cultivos primarios tratados con Gemcitabina y Deltarasin. Se observa A) el efecto de la Gemcitabina sobre la viabilidad celular a diversas concentraciones (0-8000 nM) después del tratamiento durante 72 h en los cultivos primarios de PDAC, MGKRAS003, MGKRAS004 y MGKRAS005; B) efecto de la Deltarasin a diversas concentraciones (0-8000 nM) después del tratamiento durante 72 h sobre la viabilidad celular en los cultivos primarios de PDAC, MGKRAS003MGKRAS004 y MGKRAS005 (n=3). Figura 9. P8 y C14 disminuyen la activación de K-Ras y la fosforilación de AKT y ERK en cultivos de células primarias PDAC con mutación K-Ras. Se observan A-E) imágenes de Western blot representativas de células JGCD28 (A), PBDD33 (B), MGKRAS004 (C), MGKRAS003 (D) y MGKRAS005 (E) tratadas con el IC50 de P8, C14, Gemcitabina y Deltarasin durante 3 h. Los extractos de proteínas totales se precipitaron usando perlas RAF-RBD. El RAS total (Ras-T) y GAPDH se muestran como controles de carga. Las intensidades de píxeles de K-Ras GTP se normalizaron con respecto a los controles; F-H) Western blot representativo de las células MGKRAS003 (F), MGKRAS004 (G) y MGKRAS005 (H) tratadas con el IC50 de P8, C14, Gemcitabina y Deltarasin o vehículo, para AKT y ERK total y fosforilada utilizando GAPDH como control de carga. La cuantificación de las intensidades de píxeles de pERK y pAKT en relación con los niveles totales de ERK y AKT, respectivamente, se muestran en los gráficos de la derecha. Los datos se muestran como SDM; n=5; *** p<0.001. Figura 10. Efecto sinérgico de P8 y C14 en líneas celulares y cultivos primarios de PDAC. Se observa A) la interacción sinérgica de P8 (azul oscuro), C14 (rojo) con el complejo K-Ras4Bwt (rosa y amarillo)/PDE6δ (azul); B) interacción sinérgica de P8/C14 con el complejo K-Ras4BG12D/PBDE6δ; C) interacción sinérgica de P8/C14 con el complejo K-Ras4BG12C/PBDE6δ; D) interacción sinérgica de P8/C14 con el complejo K-Ras4BG12V/PBDE6δ; E) isobolograma de la CI50 de los compuestos P8 y C14; F-J) efectos sobre la viabilidad de los compuestos P8/C14 a varias concentraciones de cada uno (5, 10, 30, 50, 100, 150 y 200 µM) en líneas celulares MIA PaCa-2 (F) y PANC-1 (G) y cultivos de PDAC primarios MGKRAS003 (H), MGKRAS004 (I) y MGKRAS005 (J); K-O) ensayos clonogénicos de MIA PaCa-2 (K), PANC-1 (L), MGKRAS003 (M), MGKRAS004 (N) y MGKRAS005 (O) tratados con IC50 P8/C14; P) análisis de muerte celular de PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004 y MGKRAS005 analizados por citometría de flujo después de la tinción con anexina-V, 7-AAD y citocalceína violeta. Los datos se muestran como SDM; n=5; *** p<0.001. Figura 11. Evaluación de citotoxicidad y genotoxicidad de P8 y C14. Se observan A-B) los efectos de P8 y C14 a diferentes concentraciones (5, 10, 30, 50, 100, 150 y 200 µM) sobre la viabilidad celular de H-PBMC estimuladas y no estimuladas (n=5); C) frecuencia de micronúcleos en eritrocitos policromáticos (PCE) aislados de médula ósea de ratones BALB/c tratados con X µM P8, C14 y Gemcitabina durante 24 h; D-G) evaluación de la presencia de proteínas (D), pH (E), bilirrubina (F) y glucosa (G) en la orina de ratones BALB/c después del tratamiento con X µM de P8, C14 y Gemcitabina durante X h; H-K) evaluación de la presencia de proteínas (H), pH (I), bilirrubina (J) y glucosa (K) en la orina de ratones BALB/c después del tratamiento con X µM de P8, C14 y Gemcitabina durante 16 días. Figura 12. La combinación de P8 y C14 reduce el crecimiento tumoral en modelos de xenoinjerto subcutáneo y ortotópico. Se observan A) los efectos de P8, C14 y C14/P8 a diferentes concentraciones (5, 10, 30 y 60 mg/kg, y la combinación de 30 mg/kg de C14 + 30 mg/kg de P8) en un modelo de xenoinjerto subcutáneo de inyección de células MIA PaCa-2 en la piel del dorso de ratones macho Nu/Nu (n=6); B) cuantificación del volumen tumoral todos los días después del tratamiento con P8, C14 y C14/P8 a diferentes concentraciones (n=6). Se muestran imágenes representativas de tumores para cada condición debajo del gráfico; C) el peso corporal se midió diariamente durante el tratamiento con P8, C14 y C14/P8; D) Western blot representativo de lisados de tumores MIA PaCa-2 tratados con P8, C14, Gemcitabina muestran una inhibición completa de la fosforilación de AKT y ERK usando AKT total, ERK y GAPDH como controles de carga. La cuantificación relativa de los resultados de la transferencia Western se muestra en los gráficos; E) efectos de P8, C14 y C14/P8 a diferentes concentraciones (30 y 60 mg/kg, y la combinación de 30 mg/kg de C14 + 30 mg/kg de P8) en un modelo de xenoinjerto ortotópico de inyección de células MIA PaCa-2 en el páncreas de ratones macho Nu/Nu (n=6); F) el peso corporal se midió diariamente durante el tratamiento con P8, C14 y C14/P8; G) imágenes representativas del efecto del tratamiento con P8, C14, P8/C14 y Gemcitabina; H) gráfico de supervivencia durante el tratamiento con P8, C14, P8/C14 y Gemcitabina. Los datos representan medias ± SD de al menos seis experimentos independientes; *** p<0.001. Figura 13. Evaluación de los marcadores de malignidad CK19, CA125 y Ki-67 en tumores derivados de xenoinjertos subcutáneos. Se observan A) imágenes representativas de hematoxilina-eosina e inmunohistoquímica de las secciones tumorales derivadas de ratones tratados con P8, C14, P8/C14, Gemcitabina o vehículo; B-D) cuantificación de intensidades de señal de CK19 (B), Ca 125 (C) y Ki-67 (D) en las secciones tumorales. Los datos se muestran como SDM; n=100 celdas por campo con 6 campos por sección; *** p<0.001. Figura 14. El tratamiento combinado con P8 y C14 reduce el crecimiento tumoral en modelos PDX. Se observan A) los efectos de P8, C14 y C14/P8 a diferentes concentraciones (5, 10, 30 y 60 mg/kg, y una combinación de 30 mg/kg de C14 + 30 mg/kg de P8) en un modelo de xenoinjerto subcutáneo utilizando células MGKRAS004; B) efecto final después del tratamiento con P8, C14 y C14/P8 a diferentes concentraciones; C) el peso corporal se midió diariamente durante el tratamiento con P8, C14 y C14/P8; D) imágenes representativas de los tumores MGKRAS004 obtenidas de cada grupo; E) efectos de P8, C14 y C14/P8 a diferentes concentraciones (5, 10, 30 y 60 mg/kg, y una combinación de 30 mg/kg de C14 + 30 mg/kg de P8) en un modelo de xenoinjerto subcutáneo (n=6) usando células MGKRAS005; F) efecto final del tratamiento con P8, C14 y C14/P8 a diferentes concentraciones para x d; G) el peso corporal se midió diariamente durante el tratamiento con P8, C14 y C14/P8; H) imágenes representativas de los tumores MGKRAS005 obtenidas de cada grupo. Los datos representan medias ± SD de al menos seis experimentos independientes. *** p<0.001. Descripción detallada de la invención. La presente invención proporciona composiciones sinérgicas que comprenden la combinación de los compuestos C14/P8, induciendo citotoxicidad en líneas celulares y cultivos primarios de cáncer pancreático mayores que los obtenidos por los compuestos evaluados individualmente, lo que las convierte en una alternativa viable, efectiva y útil para el tratamiento de cáncer de páncreas. El adenocarcinoma ductal de páncreas (PDAC) tiene el pronóstico más desalentador entre todos los cánceres humanos, ya que es muy resistente a la quimioterapia. Esto conlleva a la búsqueda de nuevas alternativas farmacológicas para mejorar la calidad de vida de los pacientes con cáncer de páncreas. Se han diseñado compuestos que puedan inhibir la vía de señalización y transporte de la oncoproteína K-Ras4B. Por otro lado, las composiciones de la presente invención que comprenden la combinación de los compuestos C14/P8 presentan efecto sinérgico, induciendo citotoxicidad en líneas celulares y cultivos primarios de cáncer pancreático mayores que los obtenidos por los compuestos evaluados individualmente. Las composiciones descritas aquí disminuyen del 90 al 95% el crecimiento tumoral en modelos de xenoinjerto Subcutáneo y Ortotópico a medida que la dosis se incrementa, así mismo no inducen efectos adversos ni genotoxicidad como lo presenta la quimioterapia de primera línea con Gemcitabina, disminuyen la activación de K-Ras4B y disminuyen los marcadores de malignidad en tumores remanentes. De este mismo modo las composiciones de la invención disminuyen hasta el 99% del crecimiento tumoral en modelos PDx de cáncer pancreático, mostrando mejores efectos antineoplásicos en los modelos de Xenoinjerto Subcutáneo, Ortotópico y PDx disminuyendo hasta un 97% el crecimiento tumoral sin inducir efectos secundarios como los presentados por Gemcitabina. Conforme a la presente invención, las composiciones descritas aquí se configuran como nuevas y eficientes soluciones quimioterapéuticas con mejores propiedades que la quimioterapia convencional. La fórmula química del compuesto C14 (fórmula I) se muestra a continuación: C14: 2-[(3-clorofenil)metil-metil-amino]-N- chroman-4-il-acetamida
Figure imgf000008_0001
Fórmula I mientras que la fórmula química del compuesto P8 (fórmula II) se muestra a continuación: P8: 2-[4-(3-clorofenil)piperazin-1-il]-N-[(4R)-chroman-4-il]acetamida
Figure imgf000008_0002
Fórmula II Conforme a la presente invención, las composiciones descritas aquí comprenden: a) El compuesto C14 (fórmula I) o sus sales farmacéuticamente activas en una concentración de 90.18 µM a 154.24 µM con respecto del compuesto P8, b) El compuesto P8 (fórmula II) (análogo al compuesto C14 el cual presenta grupos amino, bencenos, piridinas y heterociclos no aromáticos) o sus sales farmacéuticamente activas en una concentración de 18 µM a 150 µM, y c) Un vehículo farmacéuticamente aceptable, dentro de los que se incluyen aquellos que sean compatibles con los compuestos mencionados y que permitan una administración adecuada de los mismos, sin embargo, se prefiere que en dichas composiciones los compuestos mencionados se encuentren en una cantidad de C14 de 90.18 µM y de P8 de 24.18 µM; en una de sus modalidades, en cuanto a su proporción en peso (p/p), las composiciones de la presente invención comprenden a los compuestos C14 y P8 en una proporción de 1:1, tal como se describe más adelante. Para efectos de la presente invención, los compuestos C14 y P8 han sido previamente descritos en las solicitudes de patente MX/a/2018/013439 y MX/a/2020/001471; ambas solicitudes de patente, así como su contenido completo se incorporan como referencia en la presente invención. Los resultados de la presente invención muestran que las composiciones sinérgicas descritas aquí son útiles como una nueva alternativa terapéutica para pacientes con cáncer de páncreas, por lo que es parte de las modalidades de la invención el uso de dichas composiciones para el tratamiento de dicho padecimiento. Por lo tanto, es objeto de la invención proveer de composiciones farmacéuticas sinérgicas que comprendan los compuestos C14 y P8, junto con excipientes farmacéuticamente aceptables y adecuados para ser administrados al paciente que lo requiera. Es modalidad de la invención la adaptación de los principios activos para utilizarse en composiciones farmacéuticas para administración enteral, parenteral y uso tópico, incluyendo la inhalación. Las dosis efectivas para el paciente del principio activo serán también ajustadas de conformidad con estudios preclínicos y clínicos procedentes, pero tomando como base los hallazgos de la presente invención. Ejemplos de excipientes farmacéuticamente aceptables que acompañen al principio activo de la invención son por ejemplo, para la administración oral como tabletas o comprimidos, agentes que comprenden por ejemplo, diluyentes, aglutinantes, estabilizantes, agentes de carga, agentes espesantes, tales como povidona, celulosa microcristalina, lactosa, etc., agentes desintegrantes tales como por ejemplo carboximetilcelulosa entrecruzada, surfactantes como por ejemplo, laurilsulfato de sodio, agentes lubricantes o deslizantes como por ejemplo estearato de magnesio, dióxido de silicio coloidal, etc., donde dichos excipientes pueden ser formulados para liberación preferentemente lenta o prolongada para un efecto sistémico. Se pueden preparar soluciones para administración intravenosa o intraperitoneal del principio activo disueltos primero en un solvente orgánico como DMSO, etanol, o dimetilformamida y subsecuentemente en buffers acuosos, como PBS. Se tiene especial preferencia por las formas farmacéuticas diseñadas para la administración localizada al lugar donde se presenta el cáncer de páncreas, donde se pueden formular composiciones líquidas o sólidas, adecuadas para su administración local por ejemplo, y cuyos excipientes pueden seleccionarse por ejemplo, de componentes compatibles con dicha forma farmacéutica, por ejemplo de naturaleza lipídica o peptídica, o peptidomiméticos, conocidos en el estado de la técnica como no inmunogénicos, y que de preferencia puedan quedar unidos a los principios activos de la invención para mejorar su biodisponibilidad; agentes propulsores como por ejemplo, propano, butano, o clorofluorocarbonos permisibles; reguladores de pH como por ejemplo, ácido sulfúrico; agentes quelantes como por ejemplo, EDTA. También se pueden conformar los principios activos en partículas micronizadas contenidas en cápsulas de gelatina o en otros sistemas conocidos en el campo técnico, que ayuden a liberar el principio activo a su sitio blanco de acción, por ejemplo, mediante formas farmacéuticas sólidas como tabletas o grageas, incluyendo aquellas formuladas para su liberación prolongada. Conforme a la presente invención, pueden obtenerse las composiciones aquí descritas mediante la combinación de los compuestos C14 y P8 con vehículos farmacéuticamente compatibles conocidos en el arte, en las cantidades y/o concentraciones que correspondan conforme a lo descrito aquí, y pueden incluirse compuestos conocidos en el arte para la obtención de dichas composiciones. Así mismo, la administración de tales composiciones, podrá hacerse dependiendo de las condiciones del paciente, lo que determinará las dosis y frecuencia de administración necesarias para lograr un tratamiento efectivo del padecimiento cada caso en particular. Por lo anterior, es objeto de la presente invención proveer composiciones farmacéuticas sinérgicas para tratar el cáncer de páncreas, o para prevenir su progresión o su aparición en individuos que lo requieran. Las composiciones descritas logran disminuir la viabilidad de las células cancerosas sin afectar la viabilidad de las células sanas, y fueron probados por su capacidad de prevenir la aparición de tumores o de disminuir el tamaño tumoral por los que dichas composiciones son una excelente alternativa para el tratamiento de neoplasias en tejido pancreático. Se pueden utilizar las composiciones descritas aquí como antineoplásicos o anticancerígenos para tratar mamíferos, incluyendo al humano; dichas composiciones son efectivas y seguras a las dosis adecuadas, las cuales pueden ser calculadas por especialistas en el campo de la invención y para cada requerimiento individual, así como las vías de administración y las formulaciones adecuadas que permitan alcanzar al órgano y al tejido blanco, basándose en los principios que proporciona la presente invención. Una de las modalidades de la invención se refiere a un método para tratar cáncer de páncreas utilizando las composiciones que comprenden la combinación de los compuestos C14 y P8, incluyendo la eliminación/reducción de tumores causados por dicho padecimiento. Otra modalidad de la invención se refiere al método para tratar cáncer de páncreas me manera específica utilizando las composiciones que comprenden la combinación de los compuestos C14 y P8, sin que dichas composiciones causen reacciones adversas al solo afectar al tejido canceroso y no afectar al tejido sano. Realizando análisis SAR y QSAR se identificaron análogos al compuesto C14. Empleando el complejo KRas4B/PDE6δ cristalográfico y realizando ensayos de Dinámica molecular identificamos la energía de interacción de los compuestos. La viabilidad celular y tipo de muerte se evalúo empleando citometría de flujo, así como ensayo clonogénico. Se realizaron ensayos de RAS-GTP Pulldown y Western blot para medir la activación de K-Ras y sus vías de señalización. Se implantaron células de MIA PaCa-2 en modelos de xenoinjerto subcutáneo y ortotópico en ratones Nu/Nu y se realizó el modelo PDX empleando cultivos primarios de cáncer pancreático para ser tratados con los compuestos C14, P8 y la combinación C14/P8. El análogo denominado como P8 presentó mayor energía de interacción sobre los complejos mutados in silico, de este modo presenta mayores efectos citotóxicos en las líneas celulares y cultivos primarios de cáncer pancreático con K-Ras mutado sin dañar a la línea celular y cultivos primarios no cancerosos. De este mismo modo ambos compuestos C14 y P8 disminuyen la activación de K-Ras y sus vías de señalización, sin embargo el compuesto P8 presenta un IC504 veces menor que el compuesto C14, siendo hasta el momento el mejor compuesto encontrado en nuestro grupo de investigación. En la presente invención se reporta la evaluación del compuesto C14 y de su análogo P8 utilizando modelos preclínicos de cáncer de páncreas aprobados por la FDA. Los resultados mostraron que el compuesto C14 y el P8 tienen una mayor actividad citotóxica especifica que los compuestos D14 y C22 previamente reportados por nuestro grupo de trabajo. Además, la combinación de los compuestos C14 y P8 incluida en las composiciones de la presente invención presenta un efecto sinérgico, tanto en líneas celulares como en cultivos primarios y en modelos murinos. Por otra parte, reportamos que los compuestos C14 y P8 no presentan efectos secundarios como Gemcitabina, por lo tanto pueden ser considerados como nuevos agentes quimioterapéuticos. El compuesto C14 presenta mayor energía de interacción y mayor disminución de la viabilidad celular en líneas celulares de cáncer pancreático con K-Ras4B mutado. Uno de los compuestos con mayor puntaje de interacción identificados en la presente invención por medio de cribado virtual sobre el complejo heterodimérico K-Ras4B/PDE6δ, fue el compuesto denominado C14, el cual es una pequeña molécula orgánica con un peso molecular de 344.8 g/mol (Tabla 1). Tabla 1. Interacciones del compuesto C14 y P8 sobre el complejo K-Ras4B/PDE6δ. Resultados obtenidos del análisis de selección virtual en el complejo heterodimérico cristalográfico.
Figure imgf000010_0001
Para analizar las interacciones y energías de acoplamiento del compuesto C14 sobre el heterocomplejo, realizamos modificaciones para obtener los complejos K-Ras4BWT/PDE6δ, K- Ras4BG12D/PDE6δ y K-Ras4BG12C/PDE6δ (Figura 1A-C). El acoplamiento del compuesto C14 con el complejo K-Ras4BWT/PDE6δ mostró una interacción simultánea con K-Ras4B (rosa) y PDE6δ (aguamarina), donde las energías de interacción (Tabla 2) con los diferentes heterocomplejos indica que el compuesto C14 tiene mayor energía de interacción en los heterocomplejos mutados K- Ras4BG12D /PDE6δ y K-Ras4BG12C/PDE6δ que en complejos K-Ras4BWT/PDE6δ. Tabla 2. Componentes de la energía libre de unión de complejos proteína-proteína y proteína- ligando (en unidades kcal/mol).
Figure imgf000011_0001
Energías libres de unión y términos energéticos individuales de complejos a partir de conformaciones acopladas (kcal/mol). El polar (ΔEpolar=ΔEele + ΔGele, sol) y no polar Se muestran las contribuciones (ΔEnon-polar=ΔEvwd + ΔGnpol, sol). Todas las energías se promedian en 500 instantáneas a intervalos de tiempo de 100 ps desde las últimas 50 Simulaciones ns-long MD están en kcal/mol (± error estándar de la media) Dada la afinidad obtenida del complejo Ras4BG12D/PDE6δ y K-Ras4BG12C/PDE6δ a través de la interacción con el compuesto C14, y la importancia de este complejo en el mantenimiento y crecimiento del cáncer de páncreas, realizamos ensayos de viabilidad para determinar el efecto citotóxico del compuesto C14, utilizando las líneas celulares de cáncer de páncreas PANC-1 y MIA PaCa-2, la línea celular de conducto pancreático normal hTERT-HPNE y la línea celular retiniana ARPE-19 como control. Mediante análisis de microscopía de campo de las líneas celulares tratadas con 100 µM de compuesto C14 durante 72 h de incubación, se demostró que este compuesto tiene una actividad relativamente alta sobre la línea celular PANC-1 y MIA PaCa-2 sin afectar ARPE-19 y hTERT- HPENE en términos de su crecimiento y actividad, como la morfología celular, la proliferación y el número de células vivas hasta 72 h (Figura 1D). La viabilidad celular se determinó midiendo las concentraciones de ATP en la célula después de tres días de tratamiento con C14 a diferentes concentraciones (5, 10, 30, 50, 100, 150 y 200 µM), donde las líneas celulares MIA PaCa-2 y PANC -1 presentaron sensibilidad al tratamiento dosis dependiente, obteniendo una IC50 de 90.18 µM para MIA PaCa-2, de 103.5 µM para PANC-1 y de 171.4 µM en la línea celular normal hTERT-HPNE (Figura 1E-F). Estos resultados sugieren que C14 tiene una fuerte actividad específica en líneas celulares con mutaciones en K-Ras4B. La determinación del tipo de muerte celular que produce el compuesto C14 es muy importante para su posterior aprobación para tratamiento oncológico. La identificación de análogos del compuesto C14 arroja un compuesto con mayores propiedades químicas. La identificación y selección de moléculas orgánicas análogas al compuesto líder C14 se realizó utilizando una base de datos de la quimioteca ENAMINE, donde se analizaron 335 compuestos análogos del compuesto C14. Mediante el programa de bioinformática Molecular Operating Environment (MOE) 2014.09, la estructura del compuesto C14 fue sometida a un análisis SAR y QSAR para identificar el farmacóforo del compuesto, teniendo en cuenta las interacciones de esta molécula con el complejo heterodimérico K-Ras4B/PDE6δ (Tabla 1). Una vez identificado el farmacóforo del compuesto C14, se llevó a cabo un "acoplamiento" estructural en las 335 moléculas análogas al compuesto C14 para identificar aquellas moléculas que presentaban una similitud del 80 al 90% con el farmacóforo del C14. Siguiendo las reglas de Lipinski, se identificaron 20 moléculas análogas al compuesto líder C14 (Tabla 3), los cuales tienen un peso molecular menor a 500 Da, presentan menos de 5 donantes y aceptores de enlaces de hidrógeno y menos de cinco enlaces rotativos, esto para que las moléculas tengan mayor especificidad hacia el objetivo dirigido y puedan pasar libremente a través de la membrana plasmática. Las moléculas análogas que se identificaron mostraron cambios del 20% en su estructura en comparación con la estructura del compuesto líder. Los 20 análogos al compuesto líder conservan el grupo cromeno y el grupo acetamida, donde las modificaciones realizadas con respecto a la estructura del compuesto líder fueron con la adición de grupos amino, bencenos, piridinas y heterociclos no aromáticos, con el propósito de incrementar las interacciones con el complejo molecular K-Ras4B/PDE6δ. Tabla 3. Análogos al compuesto líder C14 seleccionados por medio de programas bioinformáticos.
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Para la selección de los análogos del compuesto líder C14 con mayor efecto citotóxico sobre la línea celular MIA PaCa-2, los análogos al C14 se evaluaron usando como referencia la IC50 del compuesto C14 que es 90.18 µM a las 24, 48 y 72 horas después de su tratamiento y fueron analizados por medio de microscopía de campo claro (datos no mostrados). Observamos que los compuestos P15, P16 y P19 tienen un mayor efecto sobre la proliferación celular de la línea celular hTERT-HPNE desde las primeras 24 horas, donde el compuesto P8 presentó un efecto marcado en la línea celular MIA PaCa-2 a las 24 horas y no en la línea celular control, siendo que los compuestos restantes no mostraron un efecto sobre la proliferación en ninguna de las dos líneas celulares. Teniendo en cuenta los resultados de la microscopía, se realizaron pruebas de viabilidad utilizando el kit "CellTiter-Glo" que nos permitió medir el ATP mediante luminiscencia. Las líneas celulares MIA PaCa-2 y hTERT- HPNE se cultivaron en placas de 96 pocillos, las cuales fueron tratadas con los 20 análogos a una concentración de 90.18 µM durante 24, 48 y 72 horas para seleccionar el(los) compuesto(s) que tiene(n) un mayor efecto citotóxico sobre la línea celular MIA PaCa-2 y hTERT-HPNE utilizada como control (Figura 2). Los resultados obtenidos (Figura 2) con los cinco primeros análogos del compuesto C14, muestran una disminución en la viabilidad de la línea celular de control hTERT-HPNE, pero no en la línea celular Mia-PaCa 2, donde el mismo resultado se observa en los compuestos P11 a P20 (Figura 3). Sin embargo, el compuesto P8 (Figura 1G-H) tiene un mayor efecto sobre la viabilidad celular en MIA PaCa-2. Por otro lado, en la línea celular hTERT-HPNE, se obtuvo una disminución significativa en la viabilidad celular a una concentración de 90.18 µM del compuesto P8 (Figura 1G- H), además de mostrar una disminución en la viabilidad con la presencia del compuesto líder C14, a pesar del hecho de que estos compuestos muestran un 80% de similitud con el farmacóforo, siendo que el 95% de los análogos no conserva el efecto citotóxico sobre la línea celular El análogo P8 presenta mayor energía de interacción y mayor efecto citotóxico sobre líneas celulares con K-Ras4B mutado, cuatro veces mayor que el compuesto líder C14. Una vez que se identificó al análogo P8, se realizó un acoplamiento molecular del compuesto P8 sobre los heterocomplejos K-Ras4BWT/PDE6δ, K-Ras4BG12D/PDE6δ K-Ras4BG12C/PDE6δ y K- Ras4BG12V/PDE6δ (Figura 3 A-D), obteniéndose como resultado un incremento en la energía de interacciones del compuesto P8 con respecto al reportado con el compuesto líder C14 sobre el heterocomplejo. Nuestro análisis in silico reveló que el sitio de interacción del compuesto P8 con respecto al compuesto C14 es completamente diferente, ya que el compuesto C14 tiene un ΔG de - 14.7 Kcal/mol y el compuesto P8 un ΔG de -15.7 Kcal/mol, donde el análogo P8 presenta más de 20 interacciones con diversos residuos de aminoácidos del complejo heterodimérico K-Ras4B/PDE6δ. Teniendo en cuenta estas interacciones mediante la simulación del acoplamiento molecular de los compuestos C14 y P8 en el complejo molecular K-Ras4B/PDE6δ, se realizó un ensayo de dinámica molecular del compuesto P8 sobre los complejos heterodiméricos K-Ras4BWT/PDE6δ, K- Ras4BG12D/PDE6δ K-Ras4BG12C/PDE6δ y K-Ras4BG12V/PDE6δ, para calcular los valores teóricos de afinidad sobre los heterocomplejos (Tabla 2). Las energías estimadas fueron mayores sobre los heterocomplejos mutados que con el silvestre (WT). Con estos datos teóricos de afinidad, podemos sugerir que el compuesto P8 muestra una mayor afinidad que el compuesto líder sobre los heterocomplejos de K-Ras4B/PDE6δ (Tabla 2). Dada la afinidad del compuesto P8 sobre los heterocomplejos mutados de K-Ras4B/PDE6δ, realizamos pruebas de viabilidad sobre las líneas celulares de PDAC PANC-1, MIA Paca-2 y Capan-1, así como en la línea celular hTERT-HPNE como control, comparando el efecto de las líneas celulares tratadas con los compuestos C14 y P8 (Figura 3 E-H). La viabilidad celular se determinó midiendo las concentraciones de ATP en el líneas celulares después de tres días de tratamiento con el compuesto líder y su análogo a diferentes concentraciones (5, 10, 30, 50, 100, 150 y 200 µM), donde el análogo denominado como P8 presentó mayor efecto citotóxico que su compuesto líder, presentando un IC50 de 51.18 µM en PANC-1, 24.18 µM en MIA PaCa-2 y 28.96 µM en Capan-1 de 2 a 4 veces menor que el compuesto C14, siendo que estas concentraciones de IC50 no afectan la viabilidad de la línea celular normal hTERT-HPNE con un IC50 de 103.45 µM. Una de las características más importantes de las células cancerosas es su alta proliferación celular y quimiorresistencia a la terapia, por lo que para evaluar la quimiorresistencia de las líneas celulares de cáncer de páncreas PANC-1, MIA PaCa-2 y Capan-1 se realizaron ensayos de clonogenicidad comparando los efectos entre los compuestos C14, P8, Gemcitabina y Deltarasin (Figura 3 I-K). Los compuestos C14 y P8 disminuyeron la capacidad clonogénica de las líneas celulares cancerosas en comparación con el efecto inducido por Gemcitabina, sin embargo el compuesto P8 presentó un mayor efecto en la reducción de la clonogenicidad de las tres líneas celulares, por otro lado el compuesto Deltarasin no presentó un buen efecto en la reducción de la capacidad clonogénica. La determinación del tipo de muerte celular producida por los compuestos C14 y P8 fue determinada por citometría de flujo, observándose que el compuesto P8 promueve la muerte celular por apoptosis en líneas celulares MIA PaCa-2, PANC-1 y Capan-1 con un mayor porcentaje de muestreo de células que el compuesto C14 a concentraciones de CI50 más bajas sin causar daño a las células del ducto pancreático normal hTER-HPNE. El compuesto P8 ha demostrado ser un compuesto mejor que su compuesto principal, induciendo citotoxicidad, disminución clonogénica e induciendo apoptosis con concentraciones de 2 a 4 veces menor que el compuesto líder. Los compuestos C14 y P8 disminuyen la activación de K-Ras y sus vías de señalización en líneas celulares de PDAC dependiendo de su adicción oncogénica. Para determinar si los compuestos C14 y P8 estaban afectando la activación de Ras y sus vías de señalización, realizamos ensayos RasGTP-Pulldown, utilizando la quimioterapia de primera línea Gemcitabina, Deltarasin y nuestros compuestos C14 y P8 (Figura 4 A-D). Los resultados indican que nuestros compuestos son capaces de reducir la activación de K-Ras en líneas celulares de cáncer pancreático PANC-1, MIA PACa-2 y Capan-1, que representan las 3 mutaciones más frecuentes de K-Ras oncogénico; este hallazgo es formidable, ya que no disminuye la activación de K-Ras o Ras en la línea celular normal hTERT HPNE. Posteriormente, para comprobar si esta disminución de la activación de K-Ras afectaba sus vías de señalización, realizamos ensayos de Western blot para corroborar la disminución en la activación de AKT y ERK en cada una de las líneas celulares de cáncer pancreático evaluadas previamente (Figura 4 E-G). Observamos la disminución en la activación de AKT y ERK en líneas celulares con las mutaciones G12D, G12C y G12V, tratadas con compuestos C14 y P8; dependiendo de la adicción oncogénica que presentan, los resultados observados variaron dependiendo de la mutación que presenta cada línea celular de cáncer pancreático, observándose una disminución del 100% en la activación de AKT en PANC-1 tratada con el compuesto C14 y P8, así como una disminución del 100% en la activación de AKT en MIA PaCa-2 tratada con el compuesto P8. Por otro lado, el compuesto P8 disminuyó la activación de ERK del 80 al 100% en PANC-1, MIA PaCa-2 y en Capan-1, lo que indica que los compuestos C14 y P8 están afectando directamente la vía de señalización de K-Ras en líneas celulares de cáncer pancreático con diferente adicción oncogénica. Obtención y caracterización de cultivo primario. Para la obtención de muestras de cáncer pancreático de pacientes, se acudió al Departamento de medicina genómica y al Departamento de oncología y cirugía general del Hospital 1º. de Octubre del ISSSTE en la Ciudad de México, siguiendo lo estipulado en el proyecto nacional 002.2015. Se recolectaron muestras de cáncer de páncreas en el quirófano y se transportaron al Laboratorio para su procesamiento. Se obtuvieron 19 muestras de cáncer pancreático de pacientes de 40 a 100 años, con una mayor frecuencia de 40-59 años y con una mayor incidencia en mujeres, donde dicha información contradice lo descrito anteriormente en el sentido de que la mayor incidencia de este tipo de cáncer se reporta en hombres de 60 a 80 años. Por otro lado, se obtuvieron 2 muestras de tejido epitelial de pacientes sanos PBDD33 y JGCD28 como control de nuestro estudio. Una vez obtenidas las muestras, se procesaron y se obtuvieron 3 cultivos primarios de cáncer pancreático MGKRAS003, MGKRAS004 y MGKRAS005, y 2 cultivos primarios derivados de tejido epitelial, realizándose varios pases hasta obtener los pases 5. Una vez obtenidas las células, evaluamos la presencia de células ductales neoplásicas del tejido de los pacientes, utilizando Ck19 (figura 5A) para células ductales como se muestra en la imagen de la inmunofluorescencia del tejido del paciente y nuestro cultivo primario, donde es posible que Ck19 esté presente en ambas muestras, lo que indica que nuestros cultivos primarios son células ductales. Otro marcador más utilizado en la clínica para identificar células neoplásicas es CA19-9 o MUC1 (figura 5B), como se muestra en la imagen las muestras de pacientes y nuestros cultivos primarios, presentan la expresión de MUC1, lo cual nos indica que nuestros cultivos primarios son células ductales neoplásicas del tejido del paciente. Una vez confirmado que nuestros cultivos primarios son células ductales neoplásicas, decidimos realizar una caracterización avanzada utilizamos diferentes marcadores, como marcadores de origen pancreático Ck7 y Ck19 y los marcadores de Malignidad más utilizados en varios países como CEA, MUC1, MUC4, MUC16, EFGR, VIMENTIN, B-Catenina citoplásmica y E-Caterina y Ki-67 (Tabla 4). Como control realizamos la misma caracterización en los cultivos primarios procedentes de tejido epitelial, obteniendo la presencia de marcadores epiteliales y mesenquimales en una de las muestras, por lo que se realizó la caracterización de fibroblastos y células mesenquimales (figura 6 A-C) obteniendo como resultado la identificación de células mesenquimales derivadas de tejido epitelial correspondiente a la muestra PBDD33 y fibroblastos correspondientes a la muestra JGCD28. Como resultado, nuestros cultivos primarios de cáncer pancreático pertenecen al estadio 4 y con estos resultados comprobamos el diagnóstico proporcionado por los oncólogos el cual fue, adenocarcinoma ductal pancreático altamente infiltrante e invasivo. Posteriormente identificamos por medio de PCR y secuenciación del exón-2 de KRAS la presencia de mutaciones en los cultivos primarios de cáncer pancreático 8 (Figura 7C) obteniendo como resultado MGKRAS003 WT, MGKRAS004 G12V y MGKRAS005 G12C, donde las dos mutaciones encontradas son la segunda y la tercera con mayor frecuencia a nivel mundial; por otro lado la mutación G12V representa a la mutación con mayor quimiorresistencia que se ha reportado en el adenocarcinoma ductal pancreático. Los cultivos primarios de cáncer pancreático presentan mayor sensibilidad a los compuestos C14 y P8 que a la terapia convencional. Una vez caracterizados los cultivos primarios de cáncer pancreático y de los controles, realizamos ensayos de viabilidad la cual fue determinada midiendo las concentraciones de ATP en los cultivos primarios después de tres días de tratamiento con el compuesto líder y su análogo a diferentes concentraciones (5, 10, 30, 50, 100, 150 y 200 µM) (Figura 7 A-E), obteniéndose para el compuesto líder C14 una IC50 de 15.8 µM en MGKRAS003, de 18.3 µM en MGKRAS004, de 118.9 µM en MGKRAS005, de 189 µM en PBDD33 y de 130 µM para JGCD28, mientras que para el análogo P8 se obtuvo una IC50 de 22.3 µM en MGKRAS003, de 18.03 µM en MGKRAS004, de 37.5 µM en MGKRAS005, de 192 µM en PBDD33 y de 145 µM en JGCD28. Los datos obtenidos de los dos compuestos sobre los cultivos primarios mostraron una especificidad sobre los cultivos primarios de cáncer pancreático sin tener una afectación en los cultivos primarios no cancerosos, donde el análogo P8 presenta menores concentraciones de IC50 y mayor actividad que su compuesto líder, presentando mayor sensibilidad los cultivos primarios de cáncer pancreático a los compuestos C14 y P8 que las líneas celulares ya establecidas de cáncer pancreático. Posteriormente evaluamos el efecto citotóxico de Gemcitabina y Deltarasin (figura 8) en los cultivos primarios de cáncer pancreático MGKRAS003, MGKRAS004 y MGKRAS005 obteniendo un IC50 de 1000 nM con Gemcitabina y 10 µM para Deltarasin, donde estas concentraciones de IC50 son el doble de concentración reportada para estos compuestos sobre líneas celulares de cáncer pancreático. Una vez identificadas las concentraciones de IC50 para cada cultivo primario, evaluamos la capacidad clonogénica de MGKRAS003, MGKRAS004 y MGKRAS005 empleando los compuestos C14, P8, Gemcitabina y Deltarasin (Figura 7 F-H) obteniendo como resultado una mayor disminución en la capacidad clonogénica de los tres cultivos primarios siendo tratados con el análogo P8 con respecto a su compuesto líder el compuesto C14.          
Figure imgf000017_0001
La determinación del tipo de muerte celular producida por los compuestos C14 y P8 sobre los cultivos primarios fue determinada por citometría de flujo, donde esta determinación mostró que el compuesto P8 promueve la muerte celular por apoptosis en líneas celulares MGKRAS003, MGKRAS004 y MGKRAS005, con un mayor porcentaje de muestreo de células que el compuesto C14 a concentraciones de CI50 más bajas sin causar daño a los cultivos primarios no cancerosos PBDD33 y JGCD28. El compuesto P8 ha demostrado ser un compuesto mejor que su compuesto líder C14 induciendo citotoxicidad, disminución clonogénica e induciendo apoptosis con concentraciones de 2 veces menor que el compuesto líder sobre cultivos primarios de cáncer pancreático. Los compuestos C14 y P8 disminuyen la activación de K-Ras y sus vías de señalización en cultivos primarios de cáncer pancreático. Para determinar si los compuestos C14 y P8 estaban afectando la activación de Ras y sus vías de señalización, realizamos ensayos RasGTP-Pulldown, utilizando la quimioterapia de primera línea Gemcitabina, Deltarasin y nuestros compuestos C14 y P8 (Figura 9 A-E). Los resultados indicaron que los compuestos C14 y P8 son capaces de reducir la activación de K-Ras en los cultivos primarios de cáncer pancreático MGKRAS003, MGKRAS004 y MGKRAS005, que representan 2 de las mutaciones más frecuentes de K-Ras oncogénico; este hallazgo es muy importante, ya que no disminuye la activación de K-Ras o Ras en los cultivos primarios no cancerosos PBDD33 y JGCD28, como los presenta Deltarasin. Posteriormente, para comprobar si esta disminución de la activación de K-Ras afectaba sus vías de señalización, realizamos ensayos de Western blot para corroborar la disminución en la activación de AKT y ERK en cada una de las líneas celulares de cáncer pancreático evaluadas previamente (Figura 9 F-H). Observamos la disminución en la activación de AKT y ERK en los cultivos primarios MGKRAS003, MGKRAS004 y MGKRAS005, tratados con los compuestos C14 y P8, donde los resultados observados variaron dependiendo de la mutación que presenta cada cultivo primario de cáncer pancreático; se observó una disminución del 80% en la activación de AKT en MGKRAS003 y MGKRAS004 tratadas con el compuesto P8, así como una disminución del 95% en la activación de AKT en MGKRAS005 tratada con los compuestos C14 y P8. Por otro lado, el compuesto P8 disminuyó la activación de ERK del 50 al 80% en MGKRAS003, MGKRAS004 y MGKRAS005, lo que indicó que los compuestos C14 y P8 están afectando directamente la vía de señalización de K-Ras en cultivos primarios de cáncer pancreático. Los compuestos C14 y P8 presentan efectos sinérgicos en líneas celulares y cultivos primarios de cáncer pancreático. Al realizar el análisis in silico de los compuestos C14 y P8 sobre los diferentes complejos heterodiméricos K-Ras4BWT/PDE6δ, K-Ras4BG12D/PDE6δ, K-Ras4BG12C/PDE6δ y K-Ras4BG12V/PDE6δ, observamos diferencias en el sitio de interacción de ambos compuestos, por lo que decidimos realizar docking (Figura 10 A-D) y una dinámica molecular de ambos compuestos sobre los complejos heterodiméricos WT y mutados (Tabla 2), obteniendo como resultado, -120.77 ΔG para el complejo K-Ras4BWT/PDE6δ, -153.75 ΔG para el complejo K-Ras4BG12D/PDE6δ, - 186.14 ΔG para el complejo K-Ras4BG12C/PDE6δ y -175.59 ΔG para el complejo K-Ras4BG12V/PDE6δ, obteniendo mayor energía de interacción con ambos compuestos en los complejos mutados que en el complejo WT; estos resultados sugirieron que los compuestos C14 y P8 podrían presentar efecto sinérgico si eran evaluados en conjunto. Para poder demostrar el efecto sinérgico de los compuestos realizamos un isobolograma (Figura 10E) para identificar el efecto teórico sinérgico, aditivo o antagónico, donde graficamos la concentración de IC50 de ambos compuestos y siguiendo los análisis de Chao & Talalay, obtuvimos varios puntos concordantes en la región de efecto sinérgico. Para comprobar el efecto sinérgico probamos los compuestos C14 y P8 empleando las mismas concentraciones de cada uno (5, 10, 30, 50, 100, 150 y 200 µM), las cuales fueron evaluadas en líneas celulares de cáncer pancreático tales como PANC-1 y MIA PaCa-2 (Figura 10 F-G) así como en nuestros cultivos primarios de cáncer pancreático tales como MGKRAS003, MGKRAS004 y MGKRAS005 (Figura 10 H-J), obteniéndose una IC50 de 18 µM para PANC-1, de 10.2 µM para MIA PaCa-2, de 5.8 µM para MGKRAS003, de 8.3 µM para MGKRAS004 y de 18.9 µM para MGKRAS005, siendo 10 veces menor las concentraciones obtenidas empleando ambos compuestos C14 y P8 que las obtenidas por los compuestos individualmente. Una vez identificadas las concentraciones de IC50 evaluamos la capacidad clonogénica de las líneas celulares y de los cultivos primarios para evaluar el efecto combinado de los compuestos C14 y P8 (Figura 10 K-O), obteniéndose una reducción de al menos el 99% de la capacidad clonogénica de PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004 y MGKRAS005. Posteriormente realizamos ensayos de muerte celular por medio de citometría de flujo empleando PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004 y MGKRAS005 (Figura 10P), obteniéndose la inducción de muerte o apoptosis de al menos el 90% en líneas celulares y en nuestros cultivos primarios de cáncer pancreático. Estos resultados confirmaron el efecto sinérgico de los compuestos C14 y P8, tanto en líneas celulares de cáncer pancreático como en cultivos primarios de cáncer pancreático con diferentes mutaciones en la oncoproteína K-RAS4B. Los compuestos C14 y P8 no presentan efectos secundarios en modelos murinos comparados con Gemcitabina. Para comprobar que los compuestos C14 y P8 no afectaban a células no cancerosas, realizamos un ensayo de citotoxicidad empleando PBMC´s (figura 11 A-B) humanos activados y sin activar tratados con diferentes concentraciones de los compuestos C14 y P8 (5, 10, 30, 50, 100, 150 y 200 µM) obteniéndose un IC50 de 418.3 µM de C14 para los PBMC´s estimulados y de 443 µM de C14 para los PBMC´s no estimulados; por otro lado se obtuvo un IC50 de 1144 µM de P8 para los PBMC´s estimulados y de 1371 µM para los PBMC´s no estimulados, donde estas concentraciones sobrepasan en 100 veces las concentraciones obtenidas en nuestras líneas y cultivos primarios de cáncer pancreático y de 4 a 10 veces mayor con respecto a las líneas celulares y de cultivos primarios no cancerosos, comprobando así la especificidad de los compuestos C14 y P8 por líneas celulares y cultivos primarios de cáncer pancreático. Para evaluar la genotoxicidad de los compuestos C14 y P8 sobre ratones BalB/c, realizamos la administración de 30, 60 mg/kg de C14 y P8, la combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p), 40 mg/kg de Gemcitabina, vehículos (Carboximetil celulosa al 0.05% en PBS con 0.5% de DMSO) y ratones sin tratamiento como control, administrándose una dosis por 24 h (figura 11C); transcurrido el tiempo se extrajo la medula ósea del fémur de los ratones y se cuantificó la presencia de micronúcleos para evaluar la genotoxicidad de los tratamientos, obteniéndose como resultado del 7 al 5% de micronúcleos presentes en la medula ósea de los ratones tratados con los compuestos C14 y P8 y su combinación, lo cual no es significativo con respecto al control y al vehículo, sin embargo se obtuvo más del 40% de micronúcleos presentes en los ratones tratados con Gemcitabina; estos resultados indicaron que los compuestos C14 y P8 no inducen genotoxicidad como Gemcitabina. Para evaluar los efectos secundarios de los compuestos C14 y P8 se emplearon ratones BalB/c a los cuales se les administró vía intraperitoneal 30, 60 mg/kg de C14 y P8, la combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p), 40 mg/kg de Gemcitabina, vehículos (Carboximetil celulosa al 0.05% en PBS con 0.5% de DMSO) y ratones sin tratamiento como control, administrándose una dosis por 24h (figura 11 D-G) y una vez al día por 15 días para completar el esquema de tratamiento (figura 11 H-K). Después de 24h del tratamiento se sacrificaron los ratones y se recolectó la orina para evaluar proteína, pH, bilirrubina y Glucosa, obteniéndose un aumento de 300 mg/dl de proteína, 70 mg/dl de Bilirrubina y 250 mg/dl de glucosa en orina en los ratones tratados con Gemcitabina; por otro lado no se detectó ninguna de las variaciones de los parámetros anteriores en los ratones tratados con los compuestos C14, P8 y con la combinación C14/P8, indicándonos que a las primeras 24h de tratamiento nuestros compuestos no presentan efectos secundarios. Para comprobar estos resultados se extendió el tratamiento durante 15 días, observándose durante el tratamiento varios efectos secundarios en los ratones BALB/c tratados con Gemcitabina (Tabla 5), tales como diarrea, prolapso rectal, síndrome de torsión intestinal, disminución de la masa muscular, pérdida de peso así como pérdida del apetito; debido a estos efectos secundarios los ratones tratados con Gemcitabina tuvieron que ser sacrificados, mientras que los ratones tratados con C14, P8 y la combinación C14/P8 no presentaron ningún síntoma de los mencionados. Tabla 5. Efectos secundarios obtenidos en ratones BALB/c tratados con Gemcitabina, C14, P8 y C14/P8
Figure imgf000019_0001
N. Normal; ND. No detectado De este mismo modo se realizó una biometría hemática observándose leucopenia y neutropenia en los ratones tratados con Gemcitabina, así como un aumento de las enzimas hepáticas (G1), 500 mg/dl de glucosa, 2000 mg/dl de proteína y 70 mg/dl de bilirrubina, mientras que en contraste no se observó ningún efecto secundario de los mencionados en los ratones tratados con los compuestos C14, P8 y la combinación C14/P8. Con los resultados obtenidos de Gemcitabina en los ratones nos surgió la duda si estos mismos efectos se presentan en pacientes con cáncer pancreático a los cuales se les administra esta quimioterapia. Evaluando el historial clínico de los pacientes que accedieron a participar en este estudio encontramos (Tabla 6) astenia (G2), nauseas (G2), epigastralgia, fatiga (G2), hiporexia (G1), leucopenia (G2), neutropenia (G2), baja reserva medular, aumento de plaquetas, elevación de enzimas hepáticas (G1), dolor óseo y pérdida de peso, siendo que debido a estos efectos secundarios la quimioterapia tuvo que ser suspendida y se reemplazó por medicamentos paliativos. Estos resultados nos indican que los compuestos C14 y P8 no presentan efectos secundarios ni genotoxicidad en modelos murinos y pueden usarse para su evaluación preclínica en modelos murinos de cáncer pancreático. Tabla 6. Efectos secundarios presentados en pacientes tratados con quimioterapia estándar en México.
Figure imgf000020_0001
La combinación de los compuestos C14 y P8 disminuye el crecimiento tumoral en modelos murinos de xenoinjerto heterotópico y ortotópico. Para la evaluación del efecto antineoplásico de los compuestos C14 y P8 en modelo heterotópico o xenoinjerto subcutáneo (Figura 12 A-D), injertamos 5 millones de MIA PaCa-2 en el dorso de ratones Nu/Nu hasta obtener un volumen de 150 mm3, para posteriormente administrar vía intraperitoneal diferentes concentraciones (5, 10, 30 y 60 mg/kg y una combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p),) de los compuestos C14 y P8, durante 15 días una vez al día, 40 mg/kg de Gemcitabina una vez cada 3 días por 15 días y el vehículo como control (Carboximetil celulosa al 0.05% en PBS con 0.5% de DMSO). Durante los 15 días del tratamiento medimos el volumen de los tumores antes de la inoculación (Figura 12A) observándose la disminución de más del 50% en todas las concentraciones de los compuestos C14 y P8, siendo más evidente con las concentraciones de 30 y 60 mg/kg de C14 y P8 obteniendo una disminución de más del 80% del crecimiento tumoral, siendo más evidente la disminución del crecimiento con 60 mg/kg de P8. Sin embargo, con la combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p), observamos una disminución de más del 95% del crecimiento tumoral como se muestra en la Figura 12B, en la cual se observa el volumen tumoral del ultimo día después del tratamiento y una imagen representativa del tamaño de los tumores obtenidos en cada grupo experimental. Durante el tiempo que duró el tratamiento, medimos el peso de los ratones (Figura 12 C) y no observamos pérdida de peso empleando C14, P8 ni sus combinaciones, mientras que en el grupo tratado con Gemcitabina se observó una disminución de más del 20% de su peso corporal. Una vez identificado que los compuestos C14 y P8 disminuyen el crecimiento tumoral, extrajimos el tumor y lo fragmentamos en dos porciones, donde la primera porción se utilizó para realizar la extracción de proteínas para identificar las vías de señalización de K-Ras, mientras que la segunda porción se usó para realizar inmunohistoquímica para identificar marcadores de respuesta al tratamiento. Realizamos la identificación de fosforilación de AKT y ERK en diferentes tratamientos (Figura 12 D), observándose disminuciones de fosforilación de AKR y ERK en todas las concentraciones utilizadas en tumores siendo más evidente a 60 mg/kg y con la combinación del C14/P8. La señalización de K-Ras4B desencadena proliferación y supervivencia celular, por lo que decidimos identificar la proliferación y presencia de células neoplásicas después del tratamiento en los tejidos. Realizamos inmunohistoquímica donde usamos Ck19 para células ductales, CA125 o Mucina 16 para células neoplásicas y ki-67 para proliferaciones; en la figura 13 se muestran las imágenes representativas de las diferentes concentraciones de C14, P8, Gemcitabina y Vehículo, observándose en color marrón la inmunorreacción positiva en diferentes marcadores, donde la inmunorreacción de Ck-19, CA-125 y Ki-67 disminuye conforme aumentan las concentraciones de los compuestos C14 y P8; la disminución de estos marcadores es más evidente al emplear 30 mg/kg de C14 y P8 donde se disminuye más del 70% de las células ductales neoplásicas en proliferación, sin embargo con 60 mg/kg de P8 disminuye más del 90% de las células ductales neoplásicas (figura 13 B-D). Por otro lado, logramos obtener la disminución de las células ductales neoplásicas y disminuir su proliferación a más del 95% empleando la combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p), comprobando así el efecto sinérgico de los compuestos sobre los marcadores de malignidad en PDAC. Una vez que identificamos que los compuestos C14 y P8 tienen efectos antineoplásicos en el modelo heterotópico, evaluamos los efectos antineoplásicos de los compuestos C14 y P8 en el modelo ortotópico, inoculando 1 millón de células MIA PaCa-2 directo en el páncreas de ratones cepa Nu/Nu. A los 7 días después de la cirugía se administraron por vía intraperitoneal diferentes tratamientos, vehículo, 30 y 60 mg/kg de C14, 30 y 60 mg/kg de P8, una combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p) y 40 mg/kg de Gemcitabina, donde estos tratamientos se administraron cada 24h durante 15 días (Figura 12 E-H). Como resultado de la evaluación del efecto antineoplásico de los compuestos C14 y P8, se observó una disminución del crecimiento tumoral de manera dosis-dependiente, teniendo un resultado mayor con las diferentes dosis del compuesto P8, disminuyendo el crecimiento tumoral a más del 90%. Por otro lado, en la combinación de ambos compuestos (30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p)) observamos una disminución del 95% del crecimiento tumoral, lo que indica que ambos compuestos tienen un efecto sinérgico en el modelo ortotópico (Figura 12 E); estos resultados se pueden observar con mayor claridad en la Figura 12G donde se muestra una imagen representativa de los tumores y páncreas obtenidos en cada tratamiento. Durante el experimento se realizó la medición del peso de los ratones (Figura 12F), obteniéndose una disminución de más del 20% de peso corporal de los ratones tratados con Gemcitabina por lo que fueron sacrificados, mostrando una disminución del 100% de la supervivencia con Gemcitabina a la mitad del tratamiento; por otro lado el peso corporal de los ratones tratados con los compuestos C14 y P8 no disminuyó y se obtuvo una supervivencia del 100% al terminó del tratamiento (Figura 12H). El efecto antineoplásico obtenido por los compuestos C14 y P8 resultó mejor que el obtenido por Gemcitabina, mientras que el efecto sinérgico obtenido por la combinación de los compuestos C14 y P8 fue aún mucho mejor que el que se observó para los compuestos evaluados individualmente, por lo que las composiciones de la presente invención resultan ser mejores que los compuestos de primera línea que comúnmente se administran en hospitales para el tratamiento de cáncer de páncreas. La combinación de los compuestos C14 y P8 disminuye el crecimiento tumoral en modelos PDX. Para evaluar el efecto antineoplásico de los compuestos C14 y P8 en modelos de xenoinjerto subcutáneo derivado de pacientes, se utilizaron los cultivos primarios MGKRAS004 y MGKRAS005 ya que presentan las mutaciones G12V y G12C las cuales representan a la tercera y segunda más frecuentes en cáncer de páncreas con mayor quimiorresistencia (Figura 14). Injertamos 5 millones de MGKRAS004 en el dorso de ratones Nu/Nu hasta obtener un volumen de 150 mm3, para posteriormente administrar vía intraperitoneal diferentes concentraciones (30 y 60 mg /kg y una combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p), durante 15 días una vez al día, 40 mg/kg de Gemcitabina una vez cada 3 días por 15 días y el vehículo como control (Carboximetil celulosa al 0.05% en PBS con 0.5% de DMSO). Durante los 15 días del tratamiento medimos el volumen de los tumores antes de la inoculación de cada tratamiento (Figura 14A) observándose una disminución significativa del crecimiento tumoral con ambas dosis de tratamiento, tanto del compuesto C14 como del análogo P8 reduciendo su tamaño a más de 90% en las dosis de 30 y 60 mg/kg de C14 y más de 95% con 30 y 60 mg/kg de P8, siendo más evidente a 60 mg/kg de P8; por otro lado con la combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p) observamos una reducción del 98% del crecimiento tumoral empleando el cultivo primario MGKRAS004 con la mutación G12V (Figura 14B), mientras que los tumores tratados con Gemcitabina disminuyeron solo en un 50% su crecimiento; estos efectos se pueden observar con mayor detalle en la Figura 14D donde se muestra una imagen representativa de los tumores. Durante la administración de los diferentes tratamientos se midió el peso de los ratones obteniendo una disminución de más del 20% del peso en los ratones tratados con Gemcitabina. Observando los resultados obtenidos con MGKRAS004, decidimos evaluar el efecto antineoplásico en el modelo PDX empleando el cultivo primario MGKRAS005 el cual presenta la mutación G12C. Injertamos 5 millones de MGKRAS005 en el dorso de ratones Nu/Nu hasta obtener un volumen de 150 mm3, para posteriormente administrar vía intraperitoneal diferentes concentraciones (30 y 60 mg /kg y una combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (30mg/kg de cada uno, proporción 1:1 p/p)) de los compuestos C14 y P8, durante 15 días una vez al día, 40 mg/kg de Gemcitabina una vez cada 3 días por 15 días y el vehículo como control (Carboximetil celulosa al 0.05% en PBS con 0.5% de DMSO). Durante los 15 días del tratamiento se midió el volumen de los tumores tratados con los diferentes tratamientos, observándose una disminución gradual del crecimiento tumoral conforme los días de tratamiento aumentaron hasta obtener la erradicación de los tumores en todas las concentraciones empleadas de los compuestos C14, P8 y la combinación de ambos compuestos (Figura 14E); por otro lado, en los tumores tratados con Gemcitabina no se observó una disminución significativa del crecimiento tumoral, lo cual se muestra en la Figura 14 F-H donde se observa el crecimiento tumoral del último día del tratamiento y las imágenes representativas de los ratones tratados y los tumores obtenidos del estudio. Durante la administración de los diferentes tratamientos se midió el peso de los ratones obteniendo una disminución de más del 20% del peso en los ratones tratados con Gemcitabina. Uno de los grandes problemas que nos atañen hoy en día es la falta de fármacos específicos contra el cáncer. En la actualidad se siguen empleando fármacos descubiertos en los años 60 como el 5-FU y Gemcitabina como la terapia farmacológica más adecuada para el tratamiento de diversos tipos de cáncer, entre ellos el cáncer pancreático. Estos fármacos quimioterapéuticos disminuyen la síntesis de DNA y la síntesis de proteínas, por lo que afectan tanto a las células cancerosas como a las células normales. La presencia de mutaciones en la proteína K-Ras4B en PDAC, es indispensable para el desarrollo, mantenimiento y progresión de esta neoplasia [17]. A raíz de esto han surgido varias estrategias para inhibir la activación de la proteína K-Ras4B en la membrana plasmática. Uno de los compuestos para interferir en la activación de la proteína K-Ras4B en las células cancerosas, es el compuesto denominado Deltarasin y sus análogos los cuales tienen la propiedad de inhibir a la proteína PDE6δ la cual es la proteína trasportadora de K-Ras4B hacia su sitio de activación en la membrana plasmática en las células [20, 26]. Estos compuestos interaccionan con el pocket hidrofóbico de la proteína PDE6δ, la cual es responsable del transporte de esta GTPasa a la membrana plasmática a través del reconocimiento del grupo farnesilo presente en la GTPasa Ras. Una vez que el Deltarasin interactúa con la PDE6δ, le es imposible reconocer al farnesilo presente en el extremo carboxilo de K-Ras4B, evitando así su trasporte hacia la membrana plasmática, donde ejerce su función al permitir la activación de las diferentes vías de señalización celular relacionadas con los procesos oncogénicos. Sin embargo, una de las desventajas del Deltarasin es que puede inhibir una amplia variedad de vías de señalización celular ya que Kras4B no es su único blanco para ser transportado por la PDE6δ [20]. Adicional a este compuesto, en el año 2016 se reportó el análogo del Deltarasin, presentando una mayor especificidad y con una mayor capacidad inhibitoria de la actividad de la proteína PDE6δ, ya que mostró tener un IC50 de 24 µM, siendo 5 veces mayor a la concentración que la reportada para el compuesto líder Deltarasin [23, 26] A pesar del gran progreso en la identificación de nuevas estrategias farmacológicas para el tratamiento del cáncer pancreático, hasta antes de la presente invención no se había logrado identificar compuestos que impidan e impacten la viabilidad celular de las células de cáncer pancreático, ya que el Deltarasin y sus análogos solo retardan el crecimiento tumoral en un 40%. En contraste, en la presente invención se realizó la búsqueda de pequeñas moléculas con posibles propiedades quimioterapéuticas, que mostraron actividad antineoplásica específica sobre las células provenientes de cáncer pancreático con diferentes mutaciones K-Ras4B, donde el compuesto C14 mostró ser uno de los compuestos más prometedores que obtuvimos en nuestros trabajos previos [21], esto debido a su efecto citotóxico sobre las líneas celulares PANC-1 y MIA Paca-2 a una concentración de 103.32 µM y 90.18 µM respectivamente, siendo estas concentraciones menores que las obtenidas por los compuestos D14 y C22 [21, 23]. Cabe destacar que estos compuestos fueron dirigidos no solo a una proteína sino al complejo heterodimérico K-Ras4B/PDE6δ para inhibir el transporte y activación de K-Ras4B en la membrana plasmática. Una de las estrategias usadas por diversos grupos de investigación, es la modificación de los compuestos líder, con el fin de mejorar su efecto citotóxico y aumentar su especificidad, esto mediante la modificación sutil de la estructura primaria de los compuestos líder generando así análogas [27]. En la presente invención se logró identificar al compuesto P8 el cual presenta mayores propiedades citotóxicas sobre las líneas celulares de PDAC con mutaciones en K-Ras4B que el compuesto líder C14, a una concentración cuatro veces menor. Por medio de análisis in silico, el compuesto P8 presentó una mayor afinidad de interacción con los complejos mutados que el compuesto C14. El aumento de la afinidad del compuesto P8 está dado por la presencia de una piperazina, la cual presenta dos grupos amino, aumentando los sitios de interacción con el complejo heterodimérico haciéndolo más estable al interactuar con los complejos WT y mutados de K-Ras4B/PDE6 δ. La presencia de la piperazina en el compuesto P8 le proporciona mayor solubilidad, presentando un coeficiente de partición de 3.99 y una constante de solubilidad de -4.4, por otro lado el compuesto C14 el cual no presenta en su estructura a la piperazina, tiene un coeficiente de partición de 3.63 y un coeficiente de solubilidad de -4.4. El aumento en el coeficiente de partición del P8 con respecto al C14 lo hace aún más soluble y permeable al contacto con la membrana plasmática. Esta permeabilidad que posee el compuesto P8 le permite el fácil acceso a las células blanco y debido a esto presenta un mayor efecto citotóxico sobre las células oncodependientes de K-Ras4B. Como se ha reportado, al igual que los análisis in silico de Deltasinona sobre la PDE6δ, este compuesto mostró un aumento en la afinidad de interacción sobre el pocket de PDE6δ con respecto a su compuesto líder el Deltarasin, debido a la eliminación del grupo funcional fenilbencimidazol, donde la eliminación de este grupo funcional le permitió a la Deltazinona presentar mayor especificidad hacia PDE6δ, donde sus datos in silico fueron corroborados utilizando a la proteína recombinante PDE6d, para la realización de técnicas microcalorimétricas [26]. Uno de los ensayos pendientes por realizar en la presente invención, es la obtención de los valores de afinidad del compuesto C14 y P8 mediante métodos bioquímicos como el BIACOR o por medio de técnicas microcalorimétricas, las cuales nos permitirán confirmar y obtener datos cuantitativos reales, al igual que la espectrometría de masas para identificar todos los posibles blancos de los compuestos C14 y P8. Los efectos citotóxicos obtenidos por los compuestos C14 y P8 sobre las líneas celulares de cáncer pancreático sugieren que dependiendo de la mutación presente en K-Ras4B, las células presentan diferente permeabilidad celular y por ello presentan diferentes IC50 permitiéndonos obtener mayores efectos citotóxicos en las líneas celulares con las mutaciones G12C y G12V las cuales presentan mayor quimiorresistencia en el cáncer pancreático, esto sin afectar a la línea celular no cancerosa. Uno de los hallazgos más importantes en la presente invención, es la especificidad que presenta tanto el compuesto P8 como el C14 para inducir muerte celular por apoptosis en las líneas celulares con adicción oncogénica de K-Ras4B y no sobre las líneas celulares “normales”. Este hallazgo no ha sido reportado para otros compuestos como Deltarasin, sin embargo, en el laboratorio realizamos estudios de citotoxicidad utilizando al Deltarasin sobre líneas celulares “normales”, demostrando que este compuesto afecta más a la línea celular hTERT-HPNE que a la línea celular MIA PaCa-2 [21]. Esto demuestra que los compuestos descritos aquí son más específicos hacia las líneas celulares con K- Ras4B mutado. Como ya fue mencionado, el adenocarcinoma ductal pancreático se caracteriza por la presencia de mutaciones activadoras en K-Ras4B; en la presente invención demostramos que los compuestos C14 y P8 y sus combinaciones son capaces de disminuir la activación de la oncoproteína en líneas celulares de cáncer pancreático sin afectar a la línea celular no cancerosa y por ende disminuir las vías de señalización dependiendo de la adicción oncogénica que cada línea celular presenta hacia K-Ras4B. Por otro lado, Deltarasin disminuyó la activación de K-Ras4B en la línea celular normal implicando la disminución de sus vías de señalización, dejando clara la inespecificidad que presenta este compuesto. La disminución en la activación de K-Ras4B y de sus vías de señalización, dan como resultado la disminución en la proliferación celular, disminución en la síntesis de proteínas, disminución en la transcripción y ciclo celular y un aumento en la muerte celular inducida por apoptosis, esto mostrado en ensayos in vitro. A nivel mundial el cáncer pancreático se ha identificado con mayor frecuencia en hombres de entre 60 a 80 años de edad; en nuestro caso, durante la recolección de las muestras de cáncer pancreático de los pacientes, identificamos PDAC con mayor frecuencia en mujeres de entre 40 a 60 años de edad, siendo 20 años más jóvenes que lo reportado a nivel mundial conforme a varios estudios a nivel nacional por parte de los servicios de salud [34]. Al evaluar los compuestos C14 y P8 sobre los cultivos primarios de cáncer pancreático observamos una mayor susceptibilidad al tratamiento, ya que presentaron concentraciones de IC50 de 4 a 9 veces menores que las obtenidas en las líneas celulares, sin embargo se incrementaron los IC50 de Gemcitabina y de Deltarasin al ser evaluados e nuestros cultivos primarios. Los compuestos C14, su análogo P8 y sus combinaciones disminuyeron considerablemente la activación de K-Ras4B y sus vías de señalización en cultivos primarios de cáncer pancreático sin afectar a los cultivos primarios mesenquimales y tipo fibroblastoides no cancerosos, demostrándonos y reafirmando su especificidad hacia las células de cáncer pancreático. Estos resultados son tan prometedores que podría decirse que es la primera vez que drogas con potencial quimioterapéutico no afectan a las células normales. Cuando dos o más compuestos que individualmente producen efectos similares a veces mostrarán efectos mejorados cuando se administran en combinación, como en el caso de los compuestos C14 y P8 en las líneas celulares y cultivos primarios de cáncer pancreático, se dice que la combinación es sinérgica. Una interacción sinérgica permite el uso de dosis más bajas de los compuestos, situación que puede reducir las reacciones adversas [35, 36]. La combinación de los compuestos C14 y P8 nos permitió encontrar concentraciones de IC50 en un orden de magnitud 9 veces menor que las reportadas por los compuestos individualmente administrados, esto sin inducir muerte por necrosis sino por apoptosis en más de 90%. Las combinaciones de medicamentos son bastante comunes en el tratamiento contra el cánceres, infecciones, dolores y muchas otras enfermedades degenerativas [36, 37]. Uno de los ensayos considerados para comprobar los efectos antineoplásicos del compuesto P8 y del compuesto C14 es la realización de ensayos de tumorogénesis en modelos in vivo, donde uno de los resultados nos alentó a la realización de estos ensayos en la disminución de la capacidad clonogénica del 80% empleando el compuesto C14 y 96% con el compuesto P8 en las líneas celulares y cultivos primarios de cáncer pancreático, siendo esto más evidente con la combinación de C14 y P8 donde la capacidad clonogénica disminuyó en más de 99% en ambos casos. Los modelos preclínicos aprobados por la FDA para la evaluación de drogas con posibles efectos quimioterapéuticos son Xenoinjerto subcutáneo, xenoinjerto ortotópico y xenoinjerto derivado de pacientes, con los cuales se puede observar la influencia del nicho y la heterogeneidad celular presente en cada uno de los modelos [38]. La actividad antineoplásica de los compuestos C14 y P8 disminuyó el crecimiento tumoral en modelos de xenoinjerto Subcutáneo y Ortotópico a medida que la dosis se incrementó, sin inducir efectos adversos ni genotoxicidad (como si lo presenta la quimioterapia de primera línea con Gemcitabina), disminuyen la activación de K-Ras4B y disminuyen los marcadores de malignidad en tumores remanentes. De este mismo modo los compuestos C14 y P8 disminuyen el crecimiento tumoral en modelos PDx de cáncer pancreático, mientras que la combinación de C14/P8 mostró mejores efectos antineoplásicos en los modelos de Xenoinjerto Subcutáneo, Ortotópico y PDx disminuyendo aún más el crecimiento tumoral sin inducir efectos secundarios como los presentados por Gemcitabina. Sin embargo, en un futuro próximo, la farmacocinética y estudios de biodistribución de los compuestos C14 y P8 en ratones será necesario para evaluar su uso terapéutico y así mejorar si distribución mediante el empleo de nanopartículas dirigirás recubiertas con los compuestos para obtener dosis aún más pequeñas con efectos antineoplásicos mayores que los obtenidos. Todos estos resultados muestran el gran potencial quimioterapéutico de los compuestos evaluados en la presente invención. La evaluación antineoplásica de estos compuestos demostró su especificidad hacia líneas celulares y cultivos primarios de cáncer pancreático, esto sin afectar a líneas celulares y cultivos primarios no cancerosos, disminuyendo la activación de K-Ras4B y sus vías de señalización, donde estas actividades antineoplásicas mejoraron con el efecto sinérgico antineoplásico de las combinaciones de los compuestos C14 y P8 erradicando casi por completo la presencia de tumores de cáncer pancreático en modelos preclínicos sin presentar efectos adversos ni genotoxicidad. Los compuestos C14 y P8 y su combinación son nuevas alternativas farmacológicas contra el cáncer pancreático con mejores propiedades que la quimioterapia convencional. Los siguientes ejemplos se incluyen con la única finalidad de ilustrar la presente invención, sin que ello implique limitaciones del alcance de la misma. Ejemplo 1. Simulación de acoplamiento in silico. Identificamos la estructura química de C14 y P8 usando la base de datos ENAMINE 3D Diversity set (www.enamine.net) acoplando a la región de interfaz del complejo molecular KRas4B-PDE6δ usando AutoDock 4.2.626 [19] y MOE Dock [20]. Los cálculos de acoplamiento se llevaron a cabo utilizando los ajustes de parámetros estándar recomendados. Evaluamos un máximo de 250,000 poses para C14 en el receptor objetivo (contactos cristalográficos entre KRas4B con PDE6δ, de PDB ID 5TAR). Las rejillas se calcularon utilizando Autogrid 4.2.626626 [19] con un espaciado de 0.375 Å centrándose en la interfaz de las proteínas cristalizadas. El acoplamiento molecular con MOE 2014.09 [20] se realizó utilizando la función de emparejamiento para generar las poses iniciales. Los mejores 30 resultados de la puntuación dG de Londres se refinaron aún más utilizando la minimización de energía con el campo de fuerza MMFF94x y se volvieron a puntuar utilizando la puntuación Affinity dG. Ejemplo 2. Simulación de dinámica molecular. La simulación MD del complejo proteína-ligando se realizó utilizando el paquete AMBER 16 [21] y el campo de fuerza ff14SB [22]. Las cargas de ligando para residuos no parametrizados en proteínas se determinaron utilizando el nivel de AM1-BCC y el campo de fuerza general Amber (GAFF) [23] para el complejo proteína-ligando, una caja de forma rectangular de 15 Å del modelo de agua TIP3P [24] se aplicó para solvatar el complejo; y los iones Cl- y Na+ para el sistema proteína-ligando se colocaron en el modelo para neutralizar las cargas positivas o negativas alrededor del complejo a pH 7. Antes de la simulación MD, el sistema se minimizó mediante 3000 pasos de minimización de descenso más pronunciado seguidos de 3000 pasos de minimización del gradiente conjugado. Luego, el sistema se calentó de 0 a 310 K durante 500 picosegundos (ps) de MD con restricciones de posición bajo un ensamble NVT, sucesivamente se llevó a cabo un ensamble isobárico isotérmico (NPT) de MD durante 500 ps para ajustar la densidad del solvente seguido de 600 ps de equilibrio de presión constante a 310K usando el algoritmo SHAKE [25] en átomos de hidrógeno y dinámica de Langevin para el control de temperatura. La corrida de equilibrio fue seguida por una simulación MD de 100 ns de longitud sin restricciones de posición bajo condiciones de contorno periódicas usando un ensamble NPT a 310K. Se utilizó el método de Ewald de malla de partículas para describir el término electrostático [26], y se utilizó un límite de 10 Å para las interacciones de van der Waals. La temperatura y la presión se conservaron utilizando el algoritmo de acoplamiento débil [27] con constantes de acoplamiento τT y τP de 1.0 y 0.2 ps, respectivamente (310 K, 1 atm). El tiempo de la simulación MD se fijó en 2.0 femtosegundos y se utilizó el algoritmo SHAKE [25] para restringir las longitudes de los enlaces en sus valores de equilibrio. Las coordenadas se guardaron para análisis cada 50 ps. Se utilizó AmberTools14 para examinar la dependencia del tiempo de la desviación cuadrática media de la raíz (RMSD), el radio de giro (RG) y el análisis de agrupamiento para identificar las conformaciones más pobladas durante el tiempo de simulación equilibrado. Ejemplo 3. Cálculo de energías libres de enlace. El cálculo de las energías libres de enlace se llevó a cabo utilizando el enfoque MMGBSA [28-30] proporcionado en la suite AMber16 [21]. Se eligieron 500 instantáneas a intervalos de tiempo de 100 ps de los últimos 50 ns de simulación MD utilizando una concentración de 0.1 M y el Modelo de solvente implícito de Born generalizado (GB) [31]. La energía libre de enlace del sistema proteína-ligando se determinó de la siguiente manera:
Figure imgf000025_0001
donde ΔEMM representa la energía total del campo de fuerza mecánica molecular que incluye las energías de interacción electrostática (ΔEele) y van der Waals (ΔEvdw). ΔG solvatación es el precio de la energía libre de desolvatación sobre la formación del complejo estimado a partir del modelo implícito de GB y los cálculos del área de superficie accesible al solvente (SASA) que producen ΔGele.sol y ΔGnpol.sol. TΔS es la entropía de soluto que surge de los cambios estructurales que ocurren en los grados de libertad de los solutos libres al formar el complejo proteína-ligando. Ejemplo 4. Selección de análogos del compuesto C14. Para la selección de los análogos del compuesto C14 se utilizó una lista de compuestos de la empresa ENAMIN, la cual tiene una lista de 335 compuestos derivados de C14. Los posibles análogos se analizaron mediante el programa de bioinformática Molecular Operating Environment (MOE), 2014.09, realizando pruebas de minimización de energía, similitud estructural y búsqueda de farmacóforos. Ejemplo 5. Cultivo de células. La línea celular de cáncer de páncreas humano MIA PaCa-2, PANC- 1, Capan-1, la línea celular pancreática humana hTERT-HPNE y la línea celular de retina humana ARPE-19, se obtuvieron de la American Type Culture Collection (ATCC; Manassas, VA). Las líneas celulares se cultivaron como monocapas en el medio específico sugerido por la ATCC. Ejemplo 6. Viabilidad celular. Las líneas celulares y cultivos primarios ARPE-19, hTERT-HPNE, PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004 y MGKRAS005 se sembraron en placas de 96 pocillos a una densidad de 3x104 células/pocillo y se cultivaron durante 24 h. A continuación, las células se trataron con C14 o P8 durante 72 h en medio completo. Para evaluar la viabilidad, las células se analizaron con el kit CellTiter-Glo (Promega, Madison, WI) de acuerdo con las instrucciones del fabricante. La concentración de C14 y P8 que mató al 50% de todas las células después de 72 h (IC50) se determinó aplicando análisis de ajuste de curvas con el software Prism (GraphPad Software, San Diego, CA, EE. UU.). Ejemplo 7. Ensayo clonogénico. Las líneas celulares de cáncer de páncreas se sembraron en placas de 6 pocillos a una densidad de 300 células por pocillo y se cultivaron durante la noche. Líneas celulares y cultivos primarios PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004 y MGKRAS005 fueron tratados con concentraciones finales de 0.496 µM de gemcitabina (Laboratorios PiSA, México), la concentración IC50 de C14 y P8 para 72 h, y Deltarasina 5 µM. Posteriormente, el medio se reemplazó con medio fresco suplementado cada tercer día durante un total de 10 días. Las células se fijaron con paraformaldehído (PFA) al 4% a temperatura ambiente durante 10 min y se lavaron con agua destilada. Las células se tiñeron con cristal violeta al 0.1% en ácido cítrico 0.1 M durante 30 min, se lavaron con PBS 1X, se secaron y fotografiaron. Para la cuantificación, se añadió 14% de ácido acético durante 20 min para extraer el colorante y se midió la absorbancia fotométricamente a 500 nm utilizando un Fluorómetro TECAN Infinite F500 (Tecan Austria GmbH). Ejemplo 8. Ensayo de apoptosis. Se sembraron aproximadamente 5 x 105 células en placas de 6 pocillos durante 24 h. Luego, las células se trataron con una concentración IC50 de C14 y P8 y vehículo durante 24 h. Las células se cosecharon con tripsina al 0.25%, se lavaron con solución salina tamponada con fosfato (PBS) y se recogieron juntas por centrifugación. La apoptosis se determinó utilizando el kit de detección de apoptosis/necrosis (Abcam, número de catálogo ab176749, Cambridge, Inglaterra) de acuerdo con las instrucciones del fabricante y se analizó mediante un citómetro de flujo en un instrumento FACSCalibur (BD Biosciences) seguido de un análisis de datos utilizando el software FlowJo (Tree Star Inc). Todos los experimentos fueron realizados por triplicado. Ejemplo 9. Ensayo del nivel de activación de RAS. Para la evaluación del nivel de activación de RAS después del tratamiento con Deltarasin, Gemcitabina, C14 y P8, se realizó el ensayo pull-down RAS-GTP mediante Western blot, aplicando el RAS Activation Assay Biochem Kit (BK008; Cystoskeleton, Inc.; Denver, CO), según el procedimiento estándar. Brevemente, se cultivaron células hTERT-HPNE, PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004 y MGKRAS005 para tener 3x106 células, y se lisaron en tampón de lisis helado (400 µL) suplementado con cOmplete™ Cóctel inhibidor de ultra proteasa sin EDTA y 1xPhosSTOP™ (Sigma-Aldrich). Los lisados se centrifugaron y se recogió la proteína (300 µg). Los lisados se incubaron mediante rotación de un extremo a otro con 100 µg de perlas conjugadas con Raf-RBD durante 1 h. Se eliminó el sobrenadante, y las perlas se lavaron y se hirvieron en tampón de muestra 2X Laemmli, seguido de análisis de transferencia Western, usando el anticuerpo pan-Ras. Ejemplo 10. Western blot. Las líneas celulares se privaron de suero durante 16 horas y se pre- trataron con una concentración IC50 de C14, P8, gemcitabina y deltarasina durante 3 horas después del pretratamiento. Las células se estimularon con factor de crecimiento epidérmico a 100 ng/ml durante 10 min. Los extractos de células enteras se obtuvieron mediante lisis de células PANC-1 Capan-1, MGKRAS003, MGKRAS004 y MGKRAS005 en tampón de lisis [Tris-HCl 20 mM (pH 7.5), EDTA 1 mM, NaCl 150 mM, Tritón X-100 al 1%, NaVO31 mM, NaF 1 mM, β-glicerofosfato 10 mM, fluoruro de fenilmetilsulfonilo 1 mM y 1.2 mg/ml del coctel de inhibidor de proteasa completeTM Lysis- M (Roche, Mannheim Alemania). Los extractos de proteínas se forzaron 10 veces a través de una aguja calibre 22 y se centrifugaron durante 10 min a 14,000 rpm a 4°C, y la concentración de proteínas se determinó mediante el kit PierceTM BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, EUA). Las muestras de tejido se pesaron, se congelaron rápidamente y se trituraron en nitrógeno líquido en un mortero. Las muestras se transfirieron a un tubo de microcentrífuga y se lisaron usando el reactivo de lisis de células de mamíferos ProteoJETTM, para después centrifugarse a 2,.000 x g durante 15 min y cuantificación de proteínas. Se llevó a cabo SDS-PAGE usando 30 µg de proteína de cada muestra. Las proteínas se transfirieron a membranas de PVDF (Merck Millipore) y se bloquearon durante 1 h. a temperatura ambiente usando PBS que contenía leche desnatada al 5%. Luego se incubó con los siguientes anticuerpos primarios: Total ERK (Cell Signaling-9102; 1:1000), pERK (Cell Signaling-9101; 1:1000), Total Akt (Cell Signaling-92721:1000), pAKT (Cell Signaling- 9272; 1:1000) Signaling-4060 1:1000) y anti-GAPDH (Gene Tex-GTX100118 1:100,000). La inmunodetección se realizó utilizando un sistema de imágenes ChemiDocTM (BIO-RAD). El análisis de densitometría se realizó con el software ImageJ versión 1.45 (National Institute of Health, EUA). Ejemplo 11. Tratamiento de xenoinjertos subcutáneos de carcinoma pancreático. Se mantuvieron ratones desnudos macho inmunodeficientes Nu/Nu a las 6 semanas de edad (CINVESTAV, México) en condiciones libres de patógenos con pienso irradiado. Los animales se inyectaron por vía subcutánea en el torso con 5x106 células MIA PaCa-2 por tumor en 0.2 ml de medio matrigel DMEM con alto contenido de glucosa. Cuando las células MIA PaCa-2 alcanzaron tumores palpables (>150 mm3), los ratones se dividieron aleatoriamente en cuatro grupos que recibieron vehículo [DMSO al 10%, 0.05% carboximetilcelulosa en PBS] (n=6), o C14 y P8 a 60, 30, 10 y 5 mg/kg (n=6), una combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (n=6), y Gemcitabina 40 mg/kg (n=6), administrados mediante inyección intraperitoneal una vez al día durante 15 días. El peso corporal y el tumor se midieron una vez al día. El tamaño del tumor se calculó mediante la siguiente fórmula: [(largo x ancho2)/2] en mm. Ejemplo 12. Tinción inmunohistoquímica de tumor de xenoinjerto. Un día después del último tratamiento, los ratones se sacrificaron en una cámara de CO2 y los tumores del xenoinjerto se resecaron, se fijaron en formalina tamponada al 4% y se embebieron en parafina. Los tumores se cortaron con un microtomo obteniendo cortes de 2 µm. Para la tinción con hematoxilina y eosina (H & E), los tejidos se desparafinizaron en xileno, se hidrataron en alcohol deshidratado partiendo de etanol absoluto a agua destilada, se tiñeron durante 2 minutos con hematoxilina Harris, se decoloraron con alcohol ácido al 0.5% y se fijaron para color en carbonato de litio durante 1 min, lavado en agua destilada, en etanol al 96% y teñido con Sigma Eosin, lavado y deshidratado en cambios graduales de alcohol hasta alcanzar alcohol absoluto, dejado secar a temperatura ambiente, montado y observado, para identificar el sitio de la lesión. Para la tinción inmunohistoquímica, los tejidos fueron desparafinizados en xileno, hidratados en alcoholes empobrecidos partiendo de etanol absoluto a agua destilada, los epítopos fueron desenmascarados con Tampón Citrato 10 mM de pH 6.03 en el Tender Cocker para lavado posterior con PBS pH 7.4; la peroxidasa endógena fue bloqueada con H2O2 al 0.9% durante 15 min, se bloqueó con BSA al 3% durante 1 h, mientras que los anticuerpos Ki-67 (BIOCARE MEDICAL API 3156 AA), CK 19 (GENETEX GTX110414) y CA125 (BIOCARE MEDICAL CM 101 CK) se diluyeron con PBS al 1% y BSA al 1%, donde el anticuerpo primario se incubó a temperatura ambiente durante 40 min, se lavó con PBS durante 3 min, se incubó con el anticuerpo secundario biotinilado durante 20 min a temperatura ambiente, se lavó con PBS durante 3 min, se incubó con estreptavidina durante 15 min y se lavó con PBS durante 3 min. Las reacciones se revelaron con 4% de diaminobencidina (DAB) monitoreando cada reacción bajo microscopio, para lo cual se contratiñeron con Hematoxilina de Harry 30 segundos, se lavaron con agua destilada, se deshidrataron en cambios graduales de etanol desde agua destilada a etanol absoluto, se dejó secar a temperatura ambiente, se montó y se observó. Ejemplo 13. Cultivos primarios de cáncer de páncreas de pacientes con PDAC. Los tejidos de cáncer de páncreas fueron provistos por el Hospital Regional 1º. de Octubre del Instituto de Seguridad y Servicios Sociales para los Trabajadores del Estado (ISSSTE) en el marco del proyecto 002.2015 en la Ciudad de México. Los tejidos se colectaron en el quirófano de dicho hospital, se colocaron en medio de transporte (medio base DMEM sin suero fetal bovino y antibiótico al 5%), manteniendo siempre el medio a 4°C. Se seccionó el tejido hasta obtener fragmentos de 3mm3, que se colocaron en placas de 6 pocillos con medio DMEM alto en glucosa al 20%, suero bovino fetal al 80% y antibiótico al 3%, esto hasta obtener células del tumor adheridas a la placa. El porcentaje de suero disminuyó hasta que las células pudieron sobrevivir con un 10% de suero y un 1% de antibiótico. Ejemplo 14. Modelo de xenoinjerto subcutáneo derivado de pacientes. Se mantuvieron ratones desnudos macho inmunodeficientes Nu/Nu a las 6 semanas de edad (CINVESTAV, México) en condiciones libres de patógenos con pienso irradiado. Los animales se inyectaron por vía subcutánea en el torso con 5x106 células de cultivo primario MGKRAS004 y MGKRAS005 por tumor en 0.2 ml de medio matrigel DMEM con alto contenido de glucosa. Cuando las células de los cultivos primarios alcanzaron tumores palpables (>150 mm3), los ratones se dividieron aleatoriamente en cuatro grupos que recibieron vehículo [DMSO al 10%.0.05% de carboximetilcelulosa en PBS] (n=6), C14 o P8 a 60, 30 mg/kg (n=6), combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (n=6) y gemcitabina 40 mg/kg (n=6), administrados mediante inyección intraperitoneal una vez al día durante 15 días. El peso corporal y el tumor se midieron una vez al día. El tamaño del tumor se calculó mediante la siguiente fórmula: [(largo x ancho2)/2] en mm. Ejemplo 15. Modelo de xenoinjerto ortotópico en ratones Nu/Nu. Se mantuvieron ratones desnudos macho inmunodeficientes Nu/Nu a las 6 semanas de edad (CINVESTAV, México) en condiciones libres de patógenos con pienso irradiado. Los ratones fueron anestesiados y sedados con xilazina y ketamina. El bazo del ratón se ubicó en el lado izquierdo, posteriormente se hizo una incisión de 0.5 cm en la piel y el peritoneo, se extrajo el bazo, permitiendo la visualización de las células MIA PaCa-2 después de que se inocularan 1 millón de células en 50 µl de medio mínimo esencial libre de suero y sin rojo fenol directamente en el páncreas. Los órganos se reubicaron dentro del ratón y el peritoneo, y la piel se suturó con sutura autoabsorbente. Los ratones se dividieron aleatoriamente en cuatro grupos que recibieron vehículo [DMSO al 10%. 0.05% de carboximetilcelulosa en PBS] (n=6), o C14 y P8 a 60, 30 mg/kg (n=6), combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (n=6) y gemcitabina 40 mg/kg (n=6), administrados mediante inyección intraperitoneal una vez al día durante 15 días. Se realizó una necropsia completa para obtener órganos para el estudio. Ejemplo 16. Inmunofluorescencia celular. Las células se cultivaron en cubreobjetos en placas de 24 pocillos hasta alcanzar la confluencia deseada, se fijaron con paraformaldehído durante 20 min a 37°C, posteriormente se lavaron con 1X PBS y se permeabilizaron con metanol/acetona 1:1 o tritón X100 al 0.2% durante 10 min en cámara húmeda, se lavaron y la autofluorescencia se bloqueó con NH4Cl 50 µM durante 20 min a 37°C; la proteína indeseable se bloqueó con BSA al 2% durante 30 min a 37°C, se incubó el conjunto con el anticuerpo primario durante la noche a 4°C o 90-60 min a 37°C, para posteriormente lavar e incubar el anticuerpo secundario marcado con fluorocromo durante 30-60 min a 37°C, para finalmente lavar y colocar en vectashielDapi. Ejemplo 17. Inmunofluorescencia tisular. Los tejidos se desparafinizaron en xileno, se hidrataron en alcoholes degradados partiendo de etanol absoluto a agua destilada, se desenmascararon los epítopos con Tampón Citrato 10 mM pH 6.03 en Tender Cocker, se lavaron con PBS pH 7.4, se bloqueó la peroxidasa endógena con 0.9% H2O2 (disminuye la autofluorescencia de eritrocitos) durante 5 min, se redujo la autofluorescencia con NH4Cl 0.05M durante 30 min a 37°C y se lavó con PBST tres veces; el anticuerpo primario se diluyó con PBS al 1% y BSA al 1%, de esta manera el sitio de unión no específico fue bloqueado, mientras que el anticuerpo primario (Sup M 1) se incubó a temperatura ambiente durante 60 min, para posteriormente realizar lavados con PBS durante 3 min, incubar con el anticuerpo secundario marcado con fluorocorm durante 40 min a temperatura ambiente, y lavar con PBS durante 3 min. Los núcleos se marcaron con DAPI y las muestras se analizaron con un microscopio confocal. Ejemplo 18. Extracción de ADN. Se extrajo ADN genómico de muestras humanas diagnosticadas con cáncer de páncreas (MGKRAS-003 a MGKRAS-005) de tejido congelado con el kit miniprep GenElute Mammalian Genomic DNA (Sigma-Aldrich G1N70). Ejemplo 19. PCR y secuenciación. La PCR se realizó con aproximadamente 60 ng de ADN hibridado utilizando los primers sentido y antisentido siguientes a una concentración de 10 pmol: Sentido: RASO15´-AAGGCCTGCTGAAAATGAC-3´, Antisentido: RASA25´-TGGTCCTGCACCAGTAATATG-3. La PCR se realizó en un termociclador TC-512 TECHNE con 20 ciclos de PCR de punto final (temperatura de alineación inicial de 65°C, disminuyendo 0.5°C por ciclo) y 15 ciclos a 55°C de temperatura de alineación. Los productos de la PCR se purificaron con el kit QIAprep Miniprep QIAGEN. Los productos de PCR purificados se secuenciaron en la dirección inversa. Ejemplo 20. Ensayo de genotoxicidad. Para la evaluación del efecto genotóxico de C14 y P8, se llevó a cabo el ensayo de micronúcleos con células de médula ósea según el método descrito anteriormente. Los compuestos bajo ensayo se administraron por vía intraperitoneal una vez, como solución (a una concentración de 40 mg/kg de gemcitabina (n: 5), 60 mg/kg de C14 y P8 (n: 5) y una combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (n: 5) y vehículo (n: 5)) y usando ratones sin tratamiento como control (n: 5). Se obtuvieron células de la médula ósea 24 h y 15 días después del tratamiento y se tiñeron con Giemsa-Wright (Diff-Quick; Harleco; Gibbstown, Nueva Jersey). Se contaron dos mil eritrocitos policromáticos por animal usando un microscopio óptico a 100 aumentos para determinar el número de eritrocitos policromáticos micronucleados. Ejemplo 21. Ensayo de toxicidad. Para la determinación de los pseudoefectos de los compuestos C14 y P8, la determinación se llevó a cabo en ratones BAlbc. Se administraron gemcitabina 40 mg/kg (n: 5), C14 y P860 mg/kg (n: 5), la combinación de 30 mg/kg de C14 + 30 mg/kg de P8 (n: 5) y vehículo (n: 5) una vez al día durante 15 días y usando ratones sin tratamiento como control (n: 5). Con asistencia veterinaria se realizó la necropsia, extracción de sangre y orina. Se realizó la química sanguínea con el equipo cobas c111 Roche, la biometría hemática con el equipo Xp300 Sysmex, así como un examen general de orina. Ejemplo 22. Análisis estadístico. Las comparaciones estadísticas se realizaron con un análisis de varianza de una vía (ANOVA), seguido de la prueba de comparaciones múltiples de Dunnett, utilizando el software GraphPad Prism 5.0. Los datos se muestran como media ± SEM. Se consideró estadísticamente significativo un valor de p<0.05. Referencias. 1. Soreide K, et.al. Cancer Lett 2015, 356(2 Pt A):281-288. 2. Vinay Kumar AA, et.al. Robbins and cotran pathologic basis of disease, Professional Edition: Saunders; 2009. 3. 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Cancer Chemother Pharmacol 2018, 82(3):511-519. 20. Zimmermann G, et.al. Nature 2013, 497(7451):638-642. 21. Casique-Aguirre D, BMC Cancer 2018, 18(1):1299. 22. Saliani M, et.al. Cancer Biol Med 2019, 16(3):435-461. 23. Briseño-Díaz P, et.al. KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex for the Identification and Evaluation of Nontoxic Pharmacological Compounds for the Treatment of Pancreatic Cancer. In: Pancreatic Cancer [Working Title]. edn.; 2020. 24. Baniak N, et.al. World Journal of Surgical Oncology 2016, 14(1):212. 25. Cheng J, et.al. J Med Chem 2020, 63(14):7892-7905. 26. Papke B, et.al. Nat Commun 2016, 7:11360. 27. Chuang HC, et.al. Pharmacol Res 2017, 117:370-376. 28. Simanshu DK, et.al. Cell 2017, 170(1):17-33. 29. Martin-Gago P, et.al. Angew Chem Int Ed Engl 2017, 56(9):2423-2428. 30. Chen D, et.al. Eur J Med Chem 2019, 163:597-609. 31. Siddiqui FA, et.al. ACS Omega 2020, 5(1):832-842. 32. Dharmaiah S, et.al. Proc Natl Acad Sci U S A 2016, 113(44):E6766-E6775. 33. Cox AD, et.al. Nature Reviews Drug Discovery 2014, 13:828+. 34. Gonzalez-Santiago O, et.al. Ecancermedicalscience 2017, 11:788. 35. Tallarida RJ. Genes Cancer 2011, 2(11):1003-1008. 36. Chou TC: Cancer Res 2010, 70(2):440-446. 37. Hackman GL, et.al. Cancers (Basel) 2020, 12(12). 38. Kim MP, et.al. Nat Protoc 2009, 4(11):1670-1680. Synergistic compositions of compounds against the K-Ras4B/ PDE6δ heterodimeric complex for the treatment of pancreatic cancer Field of the invention. The present invention relates to compositions with pharmaceutical activity that are useful for the treatment of diseases, particularly with pharmaceutical compositions for the treatment of pancreatic cancer, more particularly with pharmaceutical compositions that exhibit synergistic effects to reduce tumor growth, without generating adverse effects. and that comprise compounds against the K-Ras4B/PDE6δ heterodimeric complex, decreasing the activation of K-Ras4B. Background of the invention. Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers, due to its high rate of dissemination and resistance to chemotherapy. Survival of patients with PDAC is approximately 3 to 6 months in more than 95% of cases [1-3]. Approximately 99% of PDAC cases present activating mutations in the K-RAS oncogene, where these mutations correlate with the oncogenic addiction that PDAC presents for its survival, antiapoptosis and chemoresistance, which is why said activating mutations are considered as the factor most important for the progression and maintenance of pancreatic ductal adenocarcinoma [4-6]. Activating mutations in K-Ras4B consist mainly of substitutions at amino acid residues G12, G13 and Q61 of this protein, which impair GAP protein-mediated intrinsic GTP hydrolysis. These mutations induce aberrant activation of K-Ras4B, resulting in continued activation of K-Ras4B-dependent signaling pathways such as AKT and ERK [5, 7, 8]. Currently in Mexico the first-line treatment for PDAC are DNA synthesis inhibitors such as Gemcitabine, 5-FU and Oxaliplatin, which generate side effects in patients, such as elevated liver enzymes, leukopenia, neutropenia, venous collapse, pain and loss of bone mass [9]. After the findings on the presence of K-Ras4B mutations and their importance in the formation, maintenance and progression of the most deadly neoplasms such as PDAC [10, 11], studies have been carried out to discover and develop pharmacological inhibitors. against activation of oncogenic K-Ras4B [10, 11]. The search for new pharmacological alternatives directed against K-Ras4B to reduce its activation and thus improve the quality of life of patients with PDAC has taken about 40 years, where monoclonal antibodies such as Ramucirumab [12], Matuzumab [13], Trastuzumab [14] and Figitumumab [15], which target transmembrane receptor tyrosine kinases to decrease K-Ras4B activation, presenting 9 to 40% inhibition of K-Ras4B activation in pancreatic ductal adenocarcinoma [ 12]. In order to find an organic compound that was capable of inhibiting the activation of K-Ras4B, studies were carried out to direct a specific molecule towards the localization site of the K-Ras4BG12C mutation, which is the most frequent in breast cancer. lung [16]. One of the compounds studied was called SCH-54292 [17], which is capable of binding to the α2 and α3 helices of K-Ras4B, where this compound presents activity only in cell lines that present K-Ras4BG12C; With this finding, the researchers propose to identify and study SCH-54292 analogues that exhibit a greater cytotoxic effect in cancer cell lines [17]. Another group of researchers created a GDP analog called SML-8-73-1, which could covalently bind to the cysteine of K-Ras4BG12C, without taking into account the affinity of GDP with its binding site on K-Ras4B. [16]; In 2020, it was possible to obtain a compound directed to the K-Ras4B mutation site, which was called Sotorasib, presenting more than 70% inactivation of K-Ras4B in cell lines and in murine models, with these results Sotorasib was is in clinical phase-I [18, 19]. Compounds capable of blocking the transport of K-Ras4B to the plasma membrane have been identified as Deltarasin, which interacts with PDE6δ with a Kd of 38 nM, and prevents the recognition of the post-translational modification present in K-Ras4B, which would concentrate K-Ras4B in the cytosol, thus preventing its activation and tumor progression; this compound was named as the first generation of PDE6δ inhibitors [20]. However, this compound was evaluated in non-cancerous cell lines of the pancreatic duct and a high cytotoxicity was observed, considerably affecting cell viability at low concentrations [21-26]. In 2016, the analogue of the Deltarasin compound was reported, which was called Deltazinone, presenting a dissociation constant of Kd 38 nM to Kd 4 nM, proving to be a compound with better interaction energy. than the first generation. Deltazinone showed cytotoxic effects on pancreatic cancer cell lines at a concentration of 24 µM, however it took around 8 h to have an anti-proliferative effect on pancreatic cancer cell lines, while Deltarasin at a concentration of 5 µM in one hour showed the same effect as its analogue, so considering these data, the first generation of PDE6δ inhibitors have a greater effect than the second generation [27, 28]. In 2017, other analogues to Deltarasin called Deltasonamides were reported, which present higher interaction energy than the first generation, greater cytotoxic effects in pancreatic cancer cell lines at concentrations of 1 to 12 µM [29, 30]. At the beginning of 2020, new analogues called Deltaflexin appear, however despite the efforts to find analogues with greater properties than Deltarasin, they have not presented the same cytotoxic effects as the first generation of PDE6δ inhibitors [31]. Transport of the K-Ras4B protein is mediated by the PDE6δ protein from the endoplasmic reticulum to the plasma membrane for subsequent activation, thus forming the K-Ras4B/PDE6δ heterodimeric complex in the cytoplasm. [23, 32, 33]. This transport mechanism represents a great opportunity to target compounds that can stabilize the heterodimeric complex in the cytoplasm and thus prevent activation of K-Ras4B at the plasma membrane [23, 32, 33]. K-Ras4B/PDE6δ was thought to be transported as a dimer and is now known to form a cluster of 6-12 proteins or 3-6 dimers [32]. Due to this, our working group searched for a model of the heterodimer using the crystal of the heterodimeric complex in a group of 6 proteins, obtaining a representative dimer of the K-Ras4B/PDE6δ multiprotein heterocomplex, finding two compounds called D14 and C22 that bind and stabilize the K-Ras4B/PDE6δ heterodimeric complex [23]. The in vitro and in vivo evaluation of compounds D14 and C22 demonstrated a specific cytotoxic effect in pancreatic cancer cell lines with K-RAS mutations, decreasing cell viability, inducing death by apoptosis and decreasing the activation of K-Ras and its signaling pathways such as AKT and ERK by 50% [21]. On the other hand, these compounds reduced tumor growth by 50% compared to vehicle, as the doses of D14 and C22 increased [21]. Analyzing our previous results, we decided to evaluate one compound out of the 38 compounds evaluated in our first stabilization study of the K-Ras4B/PDE6δ heterodimeric complex [21]. Compound C14 presented greater cytotoxic activity at 200 µM than compounds D14 and C22, it has been shown that by making subtle modifications in the primary structure of the leading compounds, the chemical properties and their biological activity can be improved, for which the search for C14 analogue could present greater activity than its parent compound. Due to the above and in view of the solutions mentioned, they are not entirely sufficient for an effective treatment for pancreatic cancer, there is still a need for better solutions and at the same time do not generate adverse effects in the patient under treatment. . Brief description of the invention. Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis of all human cancers, as it is highly resistant to chemotherapy. This leads to the search for new pharmacological alternatives to improve the quality of life of patients with pancreatic cancer. Compounds have been designed that can inhibit the signaling and transport pathway of the K-Ras4B oncoprotein. Taking into account that the interaction of KRas4B with PDE6δ is essential for its transport and subsequent activation in the plasma membrane, in the present invention the compound C14 and its analogue P8 were identified and evaluated in preclinical models, which could stabilize the KRas4B heterodimeric complex. /PDE6δ. The analog called P8 presents higher interaction energy on the mutated complexes in silico, thus presenting greater cytotoxic effects in cell lines and primary cultures of pancreatic cancer with mutated K-Ras without damaging the cell line and non-cancerous primary cultures. . In the same way, both compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways, however the compound P8 presents an IC504 times lower than the compound C14, being up to now the best compound found in our research group. . On the other hand, compositions that comprise the combination of the C14/P8 compounds present a synergistic effect, inducing cytotoxicity in cell lines and primary cultures of pancreatic cancer that are greater than those obtained by the compounds evaluated individually. The compositions of the invention that comprise the combination of compounds C14 and P8 decrease tumor growth from 90 to 95% in subcutaneous and orthotopic xenograft models as the dose increases, do not induce adverse effects or genotoxicity as presented by first-line chemotherapy with Gemcitabine, decrease K-Ras4B activation and decrease malignancy markers in remnant tumors. In the same way, the compositions of the invention that comprise the combination of compounds C14 and P8 decrease up to 99% of tumor growth in PDx models of pancreatic cancer. The compositions of the invention showed better antineoplastic effects in the Subcutaneous Xenograft, Orthotopic and PDx models, decreasing tumor growth by up to 97% without inducing side effects such as those presented by Gemcitabine. Compounds C14 and P8 are new chemotherapeutics with better properties than conventional chemotherapy. Brief description of the figures. Figure 1. C14 stabilizes the K-Ras4B/PDEδ complex and inhibits the growth of human pancreatic cancer cell lines. A) interaction of the C14 compound (red) with the K-Ras4Bwt (violet) PDE6δ (blue) complex is observed; B) interaction of compound C14 with the K-Ras4BG12D/PBDE6δ complex; C) interaction of compound C14 with the K-Ras4BG12C/PBDE6δ complex; D) Representative bright field images of the cell lines ARPE-19, hTERT-HPNE, PANC-1 and MIA PaCa-2 treated with C14100 µM, DMSO as vehicle and untreated control cells. Bar=20 µm; E) Relative cell viability after treatment of PDAC MIA PaCa-2, PanC-1 cells and the normal pancreatic cell line hTERT-HPNE with different concentrations of C14 for 72 h.; F) percentage of viability of PDAC cell lines at 72 h.; G–H) Relative cell viability after treatment of the PDAC MIA PaCa-2 cell line (G) and the normal pancreatic cell line hTERT-HPNE (H) with different compounds known to target the K-Ras4B/PBDE6δ complex (n =5). Figure 2. Evaluation and identification of analogues of compound C14. A-B) Evaluation of cell viability after treatment with P1, P2, P3, P4 and P5 analogues of C14 at 90.18 µM in MIA PaCa-2 (A) and hTERT-HPNE (B) cells are observed; C-D) evaluation of cell viability after treatment with P6, P7, P8, P9 and P10 analogues of C14 at 90.18 µM in MIA PaCa-2 (C) and hTERT-HPNE (D) cells; E-F) evaluation of cell viability after treatment with P11, P12, P13, P14 and P15 analogues of C14 at 90.18 µM in MIA PaCa-2 (E) and hTERT-HPNE (F); G-H) Evaluation of cell viability after treatment with P16, P17, P18, P19 and P20 analogues of C14 at 90.18 µM in MIA PaCa-2 (G) and hTERT-HPNE (H). Figure 3. P8 stabilizes the K-Ras4B/PDEδ complex and inhibits the growth of PDAC cell lines better than C14. A) interaction of compound P8 (dark blue) with the K-Ras4Bwt (pink)/PDE6δ (blue) complex is observed; B) interaction of P8 with the K-Ras4BG12D/PBDE6δ complex; C) interaction of P8 with the K-Ras4BG12C/PBDE6δ complex; D) interaction of P8 with the K-Ras4BG12V/PBDE6δ complex; E-H) relative cell viability of PDAC PANC-1, MIA PaCa-2 and Capan-1 cells and the normal pancreatic cell line hTERT-HPNE treated with different concentrations of P8 for 72 h (n=5); I-K) clonogenic assays of the PDAC PANC-1, MIA PaCa-2 and Capan-1 cell lines treated with the ICfifty from P8, C14, gemcitabine and deltarasin (n=5); L-M) analysis of cell death by flow cytometry in hTERT-HPNE, PANC-1, MIA PaCa-2 and Capan-1 cells treated with ICfifty from P8 and C14, DMSO as vehicle or medium alone after staining with Annexin-V, 7-DAA and cytocalcein violet; M) Quantification of the plots shown in L) n=5. Figure 4. P8 and C14 decrease K-Ras activation and AKT and ERK phosphorylation in PDAC cell lines with K-Ras mutation. A-D) Western blot images representative of cell lines A) hTERT-HPNE, B) PANC-1, C) MIA PaCa-2 and D) Capan-1 treated with the IC50 of P8, C14, Gemcitabine and Deltarasin for 3 p.m. Total protein extracts were precipitated using RAF-RBD beads. Total RAS (Ras-T) and GAPDH are shown as loading controls. K-Ras GTP pixel intensities were normalized to total RAS and GAPDH; E-G) Western blot representative of cell lines E) PANC-1, F) MIA PaCa-2 and G) Capan-1 treated with the IC50 of P8, C14, Gemcitabine and Deltarasin or vehicle, for total and phosphorylated AKT and ERK using GAPDH as load control. Quantification of pERK and pAKT pixel intensities relative to total ERK and AKT levels respectively, are shown in the graphs on the right. Data is displayed as SDM; n=5, ***p<0.001. Figure 5. Characterization of PDAC tissues and primary cells. A) CK19 expression is observed in MGKRAS003, MGKRAS004 and MGKRAS005 PDAC tissues and primary cells by confocal immunofluorescence microscopy (Leica SP8, Barcelona, Spain). Bar=50 µm; n=3; B) MUC1 expression in MGKRAS003, MGKRAS004 and MGKRAS005 PDAC tissues and primary cells by confocal immunofluorescence microscopy. Bar=50 µm; n=3; C) sequences of nucleotides and histograms of sequencing of exon 2 containing KRAS in tissues and primary cells MGKRAS003, MGKRAS004 and MGKRAS005. Figure 6. Characterization of skin-derived primary cell cultures. A) IB-10 expression is observed in primary cultures derived from skin PBD033 and JGC028 by confocal immunofluorescence microscopy (Leica SP8, Barcelona, Spain). Bar=50 µm; n=3; B-C) Expression of CD90, CD105, CD73, CD34, CD45 and HLADR in primary cultures derived from PBD033 and JGC028 skin analyzed by flow cytometry after autofluorescence correction. Figure 7. P8 and C14 inhibit growth and induce apoptosis in PDAC primary cell cultures. A–E) Effects of P8 and C14 at various concentrations (5, 10, 30, 50, 100, 150, and 200 µM) are observed for 72 h in non-cancerous primary cultures PBD033 and JGC028, and PDAC primary cultures MGKRAS003, MGKRAS004, and MGKRAS005; F-H) clonogenic assays of the primary cultures of PDAC, MGKRAS003, MGKRAS004 and MGKRAS005 treated with the ICfifty from P8, C14, Gemcitabine and Deltarasin; I-J) Cell death analyzes of PBD033, JGC028 MGKRAS003, MGKRAS004, and MGKRAS005 were determined by flow cytometry after staining with annexin-V, 7-AAD, and cytocalcein violet; J) quantification of the percentages shown in I). Data is displayed as SDM; n=5, p<0.001. Figure 8. Evaluation of the viability of primary cultures treated with Gemcitabine and Deltarasin. A) The effect of Gemcitabine on cell viability at various concentrations (0-8000 nM) is observed after treatment for 72 h in the primary cultures of PDAC, MGKRAS003, MGKRAS004 and MGKRAS005; B) Effect of Deltarasin at various concentrations (0-8000 nM) after treatment for 72 h on cell viability in primary cultures of PDAC, MGKRAS003MGKRAS004 and MGKRAS005 (n=3). Figure 9. P8 and C14 decrease K-Ras activation and AKT and ERK phosphorylation in cultures of K-Ras mutated primary PDAC cells. A-E) Western blot images representative of JGCD28 (A), PBDD33 (B), MGKRAS004 (C), MGKRAS003 (D) and MGKRAS005 (E) cells treated with the IC are shown.fifty of P8, C14, Gemcitabine and Deltarasin for 3 h. Total protein extracts were precipitated using RAF-RBD beads. Total RAS (Ras-T) and GAPDH are shown as loading controls. K-Ras GTP pixel intensities were normalized relative to controls; F-H) Western blot representative of MGKRAS003 (F), MGKRAS004 (G) and MGKRAS005 (H) cells treated with the ICfifty of P8, C14, Gemcitabine and Deltarasin or vehicle, for AKT and total and phosphorylated ERK using GAPDH as loading control. Quantification of pERK and pAKT pixel intensities relative to total ERK and AKT levels, respectively, are shown in the graphs on the right. Data is displayed as SDM; n=5; ***p<0.001. Figure 10. Synergistic effect of P8 and C14 on PDAC cell lines and primary cultures. A) the synergistic interaction of P8 (dark blue), C14 (red) with the K-Ras4Bwt (pink and yellow)/PDE6δ (blue) complex is observed; B) synergistic interaction of P8/C14 with the K-Ras4BG12D/PBDE6δ complex; C) synergistic interaction of P8/C14 with the K-Ras4BG12C/PBDE6δ complex; D) synergistic interaction of P8/C14 with the K-Ras4BG12V/PBDE6δ complex; E) IC50 isobologram of compounds P8 and C14; F-J) effects on the viability of compounds P8/C14 at various concentrations of each (5, 10, 30, 50, 100, 150 and 200 µM) in MIA PaCa-2 (F) and PANC-1 (G) cell lines ) and primary PDAC cultures MGKRAS003 (H), MGKRAS004 (I) and MGKRAS005 (J); K-O) Clonogenic assays of MIA PaCa-2 (K), PANC-1 (L), MGKRAS003 (M), MGKRAS004 (N) and MGKRAS005 (O) treated with ICfifty P8/C14; P) Cell death analysis of PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004 and MGKRAS005 analyzed by flow cytometry after staining with annexin-V, 7-AAD and cytocalcein violet. Data is displayed as SDM; n=5; ***p<0.001. Figure 11. Evaluation of cytotoxicity and genotoxicity of P8 and C14. A-B) the effects of P8 and C14 at different concentrations (5, 10, 30, 50, 100, 150 and 200 µM) on the cell viability of stimulated and unstimulated H-PBMCs (n=5) are observed; C) frequency of micronuclei in polychromatic erythrocytes (PCE) isolated from bone marrow of BALB/c mice treated with X µM P8, C14 and Gemcitabine for 24 h; D-G) evaluation of the presence of proteins (D), pH (E), bilirubin (F) and glucose (G) in the urine of BALB/c mice after treatment with X µM of P8, C14 and Gemcitabine for X h; H-K) Evaluation of the presence of protein (H), pH (I), bilirubin (J) and glucose (K) in the urine of BALB/c mice after treatment with X µM of P8, C14 and Gemcitabine for 16 days. Figure 12. The combination of P8 and C14 reduces tumor growth in subcutaneous and orthotopic xenograft models. A) The effects of P8, C14 and C14/P8 at different concentrations (5, 10, 30 and 60 mg/kg, and the combination of 30 mg/kg C14 + 30 mg/kg P8) are observed in a model subcutaneous xenograft injection of MIA PaCa-2 cells into the skin of the back of male Nu/Nu mice (n=6); B) quantification of tumor volume every day after treatment with P8, C14 and C14/P8 at different concentrations (n=6). Representative images of tumors for each condition are shown below the graph; C) body weight was measured daily during treatment with P8, C14 and C14/P8; D) Representative Western blot of MIA PaCa-2 tumor lysates treated with P8, C14, Gemcitabine show complete inhibition of AKT and ERK phosphorylation using total AKT, ERK and GAPDH as loading controls. The relative quantification of the Western blot results is shown in the graphs; E) Effects of P8, C14 and C14/P8 at different concentrations (30 and 60 mg/kg, and the combination of 30 mg/kg C14 + 30 mg/kg P8) in a cell injection orthotopic xenograft model MIA PaCa-2 in the pancreas of male Nu/Nu mice (n=6); F) body weight was measured daily during treatment with P8, C14 and C14/P8; G) representative images of the effect of treatment with P8, C14, P8/C14 and Gemcitabine; H) Survival graph during treatment with P8, C14, P8/C14 and Gemcitabine. Data represent means ± SD of at least six independent experiments; ***p<0.001. Figure 13. Evaluation of the malignancy markers CK19, CA125 and Ki-67 in tumors derived from subcutaneous xenografts. A) Representative images of hematoxylin-eosin and immunohistochemistry of tumor sections derived from mice treated with P8, C14, P8/C14, Gemcitabine or vehicle are shown; B-D) quantification of signal intensities of CK19 (B), Ca 125 (C) and Ki-67 (D) in tumor sections. Data is displayed as SDM; n=100 cells per field with 6 fields per section; ***p<0.001. Figure 14. Combined treatment with P8 and C14 reduces tumor growth in PDX models. A) The effects of P8, C14 and C14/P8 at different concentrations (5, 10, 30 and 60 mg/kg, and a combination of 30 mg/kg C14 + 30 mg/kg P8) are observed in a model subcutaneous xenograft using MGKRAS004 cells; B) final effect after treatment with P8, C14 and C14/P8 at different concentrations; C) body weight was measured daily during treatment with P8, C14 and C14/P8; D) representative images of MGKRAS004 tumors obtained from each group; E) Effects of P8, C14, and C14/P8 at different concentrations (5, 10, 30, and 60 mg/kg, and a combination of 30 mg/kg C14 + 30 mg/kg P8) in a subcutaneous xenograft model (n=6) using MGKRAS005 cells; F) final effect of treatment with P8, C14 and C14/P8 at different concentrations for x d; G) body weight was measured daily during treatment with P8, C14 and C14/P8; H) Representative images of MGKRAS005 tumors obtained from each group. Data represent means ± SD of at least six independent experiments. ***p<0.001. Detailed description of the invention. The present invention provides synergistic compositions that comprise the combination of the C14/P8 compounds, inducing cytotoxicity in cell lines and primary cultures of pancreatic cancer greater than those obtained by the compounds evaluated individually, which makes them a viable, effective and useful alternative. for the treatment of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis of all human cancers, as it is highly resistant to chemotherapy. This leads to the search for new pharmacological alternatives to improve the quality of life of patients with pancreatic cancer. Compounds have been designed that can inhibit the signaling and transport pathway of the K-Ras4B oncoprotein. On the other hand, the compositions of the present invention that comprise the combination of the C14/P8 compounds have a synergistic effect, inducing cytotoxicity in cell lines and primary cultures of pancreatic cancer that are greater than those obtained by the compounds evaluated individually. The compositions described here decrease tumor growth from 90 to 95% in Subcutaneous and Orthotopic xenograft models as the dose increases, likewise they do not induce adverse effects or genotoxicity as presented by first-line chemotherapy with Gemcitabine, they decrease activation of K-Ras4B and decrease malignancy markers in remnant tumors. In this same way, the compositions of the invention decrease up to 99% of tumor growth in PDx models of pancreatic cancer, showing better antineoplastic effects in Subcutaneous Xenograft, Orthotopic and PDx models, decreasing tumor growth by up to 97% without inducing side effects. such as those presented by Gemcitabine. In accordance with the present invention, the compositions described herein are configured as new and efficient chemotherapeutic solutions with better properties than conventional chemotherapy. The chemical formula of compound C14 (formula I) is shown below: C14: 2-[(3-chlorophenyl)methyl-methyl-amino]-N-chroman-4-yl-acetamide
Figure imgf000008_0001
Formula I while the chemical formula of compound P8 (formula II) is shown below: P8: 2-[4-(3-chlorophenyl)piperazin-1-yl]-N-[(4R)-chroman-4-yl ]acetamide
Figure imgf000008_0002
Formula II According to the present invention, the compositions described herein comprise: a) Compound C14 (formula I) or its pharmaceutically active salts in a concentration of 90.18 µM to 154.24 µM with respect to compound P8, b) Compound P8 (formula II) (analogous to compound C14 which has amino groups, benzenes, pyridines and non-aromatic heterocycles) or their pharmaceutically active salts in a concentration of 18 µM to 150 µM, and c) A pharmaceutically acceptable vehicle, among which are included those that are compatible with the mentioned compounds and that allow their adequate administration, however, it is preferred that in said compositions the mentioned compounds are found in an amount of C14 of 90.18 µM and of P8 of 24.18 µM; In one of its modalities, in terms of their weight ratio (w/w), the compositions of the present invention comprise the compounds C14 and P8 in a 1:1 ratio, as described below. For purposes of the present invention, compounds C14 and P8 have been previously described in patent applications MX/a/2018/013439 and MX/a/2020/001471; both patent applications, as well as their complete contents, are incorporated by reference in the present invention. The results of the present invention show that the synergistic compositions described here are useful as a new therapeutic alternative for patients with pancreatic cancer, therefore the use of said compositions for the treatment of said condition is part of the modalities of the invention. Therefore, it is the object of the invention to provide synergistic pharmaceutical compositions that comprise the compounds C14 and P8, together with pharmaceutically acceptable and suitable excipients to be administered to the patient who requires them. A modality of the invention is the adaptation of the active principles to be used in pharmaceutical compositions for enteral, parenteral administration and topical use, including inhalation. The effective doses for the patient of the active ingredient will also be adjusted in accordance with preclinical and clinical studies, but based on the findings of the present invention. Examples of pharmaceutically acceptable excipients accompanying the active principle of the invention are, for example, for oral administration as tablets or tablets, agents comprising, for example, diluents, binders, stabilizers, bulking agents, thickening agents, such as povidone, cellulose microcrystalline, lactose, etc., disintegrating agents such as cross-linked carboxymethylcellulose, surfactants such as sodium lauryl sulphate, lubricating or slipping agents such as magnesium stearate, colloidal silicon dioxide, etc., where said excipients can be formulated for preferably slow or prolonged release for a systemic effect. Solutions for intravenous or intraperitoneal administration of the active ingredient can be prepared first dissolved in an organic solvent such as DMSO, ethanol, or dimethylformamide and subsequently in aqueous buffers, such as PBS. Special preference is given to pharmaceutical forms designed for localized administration to the site where pancreatic cancer occurs, where liquid or solid compositions can be formulated, suitable for local administration, for example, and whose excipients can be selected, for example, from components compatible with said pharmaceutical form, for example of a lipid or peptide nature, or peptidomimetics, known in the state of the art as non-immunogenic, and which can preferably be attached to the active principles of the invention to improve their bioavailability; propellants such as propane, butane, or permissible chlorofluorocarbons; pH regulators such as sulfuric acid; chelating agents such as EDTA. The active ingredients can also be shaped into micronized particles contained in gelatin capsules or in other systems known in the technical field, which help to release the active ingredient to its target site of action, for example, through solid pharmaceutical forms such as tablets or dragees. , including those formulated for extended release. In accordance with the present invention, the compositions described herein can be obtained by combining the compounds C14 and P8 with pharmaceutically compatible vehicles known in the art, in the amounts and/or concentrations that correspond to what is described herein, and may include known compounds in the art for obtaining said compositions. Likewise, the administration of such compositions can be done depending on the conditions of the patient, which will determine the doses and frequency of administration necessary to achieve an effective treatment of the condition in each particular case. Therefore, it is the object of the present invention to provide synergistic pharmaceutical compositions to treat pancreatic cancer, or to prevent its progression or its appearance in individuals who require it. The compositions described manage to reduce the viability of cancer cells without affecting the viability of healthy cells, and were tested for their ability to prevent the appearance of tumors or to reduce tumor size, for which said compositions are an excellent alternative for treatment. of neoplasms in pancreatic tissue. The compositions described herein can be used as antineoplastic or anticancer to treat mammals, including humans; Said compositions are effective and safe at the appropriate doses, which can be calculated by specialists in the field of the invention and for each individual requirement, as well as the routes of administration and the appropriate formulations that allow reaching the target organ and tissue, based on the principles provided by the present invention. One of the modalities of the invention refers to a method for treating pancreatic cancer using the compositions that comprise the combination of compounds C14 and P8, including the elimination/reduction of tumors caused by said condition. Another modality of the invention refers to the method for treating pancreatic cancer in a specific way using compositions that comprise the combination of compounds C14 and P8, without said compositions causing adverse reactions by only affecting cancerous tissue and not affecting healthy tissue. . Performing SAR and QSAR analysis, analogues to compound C14 were identified. Using the crystallographic KRas4B/PDE6δ complex and performing Molecular Dynamics tests, we identified the interaction energy of the compounds. Cell viability and type of death were evaluated using flow cytometry, as well as clonogenic assay. RAS-GTP Pulldown and Western blot assays were performed to measure K-Ras activation and its signaling pathways. MIA PaCa-2 cells were implanted in subcutaneous and orthotopic xenograft models in Nu/Nu mice and the PDX model was made using primary cultures of pancreatic cancer to be treated with compounds C14, P8 and the combination C14/P8. The analog called P8 presented higher interaction energy on the in silico mutated complexes, thus presenting greater cytotoxic effects in cell lines and primary cultures of pancreatic cancer with mutated K-Ras without damaging the cell line and non-cancerous primary cultures. . In the same way, both compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways, however the compound P8 presents an IC504 times lower than the compound C14, being up to now the best compound found in our research group. . In the present invention, the evaluation of the compound C14 and its analogue P8 using preclinical models of pancreatic cancer approved by the FDA is reported. The results showed that the Compound C14 and P8 have a greater specific cytotoxic activity than compounds D14 and C22 previously reported by our work group. In addition, the combination of the compounds C14 and P8 included in the compositions of the present invention has a synergistic effect, both in cell lines and in primary cultures and in murine models. On the other hand, we report that the compounds C14 and P8 do not present side effects like Gemcitabine, therefore they can be considered as new chemotherapeutic agents. Compound C14 presents higher interaction energy and greater decrease in cell viability in pancreatic cancer cell lines with mutated K-Ras4B. One of the compounds with the highest interaction score identified in the present invention by means of virtual screening on the K-Ras4B/PDE6δ heterodimeric complex, was the compound called C14, which is a small organic molecule with a molecular weight of 344.8 g/ mole (Table 1). Table 1. Interactions of compound C14 and P8 on the K-Ras4B/PDE6δ complex. Results obtained from the virtual selection analysis in the crystallographic heterodimeric complex.
Figure imgf000010_0001
To analyze the interactions and coupling energies of the C14 compound on the heterocomplex, we made modifications to obtain the K-Ras4B complexes.w.t./PDE6δ, K-Ras4BG12D/PDE6δ and K-Ras4BG12C/PDE6δ (Figure 1A-C). The coupling of the C14 compound with the K-Ras4B complexw.t./PDE6δ showed a simultaneous interaction with K-Ras4B (pink) and PDE6δ (aquamarine), where the interaction energies (Table 2) with the different heterocomplexes indicate that compound C14 has higher interaction energy in the K-Ras4B mutated heterocomplexes.G12D /PDE6δ and K-Ras4BG12C/PDE6δ than in K-Ras4B complexesw.t./PDE6δ. Table 2. Components of the binding free energy of protein-protein and protein-ligand complexes (in kcal/mol units).
Figure imgf000011_0001
Binding free energies and individual energy terms of complexes from coupled conformations (kcal/mol). The polar (ΔEpolar=ΔEele + ΔGele, sol) and nonpolar (ΔEnon-polar=ΔEvwd + ΔGnpol, sol) contributions are shown. All energies are averaged over 500 snapshots at 100 ps time intervals from the last 50 ns-long Simulations MD are in kcal/mol (± standard error of the mean) Given the obtained affinity of the Ras4B complexG12D/PDE6δ and K-Ras4BG12C/PDE6δ through the interaction with compound C14, and the importance of this complex in the maintenance and growth of pancreatic cancer, we performed feasibility assays to determine the cytotoxic effect of compound C14, using PANC pancreatic cancer cell lines. -1 and MIA PaCa-2, the normal pancreatic duct cell line hTERT-HPNE, and the retinal cell line ARPE-19 as a control. Through field microscopy analysis of cell lines treated with 100 µM of compound C14 for 72 h of incubation, it was shown that this compound has a relatively high activity on the cell line PANC-1 and MIA PaCa-2 without affecting ARPE-19. and hTERT-HPENE in terms of their growth and activity, such as cell morphology, proliferation, and the number of cells alive up to 72 h (Figure 1D). Cell viability was determined by measuring the ATP concentrations in the cell after three days of treatment with C14 at different concentrations (5, 10, 30, 50, 100, 150 and 200 µM), where the cell lines MIA PaCa-2 and PANC -1 presented sensitivity to dose-dependent treatment, obtaining an IC50 of 90.18 µM for MIA PaCa-2, 103.5 µM for PANC-1 and 171.4 µM in the normal hTERT-HPNE cell line. (Figure 1E–F). These results suggest that C14 has strong specific activity in cell lines with K-Ras4B mutations. The determination of the type of cell death produced by the C14 compound is very important for its subsequent approval for cancer treatment. The identification of analogues of the C14 compound yields a compound with higher chemical properties. The identification and selection of organic molecules analogous to the leader compound C14 was carried out using a database from the ENAMINE chemical library, where 335 analogues of compound C14 were analyzed. Using the Molecular Operating Environment (MOE) 2014.09 bioinformatics program, the structure of compound C14 was subjected to SAR and QSAR analysis to identify the compound's pharmacophore, taking into account the interactions of this molecule with the K-Ras4B/PDE6δ heterodimeric complex. (Table 1). Once the C14 pharmacophore was identified, structural "docking" was performed on the 335 C14 analogue molecules to identify those molecules that were 80-90% similar to the C14 pharmacophore. Following Lipinski's rules, 20 molecules analogous to the leader compound C14 (Table 3) were identified, which have a molecular weight of less than 500 Da, present less than 5 hydrogen bond donors and acceptors, and less than five rotary bonds, this so that the molecules have greater specificity towards the directed target and can pass freely through the plasma membrane. The analogous molecules that were identified showed 20% changes in their structure compared to the structure of the lead compound. The 20 analogues to the leader compound retain the chromene group and the acetamide group, where the modifications made with respect to the structure of the leader compound were with the addition of amino groups, benzenes, pyridines and non-aromatic heterocycles, with the purpose of increasing the interactions. with the molecular complex K-Ras4B/PDE6δ. Table 3. Analogs to the leader compound C14 selected by means of bioinformatics programs.
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
For the selection of the analogues of the C14 leader compound with the greatest cytotoxic effect on the MIA PaCa-2 cell line, the C14 analogues were evaluated using the IC50 of the C14 compound as a reference, which is 90.18 µM at 24, 48 and 72 hours later. of their treatment and were analyzed by means of bright field microscopy (data not shown). We observed that compounds P15, P16 and P19 have a greater effect on cell proliferation of the hTERT-HPNE cell line from the first 24 hours, whereas compound P8 had a marked effect on the MIA PaCa-2 cell line at 24 hours. and not in the control cell line, being that the remaining compounds did not show an effect on proliferation in either of the two cell lines. Taking into account the microscopy results, viability tests were performed using the "CellTiter-Glo" kit that allowed us to measure ATP by luminescence. MIA PaCa-2 and hTERT-HPNE cell lines were cultured in 96-well plates, which were treated with the 20 analogues at a concentration of 90.18 µM for 24, 48 and 72 hours to select for the compound(s). which has(have) a greater cytotoxic effect on the cell line MIA PaCa-2 and hTERT-HPNE used as control (Figure 2). The results obtained (Figure 2) with the first five analogues of compound C14 show a decrease in the viability of the hTERT-HPNE control cell line, but not in the Mia-PaCa 2 cell line, where the same result is observed in compounds P11 to P20 (Figure 3). However, compound P8 (Figure 1G-H) has a greater effect on cell viability in MIA PaCa-2. On the other hand, in the hTERT-HPNE cell line, a significant decrease in cell viability was obtained at a concentration of 90.18 µM of compound P8 (Figure 1G-H), in addition to showing a decrease in viability with the presence of the compound. leader C14, despite the fact that these compounds show 80% similarity with the pharmacophore, being that 95% of the analogues do not retain the cytotoxic effect on the cell line The P8 analogue presents higher interaction energy and greater cytotoxic effect on cell lines with mutated K-Ras4B, four times greater than the leader compound C14. Once the P8 analogue was identified, a molecular docking of the P8 compound was performed on the K-Ras4B heterocomplexes.w.t./PDE6δ, K-Ras4BG12D/PDE6δK-Ras4BG12C/PDE6δ and K-Ras4BG12V/PDE6δ (Figure 3 A-D), resulting in an increase in the interaction energy of the P8 compound with respect to that reported with the leader C14 compound on the heterocomplex. Our in silico analysis revealed that the site of interaction of compound P8 relative to compound C14 is completely different, as compound C14 has a ΔG of - 14.7 Kcal/mol and compound P8 a ΔG of -15.7 Kcal/mol, where the P8 analog presents more than 20 interactions with various amino acid residues of the K-Ras4B/PDE6δ heterodimeric complex. Considering these interactions by simulating the molecular docking of compounds C14 and P8 in the K-Ras4B/PDE6δ molecular complex, a molecular dynamics assay of compound P8 on K-Ras4B heterodimeric complexes was performed.w.t./PDE6δ, K-Ras4BG12D/PDE6δK-Ras4BG12C/PDE6δ and K-Ras4BG12V/PDE6δ, to calculate the theoretical affinity values on the heterocomplexes (Table 2). The estimated energies were higher on the mutated heterocomplexes than on the wild type (WT). With these theoretical affinity data, we can suggest that compound P8 shows a higher affinity than the lead compound on K-Ras4B/PDE6δ heterocomplexes (Table 2). Given the affinity of compound P8 on mutated K-Ras4B/PDE6δ heterocomplexes, we performed viability tests on the PDAC cell lines PANC-1, MIA Paca-2 and Capan-1, as well as on the hTERT-HPNE cell line as control, comparing the effect of cell lines treated with compounds C14 and P8 (Figure 3 E-H). Cell viability was determined by measuring the ATP concentrations in the cell lines after three days of treatment with the leader compound and its analogue at different concentrations (5, 10, 30, 50, 100, 150 and 200 µM), where the analogue Called P8, it presented a greater cytotoxic effect than its leading compound, presenting an IC50 of 51.18 µM in PANC-1, 24.18 µM in MIA PaCa-2 and 28.96 µM in Capan-1, 2 to 4 times lower than compound C14, being that these IC50 concentrations do not affect the viability of the normal hTERT-HPNE cell line with an IC50 of 103.45 µM. One of the most important characteristics of cancer cells is their high cell proliferation and chemoresistance to therapy, so to evaluate the chemoresistance of the pancreatic cancer cell lines PANC-1, MIA PaCa-2 and Capan-1, we performed clonogenicity assays comparing the effects between the compounds C14, P8, Gemcitabine and Deltarasin (Figure 3 I-K). Compounds C14 and P8 decreased the clonogenic capacity of cancer cell lines compared to the effect induced by Gemcitabine, however compound P8 had a greater effect in reducing the clonogenicity of the three cell lines, on the other hand the compound Deltarasin it did not present a good effect in reducing the clonogenic capacity. The determination of the type of cell death produced by the compounds C14 and P8 was determined by flow cytometry, observing that the compound P8 promotes cell death by apoptosis in cell lines MIA PaCa-2, PANC-1 and Capan-1 with a greater sample percentage of cells than compound C14 at lower IC50 concentrations without causing damage to hTER-HPNE normal pancreatic duct cells. Compound P8 has been shown to be a better compound than its parent compound, inducing cytotoxicity, clonogenic decline, and inducing apoptosis with concentrations 2 to 4 times lower than the parent compound. Compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways in PDAC cell lines depending on their oncogenic addiction. To determine if compounds C14 and P8 were affecting Ras activation and its signaling pathways, we performed RasGTP-Pulldown assays, using the first-line chemotherapy Gemcitabine, Deltarasin, and our compounds C14 and P8 (Figure 4A–D). The results indicate that our compounds are capable of reducing the activation of K-Ras in pancreatic cancer cell lines PANC-1, MIA PACa-2 and Capan-1, which represent the 3 most frequent mutations of oncogenic K-Ras; this finding is formidable, as it does not decrease K-Ras or Ras activation in the normal hTERT HPNE cell line. Subsequently, to check if this decrease in K-Ras activation affected their signaling pathways, we performed Western blot assays to corroborate the decrease in AKT and ERK activation in each of the previously evaluated pancreatic cancer cell lines (Figure 4 E–G). We observed the decrease in the activation of AKT and ERK in cell lines with the G12D, G12C and G12V mutations, treated with compounds C14 and P8; depending on the oncogenic addiction they present, the observed results varied depending on the mutation presented by each pancreatic cancer cell line, observing a 100% decrease in AKT activation in PANC-1 treated with compound C14 and P8, as well as a 100% decrease in AKT activation in MIA PaCa-2 treated with compound P8. On the other hand, compound P8 decreased ERK activation from 80 to 100% in PANC-1, MIA PaCa-2 and in Capan-1, indicating that compounds C14 and P8 are directly affecting the K signaling pathway. -Ras in pancreatic cancer cell lines with different oncogenic addiction. Obtaining and characterization of primary culture. To obtain pancreatic cancer samples from patients, the Department of Genomic Medicine and the Department of oncology and general surgery of the Hospital 1º. October ISSSTE in Mexico City, following the provisions of the national project 002.2015. Pancreatic cancer samples were collected in the operating room and transported to the Laboratory for processing. Nineteen samples of pancreatic cancer were obtained from patients between 40 and 100 years of age, with a higher frequency of 40-59 years and with a higher incidence in women, where said information contradicts what was previously described in the sense that the higher incidence of this type of cancer is reported in men aged 60 to 80 years. On the other hand, 2 epithelial tissue samples were obtained from healthy patients PBDD33 and JGCD28 as a control for our study. Once the samples were obtained, they were processed and obtained 3 primary cultures of pancreatic cancer MGKRAS003, MGKRAS004 and MGKRAS005, and 2 primary cultures derived from epithelial tissue, performing several passages until obtaining passages 5. Once the cells were obtained, we evaluated the presence of neoplastic ductal cells from patient tissue, using Ck19 (Figure 5A) for ductal cells as shown in the immunofluorescence image of patient tissue and our primary culture, where it is possible that Ck19 is present in both samples, which indicates that our primary cultures are ductal cells. Another marker more used in the clinic to identify neoplastic cells is CA19-9 or MUC1 (figure 5B), as shown in the image, patient samples and our primary cultures present the expression of MUC1, which indicates that our cultures primary are neoplastic ductal cells from the patient's tissue. Once confirmed that our primary cultures are neoplastic ductal cells, we decided to perform an advanced characterization using different markers, such as markers of pancreatic origin Ck7 and Ck19 and the Malignancy markers most used in several countries such as CEA, MUC1, MUC4, MUC16, EFGR, VIMENTIN, cytoplasmic B-Catenin and E-Caterin and Ki-67 (Table 4). As a control, we carried out the same characterization in the primary cultures from epithelial tissue, obtaining the presence of epithelial and mesenchymal markers in one of the samples, for which the characterization of fibroblasts and mesenchymal cells was carried out (figure 6 A-C), obtaining as a result the identification of mesenchymal cells derived from epithelial tissue corresponding to the PBDD33 sample and fibroblasts corresponding to the JGCD28 sample. As a result, our primary pancreatic cancer cultures belong to stage 4 and with these results we verified the diagnosis provided by the oncologists, which was highly infiltrating and invasive pancreatic ductal adenocarcinoma. Subsequently, by means of PCR and sequencing of KRAS exon-2, we identified the presence of mutations in the primary cultures of pancreatic cancer 8 (Figure 7C), obtaining as a result MGKRAS003 WT, MGKRAS004 G12V and MGKRAS005 G12C, where the two mutations found are the second. and the third most frequently worldwide; On the other hand, the G12V mutation represents the mutation with the highest chemoresistance that has been reported in pancreatic ductal adenocarcinoma. Primary cultures of pancreatic cancer show greater sensitivity to compounds C14 and P8 than to conventional therapy. Once the primary cultures of pancreatic cancer and controls had been characterized, we performed viability assays, which were determined by measuring the ATP concentrations in the primary cultures after three days of treatment with the leader compound and its analogue at different concentrations (5, 10, 30, 50, 100, 150 and 200 µM) (Figure 7 A-E), obtaining for the leader compound C14 an IC50 of 15.8 µM in MGKRAS003, 18.3 µM in MGKRAS004, 118.9 µM in MGKRAS005, 189 µM in PBDD33 and 130 µM for JGCD28, while for analogue P8 an IC50 of 22.3 µM was obtained in MGKRAS003, 18.03 µM in MGKRAS004, 37.5 µM in MGKRAS005, 192 µM in PBDD33 and 145 µM in JGCD28. The data obtained from the two compounds on the primary cultures showed a specificity on the primary cultures of pancreatic cancer without having an affectation on the non-cancerous primary cultures, where the P8 analog presents lower concentrations of IC50 and greater activity than its leading compound, presenting higher sensitivity of primary pancreatic cancer cultures to compounds C14 and P8 than already established pancreatic cancer cell lines. Subsequently, we evaluated the cytotoxic effect of Gemcitabine and Deltarasin (Figure 8) in the primary cultures of pancreatic cancer MGKRAS003, MGKRAS004 and MGKRAS005, obtaining an IC50 of 1000 nM with Gemcitabine and 10 µM for Deltarasin, where these IC50 concentrations are twice the reported concentration. for these compounds on pancreatic cancer cell lines. Once the IC50 concentrations for each primary culture were identified, we evaluated the clonogenic capacity of MGKRAS003, MGKRAS004 and MGKRAS005 using the compounds C14, P8, Gemcitabine and Deltarasin (Figure 7 F-H) obtaining as a result a greater decrease in the clonogenic capacity of the three primary cultures being treated with the analogue P8 with respect to its leader compound, compound C14.
Figure imgf000017_0001
The determination of the type of cell death produced by the compounds C14 and P8 on the primary cultures was determined by flow cytometry, where this determination showed that the compound P8 promotes cell death by apoptosis in cell lines MGKRAS003, MGKRAS004 and MGKRAS005, with a higher percentage cell sampling than compound C14 at lower IC50 concentrations without causing damage to non-cancerous primary cultures PBDD33 and JGCD28. Compound P8 has been shown to be a better compound than its lead compound C14 inducing cytotoxicity, clonogenic downregulation and inducing apoptosis with concentrations 2-fold lower than the lead compound on primary cultures of pancreatic cancer. Compounds C14 and P8 decrease the activation of K-Ras and its signaling pathways in primary cultures of pancreatic cancer. To determine if compounds C14 and P8 were affecting Ras activation and its signaling pathways, we performed RasGTP-Pulldown assays, using the first-line chemotherapy Gemcitabine, Deltarasin, and our compounds C14 and P8 (Figure 9A-E). The results indicated that compounds C14 and P8 are capable of reducing K-Ras activation in the primary cultures of pancreatic cancer MGKRAS003, MGKRAS004 and MGKRAS005, which represent 2 of the most frequent mutations of oncogenic K-Ras; this finding is very important, since it does not decrease the activation of K-Ras or Ras in non-cancerous primary cultures PBDD33 and JGCD28, as presented by Deltarasin. Subsequently, to check if this decrease in K-Ras activation affected their signaling pathways, we performed Western blot assays to corroborate the decrease in AKT and ERK activation in each of the previously evaluated pancreatic cancer cell lines (Figure 9 F–H). We observed the decrease in the activation of AKT and ERK in the primary cultures MGKRAS003, MGKRAS004 and MGKRAS005, treated with the compounds C14 and P8, where the observed results varied depending on the mutation presented by each primary culture of pancreatic cancer; an 80% decrease in AKT activation was observed in MGKRAS003 and MGKRAS004 treated with compound P8, as well as a 95% decrease in AKT activation in MGKRAS005 treated with compounds C14 and P8. On the other hand, compound P8 decreased ERK activation from 50 to 80% in MGKRAS003, MGKRAS004, and MGKRAS005, indicating that compounds C14 and P8 are directly affecting the K-Ras signaling pathway in primary pancreatic cancer cultures. . Compounds C14 and P8 exhibit synergistic effects in pancreatic cancer cell lines and primary cultures. When performing the in silico analysis of compounds C14 and P8 on the different K-Ras4B heterodimeric complexesw.t./PDE6δ, K-Ras4BG12D/PDE6δ, K-Ras4BG12C/PDE6δ and K-Ras4BG12V/PDE6δ, we observed differences in the interaction site of both compounds, so we decided to perform docking (Figure 10 A-D) and a molecular dynamics of both compounds on the WT and mutated heterodimeric complexes (Table 2), obtaining as a result, -120.77 ΔG for the K-Ras4B complexw.t./PDE6δ, -153.75 ΔG for the K-Ras4B complexG12D/PDE6δ, - 186.14 ΔG for the K-Ras4B complexG12C/PDE6δ and -175.59 ΔG for the K-Ras4B complexG12V/PDE6δ, obtaining higher interaction energy with both compounds in the mutated complexes than in the WT complex; these results suggested that compounds C14 and P8 could have a synergistic effect if they were evaluated together. In order to demonstrate the synergistic effect of the compounds, we performed an isobologram (Figure 10E) to identify the synergistic, additive or antagonistic theoretical effect, where we plotted the IC50 concentration of both compounds and following the Chao & Talalay analysis, we obtained several concordant points in the region of synergistic effect. To verify the synergistic effect, we tested the compounds C14 and P8 using the same concentrations of each one (5, 10, 30, 50, 100, 150 and 200 µM), which were evaluated in pancreatic cancer cell lines such as PANC-1. and MIA PaCa-2 (Figure 10 F-G) as well as in our primary pancreatic cancer cultures such as MGKRAS003, MGKRAS004 and MGKRAS005 (Figure 10 H-J), obtaining an IC50 of 18 µM for PANC-1, and 10.2 µM for MIA PaCa- 2, 5.8 µM for MGKRAS003, 8.3 µM for MGKRAS004 and 18.9 µM for MGKRAS005, the concentrations obtained using both compounds C14 and P8 being 10 times lower than those obtained by the compounds individually. Once the IC50 concentrations were identified, we evaluated the clonogenic capacity of the cell lines and primary cultures to evaluate the combined effect of the compounds C14 and P8 (Figure 10 K-O), obtaining a reduction of at least 99% of the clonogenic capacity. of PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004 and MGKRAS005. Subsequently, we performed cell death assays by means of flow cytometry using PANC-1, MIA PaCa-2, MGKRAS003, MGKRAS004 and MGKRAS005 (Figure 10P), obtaining the induction of death or apoptosis of at least 90% in cell lines and in our primary cultures of pancreatic cancer. These results confirmed the synergistic effect of compounds C14 and P8, both in pancreatic cancer cell lines and in primary cultures of pancreatic cancer with different mutations in the K-RAS4B oncoprotein. Compounds C14 and P8 do not have side effects in murine models compared to Gemcitabine. To verify that the compounds C14 and P8 did not affect non-cancerous cells, we performed a cytotoxicity assay using activated and unactivated human PBMC's (figure 11 A-B) treated with different concentrations of the compounds C14 and P8 (5, 10, 30 , 50, 100, 150 and 200 µM) obtaining an IC50 of 418.3 µM of C14 for the stimulated PBMC's and 443 µM of C14 for the unstimulated PBMC's; On the other hand, an IC50 of 1144 µM of P8 was obtained for the stimulated PBMC's and 1371 µM for the unstimulated PBMC's, where these concentrations exceeded by 100 times the concentrations obtained in our lines and primary cultures of pancreatic cancer and 4 to 10 times greater with respect to cell lines and primary non-cancerous cultures, thus proving the specificity of compounds C14 and P8 for cell lines and primary cultures of pancreatic cancer. To evaluate the genotoxicity of the compounds C14 and P8 on BalB/c mice, we administered 30.60 mg/kg of C14 and P8, the combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/ kg of each, ratio 1:1 w/w), Gemcitabine 40 mg/kg, vehicles (0.05% carboxymethyl cellulose in PBS with 0.5% DMSO) and untreated mice as controls, administering one dose for 24 h ( figure 11C); After time, the bone marrow of the femur of the mice was extracted and the presence of micronuclei was quantified to evaluate the genotoxicity of the treatments, obtaining as a result 7 to 5% of micronuclei present in the bone marrow of the mice treated with the compounds. C14 and P8 and their combination, which is not significant with respect to the control and the vehicle, however, more than 40% of micronuclei were present in mice treated with Gemcitabine; these results indicated that compounds C14 and P8 do not induce genotoxicity like Gemcitabine. To evaluate the side effects of the compounds C14 and P8, BalB/c mice were used, to which 30.60 mg/kg of C14 and P8 were administered intraperitoneally, the combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg each, 1:1 w/w ratio), Gemcitabine 40mg/kg, vehicles (0.05% carboxymethyl cellulose in PBS with 0.5% DMSO) and naïve mice as controls, being administered one dose for 24h (figure 11 D-G) and once a day for 15 days to complete the treatment scheme (figure 11 H-K). After 24 hours of treatment, the mice were sacrificed and the urine was collected to evaluate protein, pH, bilirubin and glucose, obtaining an increase of 300 mg/dl of protein, 70 mg/dl of bilirubin and 250 mg/dl of glucose in urine. in gemcitabine-treated mice; on the other hand, none of the variations of the above parameters were detected in the mice treated with the compounds C14, P8 and with the combination C14/P8, indicating that our compounds did not present side effects after the first 24 hours of treatment. To verify these results, the treatment was extended for 15 days, observing during the treatment several side effects in the BALB/c mice treated with Gemcitabine (Table 5), such as diarrhea, rectal prolapse, intestinal torsion syndrome, decreased muscle mass. , weight loss as well as loss of appetite; Due to these side effects, the mice treated with Gemcitabine had to be sacrificed, while the mice treated with C14, P8 and the C14/P8 combination did not present any of the aforementioned symptoms. Table 5. Side effects obtained in BALB/c mice treated with Gemcitabine, C14, P8 and C14/P8.
Figure imgf000019_0001
N.Normal; N.D. Not detected In the same way, a complete blood count was performed, observing leukopenia and neutropenia in the mice treated with Gemcitabine, as well as an increase in liver enzymes (G1), 500 mg/dl glucose, 2000 mg/dl protein and 70 mg/dl of bilirubin, while in contrast no side effects of the aforementioned were observed in the mice treated with the compounds C14, P8 and the combination C14/P8. With the results obtained from Gemcitabine in mice, the question arose whether these same effects occur in patients with pancreatic cancer who are administered this chemotherapy. Evaluating the clinical history of the patients who agreed to participate in this study, we found (Table 6) asthenia (G2), nausea (G2), epigastralgia, fatigue (G2), hyporexia (G1), leukopenia (G2), neutropenia (G2). , low bone marrow reserve, increased platelets, elevated liver enzymes (G1), bone pain, and weight loss, and due to these side effects, chemotherapy had to be suspended and replaced by palliative medications. These results indicate that compounds C14 and P8 do not present side effects or genotoxicity in murine models and can be used for their preclinical evaluation in murine models of pancreatic cancer. Table 6. Side effects presented in patients treated with standard chemotherapy in Mexico.
Figure imgf000020_0001
The combination of compounds C14 and P8 decreases tumor growth in murine models of heterotopic and orthotopic xenograft. To evaluate the antineoplastic effect of compounds C14 and P8 in a heterotopic model or subcutaneous xenograft (Figure 12 A-D), we grafted 5 million MIA PaCa-2 onto the back of Nu/Nu mice until a volume of 150 mm was obtained.3, to subsequently administer different concentrations intraperitoneally (5, 10, 30 and 60 mg/kg and a combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1:1 w/ p),) of compounds C14 and P8, for 15 days once a day, 40 mg/kg of Gemcitabine once every 3 days for 15 days and the vehicle as a control (0.05% carboxymethyl cellulose in PBS with 0.5% DMSO). During the 15 days of treatment, we measured the volume of the tumors before inoculation (Figure 12A), observing a decrease of more than 50% in all concentrations of compounds C14 and P8, being more evident with concentrations of 30 and 60 mg. /kg of C14 and P8 obtaining a decrease of more than 80% in tumor growth, the decrease in growth being more evident with 60 mg/kg of P8. However, with the combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1:1 w/w), we observed a decrease of more than 95% of tumor growth as described shown in Figure 12B, which shows the tumor volume of the last day after treatment and a representative image of the size of the tumors obtained in each experimental group. During the time that the treatment lasted, we measured the weight of the mice (Figure 12 C) and we did not observe weight loss using C14, P8 or their combinations, while in the group treated with Gemcitabine a decrease of more than 20% was observed. of your body weight. Once identified that compounds C14 and P8 decrease tumor growth, we removed the tumor and fragmented it into two portions, where the first portion was used to perform protein extraction to identify the signaling pathways of K-Ras, while the second portion was used to perform immunohistochemistry to identify markers of response to treatment. We identified AKT and ERK phosphorylation in different treatments (Figure 12 D), observing decreases in AKR and ERK phosphorylation in all concentrations used in tumors, being more evident at 60 mg/kg and with the C14/P8 combination. K-Ras4B signaling triggers cell proliferation and survival, so we decided to identify the proliferation and presence of neoplastic cells after treatment in the tissues. We performed immunohistochemistry where we used Ck19 for ductal cells, CA125 or Mucin 16 for neoplastic cells, and ki-67 for proliferations; Figure 13 shows the representative images of the different concentrations of C14, P8, Gemcitabine and Vehicle, observing in brown the positive immunoreaction in different markers, where the immunoreaction of Ck-19, CA-125 and Ki-67 decreases as the concentrations of compounds C14 and P8 increase; The decrease in these markers is more evident when using 30 mg/kg of C14 and P8, where more than 70% of the proliferating ductal neoplastic cells are decreased, however, with 60 mg/kg of P8, more than 90% of the cells are decreased. neoplastic ducts (figure 13 B-D). On the other hand, we were able to obtain the reduction of neoplastic ductal cells and reduce their proliferation to more than 95% using the combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1: 1 p/p), thus verifying the synergistic effect of the compounds on malignancy markers in PDAC. Once we identified that compounds C14 and P8 have antineoplastic effects in the heterotopic model, we evaluated the antineoplastic effects of compounds C14 and P8 in the orthotopic model, inoculating 1 million MIA PaCa-2 cells directly into the pancreas of Nu strain mice. /Wildebeest. Seven days after surgery, different treatments were administered intraperitoneally: vehicle, 30 and 60 mg/kg of C14, 30 and 60 mg/kg of P8, a combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1:1 w/w) and 40mg/kg of Gemcitabine, where these treatments were administered every 24h for 15 days (Figure 12 E-H). As a result of the evaluation of the antineoplastic effect of compounds C14 and P8, a decrease in tumor growth was observed in a dose-dependent manner, having a greater result with the different doses of compound P8, decreasing tumor growth to more than 90%. On the other hand, in the combination of both compounds (30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1:1 w/w)) we observed a 95% decrease in tumor growth. , indicating that both compounds have a synergistic effect in the orthotopic model (Figure 12 E); these results can be seen more clearly in Figure 12G where a representative image of the tumors and pancreas obtained in each treatment is shown. During the experiment, the weight of the mice was measured (Figure 12F), obtaining a decrease of more than 20% in body weight of the mice treated with Gemcitabine, for which they were sacrificed, showing a 100% decrease in survival with Gemcitabine halfway through treatment; on the other hand, the body weight of the mice treated with the compounds C14 and P8 did not decrease and a 100% survival was obtained at the end of the treatment (Figure 12H). The antineoplastic effect obtained by compounds C14 and P8 was better than that obtained by Gemcitabine, while the synergistic effect obtained by the combination of compounds C14 and P8 was still much better than that observed for the compounds evaluated individually, therefore that the compositions of the present invention prove to be better than the first-line compounds that are commonly administered in hospitals for the treatment of pancreatic cancer. The combination of compounds C14 and P8 decreases tumor growth in PDX models. To evaluate the antineoplastic effect of the compounds C14 and P8 in subcutaneous xenograft models derived from patients, the primary cultures MGKRAS004 and MGKRAS005 were used since they present the G12V and G12C mutations, which represent the third and second most frequent in pancreatic cancer. with greater chemoresistance (Figure 14). We grafted 5 million MGKRAS004 onto the back of Nu/Nu mice until a volume of 150 mm was obtained.3, to subsequently administer different concentrations intraperitoneally (30 and 60 mg/kg and a combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1:1 p/p), during 15 days once a day, Gemcitabine 40 mg/kg once every 3 days for 15 days and the vehicle as control (0.05% carboxymethyl cellulose in PBS with 0.5% DMSO).During the 15 days of treatment we measured the volume of the tumors before the inoculation of each treatment (Figure 14A), observing a significant decrease in tumor growth with both doses of treatment, both the compound C14 and the analogue P8, reducing their size to more than 90% in the doses of 30 and 60 mg/kg of C14 and more than 95% with 30 and 60 mg/kg of P8, being more evident at 60 mg/kg of P8; on the other hand with the combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each one, 1:1 w/w ratio) we observed a 98% reduction in tumor growth using the primary culture MGKRAS004 with the G12V mutation (Figure 14B), while the tumors treated with Gemcitabine decreased their growth by only 50% ; these effects can be seen in greater detail in Figure 14D where a representative image of the tumors is shown. During the administration of the different treatments, the weight of the mice was measured, obtaining a decrease of more than 20% of the weight in the mice treated with Gemcitabine. Observing the results obtained with MGKRAS004, we decided to evaluate the antineoplastic effect in the PDX model using the primary culture MGKRAS005 which presents the G12C mutation. We grafted 5 million MGKRAS005 onto the back of Nu/Nu mice until a volume of 150 mm was obtained.3, to subsequently administer intraperitoneally different concentrations (30 and 60 mg/kg and a combination of 30 mg/kg of C14 + 30 mg/kg of P8 (30mg/kg of each, ratio 1:1 w/w)) of compounds C14 and P8, for 15 days once a day, Gemcitabine 40 mg/kg once every 3 days for 15 days and vehicle as control (0.05% carboxymethyl cellulose in PBS with 0.5% DMSO). During the 15 days of treatment, the volume of the tumors treated with the different treatments was measured, observing a gradual decrease in tumor growth as the days of treatment increased until the eradication of the tumors was obtained in all the concentrations of the compounds C14, P8 used. and the combination of both compounds (Figure 14E); On the other hand, in the tumors treated with Gemcitabine, a significant decrease in tumor growth was not observed, which is shown in Figure 14 F-H where the tumor growth of the last day of treatment is observed and the representative images of the treated mice and the mice. tumors obtained from the study. During the administration of the different treatments, the weight of the mice was measured, obtaining a decrease of more than 20% of the weight in the mice treated with Gemcitabine. One of the big problems that concern us today is the lack of specific drugs against cancer. Currently, drugs discovered in the 1960s such as 5-FU and Gemcitabine are still used as the most appropriate drug therapy for the treatment of various types of cancer, including pancreatic cancer. These chemotherapy drugs decrease DNA synthesis and protein synthesis, thus affecting both cancer cells and normal cells. The presence of mutations in the K-Ras4B protein in PDAC is essential for the development, maintenance, and progression of this neoplasia [17]. As a result of this, several strategies have emerged to inhibit the activation of the K-Ras4B protein in the plasma membrane. One of the compounds that interferes with the activation of the K-Ras4B protein in cancer cells is the compound called Deltarasin and its analogues, which have the property of inhibiting the PDE6δ protein, which is the protein that transports K-Ras4B towards its activation site on the plasma membrane in cells [20, 26]. These compounds interact with the hydrophobic pocket of the PDE6δ protein, which is responsible for the transport of this GTPase to the plasma membrane through recognition of the farnesyl group present in Ras GTPase. Once Deltarasin interacts with PDE6δ, it is impossible for it to recognize the farnesyl present at the carboxyl end of K-Ras4B, thus preventing its transport to the plasma membrane, where it performs its function by allowing the activation of the different cell signaling pathways. related to oncogenic processes. However, one of the disadvantages of Deltarasin is that it can inhibit a wide variety of cell signaling pathways since Kras4B is not its only target for transport by PDE6δ [20]. In addition to this compound, in 2016 the Deltarasin analogue was reported, presenting greater specificity and greater inhibitory capacity for the activity of the PDE6δ protein, since it showed an IC50 of 24 µM, being 5 times higher than the concentration than that reported for the leading compound Deltarasin [23, 26] Despite the great progress in the identification of new pharmacological strategies for the treatment of pancreatic cancer, until before the present invention it had not been possible to identify compounds that prevent and impact the cell viability of pancreatic cancer cells, as Deltarasin and its analogues only retard tumor growth by 40%. In contrast, in the present invention the search for small molecules with possible chemotherapeutic properties was carried out, which showed specific antineoplastic activity on cells from pancreatic cancer with different K-Ras4B mutations, where compound C14 showed to be one of the most promising compounds. that we obtained in our previous works [21], this due to its cytotoxic effect on cell lines PANC-1 and MIA Paca-2 at a concentration of 103.32 µM and 90.18 µM respectively, these concentrations being lower than those obtained by compounds D14 and C22 [21, 23]. Notably, these compounds were targeted not only to a protein but to the K-Ras4B/PDE6δ heterodimeric complex to inhibit K-Ras4B transport and activation at the plasma membrane. One of the strategies used by various research groups, is the modification of the leader compounds, in order to improve their cytotoxic effect and increase their specificity, this through the subtle modification of the primary structure of the leader compounds, thus generating analogues [27]. In the present invention, it was possible to identify the compound P8, which presents greater cytotoxic properties on PDAC cell lines with mutations in K-Ras4B than the leader compound C14, at a four times lower concentration. By means of in silico analysis, compound P8 presented a higher interaction affinity with the mutated complexes than compound C14. The increase in the affinity of the P8 compound is given by the presence of a piperazine, which has two amino groups, increasing the interaction sites with the heterodimeric complex, making it more stable when interacting with the WT and mutated K-Ras4B/PDE6 complexes. δ. The presence of piperazine in compound P8 provides greater solubility, presenting a partition coefficient of 3.99 and a solubility constant of -4.4, on the other hand, compound C14, which does not present piperazine in its structure, has a coefficient partition of 3.63 and a solubility coefficient of -4.4. The increase in the partition coefficient of P8 with respect to C14 makes it even more soluble and permeable upon contact with the plasma membrane. This permeability that the P8 compound possesses allows it easy access to the target cells and due to this it presents a greater cytotoxic effect on K-Ras4B-dependent cancer cells. As has been reported, like the in silico analyzes of Deltasinone on PDE6δ, this compound showed an increase in the interaction affinity on the PDE6δ pocket with respect to its leader compound, Deltarasin, due to the elimination of the phenylbenzimidazole functional group. , where the elimination of this functional group allowed Deltazinone to present greater specificity towards PDE6δ, where its in silico data were corroborated using the recombinant protein PDE6d, for microcalorimetric techniques [26]. One of the pending tests to be carried out in the present invention is obtaining the affinity values of the compound C14 and P8 by means of biochemical methods such as BIACOR or by means of microcalorimetric techniques, which will allow us to confirm and obtain real quantitative data, by same as mass spectrometry to identify all possible targets of compounds C14 and P8. The cytotoxic effects obtained by compounds C14 and P8 on pancreatic cancer cell lines suggest that depending on the mutation present in K-Ras4B, the cells present different cell permeability and therefore present different IC50, allowing us to obtain greater cytotoxic effects in cell lines. with the G12C and G12V mutations which present greater chemoresistance in pancreatic cancer, this without affecting the non-cancerous cell line. One of the most important findings in the present invention is the specificity of both compounds P8 and C14 to induce cell death by apoptosis in cell lines with oncogenic addiction to K-Ras4B and not on "normal" cell lines. This finding has not been reported for other compounds such as Deltarasin, however, in the laboratory we performed cytotoxicity studies using Deltarasin on "normal" cell lines, demonstrating that this compound affects the hTERT-HPNE cell line more than the cell line MIA PaCa-2 [21]. This demonstrates that the compounds described here are more specific towards K-Ras4B mutated cell lines. As already mentioned, pancreatic ductal adenocarcinoma is characterized by the presence of activating mutations in K-Ras4B; In the present invention we demonstrate that the compounds C14 and P8 and their combinations are capable of decreasing the activation of the oncoprotein in pancreatic cancer cell lines without affecting the non-cancerous cell line and therefore decrease the signaling pathways depending on the oncogenic addiction. that each cell line presents towards K-Ras4B. On the other hand, Deltarasin decreased the activation of K-Ras4B in the normal cell line, implying the reduction of its signaling pathways, making clear the non-specificity that this compound presents. Decreased activation of K-Ras4B and its signaling pathways results in decreased cell proliferation, decreased protein synthesis, decreased transcription and cell cycle, and increased apoptosis-induced cell death. , this shown in in vitro tests. Worldwide, pancreatic cancer has been identified more frequently in men between 60 and 80 years of age; In our case, during the collection of pancreatic cancer samples from patients, we identified PDAC more frequently in women between 40 and 60 years of age, being 20 years younger than what has been reported worldwide according to several studies worldwide. by the health services [34]. When evaluating the compounds C14 and P8 on the primary cultures of pancreatic cancer, we observed a greater susceptibility to treatment, since which presented IC50 concentrations 4 to 9 times lower than those obtained in cell lines, however the IC50 of Gemcitabine and Deltarasin increased when evaluated in our primary cultures. Compounds C14, its analogue P8, and their combinations considerably decreased the activation of K-Ras4B and its signaling pathways in primary cultures of pancreatic cancer without affecting primary cultures of mesenchymal and non-cancerous fibroblastoids, demonstrating and reaffirming their specificity towards cells. of pancreatic cancer. These results are so promising that it could be said that it is the first time that drugs with chemotherapeutic potential do not affect normal cells. When two or more compounds that individually produce similar effects will sometimes show enhanced effects when administered in combination, as is the case with compounds C14 and P8 in primary pancreatic cancer cell lines and cultures, the combination is said to be synergistic. A synergistic interaction allows the use of lower doses of the compounds, a situation that may reduce adverse reactions [35, 36]. The combination of the compounds C14 and P8 allowed us to find IC50 concentrations in an order of magnitude 9 times lower than those reported by the individually administered compounds, this without inducing death by necrosis but by apoptosis in more than 90%. Drug combinations are quite common in the treatment of cancers, infections, pain, and many other degenerative diseases [36, 37]. One of the trials considered to verify the antineoplastic effects of compound P8 and compound C14 is the performance of tumorigenesis trials in in vivo models, where one of the results encouraged us to perform these trials in reducing the clonogenic capacity of the 80% using compound C14 and 96% with compound P8 in cell lines and primary cultures of pancreatic cancer, this being more evident with the combination of C14 and P8 where the clonogenic capacity decreased by more than 99% in both cases. The FDA-approved preclinical models for the evaluation of drugs with possible chemotherapeutic effects are subcutaneous xenograft, orthotopic xenograft, and patient-derived xenograft, with which the influence of the niche and cellular heterogeneity present in each of the models can be observed [ 38]. The antineoplastic activity of compounds C14 and P8 decreased tumor growth in Subcutaneous and Orthotopic xenograft models as the dose increased, without inducing adverse effects or genotoxicity (as if presented by first-line chemotherapy with Gemcitabine), they decrease the K-Ras4B activation and malignancy markers decrease in remnant tumors. In the same way, compounds C14 and P8 decrease tumor growth in PDx models of pancreatic cancer, while the combination of C14/P8 showed better antineoplastic effects in Subcutaneous Xenograft, Orthotopic and PDx models, further decreasing tumor growth without inducing it. side effects such as those presented by Gemcitabine. However, in the near future, pharmacokinetics and biodistribution studies of the C14 and P8 compounds in mice will be necessary to evaluate their therapeutic use and thus improve their distribution by using targeted nanoparticles coated with the compounds to obtain even smaller doses. with antineoplastic effects greater than those obtained. All these results show the great chemotherapeutic potential of the compounds evaluated in the present invention. The antineoplastic evaluation of these compounds demonstrated their specificity towards cell lines and primary cultures of pancreatic cancer, this without affecting cell lines and non-cancerous primary cultures, decreasing the activation of K-Ras4B and its signaling pathways, where these antineoplastic activities improved with the antineoplastic synergistic effect of the combinations of compounds C14 and P8 almost completely eradicating the presence of pancreatic cancer tumors in preclinical models without presenting adverse effects or genotoxicity. Compounds C14 and P8 and their combination are new pharmacological alternatives against pancreatic cancer with better properties than conventional chemotherapy. The following examples are included for the sole purpose of illustrating the present invention, without implying limitations of its scope. Example 1. Simulation of in silico coupling. We identified the chemical structure of C14 and P8 using the ENAMINE 3D Diversity set database (www.enamine.net) by docking to the interface region of the KRas4B-PDE6δ molecular complex using AutoDock 4.2.626 [19] and MOE Dock [20]. . Coupling calculations were carried out using the recommended standard parameter settings. We evaluated a maximum of 250,000 poses for C14 in the target receptor (crystallographic contacts between KRas4B with PDE6δ, from PDB ID 5TAR). Grids were calculated using Autogrid 4.2.626626 [19] with a spacing of 0.375 Å focusing on the interface of the proteins. crystallized. Molecular docking with MOE 2014.09 [20] was performed using the matching function to generate the initial poses. The top 30 London dG score results were further refined using energy minimization with the MMFF94x force field and re-scored using the Affinity dG score. Example 2. Simulation of molecular dynamics. MD simulation of the protein-ligand complex was performed using the AMBER 16 package [21] and the ff14SB forcefield [22]. Ligand charges for unparameterized residues in proteins were determined using the AM1-BCC level and the Amber General Force Field (GAFF) [23] for the protein-ligand complex, a 15 Å rectangular-shaped box of the ligand model. TIP3P water [24] was applied to solvate the complex; and the Cl- and Na ions+ for the protein-ligand system were placed in the model to neutralize any positive or negative charges around the complex at pH 7. Prior to MD simulation, the system was minimized by 3000 steepest descent minimization steps followed by 3000 minimization steps. of the conjugate gradient. Then, the system was heated from 0 to 310 K for 500 picoseconds (ps) of MD with position constraints under an NVT ensemble, successively an isothermal isobaric ensemble (NPT) of MD was carried out for 500 ps to adjust the density of the system. solvent followed by 600 ps constant pressure equilibrium at 310K using the SHAKE algorithm [25] on hydrogen atoms and Langevin dynamics for temperature control. The equilibrium run was followed by a 100 ns long MD simulation without position constraints under periodic boundary conditions using a 310K NPT assembly. The particle mesh Ewald method was used to describe the electrostatic term [26], and a 10 Å limit was used for van der Waals interactions. Temperature and pressure were conserved using the weak coupling algorithm [27] with coupling constants τT and τP of 1.0 and 0.2 ps, respectively (310 K, 1 atm). The MD simulation time was set to 2.0 femtoseconds and the SHAKE algorithm [25] was used to constrain the bond lengths to their equilibrium values. Coordinates were saved for analysis every 50 ps. AmberTools14 was used to examine the time dependence of root mean square deviation (RMSD), radius of gyration (RG), and clustering analysis to identify the most populous conformations during equilibrated simulation time. Example 3. Calculation of free bond energies. The calculation of the binding free energies was carried out using the MMGBSA approach [28-30] provided in the AMber16 suite [21]. 500 snapshots at 100 ps time intervals were chosen from the last 50 ns of MD simulation using a 0.1 M concentration and the Generalized Born (GB) Implicit Solvent Model [31]. The binding free energy of the protein-ligand system was determined as follows:
Figure imgf000025_0001
where ΔEMM represents the total energy of the molecular mechanical force field that includes the electrostatic (ΔEele) and van der Waals (ΔEvdw) interaction energies. ΔG solvation is the free energy rate of desolvation upon complex formation estimated from the implicit GB model and solvent accessible surface area (SASA) calculations yielding ΔGele.sol and ΔGnpol.sol. TΔS is the solute entropy that arises from the structural changes that occur in the degrees of freedom of free solutes in forming the protein-ligand complex. Example 4. Selection of analogues of compound C14. For the selection of the analogues of the C14 compound, a list of compounds from the company ENAMIN was used, which has a list of 335 compounds derived from C14. Potential analogues were analyzed using the Molecular Operating Environment (MOE) bioinformatics program, 2014.09, performing energy minimization, structural similarity, and pharmacophore search tests. Example 5. Cell culture. The human pancreatic cancer cell line MIA PaCa-2, PANC-1, Capan-1, the human pancreatic cell line hTERT-HPNE, and the human retinal cell line ARPE-19, were obtained from the American Type Culture Collection (ATCC ; Manassas, VA). Cell lines were grown as monolayers in the specific medium suggested by the ATCC. Example 6. Cell viability. Cell lines and primary cultures ARPE-19, hTERT-HPNE, PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004 and MGKRAS005 were seeded in 96-well plates at a density of 3x104 cells/well and cultured for 24 h. Next, cells were treated with C14 or P8 for 72 h in complete medium. To assess viability, cells were tested with the CellTiter-Glo kit (Promega, Madison, WI) according to the manufacturer's instructions. The concentration of C14 and P8 that killed 50% of all cells after 72 h (IC50) was determined by applying curve-fitting analysis with Prism software (GraphPad Software, San Diego, CA, USA). Example 7. Clonogenic assay. Pancreatic cancer cell lines were seeded in 6-well plates at a density of 300 cells per well and cultured overnight. Cell lines and primary cultures PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004 and MGKRAS005 were treated with final concentrations of 0.496 µM gemcitabine (PiSA Laboratories, Mexico), the IC50 concentration of C14 and P8 for 72 h. , and Deltarasin 5 µM. Subsequently, the medium was replaced with fresh medium supplemented every third day for a total of 10 days. Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min and washed with distilled water. Cells were stained with 0.1% crystal violet in 0.1 M citric acid for 30 min, washed with 1X PBS, dried, and photographed. For quantification, 14% acetic acid was added for 20 min to extract the dye and absorbance was measured photometrically at 500 nm using a TECAN Infinite F500 Fluorometer (Tecan Austria GmbH). Example 8. Apoptosis assay. Approximately 5 x 10 were planted5 cells in 6-well plates for 24 h. Cells were then treated with an IC50 concentration of C14 and P8 and vehicle for 24 h. Cells were harvested with 0.25% trypsin, washed with phosphate buffered saline (PBS) and collected together by centrifugation. Apoptosis was determined using the Apoptosis/Necrosis Detection Kit (Abcam, catalog number ab176749, Cambridge, England) according to the manufacturer's instructions and analyzed by flow cytometer on a FACSCalibur instrument (BD Biosciences) followed by a data analysis using FlowJo software (Tree Star Inc). All experiments were performed in triplicate. Example 9. RAS activation level assay. For the evaluation of the level of RAS activation after treatment with Deltarasin, Gemcitabine, C14 and P8, the RAS-GTP pull-down assay was performed by Western blot, applying the RAS Activation Assay Biochem Kit (BK008; Cystoskeleton, Inc.; Denver, CO), according to standard procedure. Briefly, hTERT-HPNE, PANC-1, MIA PaCa-2, Capan-1, MGKRAS003, MGKRAS004, and MGKRAS005 cells were cultured to have 3x106 cells, and lysed in ice cold lysis buffer (400 µL) supplemented with cOmplete™ Ultra Protease Inhibitor Cocktail without EDTA and 1xPhosSTOP™ (Sigma-Aldrich). The lysates were centrifuged and the protein (300 µg) was collected. Lysates were incubated by end-to-end rotation with 100 µg Raf-RBD-conjugated beads for 1 h. The supernatant was removed, and the beads were washed and boiled in 2X Laemmli sample buffer, followed by Western blot analysis, using pan-Ras antibody. Example 10. Western blot. Cell lines were serum starved for 16 hours and pretreated with an IC50 concentration of C14, P8, gemcitabine and deltarasin for 3 hours after pretreatment. Cells were stimulated with epidermal growth factor at 100 ng/ml for 10 min. Whole cell extracts were obtained by lysis of PANC-1 Capan-1, MGKRAS003, MGKRAS004 and MGKRAS005 cells in lysis buffer [20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl, Triton X-100 1%, 1 mM NaVO3, 1 mM NaF, 10 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride and 1.2 mg/ml complete™ Lysis-M protease inhibitor cocktail (Roche, Mannheim Germany). Protein extracts were forced 10 times through a 22-gauge needle and centrifuged for 10 min at 14,000 rpm at 4°C, and protein concentration was determined using the PierceTM BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Tissue samples were weighed, quick frozen, and ground in liquid nitrogen in a mortar and pestle. Samples were transferred to a microfuge tube and lysed using ProteoJET™ Mammalian Cell Lysis Reagent, followed by centrifugation at 2,000 x g for 15 min and protein quantification. SDS-PAGE was carried out using 30 µg of protein from each sample. Proteins were transferred to PVDF membranes (Merck Millipore) and blocked for 1 h. at room temperature using PBS containing 5% skim milk. It was then incubated with the following primary antibodies: Total ERK (Cell Signaling-9102; 1:1000), pERK (Cell Signaling-9101; 1:1000), Total Akt (Cell Signaling-92721:1000), pAKT (Cell Signaling- 9272; 1:1000) Signaling-4060 1:1000) and anti-GAPDH (Gene Tex-GTX100118 1:100,000). The Immunodetection was performed using a ChemiDocTM (BIO-RAD) imaging system. The densitometry analysis was performed with the ImageJ software version 1.45 (National Institute of Health, USA). Example 11. Treatment of subcutaneous xenografts of pancreatic carcinoma. Nu/Nu immunodeficient male nude mice were maintained at 6 weeks of age (CINVESTAV, Mexico) under pathogen-free conditions on irradiated chow. Animals were injected subcutaneously in the torso with 5x106 MIA PaCa-2 cells per tumor in 0.2 ml of high glucose DMEM matrigel medium. When MIA PaCa-2 cells reached palpable tumors (>150 mm3), mice were randomly divided into four groups receiving vehicle [10% DMSO, 0.05% carboxymethylcellulose in PBS] (n=6), or C14 and P8 at 60, 30, 10, and 5 mg/kg (n=6 ), a combination of 30 mg/kg C14 + 30 mg/kg P8 (n=6), and Gemcitabine 40 mg/kg (n=6), administered by intraperitoneal injection once daily for 15 days. Body weight and tumor were measured once a day. Tumor size was calculated using the following formula: [(length x width2)/2] in mm. Example 12. Immunohistochemical staining of xenograft tumor. One day after the last treatment, the mice were sacrificed in a CO 2 chamber and the xenograft tumors were excised, fixed in 4% buffered formalin and embedded in paraffin. The tumors were cut with a microtome obtaining 2 µm sections. For hematoxylin and eosin (H&E) staining, tissues were deparaffinized in xylene, hydrated in dry alcohol starting from absolute ethanol to distilled water, stained for 2 min with Harris hematoxylin, destained with 0.5% acid alcohol and were fixed for color in lithium carbonate for 1 min, washed in distilled water, in 96% ethanol and stained with Sigma Eosin, washed and dehydrated in gradual changes of alcohol until reaching absolute alcohol, allowed to dry at room temperature, mounted and observed, to identify the site of the lesion. For immunohistochemical staining, the tissues were deparaffinized in xylene, hydrated in depleted alcohols starting from absolute ethanol to distilled water, epitopes were unmasked with 10 mM Citrate Buffer pH 6.03 in the Tender Cocker for subsequent washing with PBS pH 7.4; endogenous peroxidase was blocked with H2EITHER2 0.9% for 15 min, blocked with 3% BSA for 1 h, while Ki-67 (BIOCARE MEDICAL API 3156 AA), CK 19 (GENETEX GTX110414) and CA125 (BIOCARE MEDICAL CM 101 CK) antibodies were diluted with 1% PBS and 1% BSA, where the primary antibody was incubated at room temperature for 40 min, washed with PBS for 3 min, incubated with the biotinylated secondary antibody for 20 min at room temperature, washed with PBS for 3 min, incubated with streptavidin for 15 min, and washed with PBS for 3 min. The reactions were revealed with 4% diaminobenzidine (DAB) monitoring each reaction under a microscope, for which they were counterstained with Harry's Hematoxylin for 30 seconds, washed with distilled water, dehydrated in gradual ethanol changes from distilled water to absolute ethanol, it was allowed to dry at room temperature, mounted and observed. Example 13. Primary cultures of pancreatic cancer from patients with PDAC. The pancreatic cancer tissues were provided by the 1st Regional Hospital. of October of the Institute of Security and Social Services for State Workers (ISSSTE) in the framework of project 002.2015 in Mexico City. Tissues were collected in the operating room of said hospital, placed in transport medium (DMEM base medium without fetal bovine serum and 5% antibiotic), always keeping the medium at 4°C. Tissue was sectioned until 3mm3 fragments were obtained, which were placed in 6-well plates with DMEM medium high in 20% glucose, 80% fetal bovine serum, and 3% antibiotic, this until tumor cells adhered to the plate. . The percentage of serum was decreased until the cells could survive with 10% serum and 1% antibiotic. Example 14. Patient-derived subcutaneous xenograft model. Nu/Nu immunodeficient male nude mice were maintained at 6 weeks of age (CINVESTAV, Mexico) under pathogen-free conditions on irradiated chow. Animals were injected subcutaneously in the torso with 5x10 6 primary culture MGKRAS004 and MGKRAS005 cells per tumor in 0.2 ml of high glucose DMEM matrigel medium. When cells from primary cultures reached palpable tumors (>150 mm3), mice were randomly divided into four groups receiving vehicle [10% DMSO.0.05% carboxymethylcellulose in PBS] (n=6), C14 or P8 at 60, 30 mg/kg (n=6), combination of 30 mg/kg C14 + 30 mg/kg P8 (n=6) and gemcitabine 40 mg/kg (n=6), administered by intraperitoneal injection once daily for 15 days. The weight body and tumor were measured once a day. Tumor size was calculated using the following formula: [(length x width2)/2] in mm. Example 15. Model of orthotopic xenograft in Nu/Nu mice. Nu/Nu immunodeficient male nude mice were maintained at 6 weeks of age (CINVESTAV, Mexico) under pathogen-free conditions on irradiated chow. Mice were anesthetized and sedated with xylazine and ketamine. The mouse spleen was located on the left side, subsequently a 0.5 cm incision was made in the skin and peritoneum, the spleen was removed, allowing visualization of MIA PaCa-2 cells after 1 million cells were inoculated. in 50 µl of serum-free minimal essential medium without phenol red directly into the pancreas. Organs were relocated within the mouse and peritoneum, and the skin was sutured with self-absorbing suture. Mice were randomly divided into four groups receiving vehicle [10% DMSO]. 0.05% carboxymethylcellulose in PBS] (n=6), or C14 and P8 at 60, 30 mg/kg (n=6), combination of 30 mg/kg C14 + 30 mg/kg P8 (n=6) and gemcitabine 40 mg/kg (n=6), administered by intraperitoneal injection once daily for 15 days. A complete necropsy was performed to obtain organs for the study. Example 16. Cellular immunofluorescence. Cells were grown on coverslips in 24-well plates to desired confluence, fixed with paraformaldehyde for 20 min at 37°C, then washed with 1X PBS and permeabilized with 1:1 methanol/acetone or 0.2 X100 triton. % for 10 min in a humid chamber, they were washed and the autofluorescence was blocked with 50 µM NH4Cl for 20 min at 37°C; the undesirable protein was blocked with 2% BSA for 30 min at 37°C, the pool was incubated with the primary antibody overnight at 4°C or 90-60 min at 37°C, to subsequently wash and incubate the antibody secondary labeled with fluorochrome for 30-60 min at 37°C, to finally wash and place in vectashielDapi. Example 17. Tissue immunofluorescence. Tissues were deparaffinized in xylene, hydrated in degraded alcohols starting from absolute ethanol to distilled water, epitopes unmasked with 10 mM Citrate Buffer pH 6.03 in Tender Cocker, washed with PBS pH 7.4, endogenous peroxidase blocked with 0.9% H2O2 (decreases erythrocyte autofluorescence) for 5 min, autofluorescence was reduced with 0.05M NH4Cl for 30 min at 37°C and washed with PBST three times; the primary antibody was diluted with 1% PBS and 1% BSA, in this way the non-specific binding site was blocked, while the primary antibody (Sup M 1) was incubated at room temperature for 60 min, to subsequently perform washes with PBS for 3 min, incubate with fluorocorm-labeled secondary antibody for 40 min at room temperature, and wash with PBS for 3 min. The nuclei were labeled with DAPI and the samples were analyzed under a confocal microscope. Example 18. DNA extraction. Genomic DNA from human samples diagnosed with pancreatic cancer (MGKRAS-003 to MGKRAS-005) was extracted from frozen tissue with the GenElute Mammalian Genomic DNA miniprep kit (Sigma-Aldrich G1N70). Example 19. PCR and sequencing. PCR was performed with approximately 60 ng of hybridized DNA using the following sense and antisense primers at a concentration of 10 pmol: Sense: RASO15'-AAGGCCTGCTGAAAATGAC-3', Antisense: RASA25'-TGGTCCTGCACCAGTAATATG-3. PCR was performed in a TC-512 TECHNE thermal cycler with 20 cycles of endpoint PCR (65°C initial run temperature, decreasing 0.5°C per cycle) and 15 cycles at 55°C run temperature. PCR products were purified using the QIAGEN QIAprep Miniprep Kit. Purified PCR products were sequenced in the reverse direction. Example 20. Genotoxicity test. For the evaluation of the genotoxic effect of C14 and P8, the micronucleus assay with bone marrow cells was carried out according to the method described above. Test compounds were administered intraperitoneally once, as a solution (at a concentration of 40 mg/kg gemcitabine (n: 5), 60 mg/kg C14 and P8 (n: 5) and a combination of 30 mg /kg of C14 + 30 mg/kg of P8 (n: 5) and vehicle (n: 5)) and using naive mice as control (n: 5). Bone marrow cells were obtained 24 h and 15 days after treatment and stained with Giemsa-Wright (Diff-Quick; Harleco; Gibbstown, NJ). HE Two thousand polychromatic erythrocytes per animal were counted using a light microscope at 100x magnification to determine the number of micronucleated polychromatic erythrocytes. Example 21. Toxicity test. For the determination of the pseudo-effects of the compounds C14 and P8, the determination was carried out in BAlbc mice. Gemcitabine 40 mg/kg (n: 5), C14 and P860 mg/kg (n: 5), the combination of C14 30 mg/kg + P8 30 mg/kg (n: 5) and vehicle (n: 5) were administered. : 5) once daily for 15 days and using naïve mice as control (n: 5). With veterinary assistance, the necropsy, blood and urine extraction were performed. Blood chemistry was performed with the cobas c111 Roche equipment, hematic biometry with the Xp300 Sysmex equipment, as well as a general urinalysis. Example 22. Statistical analysis. Statistical comparisons were made with a one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparisons test, using GraphPad Prism 5.0 software. Data are shown as mean ± SEM. A value of p<0.05 was considered statistically significant. References. 1. Soreide K, et al. Cancer Lett 2015, 356(2 Pt A):281-288. 2. Vinay Kumar AA, et al. Robbins and cotran pathologic basis of disease, Professional Edition: Saunders; 2009. 3. Abrams MJ, et al. Therap Adv Gastroenterol 2016, 9(2):141-151. 4. Eser S, et al. British Journal of Cancer 2014, 111(5):817-822. 5.Collins MA, et al. J Clin Invest 2012, 122(2):639-653. 6. Downward J: N Engl J Med 2009, 361(9):922-924. 7. Bryant KL, et al. Trends in Biochemical Sciences 2014, 39(2):91-100. 8. Dienstmann R, et al. Gastroenterology 2020, 158(4):806-811. 9. de Thé Bustamante-Valles F, et.al. Medical Gazette of Mexico 2006, 142:91-94. 10. Cox AD, et al. Small GTPases 2010, 1(1):2-27. 11. Buhrman G, et al. Journal of molecular biology 2011, 413(4):773-789. 12. Khan U, et al. Expert Opin Biol Ther 2019, 19(11):1135-1141. 13. Rao S, et al. Ann Oncol 2010, 21(11):2213-2219. 14. XuZ, et al. Eur J Med Chem 2019, 183:111682. 15. Becerra CR, et al. Cancer Chemother Pharmacol 2014, 73(4):695-702. 16. Cox AD, et al. Nature reviews Drug discovery 2014, 13(11):828-851. 17. Zeitouni D, et al. Cancers (Basel) 2016, 8(4). 18. Kim D, et al. Cell 2020, 183(4):850-859. 19. Furuse J, et al. Cancer Chemother Pharmacol 2018, 82(3):511-519. 20. Zimmermann G, et al. Nature 2013, 497(7451):638-642. 21. Casique-Aguirre D, BMC Cancer 2018, 18(1):1299. 22. Saliani M, et al. Cancer Biol Med 2019, 16(3):435-461. 23. Briseño-Díaz P, et.al. KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex for the Identification and Evaluation of Nontoxic Pharmacological Compounds for the Treatment of Pancreatic Cancer. In: Pancreatic Cancer [Working Title]. edn.; 2020. 24. Baniak N, et al. World Journal of Surgical Oncology 2016, 14(1):212. 25. Cheng J, et al. J Med Chem 2020, 63(14):7892-7905. 26. Papke B, et al. Nat Commun 2016, 7:11360. 27. Chuang HC, et al. Pharmacol Res 2017, 117:370-376. 28. Simanshu DK, et al. Cell 2017, 170(1):17-33. 29. Martin-Gago P, et al. Angew Chem Int Ed Engl 2017, 56(9):2423-2428. 30. Chen D, et al. Eur J Med Chem 2019, 163:597-609. 31. Siddiqui FA, et al. ACS Omega 2020, 5(1):832-842. 32. Dharmaiah S, et al. Proc Natl Acad Sci U S A 2016, 113(44):E6766-E6775. 33. Cox AD, et al. Nature Reviews Drug Discovery 2014, 13:828+. 34. Gonzalez-Santiago O, et al. Ecancermedicalscience 2017, 11:788. 35. Carved RJ. Genes Cancer 2011, 2(11):1003-1008. 36. Chou TC: Cancer Res 2010, 70(2):440-446. 37. Hackman GL, et al. Cancers (Basel) 2020, 12(12). 38. Kim MP, et al. Nat Protocol 2009, 4(11):1670-1680.

Claims

Reivindicaciones. 1. Una composición farmacéutica con actividad anticancerígena para cáncer de páncreas, caracterizada porque comprende el compuesto de fórmula I:
Figure imgf000030_0001
o cualquiera de sus sales farmacéuticamente activas, el compuesto de form
Figure imgf000030_0002
o cualquiera de sus sales farmacéuticamente activas, y un vehículo farmacéuticamente aceptable. 2. La composición farmacéutica de conformidad con la reivindicación 1, caracterizada porque tiene efecto sinérgico. 3. La composición farmacéutica de conformidad con la reivindicación 1, caracterizada porque el compuesto de fórmula I y el compuesto de fórmula II se encuentran en una proporción en peso (p/p) de 1:1. 4. La composición farmacéutica de conformidad con la reivindicación 1 a 3, para usarse en el tratamiento de cáncer de páncreas. 5. El uso de la composición de la reivindicación 1 a 3, para la fabricación de un medicamento para el tratamiento de cáncer de páncreas. 6. El uso de la composición de conformidad con la reivindicación 5, donde el compuesto de fórmula I y el compuesto de fórmula II son administrables en una proporción en peso (p/p) de 1:1 por Kg de peso. 7. Un método para el tratamiento de cáncer de páncreas, caracterizado porque comprende el paso de administrar a un paciente que lo necesite la composición de la reivindicación 1 a 3. 8. El método de la reivindicación 7, caracterizado porque la composición se administra por vía parenteral. Claims. 1. A pharmaceutical composition with anticancer activity for pancreatic cancer, characterized in that it comprises the compound of formula I:
Figure imgf000030_0001
or any of its pharmaceutically active salts, the compound of form
Figure imgf000030_0002
or any of its pharmaceutically active salts, and a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to claim 1, characterized in that it has a synergistic effect. 3. The pharmaceutical composition according to claim 1, characterized in that the compound of formula I and the compound of formula II are in a weight ratio (w/w) of 1:1. 4. The pharmaceutical composition according to claim 1 to 3, for use in the treatment of pancreatic cancer. 5. The use of the composition of claim 1 to 3, for the manufacture of a medicament for the treatment of pancreatic cancer. 6. The use of the composition according to claim 5, wherein the compound of formula I and the compound of formula II are administrable in a weight ratio (w/w) of 1:1 per kg of weight. 7. A method for the treatment of pancreatic cancer, characterized in that it comprises the step of administering to a patient in need thereof the composition of claim 1 to 3. 8. The method of claim 7, characterized in that the composition is administered by parenterally.
PCT/IB2021/062509 2021-09-14 2021-12-31 SYNERGIC COMPOSITIONS OF COMPOUNDS AGAINST THE HETERODIMERIC COMPLEX K-RAS4B/PDE6δ FOR THE TREATMENT OF PANCREATIC CANCER WO2023041978A1 (en)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020089850A1 (en) * 2018-11-01 2020-05-07 Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Pharmaceutical compositions for the effective treatment of pancreatic cancer
MX2020001471A (en) * 2020-02-06 2021-08-09 Centro De Investig Y De Estudios Avanzados Del I P N Compounds with antineoplastic effect targeted against the kras/pded molecular complex.

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Publication number Priority date Publication date Assignee Title
WO2020089850A1 (en) * 2018-11-01 2020-05-07 Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Pharmaceutical compositions for the effective treatment of pancreatic cancer
MX2020001471A (en) * 2020-02-06 2021-08-09 Centro De Investig Y De Estudios Avanzados Del I P N Compounds with antineoplastic effect targeted against the kras/pded molecular complex.

Non-Patent Citations (2)

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Title
"Challenges in Pancreatic Cancer", 19 August 2020, INTECH OPEN, ISBN: 978-1-83962-959-4, article BRISEÑO-DÍAZ PAOLA, EMMAVELEZ-URIZA DORA, PEDROCRUZ-NOVA, BELLORAMIREZ MARTINIANO, JOSECORREA-BASURTO, ROSAURAHERNANDEZ-RIVAS, : "KRas4BG12C/D/PDE6δ Heterodimeric Molecular Complex: A Target Molecular Multicomplex for the Identification and Evaluation of Nontoxic Pharmacological Compounds for the Treatment of Pancreatic Cancer", pages: 1 - 15, XP093050589, DOI: 10.5772/intechopen.93402 *
CASIQUE-AGUIRRE DIANA, BRISEÑO-DÍAZ PAOLA, GARCÍA-GUTIÉRREZ PONCIANO, LA ROSA CLAUDIA HAYDÉE GONZÁLEZ-DE, QUINTERO-BARCEINAS REYNA: "KRas4B-PDE6δ complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer", BMC CANCER, BIOMED CENTRAL, LONDON, GB, vol. 18, no. 1, 1 December 2018 (2018-12-01), LONDON, GB , pages 1299 - 16, XP093050585, ISSN: 1471-2407, DOI: 10.1186/s12885-018-5142-7 *

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