WO2023041978A1 - COMPOSITIONS SYNERGIQUES DE COMPOSÉS CONTRE LE COMPLEXE HÉTÉRODIMÈRE K-RAS4B/PDE6δ POUR LE TRAITEMENT DU CANCER DU PANCRÉAS - Google Patents

COMPOSITIONS SYNERGIQUES DE COMPOSÉS CONTRE LE COMPLEXE HÉTÉRODIMÈRE K-RAS4B/PDE6δ POUR LE TRAITEMENT DU CANCER DU PANCRÉAS 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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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 or sparfloxacin
    • 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.

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Abstract

La présente invention décrit des compositions synergiques utiles pour le traitement du cancer du pancréas, qui comprennent la combinaison des composés: de formule I, formule II ou leurs sels pharmaceutiquement actifs, qui induisent la cytotoxicité dans des lignées cellulaires et des cultures primaires de cancer du pancréas supérieures à celles obtenues avec les composés évalués individuellement, ce qui les convertit en une solution viable, efficace et utile pour le traitement du cancer du pancréas. L'évaluation antinéoplasique des compositions décrites a prouvé sa spécificité envers les lignées cellulaires et les cultures primaires du cancer du pancréas, et ce sans affecter les lignées cellulaires et les cultures primaires non cancéreuses, par diminution de l'activation de K-Ras4B et de ses voies de signalisation, ces activités antinéoplasiques s'améliorant avec l'effet synergique antinéoplasique des combinaisons de composés C14 et P8 par éradication presque complète de la présence de tumeurs du cancer du pancréas dans des modèles précliniques sans présenter d'effets secondaires ni de génotoxicité. Les compositions synergiques de l'invention sont de nouvelles solutions pharmacologiques contre le cancer du pancréas avec des propriétés supérieures à celles de la chimiothérapie conventionnelle.
PCT/IB2021/062509 2021-09-14 2021-12-31 COMPOSITIONS SYNERGIQUES DE COMPOSÉS CONTRE LE COMPLEXE HÉTÉRODIMÈRE K-RAS4B/PDE6δ POUR LE TRAITEMENT DU CANCER DU PANCRÉAS WO2023041978A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020089850A1 (fr) * 2018-11-01 2020-05-07 Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Compositions pharmaceutiques pour le traitement efficace du cancer du pancréas
MX2020001471A (es) * 2020-02-06 2021-08-09 Centro De Investig Y De Estudios Avanzados Del I P N Compuestos con efecto antineoplásico dirigidos contra el complejo molecular kras/pded.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020089850A1 (fr) * 2018-11-01 2020-05-07 Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional Compositions pharmaceutiques pour le traitement efficace du cancer du pancréas
MX2020001471A (es) * 2020-02-06 2021-08-09 Centro De Investig Y De Estudios Avanzados Del I P N Compuestos con efecto antineoplásico dirigidos contra el complejo molecular kras/pded.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
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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