ES2919898B2 - New treatment for colorectal cancer - Google Patents

Description

DESCRIPTION

New treatment for colorectal cancer

FIELD OF THE INVENTION

The present invention belongs to the field of biomedicine, and more specifically relates to a new treatment for gastrointestinal cancer, and more preferably for the treatment of gastric cancer and colorectal cancer.

STATE OF THE PRIOR ART

Colorectal cancer is the third most common cancer diagnosed in both men and women. It takes place in the colon and rectum and develops slowly from adenomatous polyps. Excessive growth of the colonic mucosa generates these polyps that have a pedunculated or sessile morphology. The development of colorectal cancer can occur through different pathways. Among them is the appearance of serrated lesions with a saw-tooth appearance of the epithelium and which occurs in the proximal colon. This pathway is detected in a third of all colorectal cancers. With respect to the adenoma-carcinoma sequence, it has been proposed that progression from small adenomas to large adenomas or advanced adenocarcinomas is mediated by multiple molecular pathways including microsatellite instability, chromosomal instability, and epigenetic pathways.

Early prevention is possible since it has been estimated that it takes at least 10 years for a polyp to transform into a tumor cell (PMID: 28842051 0ines et al. Best Pract Res Clin Gastroenterol. 2017 Aug;31(4):419-424 ). Early diagnosis through colonoscopy and detection of occult blood in feces, as well as improvements in treatments, have contributed to prolonging the survival of colorectal tumors in the healing phase. However, metastasis is present in approximately 25% of cases. patients during diagnosis and in total, 50% of colorectal cancer patients will develop metastasis (PMID: 27672422 Ohhara et al. World J Gastrointest Oncol. 2016 Sep 15;8(9):642-55). Due to the heterogeneity and multifactorial nature of colorectal cancer, studies have increased considerably in recent years to identify clinical and molecular predictors, as well as prognostic factors with the potential to improve patient management (PMID: 26964802 Aran et al. Colorectal Cancer Clin. 2016 Sep;15(3):195-203; PMID: 29139091 Bermejo et al. Drugs. 2018 Jan;78(1):1-18).

Both the incidence and mortality of colorectal cancer have decreased for both men and women since 1970 due to early detection (PMID: 29313949 Siegel et al. CA Cancer J Clin. 2018 Jan;68(1):7-30). However, although different therapies are available for metastatic colorectal cancer, the results are not optimal due to heterogeneity among patients due to molecular characteristics, response to therapy and clinical presentation (PMID: 25593032 Linnekamp et al. Cancer Res. 2015 Jan 15;75(2):245-9). The most common treatments for these tumors are based on surgery, chemotherapy, targeted therapies and hormone replacement treatment. At least 50% of patients with colorectal cancer relapse after surgical resection and eventually die of metastatic disease. Despite postoperative adjuvant chemotherapy in these patients to reduce the risk of recurrence, adjuvant treatment does not provide survival benefits, especially in patients diagnosed with stage I.

Targeted therapy is based on monoclonal antibodies and small molecular inhibitors, which selectively block cell proliferation by intervening with certain molecules and overexpressed proteins required for tumor expansion.

According to the Mesh classification, colorectal cancer is included in gastrointestinal cancers. The classification is as follows:

Neoplasms [C04]

Neoplasms by Site [C04.588]

o Digestive System Neoplasms [C04.588.274]

■ Gastrointestinal Neoplasms [C04.588.274.476]

■ Esophageal Neoplasms [C04.588.274.476.205]_

■ Esophageal Squamous Cell Carcinoma

[C04.588.274.476.205.500]

■ Intestinal Neoplasms [C04.588.274.476.411]

■ Cecal Neoplasms [C04.588.274.476.411.184] ■ Appendiceal Neoplasms [C04.588.274.476.411.184.290]

■ Colorectal Neoplasms

[C04.588.274.476.411.307],

■ Adenomatous Polyposis Coli

[C04.588.274.476.411.307.089],

■ Colonic Neoplasms [C04.588.274.476.411.307.180],

■ Colitis-Associated Neoplasms [C04.588.274.476.411.307.180.400

]

■ Sigmoid Neoplasms [C04.588.274.476.411.307.180.800

]

■ Colorectal Neoplasms, Hereditary Nonpolyposis [C04.588.274.476.411.307.190]

■ Rectal Neoplasms [C04.588.274.476.411.307.790],

■ Anus Neoplasms [C04.588.274.476.411.307.790.040

l

■ Anal Gland Neoplasms [C04.588.274.476.411.307.

790.040.040]

Therefore, it is necessary to develop new treatments for colorectal cancer.

DESCRIPTION OF THE FIGURES

Fig. 1 . Pose resulting from molecular docking between Venetoclax and hepsin.

Fig. 2 . Calculation of the IC ₅₀ value of Venetoclax on the activity of Hepsin. Maximum rate of Hepsin activity in the presence of increasing doses of Venetoclax.

Fig.3 . Effect of Venetoclax on colorectal cancer cell migration. Percentage of cell migration of Caco-2 and Caco-2-HPN cells in the presence or absence of Venetoclax.

Fig. 4 . Effect of Venetoclax on gelatin degradation of colorectal cancer cells. Percentage of cellular degradation of Caco-2 and Caco-2-HPN cells in the presence or absence of Venetoclax.

Fig. 5 . Effect of Venetoclax on thrombin generation. Values of the time until the onset of the peak (Lagtime) and the time to reach the maximum peak (ttPeak) in Caco-2-HPN cells in the presence or absence of Venetoclax.

Fig .6. Effect of Venetoclax on the cell cycle of colorectal cancer cells. Percentage of Caco-2 and Caco-2-HPN cells in the different phases of the cell cycle (G0/G1, S, G2/M) in the presence or absence of venetoclax.

Fig. 7. Effect of Venetoclax on the proliferation of colorectal cancer cells. Percentage of Caco-2 and Caco-2-HPN cells that are proliferating (EdU+) in the presence or absence of Venetoclax.

DESCRIPTION OF THE INVENTION

The present invention relates to Venetoclax and any of its salts, solid state forms and solvates for use in the treatment of colorectal cancer, and even more preferably metastatic colorectal cancer.

MEDICAL USE OF THE COMPOUND OF THE INVENTION

Therefore, a first aspect of the invention relates to Venetoclax, hereinafter compound of the invention, or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or analogues, or any of their combinations, for the prevention, improvement, relief and/or treatment of gastrointestinal cancer.

Venetoclax, is a molecule with the IUPAC name 4- (4 - {[2- (4-chlorophenyl) -4,4-dimethylcyclohex-1-en-l-yl] methyl} piperazin-1 -yl) -N -({ 3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide, of formula (I ) and CAS number 1257044-40-8

Formula (I)

Venetoclax herein also includes any of its pharmaceutically acceptable salts, esters, tautomers, solvates and hydrates, or any of its combinations, as well as all known solid-state forms of Venetoclax and pharmaceutical compositions thereof.

Venetoclax is described in several patent documents, where it is indicated that overexpression of Bcl-2 proteins correlates with chemotherapy resistance, clinical outcome, disease progression, overall prognosis, or a combination thereof in several cancers and immune system disorders. Therefore, it is necessary to develop compounds that inhibit the activity of anti-apoptotic Bcl-2 proteins, such as Venetoclax, approved for the treatment of chronic lymphocytic leukemia whose tumors have a specific genetic alteration. However, a progressive decrease in its expression during tumor progression indicates a minor role in CRC survival and resistance (Maurer CA et al., 1998. Dig Dis Sci 43(12):2641-2648; Flohil CC et al. ., 1996. J Pathol 178(4):393-397; Hawkins N et al., 1997. Tumor Biol 18(3):146-156; Kaklamanis L et al., 1996. J Pathol 179(1):10 —14; Kikuchi Y et al., 1997. Virchows Arch 431(2):111—117; Nakamura T et al., 1995. Pathol Int 45(10):721-728; Watson AJ et al., 1996. Br J Cancer 73(8):889-895; van der Heijden M et al., 2016. Nat Commun 7:10916; Ofner D et al., 1995. Br J Cancer 72(4):981-985), and a recent meta-analysis (Huang Q et al., 2017. World J Gastroenterol. 23(27):5018-5033, which is the type of study that provides the highest level of evidence, concludes that the high expression of the antiapoptotic protein Bcl-2 It is a good prognostic factor in colorectal cancer. In addition, to date, of the 344 clinical trials located in different databases as of January 2021, there are none in which Venetoclax is used for the treatment of cancer. gastrointestinal or colorectal. No expert in the field would be inclined to use Venetoclax in this type of cancer.

Pericellular proteases have been widely implicated in carcinogenesis, not only because they act as enzymes capable of degrading the extracellular matrix, thus allowing tumor cells to break the basement membrane and invade the surrounding tissue, but they are also capable of acting as proteolytic modifiers of factors. growth and protease-activated receptors that are critical for the activation of tumor signaling pathways (PMID: 27870503 Tanabe et al. FEBS J.

2017 May;284(10):1421-1436). Among these proteases is hepsin, which belongs to the super family of type II transmembrane serine proteases (TTSP). Hepsin is overexpressed in prostate cancer and its high levels are indicative of poor prognosis and relapse after radical prostatectomy (PMID: 18927246 Sardana et al. Clin Chem. 2008 Dec;54(12):1951-60). It has also been demonstrated overexpression in ovarian cancer (PMID: 18726901 Miao et al. int J Cancer, 2008 Nov 1;123(9):2041-7) and breast cancer (PMID: 21383634). It has recently been described that hepsin is elevated in the serum of patients with metastatic colorectal cancer compared to patients with primary tumor. Although there are still no data that relate its expression to metastasis in colorectal cancer.

The development of venous thromboembolic events (VTE) is a common problem in digestive tumors. The cumulative incidence of VTE in modern series varies between 12-20% (PMID: 26738412 Posch et al. Thromb Haemost. 2016 Apr;115(4):817-26), depending on the disparities in patient characteristics in each study and the context in which the evaluation is carried out. Thrombogenesis is attributed to a combination of clinical and biological factors, including activation of the hemostatic system by the tumor and vascular toxicity of chemotherapy. Thrombosis is associated with increased morbidity and mortality in these patients (PMID: 28267709 Carmona-Bayonas et al. Br J Cancer. 2017 Apr 11;116(8):994-1001, PMID: 22475313 Kline et al. Thromb Res. 2012 May;129(5):e194-9) and is one of the most important adverse events in clinical trials of anti-neoplastic therapy.

As shown in the examples of the invention, surprisingly, the inventors have demonstrated through a fluorogenic assay the ability of Veneoclax to inhibit the proteolytic activity of hepsin, as well as the ability of Veneoclax to reduce the migration and invasion of cancer cells. colorectal. They have also shown that it reduces the generation of thrombin in plasma from healthy subjects after incubation with colorectal cancer cells, and that it reduces the generation of thrombin in plasma from patients with colorectal cancer.

Therefore, another aspect of the invention relates to Venetoclax (compound of formula I) or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates, or an isomer, prodrugs, derivatives, solvates or analogues, or any of their combinations, for the prevention, improvement, relief and/or treatment of colorectal cancer in an individual, where said individual shows elevated hepsin levels with respect to normal values. More preferably the colorectal cancer is metastatic colorectal cancer.

Methods for examining hepsin protein expression levels in biological samples, and in particular tissue samples, are widely known in the art. state of the art, for example, by immunohistochemical methods and/or western blot.

The compounds of the present invention represented by formula (I) may include isomers, depending on the presence of multiple bonds, including optical isomers or enantiomers, depending on the presence of chiral centers. Individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention, that is, the term isomer also refers to any mixture of isomers, such as diastereomers, racemic, etc., including their optically isomers. active ingredients or mixtures in different proportions thereof. The individual enantiomers or diastereoisomers, as well as their mixtures, can be separated by conventional techniques.

Likewise, within the scope of this invention are prodrugs of the compounds of formula (I). The term "prodrug" or "prodrug" as used herein includes any derivative of a compound of formula (I) - for example and not limited to: esters (including esters of carboxylic acids, esters of amino acids, phosphate esters, esters of sulfonate of metal salts, etc.), carbamates, amides, etc. - which when administered to an individual can be transformed directly or indirectly into said compound of formula (I) in the aforementioned individual. Advantageously, said derivative is a compound that increases the bioavailability of the compound of formula (I) when administered to an individual or that enhances the release of the compound of formula (I) in a biological compartment. The nature of said derivative is not critical as long as it can be administered to an individual and provides the compound of formula (I) in a biological compartment of an individual. The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.

As used herein, the term "derivative" includes both pharmaceutically acceptable compounds, that is, derivatives of the compound of formula (I) that can be used in the manufacture of a medicament, and non-pharmaceutically acceptable derivatives, since these can be useful in the preparation of pharmaceutically acceptable derivatives.

The compounds of the invention may be in crystalline form as free compounds or as solvates. In this sense, the term "solvate", as used herein, includes both pharmaceutically acceptable solvates, that is, solvates of the compound of formula (I) that can be used in the manufacture of a medicament, and non-pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically acceptable solvates or salts. The nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. The solvates can be obtained by conventional solvation methods known to those skilled in the art.

For their application in therapy, the compounds of formula (I), their salts, prodrugs or solvates, will preferably be in a pharmaceutically acceptable or substantially pure form, that is, having a pharmaceutically acceptable level of purity excluding pharmaceutical additives. such as diluents and carriers, and not including material considered toxic at normal dosage levels. Purity levels for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and even more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or its salts, solvates or prodrugs.

COMPOSITIONS AND PHARMACEUTICAL FORMS OF THE INVENTION

Another aspect of the invention relates to a composition, hereinafter composition of the invention, comprising at least the compound of the invention or any of its salts, preferably any pharmaceutically acceptable salt, esters, tautomers, polymorphs, pharmaceutically acceptable hydrates. , or an isomer, prodrug, derivative, solvate or analogue, or any combination thereof, for use for the prevention, improvement, relief and/or treatment of gastrointestinal cancer in an individual.

In a preferred embodiment of this aspect of the invention is metastatic colorectal cancer. Even more preferably the individual has elevated hepsin levels with respect to normal values.

In another preferred embodiment of this aspect, the composition of the invention is a pharmaceutical composition. Furthermore, it comprises a pharmaceutically acceptable carrier, an excipient and/or a pharmaceutically acceptable vehicle.

In another preferred embodiment of this aspect, the composition of the invention comprises, as the sole active ingredient, the compound of formula (I) of the invention (Venetoclax) in a therapeutically effective amount, although it may comprise other pharmaceutically acceptable excipients and/or vehicles. .

In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of the agent or compound capable of developing the therapeutic action determined by its pharmacological properties, calculated to produce the desired effect and, in general, will be determined, among other causes, due to the characteristics of the compounds, including the age, condition of the patient, the severity of the alteration or disorder, and the route and frequency of administration.

The compounds described in the present invention, their salts, prodrugs and/or solvates as well as the pharmaceutical compositions containing them can be used together with other drugs, or active ingredients, to provide a combination therapy. Said additional drugs may form part of the same pharmaceutical composition or, alternatively, may be provided in the form of a separate composition for administration simultaneous or not with that of the pharmaceutical composition comprising a compound of formula (I), or a salt, prodrug or solvate thereof.

Therefore, in another preferred embodiment, the pharmaceutical composition further comprises another (or other) active ingredient. More preferably, the active ingredient is selected from the list consisting of: antiangiogenic agents, EGFR therapies, kinase inhibitors, chemotherapeutic agents, immunotherapeutic agents, molecular targeting agents, or any combination thereof, for the treatment of cancer. colorectal.

In another preferred embodiment, the antiangiogenic agents are selected from: Bevacizumab, Ziv-Aflibercept, Ramucirumab or any of their combinations. In another preferred embodiment, the therapies against EGFR are selected from: Cetuximab, Panitumumab or their combinations. In another preferred embodiment, the kinase inhibitor is Regofarenib. In another preferred embodiment, the chemotherapeutic agents are selected from: Trifluridine Tipiracil, Tegafur Gimeracil Oteracil, or their combinations. In another preferred embodiment, the immunotherapeutic agents are selected from: Lefitolimod (MGN-1703), Pembrolizumab, Atezolizumab or any of their combinations. In another preferred embodiment, the agents against molecular targets are selected from: Encorafenib (LGX-818) Binimetinib (MEK-162), Napabucasin (BBI-608), Vargatef (Nintedanib), Masitinib (AB-1010), Fruquintinib (HMPL-013), Famitinib (SHR-1020), Donafenib (CM-4307), or any of their combinations.

Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising the compound of the invention or the composition of the invention.

In this report, "pharmaceutical form" is understood as the mixture of one or more active ingredients with or without additives that have physical characteristics for their adequate dosage, conservation, administration and bioavailability. In another preferred embodiment of the present invention, the compositions and dosage forms of the invention are suitable for oral administration, in solid or liquid form. Possible forms for oral administration are tablets, capsules, syrups or solutions and may contain conventional excipients known in the pharmaceutical field, such as aggregating agents, fillers, disintegrants or a pharmaceutically acceptable surfactant. Other pharmaceutical forms can be colloidal systems, which include nanoemulsions, nanocapsules and polymeric nanoparticles. Compositions for oral administration can be prepared by conventional Galenic Pharmacy methods, such as mixing and dispersion. The tablets can be coated following methods known in the pharmaceutical industry.

The compositions and pharmaceutical forms can be adapted for parenteral administration, as sterile solutions, suspensions, or lyophilisates of the products of the invention, using the appropriate dosage. Suitable excipients, such as pH buffering agents or surfactants, may be used.

The aforementioned formulations can be prepared using conventional methods, such as those described in the Pharmacopoeias of different countries and in other reference texts. The term "medicine", as used herein, refers to any substance used for the prevention, diagnosis, relief, treatment or cure of diseases in man and animals.

Administration of the compounds, compositions or dosage forms of the present invention can be carried out by any suitable method, such as intravenous infusion and oral, topical or parenteral routes. Oral administration is preferred due to the convenience of patients and the chronic nature of the diseases to be treated.

The amount administered of a compound of the present invention will depend on the relative effectiveness of the compound chosen, the severity of the disease to be treated and the weight of the patient. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3 or 4 times daily, with a total dose between 0.1 and 1000 mg/Kg/day. It is important to keep in mind that it may be necessary to introduce variations in the dose, depending on the age and condition of the patient, as well as modifications in the route of administration. The compounds and compositions of the present invention can be used together with other medications in combination therapies. The other drugs may be part of the same composition or a different composition, for administration at the same time or at different times.

Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES OF THE INVENTION

Virtual screening

With the aim of finding new inhibitors for hepsin, a virtual screening was carried out using the molecular docking technique from the crystallographic structure of the protein deposited in the Protein Data Bank with code 1P57 against the DrugBank compound library (https:/ /go.drugbank.com/) in the coordinates of its catalytic site, characterized by residues HIS57, ASP102 and SER195.

Molecular docking was carried out in a high-performance computing cluster using the Autodock Vina 1.1.2 software (http://vina.scripps.edu/) and for this both the protein structure and the DrugBank ligand library were converted. to pdbqt format using MGLTools.

Once the calculations were completed, the compounds were prioritized based on docking score and the first 8 were visually examined, selecting the compound Venetoclax (DrugBank code DB11581) with a docking score of -11 Kcal/mol and hydrophobic interactions with residues PRO-60, LEU-41, GLN-73 and a hydrogen bond interaction with residue ASN143 as seen in Figure 1. In this figure we can see that venetoclax prevents access to the catalytic triad, represented in surface format for its three residues.

Venetoclax acts as an irreversible hepsin inhibitor.

Using a fluorogenic assay, the ability of Veneoclax to inhibit the proteolytic activity of hepsin was determined. Hepsin is a serine protease capable of hydrolyzing the substrate BOC-Gln-Arg-Arg-AMC (Bachem, Barcelona, Spain), so that it is possible to record the fluorescence emission using a plate reader with excitation and emission wavelengths. of 380 nm and 460 nm, respectively. To do this, hepsin (0.05 μM) (R&D Systems, Madrid, Spain) was incubated in 50 mM Tris-HCl buffer, pH 9 buffer, with 200 μM BOC-Gln-Arg-Arg-AMC and we recorded the fluorescence emitted for 5 minutes. . To check the effect of Venetoclax, the same reaction was carried out, but previously incubating hepsin with Venetoclax for 1 hour at 37°C and at different concentrations that ranged between 0.167 and 2.5 μM. In this way we calculated the IC5O value, or inhibitor concentration at which half of the maximum rate of hepsin is reached in the hydrolysis of its substrate, which was 0.48 μM.

Venetoclax reduces the migration of colorectal cancer cells

Once the cells reach confluency, they form a monolayer. By removing a strip of cells 300-500 µm wide with a 200 µL pipette tip, it is possible to evaluate the migration capacity of the cells and the effect of Venetoclax on this process. The migration capacity is evaluated based on the percentage of occupied area after 48 hours of incubation and is quantified using ImageJ software. The results show that Venetoclax at a concentration of 1.88 μM significantly reduces the migration of Caco-2 cells, both with basal expression and with hepsin overexpression, by stable transfection of a plasmid containing the HPN gene.

Venetoclax reduces invasion of colorectal cancer cells

To evaluate invasion, an assay was carried out in which the ability of the cells to degrade a gelatin matrix was evaluated. To do this, 0.2% gelatin and rhodamine (Invitrogen, Life Technologies, Madrid, Spain) were mixed in a 1:55 ratio in 61 mM NaCl buffer, 50 mM sodium borohydrate and then we dialyzed overnight in PBS.

Covers were prepared with the prepared mixture by covering them and the matrix was fixed with 0.5% glutaraldehyde for 15 min. The coverslips were washed with PBS and Caco-2 or KATO-III cell suspensions were added for 72 hours. Cells were subsequently fixed with 3.7% formaldehyde and incubated with phalloidin (0.01 mg/ml) and ProLongGold Antifade with DAPI (4',6-diamino-2-phenylindole; ThermoFisher). Images were taken with a LEICA SP8 confocal microscope and analyzed with ImageJ and processed with Adobe Photoshop. Our results showed that Venetoclax, at a concentration of 1.88 μM, significantly reduced gelatin degradation of Caco-2 cells with both basal expression and hepsin overexpression.

Venetoclax reduces thrombin generation

Since hepsin activates FVII and therefore the coagulation cascade, it could contribute to the hypercoagulable state of colorectal cancer patients with elevated hepsin levels. To test this hypothesis, plasma from 20 healthy subjects, from whom 3.8% citrated blood was extracted by venipuncture, was incubated with Caco-2 cells with hepsin overexpression. The plasma was previously centrifuged at 2500 g for 20 minutes. For the thrombin generation assay, the incubated plasma was removed and incubated with LOW® reagent (tissue factor: 1 pmol; phospholipids: 4 μmol; Diagnostica Stago) in a 96-well plate. All samples were prepared in duplicate. Coagulation in these samples was initiated by adding calcium chloride to a buffer containing the fluorogenic substrate FluCa-kit reagent® (Diagnostica Stago). A thrombin calibrator was used for each individual sample. Fluorescence was recorded for 60 min on a Fluoroskan Ascent fluorometer (Thermolab Systems) and data were analyzed using Thrombinoscope™ software (version 5.0.0.742; Diagnostica Stago). The following parameters were analyzed: (a) lagtime, which indicates the beginning of the thrombin generation phase; (b) time to reach maximum thrombin concentration (ttPeak); (c) maximum thrombin concentration (Peak); (d) mean frequency index (MRI) of the propagation phase of thrombin generation calculated by the formula Peak/(ttPeak - lag-time) and expressed in nM/min; and (e) endogenous thrombin potential (ETP) showing thrombin enzymatic activity evaluated as the area under the curve.

The results show that Venetoclax reduces endogenous thrombin potential (ETP), peak and peak speed while causing an increase in the start time of the thrombin generation phase (lag-time) and the time to reach the peak.

Table 1. Average values of the thrombin generation test.

Venetoclax does not affect cell death, cell cycle or proliferation

Since Venetoclax is a drug directed against Bcl-2, we wanted to evaluate whether the reduced cell migration or invasion could be due to greater death, altered cell cycle, or reduced cell proliferation. To do this, the cells (5x105 cells/ml) were incubated in 6-well plates in the presence or absence of Venetocláx after 48 h. The type of cell death was determined using the Annexin V/7-ADD assay (Annexin V Apoptosis Detection Kit FITC; eBiosciences, Thermo Fisher, Karlsruhe, Germany; 7-ADD BD-Biosciences) following the manufacturer's instructions. The cell cycle was analyzed after treating the cells with RNAse and propidium iodide (Invitrogen). To evaluate the proliferative activity of Caco-2 cells, 5-ethynyl-2'-deoxyuridine (EdU) (Thermo Fisher Scientific) was added for 48 h. Aliquots of the tumor cells were then used to determine the percentage of positive cells, i.e., Caco-2 or Caco-2-HPN-EdU+, detected by fluorescent azide coupling reaction with EdU according to the manufacturer's protocol ( Click-iT; Thermo Fisher Scientific).

The results showed that Venetoclax does not affect death (percentages around 5%) or cell proliferation or the cell cycle.

Claims (6)

1. The compound of formula (I) or any of its pharmaceutically acceptable salts, esters, tautomers, solvates and hydrates, or any combination thereof, for use in the prevention, improvement, relief and/or treatment of colorectal cancer in a individual where said individual has elevated hepsin levels with respect to normal levels.

Formula (I) 2. The compound of formula (I) or any of its pharmaceutically acceptable salts, esters, tautomers, solvates and hydrates, or any combination thereof, for use according to the preceding claim, wherein the colorectal cancer is metastatic colorectal cancer.345 3. A composition comprising the compound of formula (I) or any of its pharmaceutically acceptable salts, esters, tautomers, solvates and hydrates, or any combination thereof, for use according to any of claims 1-2. 4. The composition according to claim 3, which is a pharmaceutical composition. 5. The composition according to any of claims 3-4, wherein the composition further comprises excipients and/or a pharmaceutically acceptable carrier. 6. The composition according to any of claims 3-5, wherein the composition further comprises another active ingredient.