WO2018142007A1 - Nanoconjugates formed from dendritic molecules and peptides as antitumour agents against cancer - Google Patents

Abstract

The present invention relates to the formation of nanoconjugates exhibiting antitumour activity, mainly against advanced prostate cancer, but without excluding others. The nanoconjugates are formed from dendritic systems (dendrimers or dendrons) and neuropeptides. The dendritic molecules mainly have a carbosilane structure with ammonium groups on the periphery. The peptides are preferably from the glucagon/secretin family (e.g.VIP, GHRH, PACAP). The combination of these two systems leads to a nanoconjugate with anticarcinogenic properties that differ from those of their individual precursors. The antitumour activity is reflected in the growth inhibition and death of tumour cells in advanced prostate cancer (PC3) and, in addition, said nanoconjugates favour cell adhesion and prevent tumour cell migration processes, in other words metastasis processes.

Description

 NANOCONJUGADOS FORMED BY DENDRÍTIC MOLECULES AND PEPTIDES AS ANTITUMOR AGENTS AGAINST CANCER

The present invention relates to the formation of nanoconjugates that exhibit antitumor activity, mainly against advanced prostate cancer. Nanoconjugates are composed of dendritic systems (dendrimers and dendrons) and neuropeptides. The dendritic molecules are mainly of carbosilane structure and with ammonium functions in the periphery and the peptides are preferably of the glucagon / secretin family (eg, VIP, GHRH, PACAP).

STATE OF THE PREVIOUS TECHNIQUE

Prostate cancer (PC) is the second most frequent among men worldwide (Siegel, et al., Cancer J. Clin., 2014, 64, 9). It is a heterogeneous tumor with a slow but constant growth rate, which evolves from a localized stage and with androgen sensitivity to an advanced stage in which this sensitivity is lost. Treatment depends on the stage of the disease; if discovered early it can be treated satisfactorily by different procedures; Unfortunately, when tumor cells invade the area of the gland, it evolves into a phenotype of metastases and current treatments offer no cure. In addition, this type of cancer has a tendency to metastasize to bone (90%) and lung (46%) (Bubendorf, et ai, Hum Pathol, 2000, 31, 578), which are of the metastases with a lower prognosis of cure. Neuropeptides such as vasoactive intestinal peptide (VIP), growth hormone releasing hormone (GHRH), or pituitary adenylate cyclase activating peptide (PACAP), belonging to the glucagon / secretin family (Cardoso, et al., BMC Evol. Biol., 2010, 10, 13), have a wide biological activity in the organism and are very involved in the etiopathogenesis of PC. VIP has a protumoral effect on prostate tumors, increasing proliferation, protecting it from apoptosis (Gutiérrez-Cañas, et al., Br. J. Pharmacol., 2003, 139, 1050), increasing angiogenesis (Collado, et al. ., Peptides (Amsterdam, Neth.), 2007, 28, 1896), metastasis (Fernández-Martínez, et al., Prostate, 2009, 69, 774) and helping the progression of cancer towards more aggressive stages (Fernández- Martínez, et al., Cancer Lett., 2010, 299, 11). Its mechanism of action is through GPCR type receptors: PACi, VPACi, VPAC ₂ and FPRL-1 (Garcia-Fernandez, etal., Peptides, 2003, 24, 893, El Zein, et al., 2008, 83, 972 ). For its part, GHRH also acts through GPCR receptors, in this case hGHRH and its different variants of "spiicing" (SVs). Its function is to regulate the secretion of growth hormone (GH) (Martinez-Moreno, et al., Gen. Comp. Endocrinol., 2014, 199, 38), which in turn induces the activation of the IGF-1 factor ( Gan, et al., Mol. Endocrinol., 2013, 27, 1969), which plays a crucial role in malignancy, metastasis and tumorigenesis in several tumors such as CP (Bellyei, et al., Cancer Lett., 2010, 293, 31, Shevah, et al., Growth Horm. IGF Res., 2007, 17, 54, Takeuchi, et al., Mol. Cell. Endocrinol., 2014, 384, 1 17).

Both VIP and GHRH have an autocrine and paracrine action in prostate cancer cells (Gutierrez-Cañas, et al., Br. J. Pharmacol., 2003, 139, 1050, Busto, et al., Proc. Nati. Acad. Sci. USA, 2002, 99, 1 1866), so there is always a basal concentration of both inside the cell and its surroundings, which "feed" the cells. Both join their membrane GPCRs receptors and initiate a cascade of events mediated by the increase of cAMP as the second main messenger, although it can also be mediated by others such as Ca ²⁺ . Very promising antagonists have been designed for both peptides in the treatment of various human tumors (Plonowski, et al., Int. J. Cancer, 2002, 98, 624, Fahrenholtz, et al., Proc. Nati. Acad. Sci. USA , 2014, 111, 1084, Muñoz-Moreno, et al., Invest. New Drugs, 2014, 32, 871). However, antagonists continue to have problems, since although their half-life is longer than that of the original peptides, it is still short.

The dendritic systems (or molecules) (dendrimers and dendrons) are hyperbranched macromolecules of perfectly defined structure and polyfunctionalized. The dendrimers are of spherical morphology with a multivalent surface and the dendrons are of wedge type topology, also with a multivalent surface and an additional active position at the apex of this wedge called focal point. Dendrons can be considered fragments of dendrimers, and in fact, one of the synthetic procedures of dendrimers uses precisely dendrons as a building block (Sánchez-Nieves, et al., Tetrahedron, 2010, 9203). In addition, in the case of dendrimers and dendrons with the same type of skeleton, the same type of reactions for the synthesis of both topologies (Fuentes-Paniagua, et al., RSC Adv., 2016, 6, 7022). Both dendrimers and dendrons allow a large number of functional groups to be concentrated producing a unique and different effect than we would find in these same groups if they were individually (Astruc, et al., Chem. Rev., 2010, 1 10, 1857, Newkome , et al., Dendrimers and Dendrons, Wiley-VCH Verlag GmbH, 2004, 1).

Dendritic systems have been used as transporters of drugs or antitumor nucleic acids because of their ability to absorb "in vivo" in tumor areas and internalize the treatment in cancer cells due to the EPR (Enhanced Permeability and Retention) effect (Chen, et ai, World J. Surg. Oncol., 2012, 10, 3, Jain, et ai, Eur. J. Pharm. Biopharm., 2014, 87, 500, Huang, et ai, Biomacromolecules, 2014, 15, 915). This effect is based on the fact that the tumor areas with greater angiogenic activity have a greater amount of blood vessels whose endothelium has a greater permeability (the cells are usually separated between 200-600 nm) than in a "healthy" blood vessel, allowing passage of substances through it more easily. In addition, once the dendrimer leaves the blood vessel it accumulates in the tumor area, since the flow of the lymphatic system is reduced in these areas and it is more difficult to return them to the circulatory system, unlike the small molecules that do return into the bloodstream (Azzopardi, et ai, J. Antimicrob. Chemother., 2013, 68, 257, Fang, et ai, Adv. Drug Delivery Rev., 201 1, 63, 136, Konno, et ai, Cancer, 1984, 54, 2367).

To act as nucleic acid transporters, cationic systems are generally employed, such as cationic dendritic molecules, which by electrostatic interaction with them, allow these acids to be protected from degradation by nucleases (Svenson, et ai, Advanced Drug Delivery Reviews, 2012 , 64, 102). Similar strategies have been used for studies of peptide therapies, such as against human immunodeficiency virus (HIV) (lonov, et ai, Biochimica et Biophysica Acta, 2015, 1848, 907). Also in these nanoconjugates the electrostatic interaction between the dendritic system and peptide is used to stabilize the peptides and transport them to the cells, where their release facilitates their activity. DESCRIPTION OF THE INVENTION

 The present invention provides the preparation of active nanoconjugates against advanced prostate cancer. These systems are made up of dendritic molecules (dendrimers and dendrons) and neuropeptides. Preferably, the dendritic macromolecules are of carbosilane structure, mainly with cationic functions in the periphery, and the neuropeptides are of the glucagon / secretin family, mainly VIP, GHRH and PACAP. These nanoconjugates are formed by combining the dendritic molecule and the corresponding peptide in the necessary proportion. The formation of the nanoconjugate allows to create a system that presents anticancer properties in advanced prostate cancer and avoid metastasis.

Therefore, a first aspect of this invention relates to a combination of peptide and dendritic molecule comprising:

 A neuropeptide, preferably from the glucagon / secretin family. This peptide is preferably the vasoactive intestinal peptide (VIP), the growth hormone releasing hormone (GHRH) or the pituitary adenylate cyclase activating peptide (PACAP), without ruling out others having a similar activity.

 A dendritic compound, which refers in the present invention to a highly branched macromolecule where the growth units, branches or branches have carbosilane skeleton.

This dendritic compound is preferably a dendrimer, spherical in shape that has a growth nucleus of the polyfunctional molecule. The growth units, branches or branches have carbosilane skeleton and the outer layer, surface or periphery of the dendrimer incorporates preferably cationic groups. The skeleton and structure of these carbosilane dendrimers with different nuclei have been previously described (see for example ES-2444490). The dendritic compound can also be dendritic or dendron wedge type, which refers to a highly branched cone-shaped macromolecule and is defined by a focal point, the units, branches or branches of growth, which start from said focal point and the outer layer, surface or periphery of said branches that incorporates functional groups. The skeleton and structure of these dendrons carbosilanes with different focal points have been previously described (see for example ES-2444490).

In another preferred embodiment, the dendritic compound further comprises, in the outer layer of the dendrimer or dendron or at the focal point of the dendron, at least one functional group of different nature to the rest of the functional groups, and which can be selected from a molecule etiquette, a steering group or an active substance. These dendrimers and dendrons have been previously described (see for example ES-2444490; P201500669).

The term "tag molecule" refers in this description to any biorecognizable substance, chromophore, fluorophore or any other group detectable by spectrophotometric, fluorometric, optical microscopy, fluorescence or confocal, antibody and / or NMR techniques, and which easily allows detection of another molecule that alone is difficult to detect and / or quantify. For example, and without limitation, fluoroforo is selected from a list comprising cytochrome, fluorescein, rhodamine and dansyl.

By "director group" is meant a molecule or functional group capable of directing the dendritic compound specifically to a type of cells or to a specific area of a cell, such as folic acid, a signal peptide or an antibody, among other known by any subject matter expert. Said lead group can be previously functionalized to bind the dendritic compound.

By "active ingredient" or "drug" is meant in the present invention any purified chemical substance used in the prevention, diagnosis, treatment, mitigation or cure of a disease; to avoid the appearance of an unwanted physiological process; or to modify physiological conditions for specific purposes. Said active principle is able to bind directly to the dendritic compound or with a previous modification of its structure for it.

The present invention also relates to the uses in biomedicine of the peptide / dendritic molecule combinations. Preferably, this invention is for the development of drugs for the treatment of prostate cancer. However, other types of cancers are not excluded. More preferably, the present invention focuses on the treatment of advanced prostate cancer.

Another aspect of the present invention relates to the use of the nanoconjugates of the invention for the manufacture of a medicament. More preferably, the medicament is used for the prevention and / or treatment of prostate cancer. Another aspect of the present invention relates to a pharmaceutical composition comprising at least one dendritic molecule and a neuropeptide, as described above, and a pharmaceutically acceptable carrier. In addition, this pharmaceutical composition may comprise another active ingredient, preferably with antitumor properties. The antitumor may be doxorubicin, methotrexate or platinum compounds or others with antitumor properties.

The "pharmaceutically acceptable vehicles" that can be used in said compositions are the vehicles known to a person skilled in the art. Examples of pharmaceutical preparations include any solid composition (tablets, pills, capsules, granules, etc.) or liquid (gels, solutions, suspensions or emulsions) for oral, nasal, topical or parenteral administration.

In the sense used in this description, the term "therapeutically effective amount" refers to the amount of the composition calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of the composition itself, the age, condition and history of the patient, the severity of the disease, and the route and frequency of administration.

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

Figure 1 . Example of a second generation cationic carbosilane dendrimer with a polyphenolic type core ([G ₂ 03 (S-NMe ₂ R) i 2] ^{1 2+} ; (R = (CH ₂ ) ₂ OH (1), Me (2)) .

Figure 2. Example of third generation cationic carbosilane dendron. For example, X = OH, NH ₂ , SH, or others of interest.

Figure 3. Cell viability in the presence of VI P and GHRH peptides, dendrimer 1 and PC3 cell peptide / dendrimer nanoconjugate. * p <0.05; ** p <0.01; *** p <0.001.

Figure 4. Results of adhesion assays on PC3 cell lines of dendrimer 1 alone or bound to peptide VI P or GHRH. Mean values ± E.S.M. , of 6 experiments, ** p <0.01 and *** p <0.001 versus control values; # p <0.05 and ## p <0.01 with respect to the dendrimer.

Figure 5. Migration of PC3 cells at time 0, 6 and 24 hours with the different treatments. Figure 6. Migration of PC3 cells at 6 h. of treatment. Mean values ± E.S.M. , of 1 experiment, with 6 duplicates. * p <0.05 versus control values.

Figure 7. Flow cytometry test in PC3 cells 24 hours after treatment. Mean values ± E.S.M., of 2 experiments, * p <0.05 versus control values are shown.

EXAMPLES

Dendrimers and dendrons.

Two types of second generation carbosilane structure dendrimers functionalized with different ammonium groups were analyzed (G ₂ 0s (S-NMe ₂ R ⁺ ) and ₂ ; R = (CH ₂ ) ₂ OH (1), Me (2), Figure one ). Formation of nanoconjugates.

 The formation of the nanoconjugates was carried out by mixing in solution, preferably aqueous in the presence or absence of buffer, the dendritic molecules and the corresponding peptides, in the proportion of interest.

In a preferred embodiment, the mixtures were made directly on the tumor cells. In a well containing 25 x 10 ⁴ cells / ml, 445 μΙ_ of RPMI-1640 medium with 10% fetal bovine serum (FBS) with 1% antibiotic and antifungal (penicillin / streptomycin / amphotericin) were added. Then 50 μΙ_ of a 10 ^"8 M solution of the dendritic system made in the same medium mentioned above was added. Then 5 μΙ_ of peptide (VIP stock 10 ^{" 4} M or GHRH stock 10 ^"5 M) was added.

In another preferred embodiment, nanoconjugates may also be formed prior to their addition on the well containing the cells. For this, a solution is prepared containing 45 μΙ_ of RPMI-1640 medium with 10% fetal bovine serum (FBS) with 1% antibiotic and antifungal (penicillin / streptomycin / amphotericin). Then 50 μΙ_ of a 10 ^"8 M solution of the dendritic system made in the same medium mentioned above are added. Then 5 μΙ_ of peptide (VIP stock 10 ^{" 4} M or GHRH stock 10 ^"5 M) are added.

ANTITUMORAL ACTIVITY OF PEPTIDE / DENDRIMMER NANOCONJUGADOS AGAINST PROSTATE CANCER MATERIALS AND METHODS

Reagents

 As controls for cell toxicity tests, the same medium used in the dissolution of nanoconjugates was used.

Cells.

The human prostate cell line, PC3, representative of an androgen-independent stage of prostate cancer, obtained from ATCC (American Type Culture Collection) is used. The cells are maintained in RPMI-1640 medium with 10% serum Fetal bovine (FBS) with 1% antibiotic and antifungal (penicillin / streptomycin / amphotericin). The cells are stored at 37 ° C in a humid environment and with 5% C0 ₂ . Toxicity test by reduction of tetrazolium salts.

This technique is a colorimetric assay based on the selective ability of living cells to reduce 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide in formazan insoluble crystals. This method makes it possible to determine the lethal effect of the compounds under study on cell metabolism, since cellular damage results in a decrease in the mitochondrial activity of the cell and the cytotoxicity of these molecules can be measured. This test was carried out according to the manufacturer's instructions (MTT, Sigma-Aldrich, St Louis, USA) - PC-3 cells (15 x 10 ⁴ cells / ml) are plated in 24-well plates. After 24 hours the culture medium is removed and treated. The supernatant containing the treatment is removed at 24 hours and replaced by 500 μΙ of a culture medium to which 25 μΙ of MTT in PBS of a concentration of 1 mg / ml is added. After 1.5 hours of incubation under culture conditions, the supernatant is removed with the excess of unreacted MTT. Formazan crystals are subsequently dissolved with 100 μΙ of isopropanol. The absorbance is read at 570 nm by subtracting the background measured at 620 nm. The relative cell viability (%) with respect to the control (untreated cells) is calculated based on the formula: [A] test / [A] control x 100.

Adhesion tests.

The suspension of PC3 cells (25 x 10 ⁴ cells / ml) is separated into several fractions that will depend on the number of treatments to be performed and are kept in suspension for 1 hour, at 37 ° C, with agitation every 10 minutes. The plates with the collagen that acts as matrix, are prepared with 100 μΙ of type I collagen (8 μg / cm ² ) diluted in 10 mM acetic acid / well, in P-96 plates, for 1 hour, at 37 ° C, in a dry atmosphere to achieve a complete fixation to the well. After that time, the collagen is aspirated and the plate is washed four times with PBS. 100 μΙ of the previously treated cell suspension is added to each well and the medium is aspirated at 40 minutes. Finally, the evaluation of the cells adhered to the collagen is continued by means of an MTT assay, where 100 μΙ of serum-free medium and 0.1 mg / ml MTT are added to each well. After removal of the supernatant with excess MTT that does not react, Dissolve the formazan crystals with 100 μΙ of isopropanol. The absorbance at 570 nm is measured by subtracting the bottom at 620 nm. The results are expressed in% with respect to the absorbance of the control (untreated cells): [A] test / [A] control x 100. Migration test.

PC3 cells (20 x 10 ⁴ cells / ml) are seeded in 24-well plates. Cells are treated with the dendrimer and peptides 24 hours. After removing the medium, a wound is made in each well and the progression of its closure at different times is followed by photographs taken with the inverted microscope coupled to the Motic camera. The results are processed by measuring the size of the wound and the% of the progress of the wound is plotted against that of zero time.

Cell cycle tests.

PC3 cells (7.5 x 10 ⁴ cells / ml) are seeded on P-6 plates and held for 24 h to achieve adhesion, at which time they are deprived with serum-free medium for 24 h. After this time, the different treatments are performed and at 24 hours, the cells are washed with PBS, raised with trypsin and centrifuged. The cell precipitate is fixed and permeabilized with 70% cold ethanol, for 5 days, at 4 ^o C. Then, the cells are centrifuged to remove the ethanol and washed with PBS. Finally, they are resuspended in a staining solution (PBS, 50 mg / ml of propidium iodide (IP) and 10 μg / ml of RNase), before analysis by flow cytometry with FACSCalibur. The amount of DNA that is distributed in the different phases of the cell cycle is analyzed with the Cyflogic v 1.2.1 program. Statistic analysis.

 Data are expressed as the mean ± S.E.M. Statistical analyzes are performed using the Annova test and considering the results significant with P <0.05.

RESULTS

The studied dendrimer / peptide combination (1 / VIP, 1 / GHRH)) slows tumor growth and causes cancer cells to die at low concentrations, leading to anticancer behavior (Fig. 3, dendrimer results 1). It should be noted that these peptides studied (VIP / GHRH) have protumoral activity and that although the dendritic systems used (1 and 2) They showed some anti-tumor activity, the combination with VIP and GHRH is much more effective. Subsequent trials showed that these nanoconjugates favored cell adhesion and avoided tumor cell migration processes (Fig. 4, 5, 6, results of dendrimer 1). This effect was contrasted with that which only the dendrimer (1) would have and their inefficiency in preventing adhesion and migration was observed.

In order to know if the treatments were producing apoptosis or any change in the cell cycle, flow cytometry tests were performed (Fig. 7, results for dendrimer 1). As you can see, no treatment induces apoptosis, although there is a variation in the cell cycle for the treatments of peptides bound to the dendrimer. For these cases it is observed that the cycle is stopped in the synthesis phase (S), increasing the number of cells in this phase and decreasing the cells of the following phases (G2 and Mitosis).

Claims

1 A combination of peptide and dendritic molecule, dendrimer or dendron, preferably of carbosilane structure but without ruling out others, called nanoconjugate. 2. The peptide according to the preceding claim is preferably a neuropeptide, preferably from the glucagon / secretin family. 3. Nanoconjugate according to the preceding claim wherein the neuropeptide is the vasoactive intestinal peptide, VIP, growth hormone releasing hormone, GHRH, or the pituitary adenylate cyclase activating peptide, PACAP, without ruling out others having a similar activity. 4. Nanoconjugate according to claims 1 to 3, wherein the dendritic compound is a dendritic wedge or a dendrimer. Preferably these compounds are cationic of carbosilane structure. 5. Compound according to any of the preceding claims, wherein the outer layer of dendritic molecules, dendrimers or dendrons, or the focal point of dendrons further comprises an additional function. These additional functions can be selected from a tag molecule, preferably a fluorophore, a leader group or an active ingredient. 6. Method of obtaining the nanoconjugates described according to any of claims 1 to 5, comprising a reaction between the peptide and the dendritic molecule or its combination in the appropriate medium. 7. Method for obtaining the nanoconjugates described according to any of claims 1 to 6, wherein said reaction or combination is preferably carried out in the presence of a polar solvent such as water, DMSO or an alcohol R ¹ -OH, where R ¹ is a (C1-C10) alkyl group, or a mixture thereof. 8. Use of the compounds described according to any of claims 1 to 5, as active ingredients for the development of an anticancer drug. 9. Use according to the preceding claim, wherein cancer is prostate cancer, preferably advanced prostate cancer. 10. Pharmaceutical composition comprising a nanoconjugate according to any one of claims 1 to 5. 1 1. Pharmaceutical composition according to the preceding claim further comprising a pharmaceutically acceptable carrier and / or other active ingredient, preferably an antibiotic, anti-inflammatory or anticancer.