WO2015097725A2

WO2015097725A2 - Novel estrogen receptors ligands coupled to protein g

Info

Publication number: WO2015097725A2
Application number: PCT/IT2014/000345
Authority: WO
Inventors: Maria Francesca SANTOLLA; Marcello MAGGIOLONI
Original assignee: Università Della Calabria
Priority date: 2013-12-24
Filing date: 2014-12-23
Publication date: 2015-07-02
Also published as: WO2015097725A3; ITRM20130719A1

Abstract

The present invention relates to the use of nicotinic acid or nicotinamide and to compositions thereof for use in the treatment of pathological conditions that can benefit from the biological activity induced by the activation of GPR30 receptor. The invention is also related to a method for the evaluation/selection of agonist or antagonist GPR30 receptor ligands.

Description

Novel estrogen receptors ligands coupled to protein G DESCRIPTION

The present invention relates to the use of nicotinic acid or nicotinamide and compositions thereof to be used for the treatment of pathological conditions that can benefit from the biological activity induced by activation of the GPR30 receptor. The invention also relates to a method for the evaluation/selection of ligands with agonist or antagonist activity to the GPR30 receptor.

Prior Art

The GPR30 receptor, also known as GPER (G protein estrogen receptor), is an orphan receptor of about 38kDa, characterized by the presence of 7 transmembrane coils typical of G-coupled receptors. (Lappano, R. and Maggiolini M. Nat. Rev. Drug Discov. 2011, 10: 47-60). GPER, whose structure is thus not related with the classical estrogen receptors (ERa and Erp), following its interaction with a series of ligands (estrogens and different types of compounds) activates different transductions! pathways (e.g., EGFR/ERK1-2, PI3K/AKT, and others) involved with important biological responses depending on the cell context (Maggiolini, M. and Picard, D. 2010, 204: 105-14; Prossnitz, E. R. and Maggiolini, M. Mol. Cell Endocrinol. 2009, 308: 32-38).

The GPER receptor is widely distributed in different tissues, such as heart and vessels, ovary, placenta, prostate, liver, pancreas, kidney, surrenal, epithelial and bone tissue, nervous and lymphatic systems. In particular, functional studies showed that G-l, a selective GPER agonist, promotes neuronal survival, like estrogens, following to cerebral ischemia (Brailoiu E, et al. Journal of Endocrinology 2007, 193: 311-321). In the cardiovascular system, G-l exerts a protective action since it reduces the riperfusion damages and cellular necrosis after miocardial infarction, also limiting the production of pro-inflammatory cytokines (Weil BR, et al. Surgery. 2010; 148: 436- 43). Activation of GPER during nephropathies induces a reduction of proteinuria and an improvement in creatinine clearance (Gilliam-Davis S, et al. Hypertension. 2010; 56: 50-166). It was also observed that estrogens, by means of GPER and the classical estrogen receptors (ERa and ERP) are able to induce thymic atrophy with a reduction of the cortical portion with respect to the medulla and consequent reduction of thymocytes (Wang C, et al. Mol Endocrinol. 2008; 22: 636-48). In human and murine pancreatic islets the downregulation of GPER was shown to cause a decrease in insulin secretion (Sharma G, et al. Endocrinology. 2011; 152:3030-9). GPER expression has also been shown in different tumor types, in particular those responsive to estrogens and it has been associated directly with tumor size, presence of metastases and HER-2/neu overexpression (Maggiolini M, et al, 2004 J Biol Chem 279: 2700827016). In this respect, it was reported that the functional cross-talk between different GPCRs and different growth factor receptors is involved in the activation of molecular signals which, by modulating the expression of a number of genes, contributes to the development of tumors, metastases formation and neo-angiogenesis. Regarding GPER regulation, not only important growth factors, such as l'EGF and IGF-1, have been involved in its expression, but also hypoxia which activates the transcriptional factor known as "hypoxia inducible factor- la" (HIF-la) then inducing the signal cascade mediated by GPER both in tumor cells and in cardiomyocytes (De Marco P, et al. 2013 Oncogene 7, 32: 678-88). This experimental evidence is of particular interest as it points to the fundamental role that GPER can have in different cellular contexts in association with its receptor activity and its hypoxia signalling.

On the basis of the aforementioned observations, the identification of selective GPER ligands with agonist or antagonist action appears to be of importance. Finding new compounds both of endogenous and synthetic origin capable to bind and activate the transduction pathway mediated by GPER is also extremely important for setting innovative diagnostic, prognostic, and therapeutic tools in a wide range of physiopathological conditions (e.g. tumors, cardiovascular, neurodegenerative, metabolic diseases among others) wherein GPER can exert a fundamental biological role.

Summary of the Invention

The present invention is based on the finding of two new GPR30 receptor ligands which selectively activate the transduction pathway mediated by this receptor. The new ligands are nicotinic acid, also known as vitamin B3, and the amidic derivative of nicotinic acid, known as nicotinamide.

A first object of the present invention is a screening method for GPR30 receptor regulatory agents comprising the following steps:

i) exposing to GPR30 receptor a test compound and nicotinic acid and/or nicotinamide;

ii) measuring the activity of said GPR30 receptor.

A further object of the present invention is nicotinic acid and/or nicotinamide to be used for the treatment of a pathological condition that is improved by the activation of the GPR30 receptor.

Still a further object of the present invention is a composition for use in the treatment of a pathological condition that is improved by the activation of GPR30 receptor including nicotinic acid and/or nicotinamide and one or more carriers and/or excipients and/or diluents.

Description of the Figures

Figure 1. We demonstrated the ability of nicotinic acid (NA) and nicotinamide (NM) to bind GPER (Fig. 1) by using 40nM [5,6-3H] nicotinic acid in breast tumor cells SkBr3 (ATCC HTB 30) expressing GPER but not the classic estrogen receptors (ERa and ERP) and the two nicotinic acid receptor isoforms (GPR109A and GPR109B). Actually, nicotinic acid, nicotinamide and the selective ligand for GPER, called G-1, were able to compete with labelled nicotinic acid in a dose-dependent manner (Figure 1A). Silencing GPER expression in SkBr3 cells through a specific short hairpin (shGPER) transfection, nicotinic acid, nicotinamide and G-1 were not able to compete with labelled nicotinic acid (Figure 1B-C), showing that GPER is necessary for the receptor competition to take place. This experiment clearly shows that nicotinic acid and nicotinamide are GPER ligands. Figure 2. To evaluate if GPER binding ef- to nicotinic acid (NA) and nicotinamide (NM) induces the expression of some important target genes of this receptor, such as c-fos and CTGF, immunoblotting assays were carried out in SkBr3 cells transfected for 24 h with a control vector (shRNA) or with a short hairpin for GPER (shGPER) and subsequently treated with 10 μΜ nicotinic acid and 10 μΜ nicotinamide for 4 h. Figure 2 shows that both compounds significantly induce a c-fos and CTGF protein expression in SkBr3 cells transfected with shRNA (·, p<0,05), while in SkBr3 cells transfected with shGPER the induction of c-fos and CTGF was abolished, as shown in the panel that illustrates the densitometric evaluation of three independent experiments (Figure 2A-B).

Figure 3. Nicotinic acid (NA) is able to inhibit the expression of "intercellular adhesion molecule- (ICAM-1) induced by TNF-a in human umbilical venous endothelial cells (HUVECs). ICAM-1 is a protein which by mediating the leukocytes adhesion to the vessel endothelium and their gathering in the tunica intima of the vessel endothelium and contributes to start the initial process of atherosclerotic plaque formation. The endothelial cells that are under the action of pro-inflammatory cytokines such as IL-1, IFN-γ e TNF-a are characterized by high levels of ICAM-1. Based on these findings we assayed GPER for its involvement in the inhibitory action by nicotinic acid on the ICAM-1 expression induced by TNF-a. To this end, HUVECs were transfected for 24 h with a control vector (shRNA) or with a short hairpin for GPER (shGPER) and subsequently treated with 1 mM nicotinic acid for 30 h and with 50 ng/ml TNF-a at the same time, during the last 6 h. As shown in Figure 3, the inhibitory effect exerted by nicotinic acid on ICAM-1 expression induced by TNF-a is not observed by silencing GPER (Figure 3A-B, ·, p<0.05) indicating that GPER mediates the modulating action of nicotinic acid on the expression of ICAM-1. The densitometric evaluation shows the results obtained in three independent experiments.

Figure 4. The role of GPER in the angiogenetic effects induced by nicotinic acid (NA) was evaluated with in vitro Matrigel angiogenesis tests. To this end, HUVECs were transfected for 24 h with a control vector (shRNA) or with a short hairpin for GPER (shGPER) and then treated with nicotinic acid for 12 h. Figure 4 shows that the formation of tubules induced by nicotinic acid is prevented by silencing GPER expression, showing that GPER mediates the angiogenetic effects induced by nicotinic acid in this experimental model. (For the details regarding the in vitro Matrigel angiogenesis assay method, see Vinals et al., 2001).

Detailed Description of the Invention

It is the object of the present invention a screening method for GPR30 receptor regulatory agents comprising the following steps: i) contacting the— GPR30 receptor with a test compound and nicotinic acid and/or nicotinamide; ii) measuring the activity of said GPR30 receptor. With the term "regulatory agents" as herein used, we refer to compounds that influence the GPR30 activity in vitro and/or in vivo. Regulatory agents can, for example, be agonists and antagonists of GPR30 receptor and can also be, for example, compounds that exert their effect on GPR30 activity by means of expression, or by post-transductional modifications or by still different means. GPER30 agonists are molecules that, when bound to GPR30, increase or prolong GPR30 activity.

GPR30 agonists include, for example, proteins, nucleic acids, carbohydrates, small molecules, or any other molecule that activates GPR30. GPR30 antagonists are molecules that, when bound to GPR30, decrease the amount or the duration of GPR30 activity. With the term "screening method", as herein used, a method for the identification or selection of a compound is meant.

The test compound will, for example, be a peptide, organic molecules, synthetic or natural organic molecules, mixtures thereof or different molecules. Test compounds useful for the screening methods of the invention can be obtained from any suitable source, for example, from libraries of known compounds.

In the screening method according to the present invention the test compound is contacted to the GPR30 receptor in the presence of nicotinic acid and/or nicotinamide. With the term "contacting" as herein used it is also meant any competitive receptor assay suitable for use with the GPR30 receptor. The-GPR30 receptor used in the method of the present invention will be the complete GPR30 polypeptide or GPR30 isoforms or truncated forms that have the same cell function and selectively bind nicotinic acid and/or nicotinamide, e.g., GPR30 variants can be used such as biologically active fragments or a fusion protein that comprises the GPR30 polypeptide sequence.

According to one embodiment, the polypeptide sequence SEQ ID NO. l can be used or the polypeptide sequence available in the GeneBank database with the accession number AFO 15257.1 or the polypeptide sequence available in the database with the accession number Uniprot Q99527 (last modified: 5/1/1997). In addition, GPR30 can be taken from any suitable mammalian species, preferably human, rat or mouse GPR30 will be used.

Test compounds binding to GPR30 with a stimulatory or inhibitory activity on GPR30 activity are identified either with methods that use cells expressing GPR30 on their cell wall (cell assays) or in assays with isolated GPR30 (non-cell assays). In cell assays all the cell lines that express GPR30 can be used, e.g., engineered cells expressing this receptor. The method can for example, be a binding assay that comprises a direct or indirect measurement of the binding of a test compound as a GPR30 ligand in the presence of nicotinic acid and/or nicotinamide and in the absence of nicotinic acid and/or nicotinamide or an activity assay that comprises the direct or indirect measurement of GPR30 activity.

The method will provide different screening assays combined with an in vivo assay comprising a step of measuring the effect of the test compound on the symptoms of cardiovascular diseases, infections, dermatological diseases, endocrine diseases, metabolic diseases, cancer, gastrointestinal diseases, inflammation, hematologic diseases, respiratory diseases, muscle and skeletal diseases, neurological diseases and urological diseases.

The measurement of the ability of a test compound to bind to GPR30 can be carried out, for example, by coupling the test compound with a detectable marker, such as, for example, a radioisotope or an enzyme, alternatively also nicotinic acid and/or nicotinamide can be coupled, directly or indirectly, with a detectable marker. For example, radioactive isotopes such as I¹²⁵ , S³⁵ , C¹⁴ , H³ can be used as detectable markers. Alternatively, enzymatic markers can be used, such as horseradish peroxidase, alcaline phosphatase or luciferase.

According to an embodiment of the method, the test compound and nicotinic acid /and or nicotinamide are used at the same time in step i), e.g., at different concentrations of the test compound or of nicotinic acid and/or nicotinamide, for example according to any receptor competition assay known as suitable to be used with the GPR30 receptor.

According to a further embodiment, the test compound or nicotinic acid and/or nicotinamide are contacted with the receptor either separately or at the same time.

In the following step ii) the GPR30 receptor activity can be measured with any known suitable, state-of-the-art method for detecting GPR30 activity.

The method according to the present invention can also comprise non-cell assays, e.g., assays where GPR30 is in a membrane-bound form or is made to adsorb as a soluble fragment onto a solid support, such as a microplate.

According to a preferred embodiment, the method can be carried out according to the procedure described for the binding assay described in the Experimental Section as Example 1.2.

Also an object of the present invention are nicotinic acid and/or nicotinamide for use in the treatment for a pathological condition that is ameliorated by activation of GPR30 receptor.

As used herein, the term "treatment" is used to mean both prevention of the disease and treatment of pre-existent conditions. Prevention of a disease is obtained by administration of nicotinic acid and/or nicotinamide before the development of an overt disease. Treatment of a disease in progress is directed to stabilizing or improving the patient's clinical symptoms. This treatment is preferably carried out before the complete loss of function in the interested tissues.

Pathological conditions improved by GPR30 activation are for instance neurological diseases. As a matter of fact, the acute neuroprotective effect mediated by GPR30 and the neuroprotective effect mediated by GPR30 activating agents are known in the prior art. It is therefore object of the present invention the nicotinic acid and/or nicotinamide for use in the treatment of neurological diseases such as Alzheimer's disease, Parkinson's disease, Huntington's corea, peripheral diabetic neuropathy, multiple sclerosis, amiotrophic lateral sclerosis and neurodegenerative acute diseases such as, for example, ictus, cranial trauma, damages to peripheral nerves, hypoglycaemia, spinal cord lesions, epilepsy, anoxia and hypoxia.

In addition to its hypolipidemic action, nicotinic acid decreases the atherothrombotic events risk by decreasing the levels of prothrombotic factors such as fibrinogen and the expression of cell adhesion molecules (CAM) that mediate monocytic and leucocytic adhesion, recruiting and migration through vessel surfaces, essential processes for the atherosclerotic plaque formation (Tavintharan S et al. Basic Clin Pharmacol Toxicol. 2009; 104:206-10). In particular, ICAM-1 protein expression (a highly predictive factor for coronary events, also deadly ones) is induced by the proinflammatory cytokine TNF-a, but this increase can be prevented by the treatment with nicotinic acid through GPR30, as shown in the experimental assays carried out in human venous umbilical endothelial cells (HUVECs) in which GRP30 expression has been silenced (Figure 3).

Nicotinic acid, thus, due to its positive biological actions induced through GPR30, can be used in the treatment of different inflammatory and degenerative diseases of the cardiovascular system in addition to different diseases of the bone, nervous, immune, endocrine systems that can benefit from GPR30 activation by estrogens, but are not, due to the side effects of these hormones.

Therefore, particularly interesting is a therapy based on nicotinic acid or nicotinamide as estrogen substitutes, as selective activators of GPR30 that do not activate other estrogen receptors, and, thus, do not have the negative, known estrogen effects.

When nicotinic acid or nicotinamide are used according to the present invention for the treatment of the above described pathologies, the dose can be, for example, in the range from 1 mg to 1 g, preferably from 10 to 1000 mg. In the treatment method the precise dose and the frequency of administration of the compositions will depend from the particular seriousness of the condition to be treated, from the age, weight and general physical conditions of the particular patients, as well known to the expert in the field. Typical dosage regimens to be used in the treatment method can be from 1 to 3 dosage units per day, for example after meals in the amounts described above, for example administering 1 dosage unit every 8 hours.

Further object of the present invention are the compositions comprising nicotinic acid and/or nicotinamide for use in the treatment of all the pathological conditions previously described.

Compositions according to the present invention can be prepared by selecting a suitable dosage form depending on the administration route. Examples of dosage forms for use according to the present invention are, for example, tablets, powders, granules, capsules, solutions, syrups, elixirs, oils or aqueous suspensions or similar preparations for oral use. A stabilizing agent, a preservative and a solubilising agent are preferably added in case of dosage units for injections. Examples of preparations for external use are solutions, suspensions, emulsions, ointments, gels, creams, lotions, sprays among others. A solid preparation can contain, in addition to nicotinic acid and nicotinamide, other pharmaceutically acceptable vehicles. For example, fillers, diluents, disgregants, binding agents, solubilising agents, wetting agents, etcetera, can be suitably selected and mixed to give a preparation. Examples of liquid preparations are solutions, suspensions, emulsions among others. These can contain a suspension agent, emulsifier and/or similar ones.

For the preparation of the pharmaceutical compositions, the mixture of compounds is formulated in suitable dosage units with one or more pharmaceutically acceptable excipients and carriers. Pharmaceutical compositions in the form of tablets and capsules for oral administration can be in the form of dosage units and can contain conventional excipients including, for example, binders, e.g., acacia gum, gelatin, sorbitol, tragacanth gum or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example, magnesium stearate, talc, polyethyleneglycol or silica; disintegrants, for example potato starch; and pharmaceutically acceptable wetting agents, for example sodium laurylsulphate. Tablets can be coated according to methods well known in the current pharmaceutical practice.

Compositions suitable for parenteral injection can comprise aqueous or nonaqueous sterile saline, dispersions, suspensions or emulsions, or can comprise sterile powders to be reconstituted in solution or injectable sterile dispersions or emulsions, or can comprise sterile powders to be reconstituted in solutions or injectable sterile dispersions. Examples of suitable vehicles comprise water, ethanol, polyols (propylen glycol, polyethylene glycol, glycerol and others). The optimal fluidity can be maintained, for example, by means of a coating such as lecithin.

These compositions can also contain adjuvants for the preservation or prevention of microbial contamination of the compositions, for example parabens, chlorobutanol, phenol, sorbic acid among others.

The present invention has been herein described with reference to some of its embodiments. It must be understood that different embodiments that refer to the same inventive gist can exist, all of which fall within the scope of the claims herein below. Examples

1. Materials and Methods used for the Experimentation 1 Cell Cultures

Breast cancer cells (SkBr3) were cultured in 10 cm Petri dishes for cell cultures containing specific culture media, at 37°C at 5% carbon dioxide (C02) in a humidified environment. SkBr3 cells were kept in RPMI medium without phenol red with 10% FBS and 1% penicillin-streptomycin. Human umbilical venous endothelial cells (HUVECs) were cultured in EGM medium (Endothelial Growth Medium) with 5% FBS and 1% penicillin-streptomycin.

All the cell cultures manipulations were carried out in a laminar flow hood to guarantee sterility. The culture medium was changed every 48 hours, with a previous washing with the same media, in order to wash away the catabolytes accumulated during this time period and in order to avoid that they, together with pH variations in the media, could influence cell growth.

1.2 Ligand binding assays

A ligand binding assay is a test that is part of the radioanalytic techniques and takes advantage of the competitive protein binding. To determine if a compound works as a receptor agonist, the ability of a compound to displace the natural ligand from a specific receptor protein is tested.

The binding assay for GPER was carried out using SkBr3 breast cancer cells cultured in Petri dishes (10 cm diameter). Cells were washed twice and treated with 40 nM [5.6-3H] nicotinic acid (50-60 Ci/mmol), in the presence and absence of unlabeled nicotinic acid, nicotinamide and the selective ligand GPER, called G-l. In the following step, cells were incubated for two hours at 37°C. After three rinses with cold PBS, the cells were lysed using 100% ethanol and the lysate radioactivity was measured by means of a liquid scintillation counter. Each samples was mixed with 4 ml of scintillation liquid. The presence of competition for the binding to GPER is evaluated thanks to the ability of the counter to convert the invisible radioactive emissions in the samples into visible light fotons, which are easily detected by a photomultiplier. A reduction in the radioactivity emitted by the test sample with respect to the sample containing only labeled nicotinic acid, thus a reduction of the emitted light, means that the competitor molecule has a displacing potential.

1.3 Western blotting

SkBr3 and HUVEC cells were cultured in the appropriate medium in 10 cm Petri dishes until 70-80% confluent. At this point, cells were rinsed twice with cold PBS and then were kept for 24 h in serum-free culture medium. After 24 h of deprivation, cells were subjected to suitable treatments and subsequently lysed by adding 500 μΐ of lysis buffer made of 50 mM Hepes pH 7.5, 1% glycerol (to avoid protein dissociation during the dilution), 1% triton-X-100 (to avoid protein adsorption onto the container's walls), a mixture of protease inhibitors 0,6% Aprotininl, 1 mM PMSF and 0.2 mm Na-Orto-vanadate2; which allow a maximal recovery of the protein content since they inhibit the activity of the endogenous proteases.

The lysis buffer is left to work for about 7 minutes, to allow the complete cell membrane lysis, at the end of which the overall protein content is collected in sterile eppendorf tubes. The collected lysate was then boiled for 10 min to recover the protein fraction from the supernatant, separating it form the cell membranes that remains at the bottom of the eppendorf tube as a pellet. The total protein concentration was determined by a colorimetric evaluation with the Bradford method, using a standard curve obtained from the absorbance values of bovine serum albumin (BSA) at different concentrations starting from a 1 mg/ml solution. To 50 μΐ of protein lysate diluted 1:10, 1 ml of Bradford reactive was added and the thus obtained solution was read in the spectrofotometer at a wavelength of 595 nm. The amount of bound dye depends from the basic aminoacids content of the proteins in solution. Aliquots of cell lysate containing the same amount of total proteins were subjected to separation by electrophoresis by means of the reducing SDS PAGE on 10% polyacrilamide gel. The proteins were added to a reducing solution containing Laemmli buffer 2, boiled for 5 minutes and then loaded onto a gel in a running buffer 10X suitably diluted and containing 25 mM TRIS, 192 mM Glicine pH 8.3 and 0.1% SDS. Onto the gel was loaded a marker made of a protein mixture of known molecular wieght that allowed to identify the protein under study. The electrophoretic run was carried out using a potential difference between 80 and 100 V for about 2 h at room temperature. After the electrophoretic run the proteins were transferred from the gel onto a nitrocellulose membrane by means of electrotranfer with a transblot chamber in low saline content transfer buffer (25mM TRIS, 192 mM Glycine pH 8.3, 0.1% SDS, 20% Methanol). The gel and the nitrocellulose membrane, left for 10 minutes in distilled water and then in transfer buffer were arranged so that, from this system, the nitrocellulose membrane is placed in between the anode and the gel. The protein transfer was favored by applying a potential difference of about 65V for 1 hour and a half at 4°C. The actual protein transfer from the gel onto the membrane was checked by placing the membrane in a solution of Ponceau Red for about 2-3 minutes. Subsequently, the membranes was placed in a 5% solution of non-fat milk in TBST IX (100 mM Tris HCL pH 7.5, 1M NaCl, 1% Tween 20) for 1 h at room temperature to block all the non-specific sites of hydrophobic interaction. After briefly washing with TBST IX buffer the nitrocellulose membrane was incubated overnight with the primary specific antibody for the protein under exam, suitably diluted. At the end of the incubation period the solution containing the primary antibody was removed and the membrane underwent three 10-minute washes each with TBST IX buffer. Then the membrane was incubated for lh at room temperature with the secondary antibody in TBST IX and 5% milk. The secondary antibody is able to recognize the constant portion of the IgG used as the primary antibody and is also conjugated with the horseradish peroxidase enzyme. The nitrocellulose membrane was then washed again with the buffer solution and submitted to an immunodetection technique by means of the chemiluminescence ECL System Kit. The peroxidase enzyme conjugated with the secondary antibody, is able to catalyze the oxidation of a chemiluminescent substrate in basic conditions, luminol (5 amino, 2-3 dihydroftalazine, 1-4 dione). The latter emits a certain amount of light that can be visualized by exposing the treated membrane to a photographic plate (Hyperfilm ECL).

1.4 Gene Silencing Experiments

The cells were seeded in 10 cm Petri dishes, kept in sereum-free medium for 24 hours and then transfected for 24 hours or 48 hours, before being treated, with X- treme GENE 9 (Roche Molecular Biochemicals, Milan, Italy), 0.5 μΐ plate of a control vector such as the shRNA vector (control Short Hairpin) and with the shGPER (Short Hairpin for silencing GPER). The cells were subsequently lysed and the lysates were analysed by Western blotting. A short hairpin RNA, usually abbreviated as shRNA, is a sequence that, bending, makes a structure that resembles a hairpin. Thus the RNA downstrem from this hairpin structure can not be translated since all the translational complex can not read the sequences thereby present and the corresponding proteins can not be formed.

1.5 Tube formation assay

HUVEC cells were transfected with a control vector such as the shRNA vector and with the shGPER in the regular growth medium and after 8 h the cells were deprived and treated with nicotinic acid 1 mM for 18 h at 37 °C. The following day the previously defrosted growth factor-free Matrigel® was plated at the bottom of 96-well plates and left to gelatinize at 37°C for 1 h. The deprived and treated HUVECs were collected by enzymatic detachment, counted and resuspended in EBM. 10,000 cells per well were then seeded onto the Matrigel and incubated at 37°C. Tubes formation was observed starting from 2 h from the seeding.

1.6 Statistical analysis

The statistical analysis was carried out using ANOVA analysis followed by the Newman-Keuls' test to calculate the mean differences. P values <0.05 were considered statistically significant. 2. Results

Using 40nM [5.6-3H] nicotinic acid in mammalian breast cancer cells SkBr3 (ATCC number HTB 30), that express GPER but do not express the classical estrogen receptors (ERa and Ε ) and the two nicotinic acid receptor isoforms (GPR109A and GPR109B), we demonstrated the ability of nicotinic acid (NA) and of nicotinamide (NM) to bind GPER (Figure 1). As a matter of fact, nicotinic acid, nicotinamide and the selective ligand GPER, called G-l, were able to compete with labeled nicotinic acid in a dose-dependent manner (Figure 1A). Silencing GPER expression in SkBr3 cells by transfecting a specific short hairpin (shGPER), nicotinic acid, nicotinamide and G-l were not able to compete with the labeled nicotinic acid (Figures 1B-C), showing that GPER is necessary for the receptor competition to take place. This experiment clearly demonstrates that nicotinic acid and nicotinamide are GPER ligands (for the details regarding the competition assay method see Lappano et al., 2012). To evaluate if the nicotinic acid (NA) and nicotinamide (NM) binding to GPER induce the expression of some important target genes of this receptor, as c-fos and CTGF, immunoblotting assays were carried out in SkBr3 cells transfected for 24 h with a control vector (shRNA) or with a short hairpin for GPER (shGPER) and subsequently treated with 10 μΜ nicotinic acid and 10 μΜ nicotinamide for 4 h. Figure 2 shows that both compounds significantly induce the c-fos and CTGF protein expression in SkBr3 transfected with shRNA (·, p<0,05), conversely in SkBr3 cells transfected with shGPER, c-fos and CTGF induction was abrogated as shown in the panel that reports the densitometric evaluation of three independent experiments (Figures 2A-B).

In human endothelial umbilical cells (HUVECs) nicotinic acid (NA) is able to inhibit the expression of "intercellular adhesion molecule- 1" (ICAM-1) induced by TNF-a. ICAM-1 is a protein that, mediating leukocytes adhesion to vessel endothelium and their accumulation in the endothelial vessel tunica intima, contributes to determining the initial formation process of the atherosclerotic plaque. The endothelial cells subjected to the action of pro-inflammatory cytokines such as IL-1, EFN-γ and TNF-a are characterized by high ICAM-1 levels. Based on these observations, we evaluated if GPER is involved in the inhibitory action exerted by nicotinic acid on the expression of ICAM-1 induced by TNF-α. To this end, HUVEC cells were transfected for 24 h with a control vector (shRNA) or with a short hairpin for GPER (shGPER) and then treated with 1 mM nicotinic acid for 30 h and at the same time with 50 ng/ml TNF-a during the last 6 h.

As shown in Figure 3, the inhibitory effect exerted by nicotinic acid on ICAM-1 expression induced by TNF-a can not be observed silencing GPER (Figure 3A-B; ·, p<0,05), indicating thus that GPER mediates the modulating action of nicotinic acid on ICAM-1 expression. The densitometric evaluation shows the result obtained in three independent experiments. We have also evaluated, by means of in vitro angiogenesis assays on Matrigel, GPER role in the angiogenetic effetcs induced by nicotinic acid (NA). To this end, HUVECs were transfected for 24 h with a control vector (shRNA) or with a short hairpin for GPER (shGPER) and subsequently treated with nicotinic acid for 12 h. Figure 4 shows that tubules formation induced by nicotinic acid is abrogated by silencing GPER expression, showing that GPER mediates the angiogenetic effects induces by nicotinic acid in this experimental model (For the details regarding the "in vitro angiogenesis assay on Matrigel" method, see Vinals et al., 2001).

Table 1

SEQ. ID N.l

Met Asp Val Thr Ser Gin Ala Arg Gly Val Gly Leu Glu Met Tyr Pro Gly Thr Ala Gin Pro Ala Ala Pro Asn Thr Thr Ser Pro Glu Leu Asn Leu Ser His Pro Leu Leu Gly Thr Ala Leu Ala Asn Gly Thr Gly Glu Leu Ser Glu His Gin Gin Tyr Val lie Gly Leu Phe Leu Ser Cys Leu Tyr Thr lie Phe Leu Phe Pro lie Gly Phe Val Gly Asn lie Leu lie Leu Val Val Asn lie Ser Phe Arg Glu Lys Met Thr lie Pro Asp Leu Tyr Phe He Asn Leu Ala Val Ala Asp Leu He Leu Val Ala Asp Ser Leu He Glu Val Phe Asn Leu His Glu Arg Tyr Tyr Asp He Ala Val Leu Cys Thr Phe Met Ser Leu Phe Leu Gin Val Asn Met Tyr Ser Ser Val Phe Phe Leu Thr Trp Met Ser Phe Asp Arg Tyr He Ala Leu Ala Arg Ala Met Arg Cys Ser Leu Phe Arg Thr Lys His His Ala Arg Leu Ser Cys Gly Leu He Trp Met Ala Ser Val Ser Ala Thr Leu Val Pro Phe Thr Ala Val His Leu Gin His Thr Asp Glu Ala Cys Phe Cys Phe Ala Asp Val Arg Glu Val Gin Trp Leu Glu Val Thr Leu Gly Phe He Val Pro Phe Ala He He Gly Leu Cys Tyr Ser Leu He Val Arg Val Leu Val Arg Ala His Arg His Arg Gly Leu Arg Pro Arg Arg Gin Lys Ala Leu Arg Met He Leu Ala Val Val Leu Val Phe Phe Val Cys Trp Leu Pro Glu Asn Val Phe He Ser Val His Leu Leu Gin Arg Thr Gin Pro Gly Ala Ala Pro Cys Lys Gin Ser Phe Arg His Ala His Pro Leu Thr Gly His He Val Asn Leu Ala Ala Phe Ser Asn Ser Cys Leu Asn Pro Leu lie Tyr Ser Phe Leu Gly Glu Thr Phe Arg Asp Lys Leu Arg Leu Tyr He Glu Gin Lys Thr Asn Leu Pro Ala Leu Asn Arg Phe Cys His Ala Ala Leu Lys Ala Val He Pro Asp Ser Thr Glu Gin Ser Asp Val Arg Phe Ser Ser Ala Val

Claims

1. Screening method for regulatory agents of GPR30 receptor comprising the following steps:

i) contacting a compound to be tested with the GPR30 receptor and nicotinic acid and/or nicotinamide;

ii) measuring the activity of said GPR30 receptor.

2. The screening method according to claim 1, wherein said regulatory agents are GPR30 receptor antagonists or agonists.

3. The screening method according to claim 1 or 2, wherein said step i) is repeated at different concentrations of said compound to be tested.

4. The screening method according to any of the previous claims, wherein said step i) is repeated at different concentrations of said nicotinic acid and/or nicotinamide, in a receptor competition assay.

5. The screening method according to any of the previous claims comprising the following further steps:

iii) contacting the said compound to be tested with the GPR30 receptor in the absence of nicotinic acid and/or nicotinamide;

iv) determining the activity of said GPR30 receptor.

6. The method according to any of claims 1-5, wherein said compound to be tested is coupled with a detectable marker.

7. The method according to any of claims 1-6, wherein nicotinic acid and/or nicotinamide are coupled with a detectable marker.

8. The method according to claim 6 or 7, wherein said detectable marker is a radioactive isotope, preferably an hydrogen isotope.

9. The method according to any of claims 1-8, wherein said contacting step i) is in or on the surface of a cell.

10. The method according to claim 9, wherein said cell is in vitro or in vivo.

11. The method according to any of claims 1-8, wherein said contacting step is in an acellular system.

12. The method according to claim 11, wherein said GPR30 receptor is on a solid support.

13. The method according to any of claims 1-12, wherein said regulator agents are therapeutic agents suitable for use in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, infections, dermatological diseases, endocrine diseases, metabolic diseases, cancer, gastrointestinal diseases, inflammation, haematological diseases, respiratory diseases, skeletal and muscle diseases, neurological and urological diseases.

14. The method according to any of claims 1-12 to evaluate the role of GPR30 in normal and tumor tissues.

15. The method according to any of claims 1-14, wherein the GPR30 receptor has a polypeptide sequence SEQ ID NO: 1 or an isoform or a biologically active variant thereof.

16. Nicotinic acid and/or nicotinamide for use in the treatment of a pathological condition improved by activation of GPR30 receptor.

17. The nicotinic acid and/or nicotinamide for use according to claim 16, wherein said pathology is a neurological disease.

18. The nicotininc acid and/or nicotinamide for use according to claim 17, wherein said neurological disease is chosen from Alzheimer's disease, Parkinson's disease, Huntington's corea, diabetic peripheral neuropathy, multiple sclerosis, lateral amiotrophic sclerosis and acute neurodegenerative diseases.

19. The nicotinic acid and/or nicotinamide for use according to claim 18, wherein said acute neurodegenerative disease is chosen from ictus, cranial trauma, peripheral nerve damage, hypoglycaemia, spinal chord lesions, epilepsy, anoxia and hypoxia.

20. Nicotinic acid and/or nicotinamide for use as substitute of an estrogen- based therapy for the treatment of a pathological condition chosen from the group consisting of cardiovascular diseases, infections, dermatological diseases, endocrine diseases, metabolic diseases, cancer, gastrointestinal diseases, inflammation, haematological diseases, respiratory diseases, skeletal and muscle diseases, neurological and urological diseases.

21. Composition for use in the treatment of a pathological condition improved by activation of GPR30 receptor comprising nicotinic acid and/or nicotinamide and one or more vehicles and/or excipients and/or diluents.

22. The composition for use according to claim 21, wherein said pathology is a neurological disease.

23. The composition for use according to claim 22, wherein said neurological disease is chosen from Alzheimer's disease, Parkinson's disease, Huntington's corea, diabetic peripheral neuropathy, multiple sclerosis, lateral amiotrophic sclerosis, aging and acute neurodegenerative diseases.

24. The composition for use according to claim 23, wherein said acute neurodegenerative disease is chosen from ictus, cranial trauma, peripheral nerve damage, hypoglycaemia, spinal chord lesions, epilepsy, anoxia and hypoxia.

25. The composition according to the previous claims in the form of tablets, powders, granules, capsules, solutions, syrups, elixirs, oils or aqueous suspensions.