ITMI20010821A1 - USE OF COMPOUNDS DERIVED FROM SPIROLACTONE TO INHIBIT THE PRO-FIBRIGENETIC ACTION OF Hepatic star cells and Muscle cells - Google Patents

Description

DESCRIPTION

The present invention relates to the inhibition of the progression of fibrogenesis by means of an inhibition of the pro-fibrogenetic action exerted by hepatic stellate cells and by smooth muscle cells of the vascular system.

Specifically, the present invention relates to the prevention and inhibition of cytokine-mediated fibrogenesis in hepatic stellate and smooth muscle cells of the vascular system. In particular, the present invention relates to the inhibition of fibrogenesis mediated by platelet-derived growth factor (PDGF) and / or by transforming growth factor-βΐ (TGF-βΙ).

Currently, fibrogenesis is considered as a dynamic process strictly dependent on the extent and duration of parenchymal damage caused by various etiological agents. According to this conception, some fibrogenic disorders are no longer considered degenerative, but as the result of repeated events of tissue damage and chronic inflammation. The development process of hepatic fibrosis and those responsible for progressive tissue sclerosis in other organs and systems consists of three phases: acute inflammation, synthesis of extracellular matrix (ECM) and its remodeling, regeneration of parenchymal cells typical of the tissue. Under this scheme, a single event of tissue damage results in a complete return to integrity. "

On the contrary, the persistence of the initial insult causes a chronic activation of the tissue repair mechanisms. In this way, the phases of the reparative process characterized by the deposition of a fibrillar type matrix are favored, with a progression towards tissue fibrosis rather than towards tissue repair.

With particular reference to hepatic fibrosis, qualitatively the extracellular matrix has similar components in both the fibrotic and normal liver. However, liver tissue affected by active fibrogenesis shows a notable increase in ECM components, which are altered in their relative proportions and distribution. In particular, with the progression of the fibrogenic process, a notable change is observed in the composition of the ECM present along the sinusoids and between the hepatocellular laminae. There is an increased production of type I and type III collagen, the main components of the new structure, the fibrillar matrix, which replaces the pre-existing ECM typical of normal liver and characterized by a prevalence of collagen IV and laminin. These quantitative and qualitative changes at the ECM level, as well as having mechanical and physical implications, help generate a new biochemical environment.

The cellular origin of connective tissue components in the cirrhotic liver has long been debated. Although different types of liver cells are capable of synthesizing components of the ECM, it is now believed that hepatic stellate cells (HSCs, known as hepatic stellate cells) constitute the cell type mainly involved in the excessive deposition of fibrillar ECMs.

Following chronic liver damage, HSCs undergo an activation process characterized by a phenotypic transformation from the quiescent form to an actively proliferating one, morphologically similar to the myofibroblast and therefore defined as "myofibroblast-like". This event occurs gradually and includes an intermediate stage called a "transitional cell". This transformation involves morphological changes as well as the activation of many functional components, including: an increased synthesis of ECM, increased expression of growth factors and cytokines and related receptors, the appearance of well-developed intracellular contractile structures and a complex alteration of the enzymatic pathways involved in the degradation and remodeling of the newly synthesized ECM. Taken together, these modifications lead to a cellular phenotype characterized by remarkable profibrogenic and contractile capacity.

The phenomenon of "perpetuation" of HSC activation essentially consists in the definitive transformation into the "myofibroblast-like" phenotype. The characteristics of this particular phase of HSC activation, amply confirmed by morphological studies on human and rat cirrhotic liver, include a high proliferative and migratory capacity, an increase in cell contractility and ECM synthesis together with a reduced degradation capacity and remodeling of the matrix itself, and an increase in the release of cytokines and growth factors, including chemotactic agents for leukocytes (chemokines). In the sites of liver damage, a great variety of inflammatory cells are recalled which, through the production of soluble factors, cytokines and growth factors, play a key role in the activation and regulation of stellate cells, stimulating their proliferation and migration, thus such as the synthesis of ECM. These factors, individually or within a complex network of interactions, produce different effects on the biology of activated HSCs.

It is important to underline that these aspects of the biology of hepatic stellate cells are largely superimposable to those of other cells producing extracellular matrix present in other organs and systems and similarly responsible for the progression in the fibrogenic sense of diseases characterized by chronic damage and chronic inflammation. The most representative example in this sense is offered by the smooth muscle cells of the subintimal area of the arteries, responsible for the formation of the vascular lesion typical of atherosclerosis.

Numerous studies carried out in the last decade have highlighted the importance of various cytokines in the progression of the fibrogenetic process, and in particular of two polypeptide growth factors, the platelet-derived growth factor (PDGF) and the transforming growth factor-Bl ( TGF-β). These factors, with different properties, are responsible for the profibrogenic activation of the stellate cells of the liver and many other cell types responsible for sclerogenic diseases, such as the smooth muscle cells of the arterial vessels.

PDGF exerts biological effects on HSCs by promoting their proliferation and migration, while TGF-β exerts a direct profibrogenic action by increasing the synthesis and deposition of extracellular matrix and decreasing its degradation and remodeling.

An object of the present invention is to provide the use of a drug to prevent and inhibit the progression of fibrogenesis in hepatic stellate cells and smooth muscle cells of the vascular system, in particular fibrogenesis mediated by the aforementioned cytokines, by administering a drug already used for other clinical indications and for which side effects are known within the doses commonly used in the clinic.

These and other objects which will become clearer from the following description, are achieved according to the present invention by the use of a drug containing a spirolactone compound.

As used herein, the term "spironolactone" refers to a molecule comprising a lactone ring coupled to a steroid structure in a spiro configuration.

Particularly preferred spirolactone compounds for the use of the present invention are canrenone and its pharmaceutically acceptable prodrugs and salts, such as potassium canrenoate and spironolactone, and eplerenone.

The canrenone has the following structural formula

and is known as a diuretic and aldosterone antagonist.

Spironolactone has the following formula:

and is known as a diuretic and aldosterone antagonist.

Eplerenone or epoximexrenone has the following structural formula:

and is known as an antihypertensive, and aldosterone antagonist.

It has been experimentally detected by the inventors of the present invention that the spirolactone compounds have an activity of prevention and inhibition of the pro-fibrogenic role of hepatic stellate cells and smooth muscle of the vascular apparatus.

In particular, it has been found that spirolactone compounds have an inhibitory activity of the pro-fibrogenic biological effects of the cytokines described above.

It has been specifically found that spirolactone compounds, and in particular canrenone, potassium canrenoate, spironolactone and eplerenone, have an inhibitory activity of the biological effects of PDGF and TGFà. It is important to underline that, as will be further detailed, these inhibitory effects are exerted by the class of drugs in question independently of their effect as aldosterone receptor antagonist drugs, and therefore with an absolutely original mechanism, and so far not described.

As used herein the terms "prevention or inhibition of progression" of fibrosis are used interchangeably to include (1) prevention or prophylactic treatment in patients who are at high risk for hepatic, renal and vascular fibrosis or have symptoms suggesting the possible establishment of fibrosis, and (2) treatment of patients in whom the fibrosis of the aforementioned tissues has reached an advanced level of progression and / or such as to result in evident and quantifiable clinical manifestations.

An important aspect of the present invention is that the treatment can use a low dosage of spirolactone compound, and more specifically lower than the average doses commonly used in the treatment of arterial hypertension or ascitic decompensation in the patient suffering from liver cirrhosis. Thus the therapeutic effects are achievable at dosages in which the side effects are minimal and the electrolyte balance or water retention in the patient are not substantially affected.

The invention will be described in greater detail with reference to the following figures:

Figure 1 shows the effect of increasing doses of canrenone on DNA synthesis in basal conditions and after stimulation with PDGF-BB, measured by the incorporation of [<3> H] -thymidine into the DNA.

Figure 1B shows the effect of increasing doses of carnenone on PDGF-BB-induced cell migration (chemotaxis).

Figure 2A illustrates the level of phosphorylation of the β subunit of the PDGF receptor under basal conditions and after stimulation with PDGF-BB.

Figure 2B illustrates the level of PLC-γ phosphorylation after stimulation with PDGF.

Figure 3A illustrates ERK activation due to PCTF stimulation.

Figure 3D illustrates mRNA expression with c-fos under basal conditions and after the addition of PDGF.

Figure 4 shows the change in intracellular Ca <2+> concentration following stimulation with PDGF.

Figure 5 illustrates the change in intracellular pH under control conditions and in the presence of ethylisopropylamyloride (EIPA), a selective inhibitor of the Na + / H + exchanger, or canrenone.

Figure 6 illustrates the effect of PDGF on the activity of the NA + / H + exchanger and of the inhibitory effect exerted by canrenone on the activity of the exchanger itself induced by PDGF (panel A) in human hepatic stellate cells. The figure also illustrates that canrenone has no effect on PDGF-induced changes in intracellular calcium concentration (panel D). The figure also illustrates other experimental conditions aimed at demonstrating that this effect is dependent on an inhibition of the activity of the PI 3-K induced by the canrenone.

Figure 7, panel A, in fact demonstrates that canrenone induces an inhibition of PDGF-induced PI 3-K activity in human stellate cells.

Figure 8 demonstrates that ethylisopropylamiloride (EIPA), an inhibitor of the Na + / H + exchanger inhibits the proliferation and migration of human stellate cells similar to canrenone.

Figure 9 demonstrates that canrenone incubation (lanes 3-6) inhibits PDGF-induced focal adhesion kinase (FAK) phosphorylation in human stellate cells.

Figure 10 demonstrates that canrenone inhibits cell contraction coupled with thrombin-induced intracellular calcium release in human stellate cells.

Figure 11 demonstrates that canrenone is able to inhibit the de novo synthesis of procollagen type I and other components of the extracellular matrix induced by TGF- in tornane stellate cells.

Figure 12 demonstrates that canrenone is capable of inhibiting DNA synthesis and PDGF-induced migration in smooth muscle cells isolated from the iliac artery.

Figure 13 illustrates the general characteristics of the proposed direct antifibrogenic mechanism for canrenone in the cell models employed.

The effects of spirolactone compounds on biological variations exerted by cytokines were evaluated in vitro. In particular, the effects of canrenone, an active metabolite of spirolactone, on the various biological actions exerted by PGDF on human hepatic stellate cells, in particular mitogenesis and chemotaxis, were evaluated.

The effects of canrenone on the synthesis of the extracellular matrix in basal conditions and following stimulation with TGF-β were also evaluated.

The effect of canrenone on the contraction of human stellate cells in response to a powerful constricting vessel such as thrombin was also evaluated.

It must be particularly stressed that in none of the experimental conditions described was the hormone aldosterone present or was used in conjunction, and that therefore the pharmacological effects of the canrenone compound are due to a mechanism independent of the known action as a receptor antagonist drug. 'aldosterone.

MATERIALS AND METHODS USED

Materials

An anti-phosphotyrosine monoclonal antibody supplied by UBI (Lake Placid, NY, USA) was used for the Western blot. Anti-extracellular-signal regulated kinase (ERK) -l, anti-phospholipase Cyl (PLCy), and anti-FAK polyclonal antibodies were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Myelin basic protein was supplied by Sigma Chemical Co. (St. Louis, MO, USA). Protein A-sepharose was provided by Pharmacia (Uppsala, Sweden). [Γ-32P] ATP (3000 Ci / mmol), [α-32P] dCTP (3000 Ci / mmol) and Methyl- [3H] -thymidine were purchased from New England Nuclear (Milan, Italy). Recombinant human PDGF-BB was provided by Peprotech (Rock Hill, NJ, USA). Fura-2-AM was provided by Calbiochem Corp. (San Diego, CA, USA). The canrenone was supplied in micronized form by the company GN Pharma, Milan. An IO-2 M stock solution in DMSO of this drug was prepared in sterile form and subsequent experimental dilutions were made in sterile water. Human thrombin was provided by Boehringer Mannheim GmbH (Mannheim, Germany). All other reagents used were analytical grade. Human liver stellate cell isolation and culture.

Human HSCs were isolated from surgical sections (10-20 g) of normal human liver not usable for transplantation, as previously reported in the literature. Briefly, after a combined digestion of the surgical pieces with collagenase and pronase the HSCs were separated from other non-parenchymal liver cells by ultracentrifugation on stractan gradients (Cellsep ™ isotonic solution, Larex Inc, St. Paul, MN, USA) . Cells were cultured in plastic flasks (Falcon, Becton Dickinson, Lincoln, New Jersey, USA) in Iscove's modified Dulbecco's medium, supplemented with 0.6 U / ml of insulin, glutamine (2 mM), non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), antifungal and antibacterial solution (all supplied by GIBCO BRL Laboratories, Grand Island, New York, USA), and with 20% (v / v) fetal bovine serum (Imperiai Laboratories, Andover , UK). The cells were grown in an atmosphere consisting of humidified air at 95% of 02 and 5% of C02, with a constant temperature of 37 ° C. The freshly isolated and cultured cells in subsequent subcultures were characterized as previously described. The experiments described were conducted using HSCs between the third and fifth serial pass, with a distribution ratio of 1: 3. The experiments were conducted using three different cell lines.

Isolation and culture of smooth muscle cells from human femoral artery.

The muscle tunic portion was removed, placed in Hanks' Balanced Salt Solution (HBSS), and maintained at 37 ° C. After two or three washes in HBSS, the muscle tunic portion was placed on a sterile plastic disc and the residues of adventitia and calcifications have been carefully eliminated. The tissue was then fragmented, washed again with HBSS, and the resulting fragments were cultured on plastic in Iscove's complete medium with 20% fetal bovine serum (FBS) in just enough quantity to cover the surface of the flask. This medium was replaced with fresh medium the following day, taking care not to remove the pieces of artery adhering to the plastic and, thereafter, approximately every five days. The smooth muscle cells migrate from the tissue fragments onto the plastic and slowly begin to proliferate in the surrounding area. After a period of approximately one to two weeks after isolation, the artery fragments were removed and the adherent cells were kept in culture. In this way the cells proliferate with a tendency to thicken and form islets. Consequently, trypsinization must be performed before the cells reach 100% confluence. A characterization of the cell cultures obtained was performed by detecting immunohistochemical positivity for specific cell markers such as a-smooth muscle actin (a-SMA) and desmin.

Determination of DNA synthesis

The synthesis of DNA was evaluated by the method of incorporation of [methyl-3H] -thymidine in the cellular material precipitated with trichloroacetic acid. Briefly, cells were cultured in 24-well plates, at a density of 2x104 cells / well, with complete culture medium containing 20% fetal bovine serum (FBS). Once confluence was reached (approximately 1x10s cells / well), the cells were made quiescent by incubation in serum-free and insulin-free culture medium (SFIF) for 48 hours. The cells were then incubated with or without agonists, at the doses and as indicated, for 20 hours. The cells were then incubated for 4 hours with 1.0 μCi / ml of [methyl-H] -thymidine (6.7 Ci / mM). At the end of the pulsing period the incorporated [methyl-H] -thymidine was measured according to the methodology currently used in our laboratory. The number of cells was evaluated in three different wells for each disc and the final result of the experiment was expressed as cpm / 105 cells.

Cell migration analysis

The experiments were conducted using the Boyden chamber technique, equipped with polycarbonate filters with 8 μm pores. The filters were pretreated with human type I collagen (20 μg / ml) for 30 min at 37 ° C and subsequently inserted between the upper and lower chambers. The HSCs, cultured to confluence, were incubated in SFIF for 48 hours and then treated for 10 min with increasing concentrations of NO donors. The cells were then resuspended by a mild trypsinization (0.05% trypsin / EDTA) and an aliquot of the cell suspension (210 μΐ), corresponding to about 4xl04 cells, was inserted into the upper chamber and incubated at 37 ° C for 6 hours. The lower chamber contained SFIF (control) or PDGF-BB (10 ng / mL) with or without the same NO donor concentration used in the pre-incubation period. After fixation with 96% methanol and Harris hematoxylin staining, the migrated cells on the underside of the filter were quantified as cell numbers in 10 high-power fields (HPF). All experiments were performed in triplicate. The analysis of each triplicate was repeated twice, in different sessions, and with different preparations of HSC. Possible cytotoxic effects were checked with the trypan blue exclusion test. This test consistently indicated cell viability above 90%.

Determination of ERK's activity

ERK activity was evaluated by measuring the activity of myelin basic protein kinase in cell lysates immunoprecipitated with anti-ERK antibody. The cell monolayer was used with RIPA buffer (20mM Tris-HCl, pH 7.4, 150mM NaCl, 5mM EDTA, 1% Nonidet P-40, 1mM Na3V04, 1mM phenyl-methyl-sulfonyl-fluoride -PMSF-, 0.05% [w / v] aprotinin). Insoluble proteins were removed by high-speed centrifugation at 4 ° C. The protein concentration in the supernatant was measured in triplicates using a commercially available kit (Bio Rad, Hercules, CA). Aliquots of the total cell lysate (50 μg) were immunoprecipitated with an anti-ERK antibody and sepharose protein A. The measurement of ERK activity was carried out according to the methodology currently used in our laboratory.

Digital image analysis of intracellular calcium concentration in single HSCs

Cells were cultured to 60-70% confluence in complete culture medium on circular slides (25mm diameter, 0.2mm thick) for 72 hours and then incubated for 48 hours in SFIF. The cells were then loaded with 10 μΜ of Fura-2-AM (Calbiochem Corp., San Diego, CA) and 15% of Pluronic F-127 for 30 min at 22 ° C. [Ca <2> +] i was measured in a HEPES-NaHC03 buffer containing 140mM NaCl, 3mM KCl, 0.5mM NaH2P0 ”, 12mM NaHC03, 1.2mM MgCl2, 1.0mM CaCl2, 10mM HEPES, 10mM glucose , pH 7.4. Images were recorded every 3 seconds and the calibration curve was obtained for each cell preparation. PDGF-BB (10 ng / mL) was added directly into the perfusion chamber immediately after measuring [Ca2 +] i baseline. In parallel experiments the cells were preincubated with NO donors for 10 minutes before adding the PDGF-BB.

In experiments aimed at evaluating the effects of canrenone on thrombin-induced changes in [Ca2 +] i / cell surface (measurement of cell contraction), the latter agonist (0.3U / ml) was added directly into the perfusion chamber immediately after measured baseline value of [Ca2 +] i. To evaluate the variations of the cell surface, a spatial calibration was performed on a special lattice under the same optical conditions as the rest of the experiment.

Northern Blot Analysis

The RNA was extracted according to the Chomczynski and Sacchi method. Ten micrograms of total RNA were separated by 1% agarose gel electrophoresis and formaldehyde, and transferred to a nylon membrane. The procedure for DNA labeling and membrane hybridization was performed as per the method routinely used in our laboratory.

Immunoprecipitation and immunoblottinq

Cells were lysed with a buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 5 mM MgC12, 1 mM EGTA, 25 μg / ml leupeptin, 2 mM Na3V04, 10 μg / ml pepstatin A, and 2 mM of phenylmethylsulfonyl fluoride (PMSF). The lysates were then centrifuged at 14000 rpm for 10 min. The immunoprecipitation, SDS-PAGE and immunoblotting procedures were performed with the methodology currently used in our laboratory. The detection of the bands corresponding to the antibodies used was carried out by chemiluminescence using the ECL kit (Amersham Life Sciences, Little Chalfont, UK).

Measurement of the activity of the exchanger ΝΒ + / Η +

The activity of the Na + / H + exchanger was determined in HSCs loaded with the fluorescent pH sensitive indicator biscarboxyethylcarboxifluorescein (BCECF). Cells were cultured on circular slides (25mm diameter, 0.2mm thick) in complete medium up to 70% confluence and then incubated in SFIF for 48 hours; they were then loaded with 2 μΜ biscarboxyethylcarboxifluorescein acetose methylester (BCECF AM) for 30 minutes at room temperature and washed. Fluorescence in single cells was studied using an image analysis system. The fluorescence intensity of the indicator obtained at 490 nm of excitation wavelength (emission 520 nm), was divided with the fluorescence intensity obtained at 405 nm (emission 520 nm). This fluorescence intensity ratio is correlated through a suitable calibration curve to the pH of the cytosol. Cytosolic pH measurements were obtained every 3 seconds and the time course of the cytosolic pH was followed for at least 20 minutes. HSCs were acidified by adding nigericin (5 μΜ) in a buffer containing N-methylglucamine and nominally free of Na + and K +. The decrease in the calculated ratio indicates a decrease in the cytosolic pH. Once the value of this ratio had stabilized, the administration of NaCl (30 mM) caused a rapid alkalinization of the cytosol, indicating the restoration of the activity of the antiporter Na + / H +. The kinetics of alkalinization is in fact an index of the activity of this exchanger.

Ethylisopropylamiloride (EIPA), a specific antiporter inhibitor, was used as a control.

Measurement of 3-K PI activity

Preparation of cell lysates and PI 3-K activity assay were performed in accordance with standardized procedures in our laboratory. Briefly, the proteins were immunoprecipitated using antiphosphotyrosine antibodies. After washing the immunoprecipitate, a solution containing 20 mmol / L of Tris-HCl, pH 7.5, 100 mmol / L NaCl and 0.5 mmol / L EGTA was resuspended in 50 μL. 0.5 μL of phosphatidylinositol (20 mg / mL) was added and the sample was incubated at 25 ° C for 10 min. One microliter of 1 mmol / L MgCl2 and 10 μCi of [G-32P] adenosine triphosphate were added simultaneously and incubation continued for another 10 min. The reaction was stopped by adding 150 [μL of chloroform / methanol / 3 7% HCl 10: 20: 0.2. The samples were extracted with chloroform and dried. Radioactive lipids were separated with thin layer chromatography using chloroform / methanol / 30% ammonium hydroxide / water 46: 41: 5: 8. After drying the plate was subjected to autoradiography. The radioactive areas were then removed by scratching and counted in a β counter.

Metabolic labeling and immunoprecipitation of extracellular matrix components

To perform these experiments, human HSCs were metabolically labeled with 25 μCi / ml of Trans 35S-label ™ (70% 35Smethionine, 15% 35S-cysteine) in the presence or absence of the various agonists. The methodology used is that described by Rombouts et al. (J.Hepatology 2001, in press).

Statistical data analysis

The results, relating to the experiments indicated, were expressed as mean ± standard deviation. Statistical analysis was performed with the analysis of variance (ANOVA), and, when the value of F was significant, with the Duncan test.

EXPERIMENTS

In a first group of experiments, the effects of increasing doses of canrenone on PDGF-induced cell proliferation and migration were evaluated. The effects of canrenone pre-incubation on ERE activity were evaluated in a second group of experiments.

In a third group of experiments, the effect of pre-incubation with canrenone on the activity of PI3K, a kinase for lipids and proteins activated through different pathways, was analyzed.

EFFECTS OF CANRENONE ON THE BIOLOGICAL EFFECTS AND ON THE INTRACELLULAR SIGNALING INDUCED BY PDGF IN HUMATE STAR CELLS.

RESULTS

Effects of canrenone on the biological effects and intracellular signaling induced by PDGF in human stellate cells

The effect of increasing doses of canrenone on DNA synthesis under basal conditions and after stimulation with PDGF-BB, measured by incorporation of [H] -thymidine into DNA, was first evaluated.

As shown in Figure 1A, preincubation (5 minutes) with this drug causes a dose-dependent inhibition of PDGF-induced mitogenesis. This effect is statistically significant starting with the 1 μΜ dose. In basal conditions, canrenone induces a significant reduction in DNA synthesis only at maximal doses (50 μΜ). Similarly, canrenone dose-dependently reduces PDGF-BB-induced chemotase, measured in a modified Boyden chamber system; in this case the inhibitory effect is observable starting from the dose of 5 μΜ (Figure 1B).

The effect of canrenone on intracellular signaling events directly related to the interaction of PDGF with its receptor was then evaluated. Figure 2A illustrates the level of phosphorylation of the PDGF receptor β subunit under basal conditions and after stimulation with PDGF-BB. As is evident, pre-incubation with canrenone (10 and 50 μΜ) has no effect on PDGF-induced phosphorylation.

Similar observations were obtained by evaluating the level of phosphorylation of PLC-γ, a signaling molecule physically associated with the PDGF receptor. As Figure 2B shows, canrenone, at none of the tested concentrations (10, 25, and 50 μΜ), modifies PLC-γ phosphorylation due to PDGF stimulation.

The effects of canrenone on the ERK activity induced by PDGF-BB were then evaluated (Figure 3A). As is evident, the stimulation of human HSCs, maintained in SFIF, with the PDGF causes the activation of ERK after 10 minutes of incubation. Preincubation with increasing doses of canrenone (5 to 50 μΜ) has no effect on this activation. Similar results were obtained by analyzing the action of canrenone on mRNA expression for c-fos, one of the genes activated early in the PDGF cascade. As shown in Figure 3B, the mRNA expression for c-fos, undetectable under basal conditions (lane 1), becomes evident 30 minutes after PDGF addition (lane 2) and is not changed by pre-incubation of the cells with canrenone.

The changes in the intracellular concentration of Ca <2+> following stimulation with PDGF were then evaluated. As shown in Figure 4, in single human HSCs loaded with Fura-2, stimulation with the PDGF (control) causes the formation of a synchronous Ca <2+> peak on a mean baseline [Ca <2 +>] i of 230 nM, followed by a long-lasting plateau, in agreement with previously published data by our group. The pre-incubation for 10 minutes with canrenone (10 and 25 μΜ) is not associated with any significant variation in the morphology of the trace, both as regards the peak increase and the consequent plateau phase. However, a significant decrease in these components is observable as a consequence of preincubation with high doses of canrenone (50 μΜ).

Figure 5 illustrates the change in intracellular pH (expressed delta of change / min) under control conditions and in the presence of EIPA (10 μΜ, positive control) or canrenone (10 and 50 μΜ). It is evident that incubation with the Na + / H + ethylisopropylamiloride (EIPA) exchanger inhibitor blocks the activity of the exchanger in basal conditions (i.e. in the absence of stimulation with PDGF). This activity is not significantly altered by incubation with doses of canrenone in the 1-25 μΜ range, while, similarly to what has been observed for changes in [Ca <2 +>] i, an inhibitory effect is noted at a dose of 50 μΜ. However, after stimulation with PDGF, doses of canrenone equal to 10 μΜ are able to inhibit the activity of the exchanger induced by the PDGF (Figure 6A) similar to that observed with preincubation with EIPA (Figure 6B). Both experimental conditions do not alter the PDGF-induced [Ca <2 +>] i profile (Figure 6D and Figure 6E). These results suggest that canrenone has an inhibitory effect on the activity of the Na + / H + exchanger only in conditions of stimulation with the PDGF, indicating that in this case the action of the drug is expressed through a molecular communication event between the receptor of the PDGF and the Na + / H + exchanger.

Phosphatidylinositol 3-kinase (PI 3-K) is a lipid and protein kinase that can be activated by several pathways, including tyrosine kinases and heterotrimeric G proteins. Since the action of PDGF in HSCs is partly dependent on the activation of PI 3-K, and since the activity of this signaling pathway appears to have significant implications on the activity of the Na <+> / H <+> exchanger, we evaluated the effects of canrenone on the activity of this kinase in this cell type. As shown in Figure 7A, stimulation with PDGF (10 ng / ml) causes an increase in PI 3-K activity and pre-incubation of the cells with canrenone (25 and 50 μΜ) reduces this activity in a dose-dependent manner. Furthermore, as illustrated in Figure 7B, a similar inhibitory effect is exerted by pre-incubation with EIPA. The results illustrated in Figure 6C, relating to pre-incubation with the PI 3-K inhibitor, LY294002, indicate that inhibition of PI 3-K results in reduced exchanger activity similar to that observed with the canrenon. It is therefore possible to hypothesize that canrenone acts on the activity of the Na + / H + exchanger induced by PDGF through an inhibition of PI 3-K.

Following the results obtained in the previously described experiments, it was evaluated whether canrenone and EIPA had similar effects on mechanisms of cell proliferation, differentiation and migration. In particular, we evaluated the action of these substances on DNA synthesis and chemotaxis induced by PDGF in human HSCs. As Figure 8A illustrates, canrenone (10 and 50 μΜ) and EIPA (5 and 10 μΜ) do not modify basal DNA synthesis, while both cause dose-dependent inhibition of PDGF-induced DNA synthesis.

Similar results were obtained in experiments aimed at evaluating chemotaxis; in fact it appears evident from Figure 8B that the pre-incubation of cells with EIPA (5 and 10 μΜ) and canrenone (10 μΜ) causes a marked reduction in cell migration induced by PDGF. Finally, it was investigated whether canrenone and EIPA modulate the phosphorylation of FAK, a key protein of the focal adhesion complex. As illustrated in Figure 9, the adhesion of HSCs to plastic is associated with the phosphorylation of FAK; stimulation of cells with PDGF-BB (for 10 minutes) causes an increase in phosphorylation of this protein. Incubation of cells with canrenone (10 and 50 μΜ) and EIPA (10 μΜ) reduces phosphorylation of FAK both in basal conditions and after stimulation with PDGF.

Effects of canreoone as a vasodilator drug in human stellate cells

The effect of canrenone on changes in [Ca 2+] i associated with changes in the cell surface in response to stimulation with thrombin, a potent vasoconstrictor, was then evaluated. As illustrated in Figure 10A, stimulation with 0.3 U / ml of thrombin produces a rapid increase in [Ca 2+] i, which is coupled with a rapid reduction of the cell surface, a phenomenon corresponding to a reversible cell contraction coupled with changes in [Ca 2+] i. Ca 2+] i- As can be seen from Figure 10B, pre-incubation with 25 μΜ canrenone determines a net reduction of the aforementioned variations, indicating an anticontractile effect of the drug. This effect is also greater for higher concentrations of canrenone (50 μΜ), as illustrated in Figure 10C.

Effects of canrenone on the "de novo" synthesis of components of the extracellular matrix in basal conditions and after stimulation with TGF-β.

As shown in Figure HA, incubation with canrenone at a dose of 10 μΜ determines a significant inhibition of the "de novo" synthesis of type I procollagen induced by TGF-β both in the cell monolayer (celi layer) and, above all, in the supernatant of culture. A similar effect can be observed for the "de novo" synthesis of fibronectina induced by TGF-β (Figure 11), while canrenone does not appear to significantly influence the "de novo" synthesis of type IV procollagen (Figure 11C).

Effects of canrenone on PDGF-induced cell proliferation and migration in smooth muscle cells isolated from human arteries and maintained in culture

In order to extend the observations obtained in human stellate cells, a series of preliminary research has been undertaken in mesenchymal cells involved in the deposition of extracellular matrix and in progressive sclerosis in other organs and systems. Because of their involvement in the atherosclerotic process we first considered the effects of canrenone in smooth muscle cells isolated from human arteries, as illustrated in Figures 12A and B, the effects of canrenone on PDGF-induced cell proliferation and migration in this cell type. they are superimposable to those observable in human stellate cells.

CONCLUSIONS

The results of the experiments indicate that canrenone, known as a diuretic and as an aldosterone receptor antagonist, widely used in diuretic therapy in cirrhosis with ascites, is able to induce a dose-dependent inhibition of the main biological effects of PDGF in human stellate cells, considered one of the main effectors of the abnormal accumulation of fibrillar ECM as a consequence of chronic liver damage. In light of the potential role played by this growth factor in the repair of damage and in the fibrogenetic process at the level of the liver, these observations suggest that canrenone, as well as other antialdosteronic drugs of which canrenone is the final metabolite, can act as antifibrogenic drug regardless of antagonism towards aldosterone.

In a first group of experiments, the effects of increasing doses of canrenone on PDGF-induced cell proliferation and migration were evaluated. These biological actions of PDGF, although not directly involved in the deposition of excessive fibrillar ECM, are at the basis of the progressive increase of HSC in the liver site of active fibrogenesis and in the occupation, by this cell type, of areas of tissue in which the damage and therefore the repair effort is maximum. The results of this first group of experiments suggest that at least in the dose range between 1 and 10 μΜ, canrenone exerts an inhibitory effect on the action of PDGF without significantly influencing the proliferation and basal migration of HSCs. This suggests a specific effect on the biological effects induced by PDGF.

The results of the first group of experiments to evaluate the effects of increasing doses of canrenone on cell proliferation and migration induced by PGDF show that at least in the range of doses between 1 and 10 μΜ the canrenone exerts an inhibitory effect on the action of PDGF without significantly influences the profileration and basal migration of hepatic stellate cells (HSC). This suggests a specific effect on the biological effects induced by PGDF.

In order to understand at what level canrenone can interfere in the complex cascade of intracellular signal transduction events induced by PDGF stimulation, the effects of this drug on the main signaling pathways activated by this growth factor were evaluated. In this regard, the effects of canrenone on the autophosphorylation of the β receptor of PDGF and on the phosphorylation of a signaling pathway physically associated with this receptor, such as PLC-γ, following stimulation with PDGF, were first investigated.

In agreement with the observation of a lack of effect of canrenone on PLC-γ phosphorylation, are the results of the experiments aimed at evaluating the effects of this drug on changes in [Ca <z +>] i induced by stimulation with PDGF. In particular, canrenone, at doses of 10 and 25 μΜ, did not induce changes in either the peak or the plateau phase that normally follow stimulation with PDGF.

Taken together, the results of this first group of experiments suggest that the effects of canrenone are not to be ascribed to an interference with immediate post-receptor events, ie immediately following the phosphorylation of the PDGF receptor.

The experiments concerning the effects of canrenone pre-incubation on ERK activity show that the activation of this pathway located downstream of Ras is essential for the progression of the signal originating from stimulation with PDGF at the nuclear level and ultimately for induction of the mitogenic effect of this growth factor. However, the results of this group of experiments indicated that the inhibitory effect exerted by canrenone on the action of PDGF does not manifest itself through an inhibition of this fundamental pathway, as also confirmed by the absence of effect on mRNA expression. for the c-fos protooncogene, a typical nuclear target for this signaling pathway.

The lack of effect of canrenone on a fundamental pathway in the transduction of the mitogenic effect of PDGF such as Ras / ERK has therefore led to the hypothesis of a possible action of this drug on signaling events, activated following stimulation with PDGF, and able to modify the cytosolic microenvironment, such as for example the induction of changes in the intracellular pH. Based on this consideration, the effects of the canrenone on the activity of the Na <+> / H <+> exchanger were evaluated. The Na <+> / H <+> exchanger regulates the intracellular levels of the hydrogen ion and, in cooperation with other membrane exchangers, contributes to the regulation of the intracellular pH both under basic conditions and after stimulation with growth factors such as PDGF, such as recently also confirmed in rat HSC. This group of experiments showed that canrenone, despite not being able to inhibit the activity of the exchanger in basal conditions, is able to reduce the activity of the exchanger induced by PDGF. It is therefore possible to hypothesize that the effect of canrenone on the biological actions of PDGF is at least in part secondary to an alteration of this important intracellular homeostatic system.

The effect of preincubation with canrenone on the activity of PI 3-K, a kinase for lipids and proteins activated through various pathways, including tyrosine kinases and heterotrimeric G proteins, whose activation is partly responsible for the action of the PDGF in HSCs. It should also be remembered that the activity of this signaling pathway seems to have significant implications on the activity of the Na + / H + exchanger. Unlike what has been observed for ERK, canrenone has been shown to be effective in inhibiting the activation of this signaling pathway in a dose-dependent manner.

The experiments concerning the effect of canrenone pre-incubation on the activity of PE3K, a lipid and protein kinase activated through several pathways, including tyrosine kinases and heterotrimeric G proteins, whose activation is partly responsible for the action of PDGF in HSCs, showed, unlike what was observed for ERK, that canrenone is effective in inhibiting the activation of this disignaling pathway in a dose-dependent manner.

In this sense, when compared with the action of previously known inhibitors of PI 3-K activation (eg wortmannin), the effect of canrenone has the peculiarity of not inducing a parallel, even partial, inhibition of ERK. The inhibitory action of canrenone against this signaling pathway may in itself be sufficient to explain the interference with the biological effects of PDGF in HSCs.

As summarized in Figure 13, the data discussed so far lead, as a whole, to the conclusion that the inhibitory effect of canrenone on the actions of PDGF in human HSCs is mainly secondary to an inhibition of PI 3-K and exchanger activity. Na <+> / H <+>. These different actions of canrenone are potentially linked as studies carried out in other cell types have shown that the activation of the Na <+> / H <+> exchanger induced by PDGF is dependent on the activation of PI 3-K. In particular, the activation of PI 3-K would be responsible for an increased recycling of the exchanger towards the piasmatic membrane, without a significant increase in the expression of the relative protein. In this way the availability of the exchanger becomes greater for a period of time that can be evaluated in hours after stimulation with growth factors capable of activating PI 3-K. Further experimental evidence of the possible close connection between the activity of PI 3-K and that of the Na <+> / H <+> exchanger is offered by the absolutely original data of an inhibitory activity exercised by EIPA, considered a specific inhibitor of the Na <+> / H <+> exchanger, on the activity of PI 3-K.

Recently published research has shown that the activity of the Na <+> / H <+> exchanger, which appears to be coordinated with the activation of Rho, a signaling molecule of the Ras superfamily involved in the reorganization of the cytoskeleton secondary to stimulation with factors growth, is important in the regulation of cell adhesion and in the reorganization of focal adhesion complexes. According to this possibility, the pre-incubation of HSCs with canrenone results in a dose-dependent reduction of the phosphorylation of FAK, a key protein of the focal adhesion complex, indicating a possible interference with the necessary changes in cell adhesion and organization of the cytoskeleton. to ensure the complete fulfillment of the mitogenic and chemotactic action of PDGF. The possible dependence of these effects on an alteration of the Na <+> / H <+> exchanger activity is suggested by the fact that preincubation with EIPA causes similar inhibitory effects on PDGF-induced FAK phosphorylation.

Taken together, the data confirm that drugs including spierolactone compounds, in particular canrenone, potassium canrenoate, spironolactone and esplerenone antialdosterones can exert an antifibrogenic effect through an action that is expressed at the level of the intracellular signaling cascade induced by stimulation by the PDGF. An antifibrogenic effect of canrenone is also suggested by the inhibitory effect on the "de novo" synthesis of components of the extracellular matrix such as type I procollagen and on fibronectin induced by TGF-β.

Finally, given the results of the experiments in which canrenone proved capable of inhibiting the biological effects of PDGF in human smooth muscle cells, and given the wide overlap of the pathophysiological mechanisms underlying diseases characterized by the progressive formation of tissue fibrous (including the atherosclerotic process), the results of this research group provide an experimental basis for the extension of these acquisitions to other cell types responsible for the onset of sclerosis in other organs and systems.

Claims (10)

CLAIMS 1. Use of a spirolactone compound to produce a drug to inhibit the pro-fibrogenic action of hepatic stellate cells and smooth muscle of the vascular system. 2. Use according to claim 1 wherein said compound is selected from the group consisting of canrenone, potassium canrenoate, spironolactone and eplerenone. 3. Use according to claim 2 wherein said compound is canrenone. 4. Use according to claim 1 wherein said fibrogenesis is mediated by pro-fibrogenic cytokines. 5. Use according to claim 4 wherein said fibrogenesis is mediated by PDGF and / or TGF-β. 6. Use according to claim 1 wherein said hepatic cells are stellate hepatic cells. 7. Use according to claim 1 wherein said cells of the vascular apparatus are smooth muscle cells. The use according to claim 1 wherein said drug is for administration in an amount which is therapeutically effective for inhibiting the fibrogenic process and within the dosage range commonly employed in the clinic for other indications. 9. Use according to claim 8 wherein said amount is in the range of 50-100 mg / day. 10. Use of a spirolactone compound for the development of new pharmacological entities aimed at the medical treatment of pathologies mediated by cytokines and characterized by chronic inflammation and progressive fibrogenesis.