ITRM20100329A1 - MODULATION OF THE NUCLEAR RECEPTOR FOR PHARNESOIDS (FXR) WITH MOLECULES AGONIST FOR THE PREVENTION AND TREATMENT OF ATTEROSCLEROTIC PHENOMENA INDUCED BY ADMINISTRATION OF PROTEASIS INHIBITORS - Google Patents

Description

Description of the Industrial Invention entitled:

"Modulation of the nuclear receptor for farnesoids (FXR) with agonist molecules for the prevention and treatment of atherosclerotic phenomena induced by the administration of p rotease inhibitors"

on behalf of: Fiorucci Stefano, Andrea Mencarelli, Franco Baldelli, Eleonora Distrutti, Sabrina Cipriani, Daniela Francisci, Barbara Renga

all of Italian nationality, domiciled as follows:

Stefano Fiorucci Via Assisi 7, 06100 Perugia

Sabrina Cipriani Via della liberty 125, 06068 Tavernelle (PG) Andrea Mencarelli Via A. Renzini, 12, 06083 Bastia Umbra (PG) Franco Baldelli Via dei Filosofi 14, 06126 Perugia

Barbara Renga Via Sr. Madona country 4S, 06135 Collestrada (PG)

Eleonora Destroyed Via Assisi 70, 06100 Perugia

Daniela Francisci Via Pitagora 15 °, 06135 Perugia

Inventors: Fiorucci Stefano, Andrea Mencarelli,

Franco Baldelli, Eleonora Destroyed, Sabrina Cipriani, Daniela Francisci, Barbara Renga

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Camno of the invention

The present invention relates to the modulation of the farnesoid receptor (FXR) with agonist molecules for the attenuation of atherosclerotic phenomena. In particular, the invention relates to the use of the agonist compounds of the nuclear receptor for farnesoids (Farnesoid-X-Receptor; FXR) for the prevention of the formation and treatment of atheromatous plaques and for the regulation of the lipid profile in the blood, in particular in the carriers of HIV infection and undergoing therapy with antiretroviral drugs belonging to the class of protease inhibitors.

Note Art

The balanced role between the nuclear bpid receptor (LXR) and the nuclear farnesoid receptor (FXR) in maintaining the homeostasis of cholesterol, trigcerides and bibari acids is well known (Sinai et al., Celi (2000) 102: 731-744; Kalaany N. and Mangelsdorf DJ. Annu. Rev Physiol. (2006) 68: 159-191).

In short, FXR is present in the cell nucleus as a component of a dimer with the retinoic acid receptor (RXR) bound to certain regulatory gene sequences on DNA. This complex also hosts co-repressor molecules, which keep the dimer "silent". When an effector molecule binds to one of the two dimer elements, the co-repressors move apart and the downstream gene can be transcribed. Numerous genes are known that are positively or negatively regulated directly or indirectly by FXR (see Kalaany N. and Mangelsdorf DJ. Supra; Fiorucci S. et al. Progr Lipid Res (2010) 49: 171-185). FXR is expressed in enterohepatic tissue where it acts as a sensor for the intracellular concentration of bile acids. In particular, when the intracellular concentration of bile acids increases, FXR is activated, binds to its receptor and forms a complex with the partner receptor RXR Retinoid-X-Receptor). The heterodimer thus constituted binds to specific response elements (Responsive Elements; RE) located on the promoter of responsive genes. The cumulative effect of the co-ordinated modification of the expression of these genes leads to a reduced uptake of bile acids from the portal circulation, to a reduction in their endogenous synthesis and to their increased secretion through the biliary pole of the hepatocyte into the bile. Some of the effects mentioned here are mediated by the activation of a nuclear receptor called "small heterodimer partner" or SHP, which is an atypical nuclear receptor as it does not have a DNA binding domain. The SHP promoter contains a response element for FXR and is activated directly by FXR. Once activated, SHP acts as a co-repressor of genes involved in the regulation of bile acid metabolism. Indeed, SHP inhibits the transcription of two genes CYP7A1 (cholesterol 7a-hydroxylase) and CYP8B1 (sterol 12P-hydroxylase), which respectively regulate the quantity and hydrophobicity of the bile acids produced. Consequently, when the level of bile acids is high, the FXR / SHP system works as a negative regulator by restoring the amount of bile acids to the appropriate level. Furthermore, SHP negatively regulates the activity of NTCP, a protein that imports bile acids into the hepatocyte, displacing its positive inducer, HNF-1 from the promoter of this gene (Lu L., et al., Mol Celi (2000) 6 : 507-515 .; Goodwin B. et al., Mol Cell (2000) 6: 517-526).

FXR is expressed at high levels, consistent with its recognized role in regulating lipid metabolism in the liver and intestines. In these tissues, FXR directly or indirectly regulates the expression of various genes involved in the regulation of lipid and carbohydrate metabolism. One of the genes that are negatively regulated by FXR is SREPBlc (Sterol Regulatory Element-Binding Protein-lc). SREPBlc in turn controls the expression of the FAS gene for fatty acid synthase (Fatty Acid Synthase; FAS), the main gene involved in triglyceride synthesis. SREBP1 is a transcription factor that is part of the SREBP family of proteins, capable of binding specific DNA sequences called SRE (Sterol Regulatory Element). SREBP proteins are generally bound to the nuclear membrane or to that of the endoplasmic reticulum in an inactive form.

Upon binding to specific sterols, SREBP proteins undergo enzymatic cleavage in their N-terminal portion, enter the nucleus and bind to SRE sequences, thus activating the transcription of their target genes (Watanabe M. et al., J Clin Invest. (2004) 113: 1408-18).

The production of triglycerides in the liver is determined by the quantity of synthesized fatty acids, which is in turn controlled at the transcriptional level both by PPARa, which stimulates β-oxidation, and by SREBPlc, which regulates the synthesis of fatty acids. The SREBP family of transcription factors is known for its role in controlling genes that regulate the biosynthesis of cholesterol and the receptors that mediate its absorption by LDL (low density hpoproteins; low density bpoproteins).

Gb SREBP, and in particular SREBPlc, also control the expression of genes involved in lipogenesis such as acetyl-CoA carboxylase, (ACC), fatty acid synthase (FAS), acetyl-CoA synthetase (AceCS), and glycerol-3-phosphate acetyltransferase. Furthermore, the family of SREBPs can induce the expression of ATP-citrate base, deba enzyme mabco (ME), deba glucose 6-phosphate dehydrogenase and deba 6-phosphogluconate dehydrogenase, these enzymes generate NADPH and acetyl-CoA, which are essential. for bpogenesis. The expression of SREBPlc is increased by insulin, and this explains its ability to convert glucose to fatty acids. Gb bibari acids by activating FXR, induce the production of SHP, which in turn inhibits SREBPlc and consequently the synthesis of fatty acids [Watanabe M, et al. above; Brown MS, Goldstein JL. Cell (1997); 89 (3): 33 1-40.]

FXR is expressed at high hvelh also in the kidneys and adrenal glands and at lower hvelhs in the heart and vascular tissue. It has recently been shown that FXR is also present in immune cells such as macrophages. In these cells the activation of FXR negatively regulates the expression of the oxidized LDL receptor, CD36 (see Mencarelli et al., Am J Physiol Heart Ciro Physiol (2009) 296: H272-281).

For purely illustrative purposes, some natural, semi-synthetic and synthetic agonists of FXR are described in Table 1 with the respective bibliographical references alongside, the description of which is intended to be incorporated by reference in its entirety.

Table 1:

List of natural, synthetic and semi-synthetic agonist molecules or FXR modulators.

Bibliographic reference

CDCA Schoenfield et al, 198; Leiss et al, 1982

GW4064 Maloney et al, 2000; Liu et al, 2003

6ECDCA Pellicciari et al, 2002

MFA-1 Soisson et al, 2008

AGN29- AGN31 Dussalt I et al, 2003

XL335 Harnish et al. 2007; Flatt et al, 2009; Feng et al, 2009; (WAY- 362540 and FXR450) Evans et al, 2009

Fexaramine Downes et al, 2003; Cai et al, 2007

NIHS700, MeCA and MeDCA Suzuki et al, 2008

T0901317 Collins et al, 2002; Houck et al, 2004 FXRa modulators

AGN-34 Dussault I et al, 2003

Among the natural agonists of FXR are cholic acid (CA) and its derivatives of the following formula [I]:

1 CDCA R <1> = OH, R <2> = H

2 DCA R <1> = H, R <2> = OH

3 LCA R <1> = H, R <2> = H

Cholic acid corresponds to the above structure where R <3> is OH and Ri R2 is H. The structures of chenodeoxycholic (CDCA) (1), deoxycholic (DCA) (2) and lithocolic (3) acids (LCA ) are illustrated above. Said natural effectors have a low affinity for FXR, which is why new derivatives based on the central structure of natural ligands (steroid derivatives) have been synthesized. These include the derivative of chenodeoxycholic acid 6-ethyl-CDCA (6-ECDCA 0 INT-747) described in WO02072598 and in WO2005082925 (incorporated by reference in their entirety), corresponding to the following formula [II]

6-ECDCA [II]

Other steroid derivatives are described in WO2008002573 (incorporated by reference in its entirety), in particular the UPF-987 compound of the following formula [III] was found to be a particularly active FXR agonist.

UPF-987 [III]

Among the FXR agonists whose structure does not derive from bile acids, that is, which do not have a steroid structure, the best known is GW-4064 (Maloney et al. J Med Chem (2000) 43: 2971-2974) having the formula [ IV] shown below:

GW-4064 [IV]

Other derivatives of GW-4064 with agonist action on FXR are described in WO2004007521 and in US20050080064 (incorporated by reference in their entirety).

Furthermore, a whole series of studies and patents have been published in which derivative compounds of GW-4064 are described (see, for example, international patent applications W02007092751, W02007140174; W02007140183, W02009012125 and W02009 149795, incorporated by reference in their entirety).

Other known FXR agonist compounds are azepine and azepindole derivatives (see WO2005009387, WO2007070796 and US20050054634). Azepines are generically represented by the following formula [V]:

where the substituents are described in the aforementioned patent applications, which are intended as incorporated by reference in their entirety.

One of the compounds belonging to this class of azepine-derived FXR agonists is described in Lundquist et al. J Med Chem (2010) 53: 1774-1787 as a modulator of lipid levels in primates.

Other FXR agonists with a benzimidazole type structure are known and described in a series of already published international patent applications, for example WO2008000643, WO2009062874 and correspond to the following general formula [VI]:

where the substituents are described in the aforementioned patent applications and are intended as incorporated by reference in their entirety.

Furthermore, in Deng G. et al. Bioorg Med Chem Lett (2008) 18: 5497-5502 pyrazolidine-3-5-dione agonist derivatives of FXR of the following general formula [VII] are described:

Wherein the substituents Ri, R2 0 R3 are indicated in Table 2 which

follows:

Table 2:

Substituents of the compound of formula [VIII]

Ri R2 R3

3-CHs phenyl H

3-F phenyl H.

3-CFs phenyl H.

4-CFs phenyl H

3-CH32-nitrophenyl H

3-CH34-fluorobenzoyl H.

3-CH34-methoxylbenzoyl H.

3-CH3 naphthalene- 1 -carbonyl H

3-CH3 thiophene - 1 -carbonyl H

3-CH3pyrimidine-4-carbonyl H.

3-CH3CH3CH3

3-CH3H H

H phenyl H.

H benzoyl H.

2-chlorobenzoyl H

4-chlorobenzoyl H

4-bromobenzoyl H

4-methoxylbenzoyl H.

naphthalene- 1-carbonyl H

H 2-furoyl H.

H phenyl phenyl while other derivatives of the compounds are described in WO2006020680

heterocyclics of the following general formula [IX]:

where the substituents are described in the aforementioned patent applications and are intended as incorporated by reference in their entirety.

All of these FXR agonists are generically known for the treatment of dyslipidemias.

The change in lipid metabolism in the sense of the onset of dyslipidemia and a change in the levels of piasmatic lipoproteins in patients subjected to antiretroviral therapy has been known for some time and the results of some clinical studies aimed at the observation of this phenomenon (Calza et al. J. Antimicrob. Chemother. (2003) 53: 10-14; Stein JH. et al. J Clin Lipidol. (2008) 2: 464-471; Worm SW et al. J Infect Dis. (2010) 201: 318-330). In particular, in this latest work, the statistical analysis of data from patients infected with HIV and undergoing treatment with protease inhibitors (for example lopinavir, ritonavir or indinavir) showed that the risk of myocardial infarction is significantly higher in these patients than in patients treated with other antiretroviral drugs. Furthermore, this study confirmed that this risk is not fully explained by the increased risk of dyslipidemia as the time of administration of these drugs progresses. In other words, the treatment of dyslipidemia alone would not seem to lead to a corresponding reduction in the risk of heart attack.

Despite the accumulation of what is now becoming a considerable amount of clinical and epidemiological data regarding the effect of antiretroviral drugs on the development of dyslipidemia and cardiovascular diseases, it is not yet clear how it is possible to intervene as selectively as possible on this. phenomenon. In fact, it is conceivable that both the HIV infection per se and the side effects of therapy with protease inhibitors synergistically contribute to increasing the risk of serious cardiovascular events in AIDS patients.

In this regard, it is known that protease inhibitors can cause an accumulation of the protein SREBP-1 in the liver and adipose tissues which, as described above, is one of the fundamental regulatory proteins for cholesterol and lipid metabolism and that this accumulation is due to the inhibition of the mechanism of elimination of said protein by the cell (see Riddle TM et al., J Biol Chem (2001) 5: 337-343 and Parker RA et al., Mol Pharmacol (2005) 67 : 1909-1919). It is also known that these antiviral drugs affect the formation of atheromatous plaques by directly intervening on various activities of macrophages (see Zhou H et al., Mol Pharmacol (2005) 68: 690-700 and Dressman J. et al., J Clin Invest (2003) 111: 389-397), which are the main players in the formation of atheromatous plaques, as is well known to those skilled in the art.

At present, clinical practice is oriented towards the co-administration of antilipidemic drugs, such as fibrates or statins, together with antiretroviral therapy for the treatment of patients presenting with dyslipidemia secondary to treatment with antiviral protease inhibitors. This practice, however, is not without its problems and causes additional side effects, as discussed in Ray GM Cardiol Rev (2009) 17: 44-47.

From the above it emerges that there is a need to identify treatment methods that can prevent the adverse cardiovascular effects caused by antiretroviral therapy, in particular caused by HIV protease inhibitors. This would allow to maintain the use of protease inhibitor drugs, such as ritonavir and atazanavir, which have shown antiviral efficacy and which for the moment are impossible to give up in HIV therapy.

Summary of the invention

It has now been found that agonist molecules of the nuclear FXR receptor are able to protect against the side effects of therapy with HIV viral protease inhibitors, not presenting the typical side effects associated with the use of anti-lipid-lowering drugs in use up to now.

The object of the present invention is therefore to use said FXR agonist agents for the treatment of atherosclerosis and dyslipidemia which occur as a side effect of therapy with antiviral inhibitors of the HIV protease. From the pharmacological point of view, a salient feature of the FXR agonists tested so far, is the lack of side effects in primates, which makes their clinical application interesting and innovative where appropriate.

In particular, the invention relates to the use of natural, semisynthetic and synthetic derivatives of bile acids for the re-inhibition of the formation of atheromatous plaques and the reduction of cardiovascular events in the context of treatment with antiretroviral agents of HIV-infected patients.

A further object of the present invention are compositions comprising FXR agonists and HIV protease inhibitor drugs.

Further objects will become apparent from the following detailed description of the invention.

Brief Description of the Figures

Figure 1. The effects of administering an FXR agonist and in particular CDCA, chenodeoxycholic acid, in ΑροΕ <Λ> (nuli) mice treated with a protease inhibitor (ritonavir) for 12 weeks are shown diagrammatically. The experiment in question was repeated three times with different sets of animah and the data shown refer to one of the three experimental sets, the results of the other two being similar to the one shown. A: area of the plaques measured in the various experimental groups. B: Triglyceride, LDL, HDL, total cholesterol, blood glucose and ALT levels in wild type and Apo-nuU mice treated with or not ritonavir or CDCA ritonavir. As can be seen, the CDCA significantly reduces the extension of atheromatous plaques in mice exposed to ritonavir.

Figure 2. MRNA levels for different proteins that regulate lipid and cholesterol production (see Example 1) in the liver of ApoE-null mice treated with ritonavir or CDCA ritonavir for 12 weeks. The experiment in question was repeated three times with different sets of animals and the data shown refer to one of the three experiments, being the results of the other two similar to the one shown.

Figure 3. Activation of FXR protects against the development of dyslipidemia caused by ritonavir in wild type animals. Liver function parameters and blood levels of lipids, cholesterol, Uberi fatty acids, triacylglycerol, HDL, LDL and glucose are shown diagrammatically in wild type and wild type C57BL / J mice treated with ritonavir for 2 weeks (see Example 1 ) or with ritonavir in combination with the FXR agonist, CDCA.

Figure 4. Shown are hepatic UveUo results from the experiment shown in Figure 3, conducted in wild type C57BL / J mice treated with ritonavir alone or ritonavir plus the FXR agonist, CDCA for 2 weeks. A and B: change in liver weight and triacylglycerol content of livers of wild type mice treated or not with CDCA ritonavir. C: H&E coloring; D: coloring with Red Oil; E: anaphysis of the expression of genes (mRNAs) encoding proteins involved in the metabohism of Upidae in wild type mice treated with ritonavir alone or in combination with the FXR agonist, CDCA.

Figure 5. The absence of FXR in genetically modified mice exacerbates the dyslipidemia caused by ritonavir indicating an essential role of FXR in counter-regulation of adverse effects caused by protease inhibitors. Dyslipidemia in FXR <7> - (nuli) mice (see Example 1). A-F: blood levels of lipids in mice treated or not treated with ritonavir. G: effect of ritonavir treatment on liver weight. H: H&E histology of liver of wild type and FXR-null mice treated or not with ritonavir. I: analysis of the expression of genes (mRNA levels) coding for proteins involved in lipid metabolism in wild type and FXR-null mice treated or not with ritonavir.

Figure 6. CD36 expression on circulating and cultured monocytes / macrophages and its modulation following treatment with ritonavir or ritonavir in combination with the FXR agonist, CDCA. A: CD36 levels present on monocytes / macrophages in ApoE null mice. B: CD36 levels present on cultured RAW264.7 macrophages treated with scalar concentrations of ritonavir. C: effect of CDCA co-treatment of RAW264.7 macrophages cultured with ritonavir. D: Flow cytometric profiles of RAW264.7 macrophages treated with ritonavir and effect of co-treatment with CDCA. E: uptake of acetylated LDL by ritonavir-treated RAW264.7 macrophages and effect of co-treatment with CDCA.

Figure 7. Expression of genes encoding proteins involved in lipid metabolism in cultured macrophages. Exposure of macrophages (RAW264.7) to ritonavir increases the expression of CD36 and SREBP1. Co-treatment with the natural FXR agonist CDCA inhibits the induction of CD36 and SREPBlc. Furthermore, activation of CD36 with CDCA induces the expression of PPAR and SHP. Since SHP is a gene directly regulated by FXR, its induction is an index of the activation of FXR in the macrophage.

Figure 8. A different protease inhibitor (atazanavir) increases the expression of CD36 in RAW264.7 macrophages in vitro. Figure 9. Diagrammatic representation of the effect of FXR agonists on metabolic pathways modified by HIV protease inhibitors.

Figure 10. Schematic view of the assay for verifying the presence of a binding site (SRE) specific for SREBP-1 in the mouse CD36 promoter sequence of Example 2 (transactivation experiment).

Detailed description of the invention In the context of the present invention, the term "FXR agonist" refers to a natural compound, semi-synthetic or synthetic, which acts by directly activating the FXR receptor. The FXR agonists of interest according to the present invention are compounds which, characterized by a transactivation assay (see Example 2), are able to induce the transcriptional activity of the nuclear FXR receptor with an effective dose of 50% ( EC) so equal to or lower than that observed for the natural ligand CDCA (chenodeoxycholic acid) whose EC50 is equal to 10 M.

Within the scope of the invention are included the agonist agents of FXR, their corresponding derivatives and pharmaceutically acceptable salts, in their racemic or optically active forms, which can be used by sob or in admixture with each other.

The inventors, in undertaking the study of the influence of FXR modulation on the cardiovascular side effects induced by exposure to HIV virus protease inhibitor (IP) antiviral drugs, unexpectedly found that FXR agonist compounds have the ability to prevent, by modulating selectively the molecular pathways activated by protease inhibitors, the onset and formation of atheromatous plaques that develop in particular in subjects undergoing treatment with IP antiviral drugs.

Indeed, it has been found that the effect of ritonavir, an IP widely used in the treatment of AIDS, on the presence and transcription of CD36 in cultured RAW2647 macrophages, is dependent on SREBPlc (Figures 6 and 7) and that the exposure of macrophages A natural and synthetic FXR agonist ligands reduces cellular levels of the SREBPlc protein, its mRNA (see Figure 7) and its nuclear translocation by up to 50%, an event that is considered important in regulating CD36 expression. Figure 8 also shows the effect of using a different IP (atazanavir) on the expression of CD36 on the surface of cultured mouse macrophages. As can be seen, atazanavir also produces a dose-dependent increase in the presence of CD36 on the cell surface. Furthermore, a different FXR agonist, GW4064, inhibits CD36 induction caused by both ritonavir and atazanavir, demonstrating that all synthetic and natural FXR agonists inhibit macrophage activation caused by protease inhibitors (data not shown ).

Simultaneously with the reduction of SREBPlc, as illustrated in Figure 7, the use of FXR agonists on mouse macrophages in vitro causes the induction of the SHP protein proposing a situation mirroring that created by ritonavir (i.e. increased levels of SREBPlc and low levels of SHP).

Confirming the observed regulation of FXR agonists on CD36 transcription, the presence of a binding site (SRE) specific for SREBP-1 in the mouse CD36 promoter sequence was also verified (Example 2 and Figure 10).

FXR activation and protection against ritonavir-induced atherosclerosis in a specific animal model (AnoE null mice)

ApoE-null mice are a dyslipidemic mouse model that develops atherosclerosis over the course of its life. Administration of ritonavir for three months (12 weeks) to one group of these mice, alone or in co-treatment with CDCA, had no effect on the weight and mortality of the animals. However, animals treated with ritonavir alone showed a dramatic increase in aortic plaque extension, which was significantly reduced by co-treatment with CDCA (Figure 1A).

The fact that CDCA is able to protect against plaque formation in ApoE-null mice, despite having no effect in modifying the lipid profile of these animals (Figure 1B), indicates that FXR activation is able to regulate negatively the expression of pro-inflammatory mediators (CDllb, MCP-1, IL-6 and IL-Ιβ) involved in the formation of aortic plaques. The expression of these genes is increased in ApoE-null animals, is exacerbated by treatment with ritonavir and is reversed by administration with FXR agonists (data not shown). As is known to the skilled in the art, the activation of inflammatory genes in the vessel walls leads to the adhesion, chemoattraction, migration and activation of immune cells such as monocytes and T lymphocytes, inducing the formation and progression of aortic plaques.

Regarding the blood parameters of ApoE-null mice, baseline cholesterol and triglyceride levels were twice that of wild type animals, while LDL was four times normal and HDL was three times lower than normal. Neither treatment with ritonavir, nor co-treatment with CDCA or gemfibrozil were able to vary these parameters (Figure 1B). The fact that the action of FXR agonism had no effect on the lipid picture in ApoE-null animals is an unexpected observation. To verify the influence of ritonavir on this phenomenon, the effect of CDCA alone on the dyslipidemia of ApoE-null mice was observed. In this case the dyslipidemia could be corrected by the FXR ligand, indicating that the mode of action of the IP was new (results not shown). What was observed in the blood was confirmed at the level of gene expression of proteins involved in lipid and cholesterol metabolism.

In order to clarify how CDCA exerted the plaque-reducing effect of ritonavir observed in ApoE-null mice, the expression of CD36 in circulating monocytes was investigated (see Figure 6A). For this purpose, CD36 levels were detected on the surface of monocytes of ApoE-null mice and RAW2647 cells treated with ritonavir alone or together with CDCA. Co-treatment was observed to cause a return to baseline levels of the presence of CD36 on the monocyte surface both in vivo and in vitro (see Figure 6A-D) and that this drop in CD36 levels coincided with a decrease in uptake of ac-LDL by RAW2647 treated monocytes (Figure 6E). The effect exerted by CDCA was linked with a decrease in SREBP-1 levels in the nucleus (see Figure 7A). The analysis of mRNA levels for SREBPlc also showed that only treatment with CDCA was capable of reducing its levels while causing an increase in mRNA levels for PPARy and SHP (see Figure 7B).

In this experiment it was also observed that ritonavir caused a slight rise in mRNA levels for CD36 in cultured RAW2647 macrophages, and that only treatment with CDCA was able to completely abolish this increase (Figure 7B).

The studies carried out by the inventors therefore allow us to confirm that FXR agonists can be used for the prevention and treatment of cardiovascular complications and atherosclerotic phenomena caused by IP antiviral drugs in HIV infected subjects. FXR agonists according to the present invention can be used in antecedent, simultaneous or subsequent administration of said antiviral drugs.

The administrable dosage can vary between 0.1 mg / kg and 10 mg / kg / day.

The routes of administration are those known to those skilled in the art and can be the intravenous, intramuscular, intraperitoneal, oral, topical, spray, vaginal or rectal routes. The oral route is preferred.

Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers known in the art in dosages suitable for oral administration. The dosage forms can be tablets, pills, powders, hard capsules and soft capsules, lozenges, or they can be liquid formulations, such as sprays, gels, syrups, suspensions, and simih.

The formulations may also include the simultaneous presence of an IP drug and an FXR agonist.

The excipients that can be used are those known to those skilled in the art, such as carbohydrates or proteins, including, but not limited to, sugars (for example, lactose, sucrose, mannose or sorbitol); corn starch, rice starch, etc .; cellulose, such as, for example, methylcellulose and hydroxypropylmethyl cellulose; gums, proteins such as gelatin and collagen. The oral formulations can also contain solubilizing or disintegrating agents known to those skilled in the art, such as alginic acid and polyvinyl pyrrolidone or their salts.

The aqueous suspensions can contain suspending agents known to those skilled in the art, such as hydroxymethyl cellulose or sodium alginate, and wetting agents, for example natural agents such as lecithins, or of synthetic origin, such as polyoxyethylene stearate and polyoxyethylene sorbitan mono-oleate.

Pharmaceutically acceptable preservatives, such as ethyl phydroxybenzoate, or dyes, flavorings and sweeteners, such as aspartame and sucrose, may also be present in the aqueous suspensions. If necessary, the solutions can be adjusted for osmolarity.

Oily suspensions may contain oil of natural origin, such as peanut oil, or a mineral oil, such as liquid paraffin. They may also contain thickeners, such as beeswax or cetyl alcohol, sweeteners and antioxidants, such as ascorbic acid. The oil-in-water suspensions can, in addition to the components described above, contain emulsifiers such as those already described above, and sweeteners.

Syrups may contain sweeteners, preservatives and / or dyes.

Pharmaceutical formulations for intravenous administration may consist of a sterile, aqueous or oily injectable preparation. This suspension can be formulated according to methods known to those skilled in the art, using suitable dispersing or wetting agents and suspending agents already mentioned above. The sterile injectable preparation can also be a suspension or a solution in a non-toxic diluent parenterally, such as water or physiological saline. Fixed or sterih can be conventionally employed as a solvent or suspension medium, such as mono- and synthetic glycerides. Finally, fatty acids such as oleic acid can also be used in the formulation of injectable drugs.

The following examples are provided by way of example of the invention and are not to be considered as limiting its scope.

Examples

Example 1, Effects of ritonavir treatment in ApoE-null mice for 12 weeks and in wild type mice for 2 weeks. Experimental protocol.

ApoE - / - mice were divided into 3 groups:

group 1: untreated

group 2: intra-peritoneal administration of ritonavir (5mg / kg) group 3: intra-peritoneal administration of ritonavir (5mg / kg) plus intragastric administration of CDCA (15 mg / kg).

All drugs were administered once a day (5 days a week) for twelve weeks. At the end of the experiment, the animals were fasted for twelve hours, anesthetized with sodium pentobarbital. Blood was taken for the determination of the concentration of cholesterol, triglycerides, HDL, LDL, glucose, AST, ALT and for the isolation of monocytes / macrophages, in which the expression of CD36 was measured. The livers collected were used for RNA isolation and the aortas were processed for histology and specific staining of atherosclerotic lesions and for RNA isolation (see Figures 1, 2, 6A) .

Quantification of atherosclerotic lesions

The heart and aorta were removed from the animal, peripheral fat was removed, and the aorta (including the ascending arch, and thoracic and abdominal segments) was then longitudinally dissected, fixed to a support and then stained with Sudan dye. IV to highlight the lesions. The aortic lesions were photographed and the lesion area (in pixels) was calculated using the free software Image J 1.33u (Rasband W., National Institutes of Health; Bethesda, MD).

Real-time PCR analysis

The quantification of gene expression in tissues was performed through the use of the qRT-PCR technique. Total RNA was extracted from livers and aortas with Trizol (Invitrogen), incubated with DNAase I and back-transcribed with Supescript II (Invitrogen). For RT-PCR, 100 ng of cDNA in 25μ1 of reaction containing 0.3μΜ of the sense and antisense primers and 12.5μΐ of 2x SYBR Green PCR Master Mix (Bio-Rad Laboratories, Hercules, CA) were used. All reactions were performed in triplicate using the following protocol: 2 min at 95 ° C, followed by 50 cycles of 95 ° C for 10 seconds and 60 ° C for 30 seconds using iCycler iQ instrument (Bio-Rad Laboratoires).

The primers were designed using the PRIMER3-OUTPUT software using the sequences published on the NCBI database. The sequences of the primers used are shown below: mSREBPlc, SEQ ID NO.l: gatcaaagaggagccagtgc; mSREBPlc, SEQ ID NO.2: tagatggtggctgctgagtg; mSREBP2, SEQ ID NO.3: acagatgccaagatgcacaa;

mSREBP2, SEQ ID NO.4: ttcagcaccatgttctcctg;

mFAS, SEQ ID NO.5: tgggttctagccagcagagt;

mFAS, SEQ ID NO.6: accaccagagaccgttatgc;

mHMGCoAS, SEQ ID NO.7: ggtggatgggaagctgtcta; mHMGCoAS, SEQ ID NO.8: acatcatcgagggtgaaagg; mHMGCoAR, SEQ ID NO.9: ccgaattgtatgtggcactg; mHMGCoAR, SEQ ID NO. 10: ggtgcacgttccttgaagat;

PPARy, SEQ ID NO. 11: gccagtttcgatccgtagaa;

PPARy, SEQ ID NO. 12: aatccttggccctctgagat;

mFXR, SEQ ID NO.13: tgtgagggctgcaaaggttt;

mFXR, SEQ ID NO. 14: acatccccatctctctgcac;

mSHP, SEQ ID NO. 15: tctcttcttccgccctatca;

mSHP, SEQ ID NO. 16: aagggcttgctggacagtta;

mCDllb, SEQ ID NO. 17: aaggattcagcaagccagaa;

mCDllb, SEQ ID NO. 18: tagcaggaaagatgggatgg;

mIL6, SEQ ID NO. 19: ccggagaggagacttcacag

mIL6, SEQ ID NO.20: tccacgatttcccagagaac;

mCD36, SEQ ID NO.21: cggagacatgcttattgagaa

mCD36, SEQ ID NO.22: actctgtatgtgtaaggacct

mTNFa, SEQ ID NO.23: acggcatggatctcaaagac

mTNFa, SEQ ID NO.24: gtgggtgaggagcacgtagt;

mGAPDH, SEQ ID NO.25: tgagtatgtcgtggagtctac

mGAPDH, SEQ ID NO.26: gttggtggtgcaggatgcattg;

ml8S, SEQ ID NO.27: accgcagctaggaataatgga

ml8S, SEQ ID NO.28: gcctcagttccgaaaacc.

Positive modulation of FXR on ritonavir-induced dyslipidemia in wild type mice.

An attempt was made to replicate the typical clinical picture of patients undergoing IP therapy in an animal model.

Wild type C57BL6 / J mice were subjected to i.p. of 5 mg / kg of ritonavir for 2 weeks. The results obtained (see Figures 3 and 4) indicate that the administration of ritonavir increases blood levels of triacylglycerols, Free Fatty Acids (FFA), cholesterol and LDL, as expected. Also in this context, different types of liver analyzes were performed as already done with ApoE-null mice.

Ritonavir administration caused the accumulation of triglycerides within the hepatocytes (Figure 4B, C and D) and also a considerable increase in the expression levels of genes involved in lipid and cholesterol homeostasis (Figure 4E), such as SREBPlc, SREBP2, FAS and HMCoA synthase, although this was statistically significant only in the case of SREBPlc and FAS. Co-treatment with CDCA caused a significant decrease in the expression levels of FAS and SREBPlc and a significant stimulation of the expression of SHP in hepatocytes.

Ritonavir-induced dyslipidemia in FXR-null mice

FXR-null mice, i.e. lacking the FXR gene, and wild type mice were treated with ritonavir for 2 weeks (Figure 5). As shown in Figure 5A-F, the nule FXR and nule FXR mice treated with ritonavir had marked dyslipidemia, superior to that of the corresponding wild type mice. They also showed a more evident hepatic statosis (5H) and the hepatic expression profile of genes involved in the production of lipids and cholesterol was characterized by a marked dysregulation of the genes involved in the synthesis of fatty acids, in particular of the fatty acid synthase (FAS ) (51).

Example 2, Identification of an FXR ligand by means of a transactivation method

Transactivation assays are used in molecular biology to study cellular events such as the binding of transcriptional factors to particular DNA sequences called response elements (RE) which can act as enhancers or silencers of the transcription and / or to evaluate the efficacy of potential agonists in inducing the transcriptional activity of certain transcription factors (for a scheme see Figure 10).

In the present case, the transactivation assay to identify an FXR ligand was performed in human liver cells (HepG2) transfected with a viral expression vector that contains a canonical binding site for FXR represented by a defined base sequence. with the generic term of IR-1 (inverted repeat-1; inverted repeat sequence-1). The sequence in question is AGGTCA ni TGACCT.

The experimental protocol is divided into the following three main phases:

(a) Transient transfection of target cells (HepG2)

As previously described (Fiorucci et al. Gastroenterology (2004) 127: 1497-1512), HepG2 cells were transfected with:

(i) a plasmid containing human FXR cDNA (pCMVSPORT-FXR);

(ii) a plasmid containing human RXR cDNA (pSG5-RXR);

(iii) a plasmid containing the luciferase reporter gene upstream of which the responsive element of FXR is cloned;

(iv) a plasmid containing the cDNA of the β-galactosidase enzyme which is used as an internal transfection control.

(b) Stimulation of cells with potential agonists

48 hours after transfection the cells were stimulated with potential nuclear FXR receptor ligands / agonists. In particular, the transfected cells were incubated with increasing concentrations of FXR agonist starting from 1 nM (10 <9>) up to 100 μΜ (10-4);

(c) Lysis of cells and analysis of luminescence by the luminometer and normalization of luciferase values.

The luminescent assay was performed using Promega's Luciferase Assay System.

After 24 hours of stimulation, the cells were lysed with 100 1 of cold lysis buffer according to the manufacturer's instructions, vortexed and centrifuged for 1 minute at 14000 rpm. 20 1 of cell supernatant was read on the luminometer using 100 1 of luciferase substrate. The normalization of the luciferase values was carried out by analyzing the activity of the βgalactosidase enzyme on the same cellular bsates. This activity is usually tested by adding the substrate of the enzyme ortho-nitrophenyl-galacto-pyranoside (ONPG) to the used cefiulare and incubating the mixture at 37 ° C until it turns yellow. The enzyme units were measured with a spectrophotometer at a wavelength of 420 nm.

Claims (11)

CLAIMS 1. Use of FXR agonist agents for the preparation of drugs for the prevention and treatment of atherosclerosis, dyslipidemia and cardiovascular complications in subjects, such as humans or animals, undergoing treatment with antiretroviral drugs. 2. Use according to rev. 1 in which subjects are infected with HIV. 3. Use according to rev. 1-2 in which the antiretroviral drugs are HIV protease inhibitors, in particular lopinavir, ritonavir, indinavir, atanazavir and their combinations with each other and / or with other antiretroviral drugs. 4. Use according to rev. 1-3 in which the agonist agents of FXR are selected from natural, semi-synthetic or synthetic compounds, which, during a transactivation assay, are able to induce the transcriptional activity of the nuclear FXR receptor with an effective dose of 50 % (EC50) equal to 0 lower than that for the natural ligand chenodeoxycholic acid. 5. Use according to rev. 1-4 in which the activation of FXR negatively regulates the expression of the pro-inflammatory mediators CDllb, MCP-1, IL-6 and IL-Ιβ. 6. Use according to claims 1-5 wherein the agonist agents of FXR are selected from the group of natural, semi-synthetic or synthetic derivatives of bile acids, in particular chenodeoxycholic acid, steroidal and non-steroidal derivatives of bile acids, derivatives of azepines , of azepindoles, benzylimidazole and pyrazolidine derivatives and corresponding derivatives and salts, in their racemic or optically active forms, alone or in mixture with each other. Use according to claims 1-6 wherein the administrable dosage varies between about 0.1 mg / kg and 10 mg / kg / day. 8. Pharmaceutical composition comprising one or more FXR agonist agents in combination with antiretroviral drugs, in particular ritonavir, and a pharmaceutically acceptable carrier. 9. Pharmaceutical composition according to claim 8 formulated for intravenous, intramuscular, intraperitoneal, oral, topical, spray, vaginal or rectal administration. 10. Product comprising an FXR agonist agent and an antiretroviral drug as a combined preparation for simultaneous, separate or sequential use in the prevention and treatment of atherosclerosis, dyslipidemia and cardiovascular complications in subjects, such as humans or animals, undergoing to treatment with antiretroviral drugs. 11. Produced according to rev. 10 in which the antiretroviral drugs are HIV protease inhibitors, in particular lopinavir, ritonavir, indinavir, atanazavir and their combinations with each other and / or with other antiretroviral drugs.