ITRM20130167A1 - CARRIER INHIBITORS OF CITRATE IN THE TREATMENT OF INFLAMMATION - Google Patents

Description

Inhibitors of the citrate carrier in the treatment of inflammation

DESCRIPTION

The present invention relates to 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB), for use in the treatment of inflammatory pathologies.

State of the art

The inflammatory response is a complex cascade of events induced by infection or tissue damage and is associated with numerous pathologies [1,2]. For this reason, anti-inflammatory drugs are widely used in the world. For the record, it can be mentioned that NSAIDs (Non-Steroidal Anti-Inflammatory Drugs), one of the two main categories of anti-inflammatory drugs, represent the largest amount of compounds prescribed and used in the world: every day more than 30 million people take NSAIDs in order to relieve inflammatory conditions, from acute to chronic [3]. The anti-inflammatories in use include, in addition to the aforementioned NSAIDs, the best known of which is aspirin, steroids (such as cortisone). However, the anti-inflammatories marketed today have numerous side effects: for example, NSAIDs that inhibit cyclooxygenases (COX) in a more or less selective way have multiple side effects, for example by extinguishing the beneficial effects of prostaglandins on the gastric mucosa. Cortisonics also have numerous side effects, even very serious ones. Often these side effects have been of such intensity that anti-inflammatory products have been withdrawn from the market. In 2002, products based on nimesulide, a COX-2 inhibitor, were withdrawn from the market in Finland due to an increase in reports of liver toxicity and in the same year Spain cautiously suspended their sales. Nimesulide has not been approved in the United States, in some European countries, such as Germany and Great Britain, and it is also not present in Japan and Canada [4]. Precisely because of the numerous side effects, there is now a need for new drugs that can better counter the inflammatory processes associated with multiple pathologies with repeated side effects.

The discovery of more selective inhibitors against COX-2 marks the beginning of the second generation anti-inflammatories introduced on the market, the so-called COXIB: celecoxib, etoricoxib, rofecoxib, valdecoxib and lumiracoxib [5,6]. However, clinical studies have shown that these new anti-inflammatories also have serious side effects not at the gastrointestinal level like the previous ones but for example increasing the risk of cardiovascular diseases. In 2004 rofecoxib-based medicines were voluntarily withdrawn from the world market due to an increased risk of serious cardiovascular events, observed during a clinical trial and similar fate in 2005 was the turn of valdecoxib withdrawn from the European market again for severe side effects. The Italian Medicines Agency renews its invitation not to use second generation anti-inflammatory drugs still on the market in for those who have cardiovascular problems (heart attack, stroke), are obese, have high cholesterol, are a heavy smoker, have problems to the arteries or veins of the legs.

A new avenue in the treatment of inflammation has been opened by studies on the citrate carrier. The citrate carrier (CIC), encoded by the human gene SLC25A1, catalyzes the electroneutral exchange between citrate (H-citrate2-) and the non-protonated form of the malate (malato2-) [7]. The citrate exported from the mitochondria to the cytosol, through the CIC, is split by the ATP-citrate lyase (CL) into oxaloacetate and acetylCoA which will also lead to the biosynthesis of sterols and fatty acids. The oxaloacetate produced in the cytosol by CL is reduced to malate, and the latter, in turn, converted into pyruvate by the malic enzyme with consequent production, in the cytosol, of NADPH + \ H + (necessary for the synthesis of sterols and fatty acids) [7]. The citrate carrier has been functionally characterized [8]. The regulation of CIC gene expression was also studied, highlighting the involvement of transcriptional factors such as SREBP1 [9], particularly important in lipogenesis, the FOXA [10] proteins involved in the activation of genes highly expressed in liver and pancreas and epigenetic mechanisms [11]. All these studies were essentially conducted in the liver and pancreas, where CIC performs its function linked to the metabolism of fatty acids. A completely new role for CIC in the inflammatory response has recently been reported. CIC levels increase, both as mRNA and as protein, following treatment with lipopolysaccharides (LPS) in monocyte cell lines differentiated into macrophages and in monocytes isolated from peripheral blood [12]. Experimental studies show that in macrophages treated first with LPS and then with BTA (1,2,3, benzentricarboxylic acid), a classic CIC inhibitor, the production of the main inflammatory mediators drastically decreases [12] indicating that CIC can be a new target in the inflammatory process for a new approach to anti-inflammatory therapies.

However, the attempt to use BTA in the treatment of inflammation for the purpose of developing a drug was not followed up due to the fact that:

a) BTA is not lipophilic, therefore, added to cell cultures it does not cross the phospholipid bilayer of the membranes unless it is incorporated into micelles;

b) Furthermore, the minimum effective concentration that induces an anti-inflammatory effect is high: 200µM.

The need was therefore felt in the state of the art to provide new tools capable of interacting with the CIC in regulating the entire cascade of inflammation and of achieving the production of new drugs overcoming the disadvantages of the previous technique.

A specific CIC inhibitor, 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB), has recently been identified [13] by in vitro binding assays using the citrate carrier of Saccharomyces cerevisiae. The mentioned work represents the result of a generic screening on different compounds and has provided generic indications referring to a relatively large class of compounds, of which the CNFASB is only a representative. In this regard, it must be emphasized that:

a) since these are in vitro experiments these could not, by definition, be considered even only an indication for their passage to scale in vivo and, even more so, from the species S. cerevisiae to the species H. sapiens, phylogenetically very distant and completely different in the cellular structure as well as in the organization and functionality of the whole organism;

b) the alignment of the amino acid sequence of the human citrate carrier with the yeast ortholog shows an identity percentage of the amino acids equal to only 35% of the entire sequence. So it was not taken for granted that CNFASB also inhibited the human citrate carrier.

c) many residues of the amino acid serine present in the human protein are not present in the yeast protein. This is very relevant since these amino acids can be phosphorylated and thus significantly influence the activity of the protein. In any case, even if the serines had been preserved from yeast to man, the method of detection of the CNFASB inhibitor as described in ref. 13, does not allow to evaluate the effect of phosphorylations as the experiments to test the CNFASB inhibitor were carried out only by in vitro binding. This fits into the more general context of the â € œphosphoproteomeâ € which is completely different in higher eukaryotes than in unicellular yeasts [14]. However, phosphorylations are just one example of the post-translational changes that occur very differently in humans and yeast and which can drastically modify the structure of proteins and consequently the binding to any ligand, be it activator or inhibitor, and therefore biological activity.

It has now been surprisingly found that 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB) meets the above requirements and does not have the drawbacks of the state of the art.

Therefore, the object of the present invention is 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB), for use in the treatment of inflammatory pathologies according to claim 1. Further objects are better specified in the dependent claims.

The product according to the invention 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB) has the following advantages:

a) a reduction of the main inflammatory mediators such as nitric oxide (NO), reactive oxygen species (ROS) and prostaglandins E2 (PGE2), thus acting on the entire inflammation chain, in following various inflammatory stimuli, unlike BTA for which only the effect after induction of LPS-mediated inflammation was seen.

b) the effective doses are much lower, an activity is already demonstrated at 1µM achieving a high selectivity and specificity for the cells of the immune system involved in the inflammatory process

c) absence of cellular toxicity and only a specific effect on the inflammatory response d) the product does not need to be incorporated into micelles to cross the fospolipid bilayer.

The use of this inhibitor induced a reduction of the main inflammatory mediators such as nitric oxide (NO), reactive oxygen species (ROS) and prostaglandins E2 (PGE2) after stimulating a response inflammatory process with LPS, tumor necrosis factor Î ± (TNFÎ ±), interferon γ (IFNγ), TNFÎ ± and IFNγ together. The circumstance that CNFASB reduces the kynurenic pathway allows to support - without being bound to particular reaction mechanisms - that the inflammatory process of any origin, CNFASB, can block not only the production of prostaglandins, like the common anti-inflammatory drugs COX-2 on the market today, but all the inflammatory cascade, blocking inflammation in a more solid way. This can be of considerable importance for diseases associated with inflammation.

Brief description of the figures

Four tables are attached to this description showing:

figure A the dosage of NO in cell cultures of human macrophages U937 variously stimulated and treated with CNFSAB;

figure B the dosage of ROS variously stimulated and treated with CNFSAB;

figure C the dosage of PGE2 variously stimulated and treated with CNFSAB and figure D the chemical formulas of BTA and CNFASB.

Description of the invention

The present invention overcomes the previous limitations in that the inhibition of CIC by CNFASB, at a concentration of only 1µM, induces a drastic reduction not only of the synthesis of prostaglandins (effect of the anti-inflammatories currently on the market) but also of other mediators of the Inflammation such as nitric oxide (NO) and radical oxygen species (ROS).

It should be reiterated that another problem overcome by the present invention is the poor selectivity of anti-inflammatory drugs belonging to the state of the art. In this regard, the expression of CIC in macrophages is extremely high even compared to liver cells, where until now it was thought to carry out its main function in the biosynthesis of fatty acids. This situation makes CIC inhibitors very selective and specific for the cells of the immune system. Furthermore, at the concentrations used, CNFASB does not show cellular toxicity but only a specific effect on the inflammatory response.

CNFASB is used as a pure molecule for research purposes and is produced by Calbiochem cod. 475877 and distributed by Merck Chemicals. The CNFASB is used at a dosage of active substance between 1 and 100mg per day depending on the body weight, advantageously between 20 and 30mg per day.

Studies on the inflammatory response

The effect of 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB) as a CIC inhibitor in the inflammatory response was tested at different concentrations, then choosing the lowest concentration at which the effect on inflammatory molecules was observed and there was no cell toxicity, ie 1µM. This inhibitor was recently discovered through an in silico screening of a compound database. In this way it has been found that CNFASB is a specific competitive inhibitor of CIC activity [13]. However, these studies were conducted using the CIC protein in liposomes, an approach completely foreign to the cellular environment. Also, as mentioned, the protein used is yeast CIC. On the contrary, according to the present invention, it is proposed to use the CNFASB in human cells metabolically and phylogenetically very distant from Saccharomyces cerevisiae from which CNFASB has been isolated. The human cell line U937 was used for the experiments, a line widely used for studies on the inflammatory response [15]. In U937 cells, the inflammatory response was induced by: LPS of Salmonella enterica serotype typhimurium (200ng / ml), TNFÎ ± (5ng / ml), IFNƳ (10ng / ml), TNFÎ ± (5ng / ml) IFNƳ (10ng / ml). Following these treatments, the inflammatory response was monitored by measuring NO (figure A), ROS (figure B) and PGE2 (figure C). When U937 induced with LPS, TNFÎ ±, IFNƳ, TNFÎ ± IFNƳ were treated with CNFASB, there was a drastic reduction in the production of the main inflammatory mediators such as NO (figure A), ROS (figure B) and PGE2 (figure C).

The CNFASB was dissolved in dimethyl sulfoxide at a concentration of 1mM and then added to the culture medium making a 1: 1000 dilution. In the control samples, therefore, dimethyl sulfoxide was used as a blank. The addition of CNFASB did not induce changes in the pH of the soil, as is the case with BTA. In fact, BTA has three carboxylic groups resulting in a highly acidic, while in the CNFASB there is only one carboxylic group (figure D). Without being bound to particular scientific explanations, it can be said that these structural differences between the two inhibitors are also at the basis of the different delivery of the two molecules. BTA, being very acidic, cannot cross the plasma membrane; consequently, to treat the cells, a pre-incubation of the molecule with liposomes is required. On the other hand, the CNFASB is more lipophilic and is able to reach the inside of the cell without difficulty. Finally, the CNFASB appears to be sterically more bulky than the BTA (figure D), suggesting an inhibition of CIC through the occupation of both binding sites of the protein [13]. Probably for this reason it turns out to be a more effective inhibitor of CIC activity than BTA.

The cellular mechanism underlying the results obtained depends on the role of CIC in transferring substrates and energy from the mitochondria to the cytosol. In fact, CIC, highly expressed in activated macrophages, by transporting the citrate into the cytosol makes it available for ATP citrate lyase. Then acetylCoA and oxaloacetate are formed. AcetylCoA is necessary for the elongation of polyunsaturated fatty acids that will reform arachidonic acid used in the biosynthesis of prostaglandins during the inflammatory process. The oxaloacetate instead by the malic dehydrogenase enzyme is transformed into malate which in turn will give pyruvate and NADPH thanks to the malic enzyme. NADPH is essential - for the activity of the inducible nitric oxide synthetase (iNOS) enzyme which will produce NO;

- for the NADPH activity which will produce the ROS.

Therefore, by blocking the activity of CIC through the use of CNFASB, the production of prostaglandins is simultaneously inhibited, as the acetylCoA produced by the action of CIC, NO and ROS, whose synthesis depends on the intracellular availability of NADPH always supplied following the biological activity of CIC. Through this mechanism, the CNFASB is undoubtedly more effective in blocking the inflammatory cascade not only by reducing the production of prostaglandins (mechanism of action of the anti-inflammatories on the market today) but also of other inflammatory mediators such as NO and ROS .

Cell cultures and treatments

Cell cultures of U937 human macrophages were set up in RPMI 1640 (Roswell Park Memorial Institute 1640), supplemented with 10% (v / v) fetal bovine serum, 2 mM L-glutamine, 100 U penicillin and 100 µg / ml streptomycin in CO2 incubators (5%) at a controlled temperature of 37 ° C.

Human U937 cells are monocytes which after seeding were differentiated into macrophages by 10ng / ml of PMA (phorbol-myristate-acetate) for 24h. Differentiation involves cell adhesion. Only after cell differentiation, the macrophages were treated with the different inducers of inflammation: LPS (200ng / ml), TNFÎ ± (5ng / ml), IFNγ (10ng / ml), TNFÎ ± IFNγ (5ng / ml 10ng / ml).

Dosage of NO, ROS, PGE2

In cell cultures of human macrophages U937 stimulated with LPS (200ng / ml), TNFÎ ± (5ng / ml), IFNγ (10ng / ml), TNFÎ ± IFNγ (5ng / ml 10ng / ml) and treated with CNFASB, the CIC inhibitor, the levels of the following inflammatory mediators were quantified: nitric oxide (NO), radical oxygen species (ROS), prostaglandin E2 (PGE2). The CNFASB was tested at different times and concentrations. Cytotoxicity was evaluated for each time and concentration of inhibitor by means of the colorimetric assay MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium). Therefore, the lowest concentration at which the anti-inflammatory effect was observed and there was no cellular toxicity was chosen: 1µM. The quantification of ROS and NO was evaluated after 24h from the addition of the CIC inhibitors.

NO production was determined by measuring nitrite levels in the cell culture supernatant. This measurement was initially performed with a spectrophotometric method, with Griess's reagent (Promega) following the protocol suggested by the company. Cell culture supernatants were collected and centrifuged at 2000 rpm for 10 min. The absorbance was determined at 540 nm using a Victor3 model plate reader (PerkinElmer).

The ROS measurement was carried out with the fluorimetric method which exploits the transformation of dichlorofluorescein diacetate (DCFH-DA, Molecular Probes) into dichlorofluorescein (DCF) in the presence of ROS. The fluorescence of the DCF was measured by means of a fluorometer at an excitation wavelength of 495 nm and an emission wavelength of 525 nm.

The quantification of secreted PGE2 was carried out by means of an ELISA assay conducted on a 96-well plate following the protocol recommended by the company Arbor Assays. The amount of PGE2 secreted was determined by reading the optical density at 450 nm to a plate reader. This made it possible to quantitatively measure the prostaglandins secreted in the cell culture medium. All samples were referenced to a standard PGE2 curve. The secretion of PGE2 was evaluated 48h after the addition of the inhibitor.

Also object of the present invention are pharmaceutical compositions containing 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid (CNFASB) and usual additives and / or carriers and / or pharmaceutically tolerable diluents.

According to a variant of the invention, the CNFASB is advantageously administered by means of gastro-resistant tablets which release the active principle after passing the stomach. The release beyond the stomach is useful for combating inflammatory intestinal diseases such as Crohn's disease, since the residence time in the intestine is greater compared to a modest reduction in absorption as the conditions of Intestinal pH (basic) are less favorable (it is the undissociated form that is easily absorbable) from the other

A further variant of the compositions according to the invention provides for the administration of CNFASB in soft capsules for the immediate treatment of inflammation, since the active principle already in solution is absorbed more quickly or by means of coated tablets.

In a further variant of the present invention, the gastro-resistant tablets and soft capsules contain further components selected from the class formed by prostaglandin analogues (for example Misoprostol) and, or proton pump inhibitors (for example: Pantoprozole) capable of limiting aggression of the gastric mucosa.

Bibliography

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Claims (8)

CLAIMS 1. 4-chloro-3 - {[(3-nitrophenyl) amino] sulfonyl} benzoic acid compound (CNFASB) for use as a drug. Compound according to claim 1 for use in the treatment of inflammatory pathologies. 3. Pharmaceutical compositions comprising a compound as claimed in claims 1 or 2 and usual additives and / or carriers and / or pharmaceutically tolerable diluents. 4. Compositions according to claim 3 formulated as gastro-resistant tablets for the treatment of inflammatory intestinal pathologies. 5. Compositions according to claim 4 wherein said intestinal inflammatory pathologies are Crohn's disease. 6. Compositions according to claim 3 formulated as soft capsules. 7. Compositions according to claim 6 for the immediate treatment of inflammation. 8. Compositions according to at least one of claims 3 to 7 further comprising compounds selected from the class formed by prostaglandin analogs and / or proton pump inhibitors.