Pregnane x receptor (PXR) antagonists for the treatment of dengue fever

Abstract

The invention describes compounds useful for the treatment of viral infection caused by Dengue virus, which comprise compounds that inhibit viral replication of the virus, such as pregnane X receptor (PXR) antagonists, as well as pharmaceutical compositions comprising them, which are useful for the treatment of said viral infection, as well as methods of treatment for viral infection using said compounds and the compositions comprising them.

Description

[0001] Pregnane X receptor (PXR) antagonists for the treatment of Dengue fever.

[0002] Field of the invention.

[0003] The present invention relates to the treatment of viral infections, particularly to molecules useful for inhibiting viral replication of pathogenic viruses, such as, for example, antagonists of the X receptor for pregnane (PXR) for inhibiting viral infection caused by Dengue virus; likewise to pharmaceutical compositions useful for the treatment of said viral infection comprising said antagonists and to methods of treatment for viral infection using said antagonists and compositions comprising said antagonists.

[0004] Background of the invention.

[0005] Dengue virus (DENV) belongs to the family Flaviviridae and the genus Flavivirus. It is transmitted by mosquitoes, mainly of the Aedes aegypti species. Currently, four antigenically differentiated serotypes of the virus (DENV-1 , DENV-2, DENV-3 and DENV-4) are known [1], DENV is considered the most common mosquito-borne viral infection, with approximately 390 million people infected worldwide. Of these, 500,000 develop severe forms of dengue and there is a mortality of over 25,000 people per year [2], Usually a first infection with dengue results in an asymptomatic picture or dengue fever (DF); however, with a second infection, there is a high probability of contracting severe forms of dengue such as dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF), being more acute when it occurs with a different serotype due to the presence of non- or sub-neutralizing antibodies originated during the first infection [3], DENV is a single-stranded, positive-sense (5 - 3') RNA virus with 10,600 nucleotides and 3'and 5'untranslated regions (UTR) [6], It is a spherical virus, with a diameter of 50 nm, which contains three structural proteins that constitute the envelope (E), membrane (M) and capsid (C), in addition to a membrane precursor protein (prM) and seven non- structural proteins (NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5) that play roles during viral RNA translation [7], Infection with DENV is initiated when the virus enters a cell by receptor-mediated endocytosis, the most susceptible being macrophages, lymphocytes, hepatocytes and endothelial cells [4], The main receptors used by DENV to enter the cell are C-type lectin receptors (e.g. DC- SIGN / CD209, mannose receptor / CD206) and phosphatidylserine receptors (e.g. TIM, TAM) [5], Once inside the cell, there is a fusion between the E-glycoprotein and endosomal vesicles, which allows the release of the viral genome into the cytoplasm. Subsequently, the open reading frame (ORF) is translated into the proteins mentioned above. The NS5 protein, the longest of these, functions as an RNA-dependent RNA polymerase (RpRd), which synthesizes a complementary negative-stranded copy from the RNA template. RNA replication then takes place in replication complexes (RCs), which are formed in the endoplasmic reticulum (ER) membrane and where virion assembly takes place [8], The immature virion buds into the ER lumen and migrates to the transGolgi network where maturation occurs. Finally, the virus is released from the cell by exocytosis [9], In vitro and autopsy studies reveal that the main system involved in the pathogenesis of DENV is the immune system, as well as the liver and endothelial cells of blood vessels [1], As the virus enters the bloodstream through the skin, it infects Langerhans cells (DCs) which migrate to the lymph nodes whereby the virus spreads to the lymphatic system and begins to infect mononuclear cells, which enter apoptosis

[0010] , Various factors, including nonstructural proteins such as NS1 , as well as levels of viremia, determine the ratio of cytokines, chemokines and other proinflammatory and antiinflammatory mediators, as well as how the inflammatory response affects the hemostatic system of the infected patient

[0011] , The pathogenesis of DENV infection also affects the liver, characterized by high enzyme levels and spontaneous bleeding, as well as increased hepatocyte and Kupffer cell death by apoptosis

[0012] , A tropism for endothelial cells is also observed, mainly in those surrounding the microvasculature of the dermal papilla, pulmonary and intestinal vascular endothelium, while its selective apoptosis, generates the pictures of DHF and DSS, causing plasma permeability in the peritoneal and pleural cavity

[0013] ,

[0006] RNA viruses are commonly identified by cells of the innate immune system through their pattern recognition receptors (PRR), which recognize different structures of various pathogens. Among the main PRRs are Toll-like receptors (TLRs) such as TLR3, TLR7 and TLR8, as well as cytosolic receptors such as retinoic acid-inducible gene I [RIG-I] and melanoma differentiation-associated gene 5 (MDA5). In general, they activate the phosphorylation of signaling cascades in order to produce interferon (IFN) and proinflammatory cytokines

[0014] , Among IFNs, type I is the most common and is the most active during infection with DENV. By binding to its receptor (IFNAR) in an autocrine or paracrine manner, IFN-I induces phosphorylation of activators of signal transcription and translation 1 and 2 (STAT1 and STAT2) which in turn stimulate the formation of IFN-stimulated gene factor 3 (ISGF3) with IFN regulatory factor 9 (IRF9) thus forming the ISGF3 complex which translocates to the nucleus and acts as a transcriptional activator by binding to IFN-stimulated response elements (ISRES) in target genes important for antiviral activity against DENV

[0015] , Expression of cytokines and other mediators such as soluble receptors is also very common and increases significantly during severe dengue infection. High levels of IL-1 p, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-18, TGF-i p, TNF-a, and IFN-y have been identified in plasma from patients with DSS [1], In addition, other mediators have been identified such as vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage migration inhibitory factor, thrombopoietin, vascular cell adhesion molecule 1 (VCAM-1), ICAM-1 , thrombomodulin, selectin E, among others; which vary depending on the tissue and the degree of infection [1], On the other hand, in the serum of mice infected with DENV, an increase has been observed, mainly, of TNF-a, IL-1 J3, IL-6 and IL-10

[0017] , DENV also participates in the regulation of the complement system as antibodies generated to the viral NS1 protein are generally over-activated, thus allowing recruitment of the C5b-C9 complex, stimulating inflammatory cytokine production, intravascular coagulation and the development of DHF / DSS

[0016] , DENV infects various types of human cells, but those of the monocytic lineage such as macrophages and dendritic cells are considered the major target for viral replication. The way in which the virus enters these cells is through glycoprotein E, which interacts with macrophage membrane receptors such as C-type lectins, TIM and TAM. After binding, the virus enters the cell by clathrin-mediated endocytosis. In the late endosome, due to low pH and anionic lipids, conformational changes occur in glycoprotein E that allow membrane fusion and release of viral RNA into the cytoplasm

[0021] , When infection with DENV occurs, the number of macrophages in the mouse spleen and peritoneal cavity is reduced, as well as their phagocytic activity and migration

[0022] , In addition to the aforementioned receptors used by DENV to internalize into the cell, others have been described such as Fc receptors, which recognize the Fc region (crystallizable fragment) of DENV-specific antibodies

[0023] ; and the heat shock protein 90 and 70 (HSP90 / 70) that acts as a receptor complex that associates with membrane microdomains in response to DENV infection

[0024] , DENV-infected macrophages present the antigens to B lymphocytes, thus generating specific IgM antibodies to the virus, although these antibodies are often not neutralizing, and cause severe dengue disease by antibody-dependent enhancement (ADE)

[0025] , On the other hand, key macrophage cytokines and mediators have been identified that, upon DENV infection, promote a mainly Th1-type response. Among these are IL-8, mainly induced by the viral protein NS5, TNF-oc, IFN-a, IL-1 p, IL-6, IL-12, MIP-1a and RANTES

[0026] , Cytotoxic factor (CF), produced by CD4+ T cells upon DENV infection in both mouse and human, causes cell death of the same CD4+ T cells and induces macrophages to produce a protein called CF2, which amplifies the effect of CF resulting in DHF. In addition, free radicals such as nitrite, reactive oxygen and peroxynitrite are released, which ultimately induce macrophage apoptosis

[0027] , On the other hand, DENV-infected macrophages induce signals to recruit suppressor T cell (ST) populations and in turn these express soluble suppressor factors (SF), which suppress the production of DENV-specific antibodies and helper T cells (Th), thereby decreasing ADE

[0028] , This is how, roughly speaking, the interaction between macrophages and DENV occurs, with both executing their own strategies to, in the case of macrophages, maintain immune homeostasis and virus clearance, while in the case of DENV, allowing its replication

[0029] ,

[0007] In addition to the role played by the immune system in the response to DENV infection, there are genetic factors that determine the course of the disease. Through genomic studies, genetic determinants that generate susceptibility to this virus have been identified

[0030] ,

[0008] Among the genetic determinants that make a population susceptible to severe forms of DENV is the AB blood type associated with having DHF

[0031] , Another gene, important in the elimination of DENV, is 2', 5' oligoadenylate synthetase (OAS). OAS, induced by IFN, produces the 2', 5' oligo A, which binds to the double-stranded RNA of the virus. It is also capable of inducing an RNAase L that degrades DENV viral RNA. OAS has been found to be less expressed in patients with DSS

[0032] , Another feature of infection with DENV is the manifestation of bleeding due to plasma extravasation resulting in thrombocytopenia and thrombocytopathy, in addition to coagulation abnormalities. It has been reported that in patients with FHD / SSD, there is a base pair insertion / deletion polymorphism (4G / 5G) in the promoter region of the plasminogen activator inhibitor type I (PAI) gene, which is associated with increased plasma concentrations, hindering blood vessel coagulation

[0033] ,

[0009] Thanks to different databases compiled around the world, single nucleotide polymorphisms (SNPs) associated with the development of the disease have also been identified. Among them is the vitamin D receptor (VDR). The VDR is involved in the control of mineral metabolism, but it is also involved in activating monocytes and inhibiting the activation of T and B lymphocytes, favoring viral replication. The SNP at position 352 of the VDR gene, (a change of arginine (R) to histidine (H)), has been associated with protection against DSS

[0034] ,

[0010] Genome-wide association studies (GWAS) have recently identified other SNPs that are possibly important in the development of DENV infection. Among these genes are MICB and PLCE1, which were associated with the degree of thrombocytopenia and clinical shock resulting from dengue. This is because MICB encodes an activation ligand for the type II receptor NGK2D found on NK and CD8+ T cells, which stimulates antiviral effector functions of these cell types against DENV. The ACC genotype of MICB generates susceptibility to severe forms of dengue due to a dysfunction of NK and CD8+ T cells towards viral elimination. On the other hand, the PLCE1 gene is associated with nephrotic syndrome, a kidney disorder caused by glomerular basement membrane dysfunction resulting in proteinuria and hypoproteinemia. The AA genotype of PLCE1 also causes susceptibility to severe forms of dengue as it disrupts the vascular endothelial barrier

[0035] ,

[0011] Recently, haplotypes with SNPs that make them more susceptible to severe forms of dengue were identified in groups of Thai FD patients compared to a control group. Of these, four genes are involved in the xenobiotic metabolism signaling pathway: CHST10 which codes for a carbohydrate sulfotransferase and is a xenobiotic metabolizing enzyme, AHRR which codes for the aryl hydrocarbon receptor repressor (AHR), a ligand-dependent transcription factor that is responsible for transcribing xenobiotic metabolizing enzymes, GRIP1 which codes for protein 1 that interacts with the glutamate receptor and allows entry into the cell of different xenobiotics, and finally the PPP2R5E gene which codes for the epsilon isoform of the regulatory B subunit of protein phosphatase 2A (PP2A), which activates, through dephosphorylations, the pregnane x receptor (PXR) and the constitutive androstane receptor (CAR), xenosensors and transcription factors of xenobiotic metabolizing enzymes.

[0012] In particular, through a transcriptome database performed on patients with FD, it was identified that there is an increase in the levels of PPP2R5E, so not only is it a haplotype susceptible to severe forms of DENV, but also, in some way not yet elucidated, the virus increases the expression of that gene

[0037] ,

[0013] So far, multiple solutions for the treatment of dengue fever have been reported, some of which are mentioned below:

[0014] Patent application MX2022003160 A describes benzimidazole compounds for the treatment of viral diseases, such as hepatitis C, Zika, dengue, Powassan, Chikungunya, RSV enteroviruses and others; Patent application WO2023 / 187599 A1 describes methods and compounds for the treatment of viral infections by administering highly permeable and bioavailable dispersions or solutions based on avermectin, which are useful for the treatment of SARS-CoV-2, dengue, chikungunya, yellow fever, Zika and other viral infections; Patent application MX2022005879 A describes substituted indole derivatives and substituted indoline derivatives for the treatment of dengue fever, as well as methods of treatment of such viral infection; Patent application US2023 / 0265136 A1 describes the use of CNPY3 protein for the treatment of dengue fever, as its positive regulation has been found to be useful for the treatment of such infection.

[0015] Despite the above, there is still a need for more and better solutions for the treatment of Dengue.

[0016] Brief summary of the invention.

[0017] Dengue virus is the world's leading mosquito-borne virus. It infects approximately 390 million people annually, mainly in tropical areas, most of whom lack access to health systems. Currently, four serotypes are known (DENV-1 , DENV-2, DENV-3 and DENV-4), which can develop severe forms of dengue such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The probability of developing these severe forms increases when there is a re-infection with a different serotype, due to the formation of non-neutralizing antibodies. This situation also hinders the development of vaccines. Among the strategies to neutralize this disease, it has been tried to interfere with the different signaling pathways used by the virus to infect its target cells, the professional phagocytic cells (macrophages and dendritic cells). In accordance with the present invention, it has been determined that after infecting mouse peritoneal macrophages with dengue virus serotype 2 (DENV- 2), an increase in the expression of cytochrome P450 3a11 (Cyp3a11), a Pregnane X Receptor (PXR) target gene, is observed. PXR is an important xenosensor in the induction of xenobiotic metabolizing enzymes and a negative regulator of the inflammatory response. This suggests that the virus is able to activate PXR, resulting in a suppression of the inflammatory response and a more acute infection. Accordingly, in the present invention we determined that the administration of inhibitors of PXR activity, for example PXR antagonists, during an active virus infection, inhibits viral replication of Dengue virus, making such inhibitors useful for the treatment of such viral infection.

[0018] Therefore, one of the main objectives of the invention is to provide PXR antagonists, which are useful to reduce the infective capacity of DENV.

[0019] It is another objective of the invention to provide pharmaceutical compositions comprising PXR antagonists to decrease the infective capacity of DENV.

[0020] It is another objective of the invention to provide methods of treatment to decrease the infective capacity of DENV, through the use of PXR antagonists and pharmaceutical compositions containing them.

[0021] It is yet another objective of the invention to provide pharmaceutical compositions comprising PXR antagonists for use in decreasing the infective capacity of DENV.

[0022] It is yet another objective of the invention to provide novel uses of PXR antagonists for the manufacture of pharmaceutical compositions to decrease the infective capacity of DENV.

[0023] It is another objective of the invention to determine the mechanism by which inhibitors of PXR activity, e.g. PXR antagonists, are able to decrease the infective capacity of DENV: a) The evaluation of whether the induction of cyp3a11 expression, canonical target gene of PXR, caused by DENV is mediated by PXR, which would imply the direct relationship of the interaction of these molecules in the life cycle of the virus; b) Determine whether PXR activation by DENV is PP2A-dependent; c) To evaluate the role of PXR on the inflammatory response elicited by DENV in macrophages, and d) To evaluate the role of PXR on the development of DENV infection in animal models, such as mice.

[0024] Brief description of the figures.

[0025] Figure 1. Cyp3a11 expression in Mips. Mcps (3 x 106) were treated with PCN (50 pM), KTZ (10 pM) and OA (10 nM) and infected with DENV2 (0.1 MOI) for 72 h. After treatment time, mRNA was obtained and Cyp3a11 transcript levels were assessed. Vehicle (DMSO <0.01 %). n = 3 ± S.D. ANOVA-Tukey. (*p<0.05), (**p<0.01), (***p<0.001).

[0026] Figure 2. Effect of DENV2 on the induction of inflammatory cytokines and the role of negative modulation of PXR. Mcps (3 x 106) were treated with KTZ (10 pM) and OA (10 nM) and infected with DENV2 (0.1 MOI). 48 h later, Poly l:C (100 pg / ml) was added for 24 h. After treatment time, mRNA and supernatants were obtained, and expression levels (A) and inflammatory cytokine release (B) were evaluated. Vehicle (DMSO <0.01%). qRT-PCR n = 3 ± S.D. ELISA n = 3 ± S.D. ANOVA-Tukey. (****p<0.0001), (***p<0.001), (**p<0.01), (*p<0.05).

[0027] Figure 3. Lipid gene expression following PXR activation, infection with DENV2 and negative modulation of the PXR pathway. Mcps (3 x 106) were treated with PCN (50 pM), KTZ (10 pM), and OA (10 nM) and infected with DENV2 (0.1 MOI) for 72 h. After treatment time, mRNA was obtained and expression levels were evaluated. Vehicle (DMSO <0.01%). qRT-PCR n = 3 ± S.D. ANOVA- Tukey. (*p<0.05), (**p<0.01), (***p<0.001), (****p<0.0001).

[0028] Figure 4. Lipid droplet formation in Mcps caused by infection with DENV2 and the effect of negative modulation of PXR. Se Mcps (3 x 106) were treated with KTZ (10 pM) and OA (10 nM) and infected with DENV2 (0.1 MOI) for 72 h. Lipid droplet formation was assessed by 63X confocal microscopy (Optical Section 16), and by flow cytometry using Nile red fluorescence intensity (0.1 pg / ml). n = 3. Blue - DAPI 1 :2000. Green - C protein 1 :300, Alexa Fluor 488 1 :500. Scale 10 pm.

[0029] Figure 5. Viral genome copy number and titer. Mcps (3 x 106) were treated with KTZ (10 pM) and OA (10 nM) and infected with DENV2 (0.1 MOI) for 72 h. After treatment time, mRNA was obtained and expression levels of the DENV2 capsid protein C gene (A) were evaluated. From the recovered supernatants, serial dilutions were performed and determined by DENV2 membrane protein M fluorescence for foci formation (B). n = 3 ± S.D. ANOVA-Tukey. (****p<0.0001).

[0030] Detailed description of the invention.

[0031] The present invention provides antagonists of the X receptor for pregnane (PXR) as well as pharmaceutical compositions comprising them, for inhibiting Dengue virus viral infection, thus allowing to additionally provide effective treatments for such infection.

[0032] For purposes of the invention and as described herein, the term "antagonist" refers to and / or also includes the term "inhibitor", as well as any other concept describing the decrease and / or elimination of the activity and / or function of the molecule referred to. Until prior to the present invention, there were no reports related to the potential use of pregnane X receptor (PXR) antagonists, as the existing ones relate to the use of such compounds for the treatment of cell proliferation disorders, such as for example cancer (US2020 / 0071281 , US2017 / 0226115, US8669260).

[0033] PXR is a ligand-dependent transcription factor that regulates the expression of xenobiotic metabolizing enzymes as well as drug transporters critical in the elimination of xeno- and endobiotics. In humans, it is the main regulator of CYP3A4 induction, which is involved in the metabolism of approximately 50% of currently used drugs. Among the main ligands of PXR are drugs such as antibiotics (rifampicin), anti-inflammatory drugs (dexamethasone), antiglucocorticoids (pregnelonone 16a-carbonitrile), as well as compounds derived from steroid metabolism. Its expression is most abundant in liver and intestine, although it is also expressed to a lesser extent in other organs

[0038] ,

[0034] The protein structure of PXR has a DNA-binding domain (DBD) at the amino terminus which contains two zinc fingers. This domain binds to the promoter of its target genes in a region known as the xenobiotic response enhancer module (XREM). At the carboxyl terminus is the ligand binding domain (LBD), dimerization domains, transactivation domains, and the central H (hinge) region, which is a flexible domain that connects the DBD to the LBD allowing it to present multiconformations to bind different ligands

[0039] ,

[0035] The association of PXR with HSP90 and cytoplasmic CAR retention protein (CCRP) maintains it in the cytosol. Upon ligand binding, it dissociates and translocates to the nucleus, where it forms a heterodimer with the X receptor for retinoids (RXR) leading to recruitment of the machinery to transcribe its target genes. PXR can also be activated, in a ligand-independent manner, by PP2A, which is able to phosphorylate it and cleave it from its co-repressors allowing its translocation to the nucleus

[0040] ,

[0036] It has been reported that PXR not only forms heterodimers with RXR, but can also form PXR-PXR homodimers. Due to this versatility, it is that it can regulate the transcription of a wide variety of genes and participate in several cellular mechanisms

[0039] , Regarding the immune response, PXR has an anti-inflammatory function. Its activation in the liver and intestine inhibits the action of NF-KB and the consequent expression of inflammatory cytokines

[0041] , In accordance with the present invention, the above suggests that PXR has an important role in the negative regulation of the inflammatory response. Indeed, some pathogenic microorganisms have taken advantage of this feature and are able to modify PXR signaling. For example, it has been observed, in M<t>'s, that lipids from Mycobacterium tuberculosis activate PXR

[0042] ,

[0037] Considering the above, some studies have identified that different viral proteins, which are mentioned below, can modify the regulation of an important enzyme in the PXR signaling pathway, namely protein phosphatase 2A (PP2A). PP2A are Ser / Thr phosphatases, which have the ability to dephosphorylate multiple proteins involved in different signaling pathways, among which one of their targets is PXR, which allows their ligand-independent activation (43, 50). PP2A is a heterotrimeric protein composed of three subunits: A - structural, B - regulatory and C - catalytic. Of these three subunits, subunit B is the most diversified because it is the one that gives specificity to the substrate, therefore, there are approximately 23 isoforms, which are: PPP2R2 (A to D), PPP2R5 (A to E), PPP2R3 (A to C) and the stratin isoforms - STRN, STRN3 and STRN4

[0051] ,

[0038] Some viral proteins have been identified that can form complexes with PP2A that alter different cellular pathways in order to successfully complete the viral life cycle of pathogenic viruses. For example, Epstein-Barr virus (EBV) interacts, through its EBNA-LP protein, with PP2Ac inhibiting apoptosis as a method of virus replication

[0044] , Human polyomavirus 2, through its small T antigen protein, binds to the dimer of the A / C subunits of PP2a and inhibits the dephosphorylation of the agnoprotein responsible for regulating the life cycle of this virus

[0045] , It has also been reported that human papillomavirus (HPV) through its HPV-E7 protein binds to A / C and inhibits the dephosphorylation of Akt / PKB, thus allowing cell survival

[0046] , Similarly, human immunodeficiency virus (HIV) also modifies the A subunit of PP2A through the Vpr protein, which exerts a role in cell arrest and apoptosis

[0047] ,

[0039] As for DENV, proteins of the Flaviviridae family have been identified that interact with PP2A modifying signaling pathways that favor the life cycle of these viruses. East Nile virus (WNV) through its capsid protein, binds and inactivates the PP2A inhibitor (l2PP2A) causing an increase in PP2A activity

[0048] , On the other hand, hepatitis C virus (HCV), through its NS5 protein - an RdRp like that possessed by dengue virus - binds to the A / C dimer that overregulates PP2A activity resulting in inhibition of IFN-oc signaling

[0049] ,

[0040] These studies demonstrate that different viral proteins modify different signaling pathways through their interaction with PP2A, allowing it to successfully carry out its life cycle.

[0041] DENV is the leading mosquito-borne virus in the world. This disease can develop into severe forms that claim approximately 25,000 lives per year. It is a major health problem in tropical areas, which generally lack access to health care. Faced with this problem, attempts have been made to mitigate the infection through various strategies that interfere with different signaling pathways used by the virus to carry out its life cycle. In this sense, in the present invention we demonstrate that when mouse peritoneal macrophages (M<t>'s) are treated with inactivated DENV-2, the expression of Cyp3a11, a PXR target gene, is induced. Moreover, the observed induction is greater than that elicited by its canonical agonist, pregnenolone 16a-carbonitrile (PCN). As discussed above, an increase in PXR activity results in an immunosuppressive effect and it is therefore possible that DENV-2 utilizes this pathway to decrease the inflammatory response dependent on M<t>'s.

[0042] In accordance with the present invention, we determined that surprisingly DENV-2, through PP2A- mediated activation of PXR, decreases the inflammatory response, with which the virus acquires the necessary conditions for its replication, thus increasing and facilitating viral infection in the host; Consequently, and in accordance with what is described here, the inhibition of PXR results in the activation of an inflammatory response to infection with DENV-2, with which viral replication is significantly reduced and therefore viral infection is also reduced, thus providing an effective therapeutic treatment for such infection. For purposes of the invention, PXR antagonists or their pharmaceutically acceptable salts, which can directly bind to PXR and inhibit its activity, are molecules selected from: a) ET-743, the first reported antagonist

[0054] , b) Polychlorinated biphenyls, camptothecin, fluconazole, enilconazole [55-62], c) Ketoconazol (1-[4-(4-'[2R,4S)-2-(2,4-Diclorophenil)-2-(1 H-imidazol-1 -ilmetil)-1 ,3-dioxolan-4- il]metoxi-phenil)piperazine-1-il]etanone, No. CAS: 65277-42-1), [63-64] d) Sulforaphane

[0061] , e) A792611 , HIV protease inhibitor

[0065] , f) Metformin

[0066] , g) Sesamin (natural lignan)

[0067] , h) Fucoxanthin

[0068] , i) Coumestrol

[0069] , j) SPA70

[0070] , k) Resveratrol

[0071] , and / or l) Mixtures of the above compounds.

[0043] Within the PXR antagonist compounds or their pharmaceutically acceptable salts, additionally the 1 ,4,5-substituted 1 ,2,3-triazole analogues described in patent application US2020 / 0071281 A1 , the CAR antagonist compounds described in patent application US2017 / 0226115 A1 , the ketoconazolederived compounds described in patent US8669260 B2, as well as those compounds exhibiting PXR antagonist activity may additionally be occupied.

[0044] The present invention comprises pharmaceutical compositions comprising as active principles, PXR antagonists and / or mixtures thereof.

[0045] Therefore, in accordance with the present invention, it is one of its objectives to provide pharmaceutical compositions comprising PXR antagonists and / or mixtures thereof together with pharmaceutically acceptable excipients suitable for administration to the patient requiring it, for example those patients affected by Dengue. It is modality of the invention to adapt the active principles for use in pharmaceutical compositions for enteral administration, parenteral and topical use, including inhalation. The effective doses for the patient of the active principle will also be adjusted in accordance with preclinical and clinical studies, but taking as a basis the findings of the present invention.

[0046] It is one mode of the invention to provide pharmaceutical compositions comprising at least from 5 pg to 100 pg of PXR antagonists or their pharmaceutically acceptable salts, preferably from at least from 5 pg to 50 pg of PXR antagonists, more preferably from at least from 5 pg to 15 pg of PXR antagonists, and still more preferably from at least from 10 pg of PXR antagonists.

[0047] The treating physician can adjust the unit dose, as well as the maximum daily dose, which has an optimized effect for the treatment of dengue and which he / she considers to be the best, depending on the circumstances of each patient.

[0048] It is another embodiment of the invention to use pharmaceutical compositions comprising PXR antagonists or their pharmaceutically acceptable salts for the effective treatment of dengue fever, preferably dengue caused by serotype DENV2, which can be administered subcutaneously, orally, intraperitoneally, intramuscularly, intravenously or topically, in doses that are synergistically effective for the treatment of the disease.

[0049] It is another of the modalities of the invention, the possibility of formulating pharmaceutical forms comprising as active principles antagonists of the PXR or its pharmaceutically acceptable salts, to make them administrable by different routes to the patient who requires it, being a mammal including the human, and being able to be adapted to provide the unitary doses that allow exerting the antiviral effect described in the present invention for the treatment of dengue, where the pharmaceutical forms are obtained by a person from the technical field of pharmacology without being limited to any specific one, and seeking to preserve the efficacy of the drugs in terms of the described antiviral effect, either by releasing the active principle quickly or slowly, or instead seeking the greatest efficiency in the target tissue, avoiding damage to the patient by chemical interaction, solubilizing insoluble substances, improving flavors, improving aspects and facilitating to the physician the posology to perform the treatment, the different existing pharmaceutical forms being able to be solid, semisolid or liquid, for example: powders (which may be encapsulated), granules and tablets or tablets, pills, and suppositories. Also its administration in patches and in semi-solid pharmaceutical forms: ointments, pastes, gels, or liquid pharmaceutical forms: solutions, injections, syrups, suspensions or eye drops.

[0050] Examples of pharmaceutically acceptable excipients accompanying the active principle of the invention are for example, for oral administration as tablets or tablets, agents comprising for example, diluents, binders, stabilizers, bulking agents, thickening agents, such as povidone, microcrystalline cellulose, lactose, etc., disintegrating agents such as for example cross-linked carboxymethylcellulose, surfactants such as for example sodium lauryl sulfate, lubricating or sliding agents such as for example magnesium stearate, colloidal silicon dioxide, etc., wherein said excipients may be formulated for preferably slow or prolonged release for a systemic effect.

[0051] Solutions for intravenous or intraperitoneal administration of the active substance can be prepared by first dissolving them in an organic solvent such as DMSO, ethanol, or dimethylformamide and subsequently in aqueous buffers, such as PBS.

[0052] Particular preference is given to dosage forms designed for systemic administration, wherein liquid or solid compositions can be formulated, suitable for administration and whose excipients can be selected for example from components compatible with said dosage form, for example of a lipidic or peptidic nature, or peptidomimetics, known in the prior art as non-immunogenic, and which preferably can be bound to the active principles of the invention to improve their bioavailability; propellant agents such as, for example, propane, butane, or permissible chlorofluorocarbons; pH regulators such as, for example, sulfuric acid; chelating agents such as, for example, EDTA. The active ingredients can also be formed into micronized particles contained in gelatin capsules or other systems known in the technical field, which help to release the active ingredient to its target site of action, for example, by means of solid dosage forms such as tablets or dragees, including those formulated for prolonged release.

[0053] According to the present invention, the compositions described herein may be obtained by combining PXR antagonists and / or mixtures thereof with pharmaceutically compatible vehicles known in the art, in the amounts and / or concentrations that correspond as described herein, and compounds known in the art may be included to obtain such compositions. Likewise, the administration of such compositions, may be made depending on the conditions of the patient, which will determine the doses and frequency of administration necessary to achieve an effective treatment of the ailment in each particular case.

[0054] In another embodiment of the invention, methods of treatment for patients affected with Dengue are included, comprising administering to the patient with said ailment the pharmaceutical compositions of the invention, in the amounts, dosage frequency and treatment time indicated by the treating physician and based on the results obtained in the present invention. As can be seen from the results described herein below, said compositions will have an effective therapeutic effect for the treatment of Dengue fever in patients affected by said disease and at the same time will prevent a considerable drop in the effectiveness of the patient's immune system, which will make it possible to enhance the treatment administered.

[0055] The following examples are included below, which are only intended to illustrate the different modalities of the invention, without implying limitations to its scope. Likewise, these examples include possible variations that may be detected by a person skilled in the technical field of the present invention.

[0056] Example 1. Experimental strategy and methods.

[0057] Obtaining peritoneal macrophages. Peritoneal M<t>'s were obtained from wild-type C57BL / 6 strain (WT) or PXR-null mice (53), male, 8 -13 weeks old. They were sacrificed by cervical dislocation according to NOM-062-ZOO-1999. Subsequently, a skin incision was made to expose the peritoneum where 10 mL of cold 1X phosphate buffer solution (PBS) were injected with a 22G syringe. Massaging was performed for 10 min, and the PBS was obtained inside the peritoneum to collect it later in a tube, thus performing a "pull" of peritoneal M<t>'s from an n=3. The sample was centrifuged at 16,773 g for 10 min at 4°C, the supernatant was removed and the cell button was diluted in 1 mL of supplemented RPMI-1640 medium (Thermofisher MA, USA). From the cell resuspended, a 10 pL sample was taken, diluted in 190 pL of trypan blue and cell number and viability were determined by the trypan blue exclusion method under an inverted microscope with the aid of a Neubauer chamber. Cell culture. Cell cultures were grown in 1 mL of supplemented RPMI medium in a 3.5 mm2Petri dish. Subsequently, the M<t>'s were incubated for at least 3 h to allow them to adhere to the surface of the box. After the adhesion time had elapsed, to remove erythrocytes and unattached cells, a wash was performed with 1X PBS, and RPMI medium supplemented with the corresponding treatment was added. The culture conditions were 5% CO2 and 37 °C temperature.

[0058] Treatments. The treatments used were as follows:

[0059] 1) 5-pregnane-3p-ol-20-one-16a-carbonitrile (PCN) (Sigma-Aldrich MO, USA), prototype ligand of PXR, at a concentration of 50 nM;

[0060] 2) Okadaic acid (OA), a PP2A antagonist at different concentrations from 10 to 100 pM. DMSO at 0.008% was used as a vehicle for these compounds.

[0061] 3) Ketoconazole (KTZ) at different concentrations from 10 to 100 pM.

[0062] On the other hand, a sonicate of dengue virus serotype 2 (DENV-2) was used as the infectious agent, which was previously inactivated and used in vitro at a concentration of 5 plaque-forming units (PFU) / cell for 72 h and in vivo at a concentration of 7 x 107PFU / animal for 15 days. RPMI medium was used as a vehicle for these treatments.

[0063] MTT cytotoxicity assay. A 98-well plate, 2x105M<t>'s / well were seeded in a 98-well plate and incubated as described above. Treatments used were RPMI medium as control, DMSO 0.008% as vehicle, PCN (10-50 pM) for 24 h, OA (10 - 100 pM) for 24 h, KTZ (10 - 100 pM) for 24 h and a sonicate of DENV-2 (1 , 2, 5 and 10 PFU / cell). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-bromide) diluted in 1X PBS to a final concentration of 5 mg / mL was prepared and filtered with a 0.22 pm membrane. Three h before the end of the 24 h treatment, 50 pL of the MTT solution was added to the cultures. After treatment, the medium was removed and allowed to dry for 5 min. Subsequently, the crystals formed were diluted with DMSO and read on a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific MA, USA) at a wavelength of 590 nm.

[0064] RNA extraction. Once the treatments were finished, the medium was removed and washed with 1X PBS, the Petri dish was placed on ice, and the corresponding RNA was extracted using the RNeasy® kit (Quiagen DE-NW, GER). In this procedure the cells were lysed and homogenized, and then 70% ethanol was added and centrifuged in RNeasy mini-spin columns at 12000 rpm for 15 s, washed with washing buffer diluted 1 :4 with absolute ethanol, and centrifuged under the same conditions. The RNA obtained was diluted in 30 pL of RNAse-free water. Subsequently, the RNA concentration was determined in a NanoDrop 2000 (Thermo Fisher Scientific MA, USA). To verify the integrity of the RNA, 2 pL per well were loaded in a 1 % agarose gel and run at 100 V for 30 min. At the end of the time, the resulting bands were visualized in a transilluminator Gel Doc™ XR+ Imager (Bio-Rad CA, USA), where a photographic record was taken.

[0065] Real-time quantitative PCR (RT-qPCR). cDNA was obtained from the RNA template strand using a reverse transcriptase (Superscript III, Thermo Fisher Scientific MA, USA). It was started from a concentration of 500 ng of RNA and using a GeneAmp PCR System 2700 thermal cycler (Applied Biosystems CA, USA), the sample was heated to 65°C for 5 min, incubated on ice for 1 min, 0.4 pL per sample of reverse transcriptase was added and incubated at 25°C for 5 min at room temperature (RT). Retrotranscription was performed at 65°C for 40 min, the reaction was inactivated at 70°C for 15 min, and samples were stored at 4°C.

[0066] After obtaining the cDNA, qPCR was performed using TaqMan™ probes coupled to the FAM reporter fluorophore, corresponding to the gene of interest to be amplified. For Cyp3a11 the probe ID: Mm00731567_m1 (Thermo Fisher Scientific MA, USA), and as endogenous gene the probe for the 18s ribosomal unit ID: Mm00507222_s1 (Thermo Fisher Scientific MA, USA) was used. 2 pL of cDNA per well was placed in a 48-well plate, and 13 pL of a mixture of the gene of interest probe with TaqMan™ Universal PCR Master Mix (Thermo Fisher Scientific MA, USA), which contains the DNA polymerase, was subsequently read on a StepOne Real-Time PCR System (Applied Biosystem CA, USA). The amplification conditions were: one cycle of incubation at 50°C for 2 min, one cycle of DNA polymerase activation at 45°C for 10 min, 40 cycles of denaturation at 95°C for 15 s followed by alignment and extension of DNA synthesis at 60°C for 1 min. Upon completion of qPCR, the relative expression of the gene of interest was determined, using the comparative Ct method (AACt) (Life technologies, 2014).

[0067] Western blot. At the end of the treatments, total proteins were extracted by adding to each box 500 pL of extraction buffer at pH 7.5 (HEPES 50 mM, NaCI 0.5 M, MgCh 1.5 mM, EDTA 1 mM, Glycerol (v / v) 10%, Triton-X-100 1%, 1 protease inhibitor tablet per 10 mL of solution). Subsequently, a scraping was performed with a gendarme to harvest and place the resulting solution in an Eppendorf tube, which were subjected to constant vortexing at 4°C for 30 min. Subsequently, they were centrifuged at 12,000 rpm at 4°C for 15 min. The resulting supernatant was preserved in Eppendorf tubes and the protein concentration was quantified by Bradford's method (Bradford, 1976).

[0068] A 10% polyacrylamide gel was prepared and mounted in an electrophoresis chamber with 1X running buffer (0.192 M glycine, 25 mM Tris-base and 3.5 mM SDS). In Eppendorf tubes, the volume corresponding to 20 pg of protein from each cell extract was placed, added with 4X loading buffer (60 mM, 3.42 M glycerol, 68 mM SDS, 1.48 mM bromophenol blue and 0.708 M p-mercaptoethanol). The resulting solution was boiled for 5 min and subsequently each sample was placed in a different well of the polyacrylamide gel. In a different well, 2 pL of Bio Rad® molecular weight marker was placed. Subsequently, the gel was run at 100 V for 30 min, followed by 80 V for 2.5 h.

[0069] At the end of the run time, the resulting gel was transferred semi-dry at 20 V for 1.5 h to a nitrocellulose membrane moistened in transfer buffer (48 mM Tris-HCI, 39 mM glycine and 20% methanol). Once the transfer was completed, staining with Ponceau red was performed to visualize the efficiency of the transfer. Subsequently, the membrane was washed with PBS until the dye was removed and blocked with a 5% solution of low fat milk in TBS Tween (0.1%) for 1 h at room temperature (RT). Then a quick wash was performed with TBS Tween and incubated with the respective rabbit anti-mouse primary antibodies, which are Cyp3a11 (Cat. No. sc-271033, Santa Cruz Biotechnology), and as a loading control actin (Cat. No. ab8226, Abeam), for 16 h at 4°C. Subsequently, washes were performed with TBS Tween at 15, 10, and 5 min, followed by a wash for 5 min with 1X TBS. HRP-conjugated anti-rabbit HRP secondary antibody was added at a dilution of 1 :2000 for 1 h. Subsequently, the washing pattern described previously was repeated and the membrane was exposed with luminol, and then Clarity Bio Rad solution was used in a 1 :1 ratio with developer solution, where finally it was exposed to a photographic plate and the plate was developed in complete darkness.

[0070] Immunofluorescence. To perform immunofluorescence assays, 5 x 105M 's / mL were seeded onto a coverslip inside a 3.5 mm2Petri dish. The respective treatment was added and adjusted to a final volume of 1.5 mL with supplemented RPMI medium. After the treatment time had elapsed, the medium was removed, washed with 1X PBS and the cells were fixed with 4% paraformaldehyde (PFA) for 30 min at RT. Cells were then washed twice with 1X PBS, permeabilized with 0.05% triton X-100 cooled to 4°C for 5 min at RT, washed and proceeded to block nonspecific antibody binding with blocking medium (1% BSA, 22.52 mg / mL glycine diluted in 1X PBS at 0.1% tween 20) for 30 min. Subsequently, the blocking solution was removed and without washing the primary antibody for PXR (bs-2334R, Bioss antibodies MA, USA) was placed at a dilution of 1 :150, and incubated overnight at 4°C. At the end of the incubation time, 3 washes were performed with 0.1% PBS-T for 5 min each, the secondary antibody coupled to Alexa Fluor® 488 (ab150077, Abeam CAM, UK) was added at a dilution of 1 :1000, and incubated for 1 h. at RT in complete darkness. Subsequently, 3 washes were performed with 0.1% PBS-T for 5 min each and rhodamine-phalloidin R415 (Thermo Fisher Scientific MA, USA) was added at a 1 :20 dilution for 15 min at RT, protecting the samples from light. Two washes were performed with 1X PBS for 3 min each, the coverslip was mounted on a slide with 5 pL of Vectashield with DAPI H-1200-10 (Vector Laboratories CA, USA), and photographs were taken, using a Leica TCS SP5 confocal microscope (Leica Microsystems DE-HE, GER).

[0071] ELISA. Supernatants were obtained from treated cell cultures with their respective treatments and the amount of inflammatory factors IL-1 p, IL-6, TNF-a and IL-12, and anti-inflammatory factors such as IL-10 were quantified. For each cytokine, a capture antibody concentration of 2 pL / mL dissolved in 0.1 M sodium dibasic phosphate binding buffer at pH 6 was used. 50 pL per well was plated and incubated overnight at 4°C. The plates were washed four times with 1X PBS solution and 0.01% Tween 80. The plate was blocked with 200 pL of blocking solution (1 N NaOH diluted in 1X PBS, pH 7) with 5 g of casein for 30 min at RT. Afterwards, the plate was washed 3 times, 100 pL per well were placed for the standard curve as well as the samples. The standard curve consists of a dilution series of recombinant cytokine and serves as a positive control. It was incubated at 4°C overnight. After the incubation time had elapsed, the plate was washed 4 times, the detection antibody (diluted in 1 % bovine albumin with 0.05% Tween 20) was placed, 100 pL per well of detection antibody was placed and incubated 1 h. at RT. The plate was washed 6 times, 100 pL of streptavidin-alkaline phosphatase at a 1 :2000 dilution in 1% bovine albumin with 0.05% Tween 20 was placed, incubated for 30 min at RT, the plate was washed 8 times. Finally, the developer solution (composed of substrate buffer and pNPP phosphatase substrate) was prepared, 100 pL per well was placed, incubated at TA and in the dark. The plate was analyzed in a spectrophotometer at a wavelength of 405 nm. Finally, the standard curve was performed obtaining the concentrations for the cytokines in pg / mL. Electrophoretic mobility shift assay (EMSA). After obtaining nuclear extracts from mouse peritoneal M 's, they were incubated (2 pL of these nuclear extracts) for 10 min at RT with a32P-labeled probe containing the PXRE (PXR Response Element) in Binding Buffer (10 mM Tris (pH8), 100mM KCI, 6% glycerol, 1 mM PMSF (Phenylmethylsulfonyl fluoride), 1 mM DTT (Dithiothreitol) and 100 ng / mL poly[d(l-C)]. Subsequently, 6% polyacrylamide gel electrophoresis was performed in 0.5X TBE (45 mM Tris-base, 45 mM boric acid, 1 mM EDTA) at 4°C. Supershift was also performed by incubating the nuclear extracts with the antibody for PXR (bs-2334R, Bioss antibodies MA, USA). The gel was transferred to a membrane which was used to expose to a photographic plate for development.

[0072] In vivo infection. Mice of the C57BL / 6 WT and PXR-null strain were used, weighed before treatments, and fed ad libitum. Two main groups were generated, the group treated intraperitoneally with DENV-2 at a concentration of 7 x 107PFU per animal and the control group treated with unsupplemented RPMI medium at the same volume as the DENV-2 suspension for 15 days. Survival of the animals during the treatments was determined. Finally, endothelial vessels were obtained from the peritoneum and liver.

[0073] Histological analysis. After extraction, the blood vessels surrounding the site of inoculation of the DENV-2 sonicate and liver were fixed for at least 12 h with 4% PFA. Subsequently, they were dehydrated in ethyl alcohol at increasing concentrations (70%, 80%, 96% and 100%). After dehydration, they were clarified using xylol. Finally, the samples were embedded in kerosene (Paraplast®). Cuts of 5 pm were made using a microtome. To prevent tissue detachment, the slides were treated with a solution of tespa (3-aminopropyltriethoxysiline). Slides were deparaffinized at 56°C, subsequently passed through xylol and rehydration was performed at descending concentrations of ethyl alcohol starting from 100%, 96%, 80% and 70% for 30 s in each, subsequently with ddH O.2

[0074] The histological sections after rehydration, were stained with Harris hematoxylin (Merck) for 10 min, the excess dye was washed with running water, it was taken to acid alcohol solution, to differentiate the nuclei, rinsed with running water, then to lithium carbonate (IJ2CO3) for 5 s, to turn the nuclei to blue, the slides were dehydrated with ethyl alcohol at increasing concentrations of 70%, 80%, 96%, 100% and xylol for 30 s and mounted with resin (Merck).

[0075] Statistical analysis. For statistical analysis, analysis of variance (ANOVA) tests were used to determine significant differences in the effect of the different treatments and their controls. A *P<0.05 was considered significant.

[0076] Example 2. Pregnenolone 16-a carbonitrile (PCN) and infection with DENV2 promote PXR activation, whereas KTZ and OA inhibit it. We initially evaluated whether PXR could be activated by its agonist - PCN - and by DENV2 infection in mouse peritoneal macrophages (Mas), and whether such activation could be inhibited by an antagonist such as ketoconazole (KTZ), and by an inhibitor of PP2A, which dephosphorylates and activates PXR, okadaic acid (OA). PXR activation was assessed through the expression of its canonical gene Cyp3a11. It was observed that both PXR agonist and DENV2 promote Cyp3a11 induction. On the other hand, both KTZ and OA block PXR activation promoted by CNP and DENV2 (Fig. 1). From the above, it is concluded that the inducing effect of DENV2 on Cyp3a11 expression is PXR-dependent.

[0077] Example 3. Infection with DENV2 suppresses the inflammatory response, but this is restored by inhibiting PXR. Poly l:C was used as a positive control for the induction of inflammatory mediators. As seen in Fig. 2, Poly l:C promotes the expression (Fig. 2A) and release of inflammatory cytokines (Fig. 2B) (IL-1 p, IL-6, TNF-a, IL-12 and IFNy). In contrast, when infection with DENV2 was carried out the induction of the inflammatory response decreases considerably. However, when PXR is inhibited with KTZ or OA, the levels of inflammatory cytokines recover. The above indicates that DENV2 uses the PXR pathway to suppress the inflammatory response and that PXR inhibition blocks DENV2-induced immunosuppression.

[0078] Example 4. PCN agonist and DENV2 infection promote the expression of genes involved in lipid metabolism and lipid droplet formation, whereas PXR inhibition inhibits this effect. PXR has been reported to promote the expression of genes involved in lipid metabolism necessary for DENV2 replication. These include sterol regulatory element binding protein 2 (Srebp2), peroxisome proliferator-activating receptor gamma (PPARy), differentiation cluster 36 (CD36), and squalene epoxidase (Sqle). Upon activation of PXR with PCN or infecting M s with DENV2, a significant induction of these genes was observed with respect to vehicle. On the other hand, KTZ and OA block such induction, confirming that DENV2 modulates PXR activation to promote the expression of these genes involved in fatty acid influx and cholesterol synthesis (Fig. 3).

[0079] Example 5. DENV2 infection promotes lipid droplet formation, while PXR inhibition decreases their formation and size. Subsequently, we evaluated the formation of lipid droplets, which are necessary for the assembly of DENV virons. Figure 4 shows the basal presence of lipid droplets in control cultures. In contrast, when infection with DENV2 is performed, an increase in the formation and size of these droplets is observed. In addition, DENV2 protein C is observed, indicating the presence of the virus in the cells. When PXR is inhibited, a decrease in the number and size of lipid droplets, as well as the presence of DENV2 protein C, is observed (Fig. 4). These data show that inhibition of PXR decreases the formation of lipid droplets, which prevents an environment conducive to virus assembly and decreases the presence of DENV2 C protein.

[0080] Example 6. PXR inhibition decreases DENV2 titer and viral load. Because PXR inhibition, upon infection with DENV2, positively modulates the inflammatory response and negatively modulates the expression of lipid metabolism genes and lipid droplet formation, we proceeded to evaluate the effect of PXR inhibition on viral load by assessing the number of transcripts of the capsid protein C gene (Fig. 5A) and viral titer through the formation of foci (UFF) of the DENV2 membrane protein M (Fig. 5B). In both cases, inhibiting PXR resulted in a one-log decrease from 1 x 106to 1 x 105in both copy number and viral titer compared to untreated infected cells. This demonstrates that PXR inhibition decreases viral replication.

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Claims

Claims.1 . A pharmaceutical composition for use in the treatment of viral infection caused by Dengue virus, characterized in that it comprises a therapeutically effective amount of a pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable vehicle.

2. The pharmaceutical composition for use according to claim 1 , characterized in that the pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof are selected from the group comprising compound ET-743, polychlorinated biphenyls, camptothecin, fluconazole, enilconazole, ketoconazole, sulforaphane, HIV protease inhibitor, A792611 , metformin, sesamin, fucoxanthin, coumestrol, SPA70, resveratrol and mixtures thereof.

3. The pharmaceutical composition for use according to claim 2, characterized in that the pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof is ketoconazole.

4. The pharmaceutical composition for use according to claim 1 to 3, characterized in that the pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof is in a concentration of at least 5 pg to 100 pg.

5. The pharmaceutical composition for use according to claim 1 to 4, characterized in that it is orally or parenterally administrable.

6. The pharmaceutical composition for use according to claim 1 to 5, characterized in that it is administrable at least once daily.

7. The pharmaceutical composition for use according to claim 1 to 6, characterized in that the dengue virus causing viral infection is selected from serotype DENV-1 , DENV-2, DENV-3, DENV-4 or mixtures thereof.

8. The pharmaceutical composition for use according to claim 7, characterized in that the serotype of the dengue virus causing the viral infection is serotype DENV-2.

9. The use of a pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of viral infection caused by Dengue virus.

10. The use of claim 9, wherein the X receptor antagonist for pregnane (PXR) or its pharmaceutically acceptable salts are selected from the group comprising compound ET-743, polychlorinated biphenyls, camptothecin, fluconazole, enilconazole, ketoconazole, sulforaphane, HIV protease inhibitor, A792611 , metformin, sesamin, fucoxanthin, coumestrol, SPA70, resveratrol and mixtures thereof.11 . The use of claim 10, wherein the X receptor antagonist for pregnane (PXR) or pharmaceutically acceptable salts thereof is ketoconazole.

12. The use of claim 9 to 11 , wherein the X receptor antagonist for pregnane (PXR) or pharmaceutically acceptable salts thereof, is administrable at a concentration of at least 5 pg to 100 pg.

13. The use of claim 9 to 12, wherein the pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof is orally or parenterally administrable.

14. The use of claim 9 to 13, wherein the X receptor antagonist for pregnane (PXR) or pharmaceutically acceptable salts thereof is administrable at least once daily.

15. The use of claim 9 to 14, wherein the dengue virus causing the viral infection is selected from serotype DENV-1 , DENV-2, DENV-3, DENV-4 or mixtures thereof.

16. The use of claim 15, wherein the dengue virus serotype causing the viral infection is DENV-2 serotype.

17. The use of a composition comprising a pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable vehicle, for the manufacture of a medicament for the treatment of viral infection caused by Dengue virus.

18. The use of the composition of claim 17, wherein the X receptor antagonist for pregnane (PXR) or its pharmaceutically acceptable salts are selected from the group comprising compound ET- 743, polychlorinated biphenyls, camptothecin, fluconazole, enilconazole, ketoconazole, sulforaphane, HIV protease inhibitor, A792611 , metformin, sesamin, fucoxanthin, coumestrol, SPA70, resveratrol and mixtures thereof.

19. The use of the composition of claim 18, wherein the pregnane X receptor (PXR) antagonist or pharmaceutically acceptable salts thereof is ketoconazole.

20. The use of the composition of claim 17 to 19, wherein the X receptor antagonist for pregnane (PXR) or pharmaceutically acceptable salts thereof, is administrable at a concentration of at least 5 pg to 100 pg.

21. The use of the composition of claim 17 to 20, wherein the X receptor antagonist for pregnane (PXR) or its pharmaceutically acceptable salts is orally or parenterally administrable.

22. The use of the composition of claim 17 to 21 , wherein the X receptor antagonist for pregnane (PXR) or pharmaceutically acceptable salts thereof is administrable at least once daily.

23. The use of the composition of claim 17 to 22, wherein the dengue virus causing viral infection is selected from serotype DENV-1 , DENV-2, DENV-3, DENV-4 or mixtures thereof.

24. The use of the composition of claim 23, wherein the dengue virus serotype causing the viral infection is serotype DENV-2.

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