WO2021003543A1 - Composés immunomodulateurs et/ou leurs sels pharmaceutiquement acceptables, composition pharmaceutique comprenant des composés immunomodulateurs et utilisation de composés immunomodulateurs dans la préparation d'une composition pharmaceutique pour le traitement de maladies virales - Google Patents

Composés immunomodulateurs et/ou leurs sels pharmaceutiquement acceptables, composition pharmaceutique comprenant des composés immunomodulateurs et utilisation de composés immunomodulateurs dans la préparation d'une composition pharmaceutique pour le traitement de maladies virales Download PDF

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WO2021003543A1
WO2021003543A1 PCT/BR2020/050248 BR2020050248W WO2021003543A1 WO 2021003543 A1 WO2021003543 A1 WO 2021003543A1 BR 2020050248 W BR2020050248 W BR 2020050248W WO 2021003543 A1 WO2021003543 A1 WO 2021003543A1
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immunomodulatory compounds
viral
infections
ifn
acetate
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Portuguese (pt)
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Ana Paula DUARTE DE SOUZA
Krist HELEN ANTUNES
Marco AURELIO RAMIREZ VINOLO
Renato TETELBOM STEIN
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Universidade Estadual De Campinas
União Brasileira De Educação E Assistência - Ubea
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present invention describes immunomodulatory compounds, a pharmaceutical composition comprising such compounds and the use of these compounds in the preparation of a pharmaceutical composition for treating viral diseases.
  • the present invention is in the fields of chemistry, pharmacy and medicine.
  • Viral infections are a major cause of death worldwide. Viruses such as Influenza, Ebola, Zica virus and Chikungunya are responsible for emerging epidemics that affect the population worldwide. The immune response against viruses is an important tool for controlling these infections.
  • the immune system reacts to a viral infection by recruiting components of the innate immune response (NK cells, macrophages and type 1 interferon) and the adaptive immune response (T and B cells).
  • Type 1 interferon Induction of type 1 interferon by viruses is crucial for immune response and is mediated by standard receptors, such as toll, RIG or MDA-5 receptors, through the activation of NF-kB and regulatory factors of interferon (IRFs).
  • IRFs interferon
  • the type 1 interferon response is controlled from the interferon responsive genes after activation of its IFNAR receptor.
  • Type 1 interferon prevents virus replication within the host cell, in addition, it acts as a signaling molecule warning the neighboring cells of the presence of the virus. It can also act by recruiting cells from the innate and adaptive immune system to the site of infection.
  • Viral infections of the upper airways IVAS
  • acute viral otitis media CAD
  • rhinosinusitis CAD
  • inflammatory respiratory diseases which may also be related to viral infections, such as exacerbation of asthma, bronchitis, bronchiolitis and pneumonia, make more than 4 million victims a year and affect hundreds of millions of people worldwide (Bousquet, J and Khaltaev, N. et al. ISBN 978 92 4 156346 8, World Health Organization 2007).
  • These diseases affect the health and well-being of patients and have a negative impact on the family and society.
  • bronchiolitis is a respiratory disease that affects children under 2 years of age and is characterized by vast inflammation and edema of the airways, causing an increase in mucus production and necrosis of pulmonary epithelial cells, specifically bronchioles.
  • RSV respiratory syncytial virus
  • RSV infection can also lead to other respiratory diseases, such as pneumonia and asthma exacerbation.
  • THE pneumonia is an infection of the lung parenchyma that can be caused by different organisms (Grant Mackenzie The definition and classification of pneumonia Pneumonia 2016 8: 14).
  • the highest incidence of pneumonia associated with bronchiolitis is caused by RSV infection, which significantly contributes to worsening the patient's clinical condition (Turner C, Turner P, Cararra V, Eh Lwe N, Watthanaworawit W, Day NP, White NJ, Goldblatt D, Nosten F.
  • Asthma exacerbation is a heterogeneous, multifactorial disease, which can also be associated with RSV infection or other agents, with reversible airway obstruction due to a chronic bronchial inflammatory reaction. Symptoms are cough, snoring, wheezing, difficulty breathing (Fritz Florak. Diagnosis and management of asthma - Statement on the 2015 GINA Guidelines Wien Klin Klin Klin Klin Klin. 2016; 128 (15): 541-554).
  • viruses that can trigger respiratory diseases, but to a lesser extent when compared to RSV, such as bronchiolitis. They are rhinovirus, parainfluenza virus (frequency of 5-25%), metapneumovirus, coronavirus, adenovirus (frequency of 5-10%), influenza virus and enterovirus (frequency of 1-5%) (Meissner HC. Virai Bronchiolitis in Children. The New England journal of medicine. 2016; 374 (18): 1793-4).
  • RSV binds to the epithelial cells and begins their replication, resulting in necrosis of the epithelial tissue and ciliary destruction.
  • Cellular destruction due to infection results in an intense inflammatory response with the proliferation of polymorphonuclear cells and lymphocytes.
  • the submucosa and other lung tissues become edematous with increased mucus secretion in response to pro-inflammatory cytokines (Aherne W, Bird T, Court SD, Gardner PS, McQuillin J. Pathological changes in virus infections of the lower respiratory tract in children. Journal of clinicai pathology. 1970; 23 (1): 7-18.
  • RSV disease A unique feature of RSV disease is that the host can be reinfected with the same pathogen from the same viral strain and exhibit the same symptoms. RSV reinfections are observed throughout life, despite the induction of both antibody and T cell responses after a primary infection and the absence of detectable antigenic change in the virus's surface glycoproteins (Collins PL, Melero JA. Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years. Virus research. 201 1; 162 (1 -2): 80-99).
  • Short-chain fatty acids are organic compounds that have a carboxylic group linked to a carbonyl chain of 1 to 5 carbons in its structure. Between 90 to 95% of AGCCs formed in the body are acetate, propionate and butyrate, these with two, three and four carbons respectively. The remaining 5-10% are represented by isobutyrate, valerate, isovalerate and caproate (Ambroze WL, Jr., Pemberton JH, Phillips SF, Bell AM, Haddad AC. Fecal short-chain fatty acid concentrations and effect on ileal pouch function. Diseases of the colon and rectum. 1993; 36 (3): 235-9).
  • AGCCs are products mainly derived from the metabolism of undigested carbohydrates by bacteria present in the microbiota, such as lactobacilli, bifidobacteria and proteobacteria (Flint HJ, Duncan SH, Scott KP, Louis P. Links between diet, gut microbiota composition and gut metabolism The Proceedings of the Nutrition Society. 2015; 74 (1): 13-22).
  • Carbohydrates including soluble dietary fibers are important components in the intestinal production of AGCCs (Flint HJ, Bayer EA, Rincon MT, Lamed R, White BA. Polysaccharide utilization by gut bacterium: potential for new insights from genomic analysis. Nature reviews Microbiology. 2008; 6 (2): 121 -31).
  • AGCCs are also absorbed by the cells of the intestinal epithelium performing functions both local (in the intestinal epithelium) and in the rest of the body. Most of the metabolized butyrate is used as an energy source by colonocytes, while propionate is used in liver metabolism. Acetate, on the other hand, can be found in higher concentrations in the blood, with around ⁇ 79 mmol / L in the peripheral circulation (den Besten G, van Eunen K, Groen AK, Venema K, Reijngoud DJ, Bakker BM. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. Journal of lipid research.
  • Formulas enriched with these prebiotics increase concentrations of AGCCs, mainly acetate, up to 3 times in the feces of patients who received the intervention compared to controls (Majid HA, Emery PW, Whelan K. Faecal microbiota and short -chain fatty acids in patients receiving enteral nutrition with Standard or fructo-oligosaccharides and fiber-enriched formulas. Journal of human nutrition and dietetics: the official journal of the British Dietetic Association. 201 1; 24 (3): 260-8; Vandenplas Y, De Greef E, Veereman G. Prebiotics in infant formula. Gut microbes.
  • breastfeeding milk is rich in oligosaccharides
  • infant formulas rich in soluble fibers may play a role in protective immunity against viruses and in preventing respiratory infections (Luoto R, Ruuskanen O, Waris M, Kalliomaki M, Salminen S , Isolauri E.
  • Prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebo-controlled trial. The Journal of allergy and clinicai immunology. 2014; 133 (2): 405-13).
  • G protein-coupled receptor ligands GPCR - GPR43, GPR41, GPR109a, Olfr78
  • receptor ligands can be classified into: total agonists, partial agonists, neutral antagonists and inverse agonists. This classification is usually done under specific experimental conditions, because the classification can be affected by the amount of expressed receptor and the strength of the stimulus-response coupling.
  • cumulative evidence suggests that agonists may induce different changes in the conformation of a particular GPCR, which leads to the activation of different signaling pathways, even when the level of receptor expression and the strength of the stimulus-response coupling are the same .
  • histone acetylation and deacetylation play an important role in regulating the gene transcription of eukaryotic cells.
  • the acetylation status of histones is determined by histone deacetylases (HDACs) and histone acetyl transferases (HATs).
  • HATs add acetyl groups to lysine residues in proteins, while HDACs do the reverse, that is, remove acetyl groups.
  • histone acetylation produces a more relaxed chromatin structure, allowing transcriptional activation.
  • HDACs can be transcriptional repressors, due to histone deacetylation and consequently by promoting chromatin condensation.
  • HDAC inhibitors HDAC inhibitors (HDACi) alter transcription by remodeling chromatin and modifying the protein structure in transcription factor complexes.
  • viral inflammatory diseases mainly respiratory ones, such as bronchiolitis, pneumonia, exacerbation of asthma, upper airway infections (URTI), viral acute otitis media and / or rhinosinusitis, caused by infections and / or co-infections comprising pneumoviruses, rhinoviruses, parainfluenza viruses, metapneumoviruses, coronaviruses, adenoviruses, influenza viruses and / or enteroviruses, in particular the Respiratory Sinus Virus (RSV).
  • RSV Respiratory Sinus Virus
  • Palivizumab is a humanized anti-RSV Protein F monoclonal antibody that inhibits the entry of the virus into cells, being recommended as a preventive conduct for severe bronchiolitis caused by RSV, protecting 50% of children who use it.
  • this antibody has been shown to reduce hospitalizations and the clinical severity of RSV infection in only 55% of high-risk children (Palivizumab, the Humanized Respiratory Syncytial Virus Monoclonal Antibody, Reduces Hospitalization From Respiratory Syncytial Virus Infection in High-risk Infants. Pediatrics. 1998; 102 (3): 531 -7).
  • Ribavirin is a synthetic antiviral drug analogous to guanosine that is on the list of essential medicines of the World Health Organization (WHO). It acts by inhibiting the enzyme inosine monophosphate dehydrogenase, however, it is recommended only in special cases because it is toxic to young children.
  • the present invention differs from Ribavirin in that the short chain fatty acids are present in the body and, when tested in vitro on A549 cells or in vivo in mice, in the inhibitory concentrations of viral replication and different from the physiological ones, did not induce cytotoxicity.
  • the present invention proposes a new approach based on immunomodulatory compounds for the treatment of diseases viral.
  • the existing preventive and treatment conducts are inefficient and demand alternatives at public health levels.
  • inflammatory viral diseases more precisely the respiratory tract
  • the main diseases that point out in the statistics, not limited to these, are: bronchiolitis, pneumonia, exacerbation asthma, upper airway infections (URTI), viral acute otitis media and rhinosinusitis.
  • the viral agents commonly involved are pneumoviruses, rhinoviruses, parainfluenza viruses, metapneumoviruses, coronaviruses, adenoviruses, influenza viruses and enteroviruses, with the Pneumoviruses being the most important, mainly the Respiratory Syncytial Virus (RSV).
  • RSV Respiratory Syncytial Virus
  • the present invention provides immunomodulatory compounds selected from the group among short chain fatty acids (AGCCs), synthetic agonists of AGCC receptors and / or interferon beta (INF-b), histone modulators deacetylase (HDAC) and / or histone acetyl transferase (HAT) modulators. More precisely, immunomodulatory compounds are selected from acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate, valproate, tributyrin, 4CMTB, AR420626, MS275, CI994, RGFP966, PCI-34051, TTK21, CTPB and / or their pharmaceutically salts acceptable.
  • AGT immunomodulatory compounds selected from the group among short chain fatty acids (AGCCs), synthetic agonists of AGCC receptors and / or interferon beta (INF-b), histone modulators deacetylase (HDAC) and / or histone acetyl transferase (HAT
  • the present immunomodulatory compounds act as activators of AGCC receptors, activators of INF-b receptors, HDAC inhibitors and / or activators of HAT, triggering anti-viral and anti-inflammatory response mediated by INF-b.
  • the activated receptors comprise GPR43, GPR41, GPR109a, O If r78 and / or IFNAR; histone deacetylases comprise HDAC 1 and / or 3.
  • the mechanism of action occurs through epigenetic modifications such as histone methylation and control of gene transcription. More specifically, the present compounds act by triggering an anti-viral and anti-inflammatory response, through the use of signaling by means of nuclear transcription factor kappa beta (NFkb), production of interferon beta (IFN-b) and consequent activation of responsive genes of the IFN (ISGs) such as ISG15, OAS, RIG-1 and MAVS. The consequence of activating these pathways is to reduce viral load.
  • NFkb nuclear transcription factor kappa beta
  • IFN-b interferon beta
  • ISGs responsive genes of the IFN
  • the present invention provides a pharmaceutical composition comprising such immunomodulatory compounds and / or their pharmaceutically acceptable salts and vehicles.
  • the immunomodulatory compounds can be used alone or in combination, preferably isolated.
  • the present invention provides the use of such immunomodulatory compounds and / or their pharmaceutically acceptable salts in the preparation of a pharmaceutical composition for treating viral diseases.
  • the evidence demonstrates better results in respiratory viral diseases, such as bronchiolitis, pneumonia, exacerbation of asthma, upper airway infections (URTI), acute viral otitis media and / or rhinosinusitis, caused by infections and / or co-infections of two or more viruses, including pneumoviruses, rhinoviruses, parainfluenza viruses, metapneumoviruses, coronaviruses, adenoviruses, influenza viruses and / or enteroviruses, especially by the Respiratory Syncytial Virus (RSV).
  • RSV Respiratory Syncytial Virus
  • the present invention provides a method of treating infections and / or co-infections of two or more viruses, comprising selecting and administering a sufficient amount of the immunomodulatory compounds acetate, propionate, butyrate, isobutyrate , valerate, isovalerate, caproate, valproate, tributyrin, 4CMTB, AR420626, MS275, CI994, RGFP966, PCI-34051, TTK21 and / or CTPB, or their mixture, triggering an anti-viral and anti-inflammatory response, through signaling of nuclear transcription factor kappa beta (NFkb), production of interferon beta (IFN-beta) and consequent activation of IFN-responsive genes (ISGs) such as ISG15, OAS, RIG-1 and MAVS and consequent reduction in viral load by at least 80%.
  • Such a method of treatment comprises such immunomodulatory compounds being AGCC receptor agonists, INF-b receptor agonists, FIDAC and
  • Figure 1 demonstrates that prophylaxis with AGCCs in vitro on human lung epithelial cells A549 decreases viral replication.
  • A- A549 cells were treated with propionate, butyrate and acetate (260 ⁇ M) for 24 hours before infection with 10 4 PFU / mL of RSV (strain A2) and were evaluated for cell viability after 96 hours.
  • ( * ) represents the difference between the control; (#) represents the difference between VSR; statistical differences between groups were determined using the two-way ANOVA test followed by Bonferroni's post-test; * # p ⁇ 0.05, ** p ⁇ 0.01, *** $ ** p ⁇ 0.001.
  • FIG. 2 shows the treatment with AGCCs (propionate, butyrate and acetate) in vitro after infection with RSV, in human lung epithelial cells A549: the cells were infected and subsequently treated with AGCCs 260uM, for 4 days and Cell viability was analyzed using MTT. Treatment with AGCCs has also been shown to have a protective effect against cell death mediated by RSV.
  • AGCCs propionate, butyrate and acetate
  • FIG. 3 demonstrates in vivo treatment with AGCCs (propionate, butyrate and acetate) protects against RSV infection.
  • AGCCs propionate, butyrate and acetate
  • Balb / c mice were infected intranasally with 10 7 PFU of RSV and treated in drinking water with AGCCs.
  • A- Viral load in the lungs of non-infected and RSV-infected animals treated with AGCCs obtained through real-time PCR.
  • ND not detected B- Weight loss percentage in relation to the original weight (day 0). ⁇ 0.05; * > ⁇ 0.01; " > ⁇ 0.001.
  • Figure 4 shows the in vivo analysis of IFN-b production in Balb / c mice: lung homogenate of animals treated with acetate and infected for 5 days infection induces an increase in IFN-b.
  • the animals started treatment with 200 mM acetate in drinking water and were infected with RSV intranasally on the same day. After 5 days, the lung was collected and the amount of IFN-b in the lung homogenate was analyzed.
  • Figure 5 demonstrates that protection against RSV infection mediated by acetate is dependent on the IFNAR and GPR43 receptors.
  • Balb / c mice were infected intranasally with 10 7 PFU RSV and treated with drinking water with acetate, propionate or butyrate.
  • FIG. 6 demonstrates that specific activation of GPR43 protects in vitro against RSV infection.
  • A549 cells were treated with 260 ⁇ M acetate or a synthetic GPR43 agonist (10 ⁇ M 4-CMTB) for 24 h and then infected with RSV (10 4 PFU / ml). Four days later, the response parameters were analyzed.
  • Figure 7 demonstrates that inhibition of HDAC 1 and 3 protects against RSV infection in vitro.
  • A549 cells were plated and pretreated with butyrate (260 ⁇ M) or with different HDAC inhibitors (10 ⁇ M) for 24 hours and infected with RSV for 96 hours. Afterwards, the viability of the cells was performed by MTT. * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • Figure 8 shows a schematic representation of a high fiber diet or administration of acetate in response to RSV infection.
  • a diet rich in fiber increases the production of acetate in the intestinal lumen.
  • the acetate administered orally enters the bloodstream and reaches the lungs through connection to its receptor (GPR43).
  • GPR43 receptor
  • lung acetate it increases the genes stimulated by interferon (Oas1 and Isg15) and the interferon-beta gene (Ifnb), which contributes to decrease the viral load of RSV.
  • AGCCs and other immunomodulatory compounds such as AGCC receptor agonists, INF-b receptor agonists, HDAC and HAT modulators, and their respective mechanisms of action, associated with the protective role of compounds such as acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate, valproate, tributyrin, 4CMTB, AR420626, MS275, CI994, RGFP966, PCI-34051, TTK21 and CTPB have shown remarkable efficiency for treating viral infections.
  • the viral strain of VSR A2 was identified by unit-forming viral plaques (PFU) using an anti-VSR antibody (Millipore, Billerica, MA, USA).
  • PFU unit-forming viral plaques
  • female BALB / c mice, male and female 129 / Sv mice deficient in type 1 (lfnar-1) interferon receptor and wild type female mice deficient in Rag-1 (Rag1 - / - ), C57BL / 6 female mice deficient in GPR43 (Gpr43 - / -), all aged 6-8 weeks.
  • the animals were housed in the vivarium of the Biology Institute of the State University of Campinas.
  • mice were anesthetized with 5% isoflurane and infected intranasally with 10 7 PFU / mL of the RSV A2 strain. All animals were weighed daily. Data analysis was performed 5 days after infection. The bronchoalveolar lavage (BAL) was collected or, after perfusion with formalin, the left lung was removed for histopathological and immunohistochemical analysis.
  • BAL bronchoalveolar lavage
  • mice fed a high fiber diet received a mixture of antibiotics (kanamycin (0.4 mg / ml), gentamicin (0.035 mg / ml), metronidazole (0.045 mg / ml), vancomycin (0.045 mg / ml) and colistin (0.035 mg / mL) diluted in drinking water for 3 days before infection. All antibiotics were purchased from Sigma-Aldrich.
  • mice received AGCCs (sodium acetate, sodium propionate or sodium butyrate; Sigma-Aldrich, St. Louis, MO, USA) in sterile drinking water at a final concentration of 200 mM for 3 weeks before RSV infection. treatment with AGCCs was carried out starting simultaneously with the infection at the same final concentration. Water volume consumption was measured daily and no difference in water intake was observed between groups.
  • AGCCs sodium acetate, sodium propionate or sodium butyrate
  • Flow cytometry was performed with cells isolated from the spleen or axillary and mesenteric lymph nodes. The cells were incubated with Mouse Fc Block (BD Biosciences®) for 20 min and then stained with anti-CD1 1 c (HL3 clone), anti-l-Ad / l-Ed (clone 2G9), anti-CD86 (GL1 clone), anti-CD8 (clone 53-6.7), anti-CD4 (RM4-5 clone) and anti-CD62L (MEL-14 clone) (BD Biosciences®). For intracellular staining, cells were fixed with cytofix / cytoperm (BD Biosciences®) and stained with anti-IL-4 antibody. The samples were acquired on a Gallios flow cytometer (Beckman Coulter®). The data were analyzed using FlowJo software (version 7.5, Tree Star Inc., MA, USA).
  • Microbiological analysis in feces of mice fed with diet rich in fiber or control diet and infected with RSV were performed with samples collected in sterile conditions. They were stored at -80 ° C.
  • the bacterial DNA was then isolated using the QiaAMP DNA Stool Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
  • the DNA was amplified using primers selected to cover the V4 region of the bacterial 16s rRNA with the enzyme Phusion.
  • the amplicons were purified with a magnetic sphere capture kit (AMPure XP; Agencourt) indexed with Nextera XT indexes and normalized according to the llumina protocol.
  • AMPure XP Agencourt
  • the sequencing of the 16s rRNA gene was performed on the Miseq platform, with a v2 500 cycle kit (Ilumina, CA, USA), which generates a final paired reading (2 x 250 bp).
  • the analytical pipeline used incorporates approaches based on phylogeny and alignment to maximize data resolution.
  • the read pairs were analyzed based on unique molecular bar codes added via PCR during the generation of the library using USEARCH v7.0.1090.
  • Subsequent analysis steps in the pipeline leverage the custom analytical packages developed at the Alkek Center for Metagenomics and Microbiome Research (CMMR) at Baylor College of Medicine to produce summary statistics and quality control measures to characterize microbial communities in large numbers of samples or groups of samples.
  • CMMR Alkek Center for Metagenomics and Microbiome Research
  • the 16S v4 rDNA sequences were grouped into Operational Taxonomic Units (OTUs), at a similarity cutoff value of 97%, using the UPARSE algorithm.
  • OTUs Operational Taxonomic Units
  • the OTUs were subsequently mapped to an optimized version of the SILVA database containing only sequences from the v4 region of the 16S rRNA gene to determine taxonomies.
  • the abundances were recovered by mapping the readings to UPARSE OTUs.
  • a custom script builds an OTU table from the output files generated in the previous two steps for further analysis using a visualization toolkit also developed in the CMMR called ATIMA (Agile Toolkit for Incisive Microbial Analyzes).
  • Chromatographic analyzes were performed using a gas chromatograph mass spectrometer (model GCMS-QP2010 Ultra; Shimadzu) and a capillary Stabilwax column with fused silica (Restec Corporation, USA) with dimensions of 30mx0.25mm internal diameter (id) with filter 0.25pm polyethylene glycol thickness.
  • the samples (1 pL) were injected at 250 ° C using an approximately 25: 1 ratio.
  • High quality pure helium (He) was used as carrier gas with a constant flow rate of 1.0 mL / min.
  • the mass conditions were as follows: ionization voltage, 70 eV; ion source temperature 200 ° C; full scan mode in mass range 35-500 with 0.2 s / scan speed.
  • the execution time for each analysis was 11.95 min.
  • lung cDNA was used to perform the relative expression of IFN-a and IFN-b using primers and probes for TaqMan (Mm03030145_gH Ifnal, Mm00439552_s1 Ifnbl Thermo Fisher Scientific) and b-Actina of mouse (Mm02619580_g1 Actb) as endogenous control.
  • TaqMan Mm03030145_gH Ifnal, Mm00439552_s1 Ifnbl Thermo Fisher Scientific
  • b-Actina of mouse Mm02619580_g1 Actb
  • PCR conditions were recommended by the GoTaq TM Probe qPCR Master Mix protocol (Promega TM, Madison, Wl, USA) or Master Mix SYBR Green / ROX qPCR (Thermo Fisher Scientific, Waltham, MA, USA). The quantification of gene expression was performed using StepOneTM (Applied Biosystems). AACt or 2 _ACt analysis was used to calculate the fold change. Levels of IFN-a and IFN-b proteins were quantified in the BAL supernatant using an IFN-a / IFN-b 2-Plex Mouse ProcartaPlex assay kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The tests were performed according to the manufacturer's instructions and read on the MAGPIX b350 Multiplexing Instrument (Merck Millipore, Billerica, MA).
  • Viral load was quantified by real-time PCR and viral plaque assay.
  • the viral plaque assay was conducted with A549 and MRC-5 cells, cultured in a 6-well plate, pretreated with 260mM acetate for 24h and infected with 10 4 PFU / mL RSV for 96h. The cells were collected and subjected to freeze-thaw and inoculated into monolayers of Vero cells. Viral plaque forming units (PFU) were identified using an anti-VSR antibody (Millipore, Billerica, MA, USA) after 4 days.
  • NFkb nuclear transcription factor kappa beta
  • IFN-b interferon beta
  • ISGs responsive IFN genes
  • Immunomodulatory compounds are short-chain fatty acids (AGCCs), AGCC receptor agonists and / or interferon beta (INF-b), histone deacetylase (FIDAC) modulators, histone acetyl transferase (FIAT) modulators or their salts, being preferably chosen from acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, caproate, valproate, tributyrin, 4CMTB, AR420626, MS275, CI994, RGFP966, PCI-34051, TTK21 and CTPB.
  • AGCCs short-chain fatty acids
  • INF-b interferon beta
  • FIDAC histone deacetylase
  • FIAT histone acetyl transferase
  • the immunomodulatory compounds of the present invention act as activators of AGCCs or INF-b receptors (GPR43, GPR41, GPR109a, Olfr78 and / or IFNAR), FIDAC inhibitors (FIDAC 1 and / or 3) and / or FIAT activators, triggering anti-viral and anti-inflammatory responses, by signaling by nuclear transcription factor kappa beta (NFkb), production of interferon beta (IFN-b) and consequent activation of IFN responsive genes (ISGs) such as ISG15, OAS, RIG-1 and MAVS, with consequent load reduction viral.
  • AGCCs or INF-b receptors GPR43, GPR41, GPR109a, Olfr78 and / or IFNAR
  • FIDAC 1 and / or 3 FIAT activators
  • IFN-b nuclear transcription factor kappa beta
  • IFN-b interferon beta
  • ISGs IFN responsive genes
  • the present invention further proposes a pharmaceutical composition comprising such immunomodulatory compounds, used alone or in combination, preferably isolated, and pharmaceutically acceptable vehicles.
  • the present invention also proposes the use of immunomodulatory compounds in the preparation of a drug for the treatment of viral diseases, especially respiratory diseases, such as bronchiolitis, pneumonia, exacerbation of asthma, infections of the upper airways (IVAS), acute otitis media viral and / or rhinosinusitis, caused by infections and / or co-infections comprising pneumovirus, rhinovirus, parainfluenza virus, metapneumovirus, coronavirus, adenovirus, influenza virus and / or enterovirus, especially by Pneumovirus, more specifically by the Respiratory Syncytial Virus (RSV).
  • respiratory diseases such as bronchiolitis, pneumonia, exacerbation of asthma
  • IVAS infections of the upper airways
  • acute otitis media viral and / or rhinosinusitis caused by infections and / or co-infections
  • pneumovirus pneumovirus
  • rhinovirus parainfluenza virus
  • the present invention provides a method of treating infections and / or co-infections of two or more viruses, comprising selecting and administering a sufficient amount of the immunomodulatory compounds acetate, propionate, butyrate, isobutyrate, valerate , isovalerate, caproate, valproate, tributyrin, 4CMTB, AR420626, MS275, CI994, RGFP966, PCI-34051, TTK21 and / or CTPB, or their mixture, triggering an anti-viral and anti-inflammatory response, through factor signaling nuclear transcription kappa beta (NFkb), production of interferon beta (IFN-beta) and consequent activation of IFN-responsive genes (ISGs) such as ISG15, OAS, RIG-1 and MAVS and consequent reduction in viral load by at least 80% .
  • Such a method of treatment comprises such immunomodulatory compounds being AGCC receptor agonists, INF-b receptor agonists, FIDAC and FIAT modulators
  • the concentrations of AGCCs used in vitro were determined in a way that would not result in cytotoxicity and, even so, its protective effect against the virus remaining, establishing a concentration range of 30 to 260 mM. We opted for the higher concentration to offer greater protection.
  • the concentrations of AGCCs used in vivo were determined through previously published articles that used the same treatment in drinking water at a concentration of 200 mM.
  • Type 1 interferons are cytokines that constitute the first line of defense against viral infections and the increase in these cytokines is responsible for the phenomenon of protection of AGCCs against RSV infection.
  • VSR has previously been shown to inhibit the production of type 1 interferons primarily through NS1 and NS2 proteins (Hall CB. Respiratory syncytial virus and parainfluenza virus. The New England journal of medicine. 2001; 344 (25): 1917- 28 ).
  • IFN-b production was also demonstrated in the lung homogenate of animals that were treated with acetate and infected with RSV for 5 days and it was demonstrated that acetate induces IFN-b production (Figure 4).
  • AGCCs the mechanism of action of acetate was first investigated and demonstrated to be dependent on its receptor (GPR43) and type I interferon receptor - IFNAR ( Figure 5).
  • the other AGCCs are having their mechanisms of action confirmed in relation to the soluble receptors and / or molecules GPR43, GPR41, GPR109a, type I interferon (IFNAR) and / or their synthetic agonists, and the inhibition of FIDAC (histone deacetylase).
  • HDAC inhibitor compounds such as MS 275 (iHDACs 1 and 3), PCI-34051 (iHDACs 8), RGFP966 (iHDAC 3) and CI994 (iHDACs 1, 2 and 3) protect against cytotoxicity caused by RSV ( Figure 7).
  • HDAC inhibitor compounds such as MS 275 (iHDACs 1 and 3), PCI-34051 (iHDACs 8), RGFP966 (iHDAC 3) and CI994 (iHDACs 1, 2 and 3
  • Histones acetyl transferases (HAT) and Histones deacetylases (HDAC) are known to modulate physiological processes like genetic transcription, cell cycle, gene silencing, differentiation, DNA replication and HAT activity is contrary to HDAC, the effect of fatty acids is expected to directly interfere in the action of these two enzymes, that is, inhibition of HDACs and activation of HATs .

Abstract

La présente invention concerne des composés immunomodulateurs, une composition pharmaceutique comprenant ces composés et l'utilisation de ces composés dans la préparation d'une composition pharmaceutique et pour le traitement de maladies virales. Plus particulièrement, la présente invention concerne des composés immunomodulateurs, une composition pharmaceutique et son utilisation dans la préparation d'une composition pharmaceutique pour le traitement de maladies virales respiratoires, notamment d'infections à VRS.
PCT/BR2020/050248 2019-07-08 2020-07-07 Composés immunomodulateurs et/ou leurs sels pharmaceutiquement acceptables, composition pharmaceutique comprenant des composés immunomodulateurs et utilisation de composés immunomodulateurs dans la préparation d'une composition pharmaceutique pour le traitement de maladies virales WO2021003543A1 (fr)

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Citations (3)

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WO2008143647A2 (fr) * 2006-11-29 2008-11-27 The Board Of Trustees Of The Leland Stanford Junior University Procédés de traitement d'infection virale
WO2012131069A1 (fr) * 2011-03-31 2012-10-04 Proponent Biotech Gmbh Acides gras à chaîne courte et leurs dérivés à utiliser dans le traitement de troubles immunogènes
WO2014053592A1 (fr) * 2012-10-03 2014-04-10 Proponent Biotech Gmbh Esters d'acides gras à chaînes courtes pour l'utilisation dans le traitement de troubles immunogènes

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WO2008143647A2 (fr) * 2006-11-29 2008-11-27 The Board Of Trustees Of The Leland Stanford Junior University Procédés de traitement d'infection virale
WO2012131069A1 (fr) * 2011-03-31 2012-10-04 Proponent Biotech Gmbh Acides gras à chaîne courte et leurs dérivés à utiliser dans le traitement de troubles immunogènes
WO2014053592A1 (fr) * 2012-10-03 2014-04-10 Proponent Biotech Gmbh Esters d'acides gras à chaînes courtes pour l'utilisation dans le traitement de troubles immunogènes

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MOHAMMAD JAMALUDDIN: "Respiratory Syncytial Virus-Inducible BCL-3 Expression Antagonizes the STAT/IRF and NF-B Signaling Pathways by Inducing Histone Deacetylase 1 Recruitment to the Interleukin-8 Promoter", JOURNAL OF VIROLOGY, vol. 79, no. 24, December 2005 (2005-12-01), pages 15302 - 15313, XP002632621, DOI: 10.1128/JVI.79.24.15302-15313.2005 *
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