[go: up one dir, main page]

WO2019126860A1 - Pharmaceutical composition for treating infection caused by viruses from the flaviviridae family and use of the composition - Google Patents

Pharmaceutical composition for treating infection caused by viruses from the flaviviridae family and use of the composition Download PDF

Info

Publication number
WO2019126860A1
WO2019126860A1 PCT/BR2018/050489 BR2018050489W WO2019126860A1 WO 2019126860 A1 WO2019126860 A1 WO 2019126860A1 BR 2018050489 W BR2018050489 W BR 2018050489W WO 2019126860 A1 WO2019126860 A1 WO 2019126860A1
Authority
WO
WIPO (PCT)
Prior art keywords
virus
viruses
flaviviridae family
infection caused
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/BR2018/050489
Other languages
French (fr)
Portuguese (pt)
Inventor
Lucio Holanda Gondim de FREITAS JUNIOR
Carolina Borsoi Moraes Holanda DE FREITAS
Denise Regina Bairros DE PILGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
INSTITUTO BUTANTAN
Original Assignee
INSTITUTO BUTANTAN
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from BR102017028623-1A external-priority patent/BR102017028623B1/en
Application filed by INSTITUTO BUTANTAN filed Critical INSTITUTO BUTANTAN
Publication of WO2019126860A1 publication Critical patent/WO2019126860A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • 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
    • 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
    • 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
    • A61K31/69—Boron compounds
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is in the field of biochemistry and pharmacology.
  • the present invention relates to a pharmaceutical composition for the treatment of infection caused by Flaviviridae family viruses and to the use of said pharmaceutical composition for the manufacture of a medicament for the treatment of infection caused by Flaviviridae family viruses.
  • the Flaviviridae family is composed of the genera Flavivirus, Pestivirus and Hepacivirus.
  • the genus Flavivirus comprises more than 70 viruses circulating on virtually all continents except Antarctica.
  • Mosquito-borne flaviviruses such as yellow fever virus, serotypes of dengue virus 1-4, Japanese encephalitis virus and West Nile virus cause endemic and epidemic diseases with a high mortality rate around the world.
  • Flaviviruses are encoded by a single-stranded, single-stranded sense genome RNA of approximately 11 kb in length.
  • the genome is a single open reading frame coding for 10 viral proteins that are cleaved co- and post-translationally from the polyprotein, the capsid (C), membrane (M) and envelope (E) structural proteins and non-structural proteins (NS) 1, 2A, 2B, 3A, 4B and 5.
  • the polyprotein is flanked by 5 'and 3' non-coding regions (Lindenbach & Rice, 2003) Syndromes that cause human infection with flaviviruses range from asymptomatic infections to serious and sometimes fatal diseases, including haemorrhagic manifestations of severe yellow fever and dengue virus infection and encephalitis caused by infection with Japanese encephalitis virus and virus (Barrett & Higgs, 2007; Barrett & Monath, 2003; Gubler, 2004).
  • Yellow fever is endemic in several regions of South America and Africa and is spread by the Aedes aegypti, the mosquito that also carries the dengue virus and Zika.
  • Early symptoms - fever, nausea, vomiting and muscle pain - are fairly mild and usually last only 3 to 4 days. About 15% of those affected by the disease, however, present a second most toxic phase 24h after recovery from the initial symptoms. Patients experience severe abdominal pain, become jaundiced, and bleeding from the mouth, nose, eyes, or stomach may occur. About half of people who enter the toxic phase die within 7 to 10 days.
  • the present invention relates to the screening of a library of pharmacologically active compounds containing various experimental and clinically useful drugs for the detection of inhibitors of yellow fever virus infection.
  • the work utilized the High Content Screening (HCS) or High Content Screening strategy. This technology allows collections of chemical compounds, both of synthetic and natural origin, to be rapidly triadic against the virus in a physiological environment, with the virus infecting human cells.
  • HCS High Content Screening
  • This technology allows collections of chemical compounds, both of synthetic and natural origin, to be rapidly triadic against the virus in a physiological environment, with the virus infecting human cells.
  • composition of the present invention comprising the compound of formula (I) defined herein and a pharmaceutically acceptable carrier for the treatment of infection caused by Flaviviridae family viruses and nor their use for the production of a medicament for the treatment of infection caused by viruses of the Flaviviridae family.
  • Vaccination is an important way of preventing yellow fever.
  • risk groups that can not benefit from such prevention, such as people over 60, babies under 9 months, pregnant women or people with immunodeficiency.
  • there has already been an epidemic of the disease in Angola and, apparently, there are risks of the disease spread throughout Asia, specifically high population rate such as China and India.
  • There are many Chinese workers in Angola and there is probably not enough vaccine for everyone if the virus reaches these high-index countries. In this sense, it is important to treat the disease so that there is an increase in the rate of survivors.
  • At the moment there is no specific antiviral treatment for yellow fever only care to treat dehydration, liver and kidney failure and fever. This fact demonstrates the importance of this patent application.
  • the present invention relates to a pharmaceutical composition for the treatment of infection caused by Flaviviridae family viruses comprising the compound defined by formula (I)
  • the compound defined by formula (I) can be used in the concentration of 12.5 mM to 25 mM. In a further embodiment, the compound defined by formula (I) is preferably used at the concentration of 12.5 mM.
  • the invention relates to the use of the composition as defined above for the manufacture of a medicament for the treatment of infection caused by viruses of the Flaviviridae family.
  • viruses of the Flaviviridae family include viruses selected from the group consisting of: West Nile virus, dengue virus, Zika virus, encephalitis virus transmitted by tick, St. Louis encephalitis virus, hepatitis C and yellow fever virus.
  • the virus infection caused by the Flaviviridae family is caused by the yellow fever virus.
  • FIG. 1 High Content Analysis representative images of Huh7 (blue) cells infected with YFV-YFP (yellow) stained with DAPI 72 h after infection.
  • the images show the process of automated image analysis: A) raw data; B) segmentation of host cell nuclei; C) segmentation of host cell cytoplasm; D) YFP intensity; E) Infected cells (green).
  • FIG. 2 Screening of LOPAC1280 library against YFV-YFP. Correlation between cellular rate (Y axis) and normalized activity (X axis). Negative control represented by red dots [(-) DMSO]; blue spots indicate wells treated with 6.25 ng / mL of Interferon alfa 2A [positive control - (+) EC100]; green dots indicate wells infected with mock [positive control - (+) MOCK]; the orange dot represents the sample selected for secondary screening [hit] - normalized activity of more than 70% and cell rate close to 0.8.
  • Negative control represented by red dots [(-) DMSO]; blue spots indicate wells treated with 6.25 ng / mL of Interferon alfa 2A [positive control - (+) EC100]; green dots indicate wells infected with mock [positive control - (+) MOCK]; the orange dot represents the sample selected for secondary screening [hit] - normalized activity of more than 70% and cell rate close to
  • Figure 3 Representative images obtained from the Operetta High Content System (objective of 20x WD - DNA staining with DAPI) showing the controls, the reference compound Interferon alpha 2A (at 6.25 ng / mL) - ECiocv control negative (DMSO) and positive (mock-infected) control, in addition to various concentrations of compound HA155-LOPAC-886.
  • Figure 4 Antiviral activity of compound HA155-LOPAC-886 YFV-YFP infecting host cell Huh7.
  • Left-Y axis normalized activity values (black points and curves);
  • Right-Y axis normalized cell rate values (squares and red curves);
  • X axis Compound log / sample concentration. The mean values (points and squares) and the standard deviation (bars) are shown. Data from two independent experiments.
  • the present invention relates to a pharmaceutical composition for the treatment of infection caused by Flaviviridae family viruses comprising the compound as defined by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the compound defined by formula (I) may be used in the concentration of 12.5 to 25 mM.
  • the compound defined by formula (I) is preferably used at the concentration of 12.5 mM.
  • the compound of formula (I) is an inhibitor of autotaxin IV.
  • Autotaxin converts lysophosphatidylcholine to lysophosphatidylic acid (LPA), which can mediate changes in cell proliferation, angiogenesis and cytokine secretion.
  • LPA lysophosphatidylic acid
  • Dose-dependent blockade of thrombin-induced LPA secretion in platelets has been demonstrated (Fulkerson Z et al, 2011).
  • ATX Autotaxin
  • LPC lysophosphatidylcholine
  • LPA lysophosphatidic acid
  • HA155-LOPAC-886 Molecular coupling indicated a remarkable binding form for one of the isomers, which differed from the original binding form of HA155-LOPAC-886 to ATX.
  • the thrombin-mediated increase in platelet-derived LPA was completely attenuated in a dose dependent manner by HA155-LOPAC-886.
  • HA155 - LOPAC-886 could inhibit autotaxin by selective binding to its catalytic threonine.
  • HA155-LOPAC-886 has been shown to dose-dependently block platelet-mediated thrombin-induced LPA secretion (Albers, H. M. H. et al., 2011 and Fulkerson Z et al, 2011).
  • the use of the term "pharmaceutically acceptable” essentially means being nontoxic to the subject to which the Pharmaceutically acceptable material is administered.
  • the use of the term "pharmaceutically acceptable carrier” means a nontoxic, inert solid, semisolid liquid excipient, diluent, auxiliary formulation of any type, such as saline, except water.
  • materials that may serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, cyclodextrin; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols, such as glycerinaglycol, sorbitol, mannitol and polyethylene; esters, such as ethyl laurate, ethyl oleate, agar; buffering agents, such as aluminum hydroxide and magnesium hydroxide; alg
  • Flaviviridae family virus is used in this application as a synonym for "flavivirus” and “flavivirose” as synonymous with “infection caused by Flaviviridae family viruses”.
  • composition for the production of a medicament for the treatment of infection caused by the family virus Flaviviridae
  • the present invention relates to the use of the composition as defined above for the manufacture of a medicament for the treatment of infection caused by Flaviviridae family viruses.
  • the virus infection of the Flaviviridae family includes viruses selected from the group consisting of: West Nile virus, dengue virus, Zika virus, tick-borne encephalitis virus, St. Louis encephalitis virus, hepatitis C virus and yellow fever viruses.
  • the virus infection caused by the Flaviviridae family is caused by the yellow fever virus.
  • the reference compound Interferon alpha 2A was obtained from Sigma-Aldrich.
  • the LOPAC1280 library containing 1280 samples was purchased from Sigma-Aldrich.
  • Huh7 The Huh7 cell line was derived from a human hepatic cell carcinoma. The cells form a contiguous monolayer that can be maintained for several weeks. This inventory was purchased from the Japanese Bioressources Research Collection (JCRB). This cell line was maintained in DMEM F12 medium (Sigma-Aldrich), supplemented with 10% FBS (Sigma Aldrich), 100 U / ml penicillin (GIBCO) and 100 ⁇ g / ml streptomycin (GIBCO), hereinafter described as " DMEM F12 ".
  • C6 / 36 The C6 / 36 cell line was derived from incubated larvae of Aedes albopictus. C6 / 36 cells exhibit adherent epithelial morphology. This inventory was provided by Prof. Dr. Amilcar Tanuri, from the Federal University of Rio de Janeiro. This cell line was maintained in L15 medium (Sigma-Aldrich), supplemented with 10% FBS
  • Yellow fever virus YDV-YD virus (YFV) 17D virus expressing the yellow fluorescent reporter protein (YFP) (YFV-YFP) was donated by Laura Gil (Aggeu Magalh ⁇ es Research Center, Fiocruz, Pernambuco , Brazil) . Yellow fever viruses were obtained from the supernatant of C6 / 36 tissue cultures infected with the virus. Infected cultures were maintained in L15 medium and frozen in 250 pL aliquots. Preparation of the compound solution
  • the sample library was sent into 96-well plates (25 ⁇ l / well) at a stock concentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Samples were transferred manually to 384 well polypropylene stock plates (Mother plates) (Greiner Bio-One). These plates were used to prepare new plates in a lower concentration. Samples were transferred manually to new 384-well polypropylene plates (10 ⁇ l / well) at a final concentration of 1 mM in 100% DMSO. Prior to performing the assay, intermediate plates were prepared. Samples were diluted in the buffered saline solution (PBS IX) to a final concentration of 600 pM compound and 6% DMSO. The selected hits were collected from the 384-well motherboard.
  • PBS IX buffered saline solution
  • HCS allows the evaluation of samples having antiviral activity against infected Huh7 cells.
  • 10 ⁇ l of each sample were manually transferred from Greiner Bio-One plates to 384-well black polystyrene assay plates (Greiner Bio-One) by means of a multichannel pipette which yields a final concentration of 10 mM with 1.0% DMSO (100 fold dilution).
  • Infections were then performed with 3,000 Huh7 / well cells co-plated with MOI 2.5 of YFP-YFP, all resuspended in F12 DMEM and dispensed with the aid of a multichannel pipette.
  • the plates were incubated for 72 h at 37 ⁇ C / 5% CO 2 under a humidified atmosphere.
  • the plates were fixed with 4% paraformaldehyde (PFA), stained with DAPI (Sigma-Aldrich) for 30 minutes in the dark at room temperature and four images of each well were purchased from the High Content Imaging System Operetta (PerkinElmer) with 20X magnification.
  • the images were analyzed by Harmony's high content analysis software (HCA) (PerkinElmer) for identification, segmentation and quantification of the host cell nucleus, cytoplasm and intracellular parasites ( Figure 1).
  • HCA Harmony's high content analysis software
  • the HCA provided as output data for all images from one well the total number of cells, total number of infected cells and YFP intensity of infected cells.
  • IR Infection Rate
  • Activity Norrralizacfe (A) [1- (ft H3 ⁇ 4-A / P3 ⁇ 4) / (ft H3 ⁇ 4-A / P3 ⁇ 4 > )] x 100
  • Av. IR N mean infection rate of negative control wells.
  • Av. IRp mean infection rate of positive control wells.
  • Av. IR T mean infection rate of test compound wells (at a given concentration).
  • Av.IRp mean of infection rate of positive control
  • AV.IR N mean of infection rate of negative control
  • EC 50 was defined as the concentration of the compound corresponding to 50% normalized activity after 72 h incubation of the compound. Power refers to the EC50 - the more potent the compound, the lower its EC50 - while the efficacy is related to the maximum observed activity of a compound (in%) - the more effective the compound, the closer its maximum activity of 100%.
  • the same rationale is used for the CC50 definition: the compound concentration, which reduces the cell rate by 50%, when compared to the mean of negative control.
  • the LOPAC1280 library was screened at a 100-fold dilution, and the assay plates were considered approved with factor Z'30.5; the mean Z factor of the assay was 0.84 ⁇ 0.06.
  • the cell rate values of all samples tested in the library revealed that only 9.61% had host cell toxicity greater than 50% at the concentration tested ( Figure 2).
  • the primary hits were selected as the best twenty-eight more active samples yielding a host cell toxicity of 20% and normalized activity equal to or approximately 70% as the individual images of the wells tested were confirmed and confirmed the reduction of rate of infection. Selection of samples at primary screening resulted in a global hit rate of 2.18% ( Figure 2). The results stimulate the process with subsequent trials, since the concentration tested for hits on primary screening is considered low (10 mM).
  • selectivity index rate of CC 50 value by EC 50 value (CC 50 / EC 50 ). Whenever the CC 50 is not generated by curve fitting, the highest concentration tested is used to estimate the SI (Max [] tested / ECso). Whenever the EC 50 is not generated by curve fitting, the lowest concentration tested is used to estimate the SI (CC 50 / Min [] tested). The maximum activity was obtained from the value by the nonlinear regression curve.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a pharmaceutical composition for treating infection caused by viruses from the Flaviviridae family, comprising the compound as defined by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The present invention also relates to the use of the previously defined composition for producing a medicament for treating infection caused by viruses from the Flaviviridae family.

Description

"COMPOSIÇÃO FARMACÊUTICA PARA O TRATAMENTO DE INFECÇÃO CAUSADA POR VÍRUS DA FAMÍLIA FLAVIVIRIDAE , E, USO DA  PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF INFECTION CAUSED BY VIRUSES OF THE FLAVIVIRIDAE FAMILY, AND USE OF THE

COMPOSIÇÃO"  COMPOSITION"

CAMPO DA INVENÇÃO  FIELD OF THE INVENTION

[0001] A presente invenção encontra-se no campo da bioquímica e farmacologia. A presente invenção se refere a uma composição farmacêutica para o tratamento de infecção causada por vírus da família Flaviviridae e ainda ao uso da referida composição farmacêutica para a produção de um medicamento para o tratamento de infecção causada por vírus da família Flaviviridae.  The present invention is in the field of biochemistry and pharmacology. The present invention relates to a pharmaceutical composition for the treatment of infection caused by Flaviviridae family viruses and to the use of said pharmaceutical composition for the manufacture of a medicament for the treatment of infection caused by Flaviviridae family viruses.

ANTECEDENTES DA INVENÇÃO  BACKGROUND OF THE INVENTION

[0002] A família Flaviviridae é composta pelos gêneros Flavivirus, Pestivírus e Hepacivírus. O gênero Flavivirus compreende mais de 70 vírus que circulam por praticamente todos os continentes, exceto Antártica. Os flavivirus transmitidos por mosquitos, como o vírus da febre amarela, os sorotipos de vírus da dengue 1-4, o vírus da encefalite japonesa e o vírus do Nilo Ocidental causam doenças endémicas e epidêmicas com alta taxa de mortalidade ao redor do mundo.  [0002] The Flaviviridae family is composed of the genera Flavivirus, Pestivirus and Hepacivirus. The genus Flavivirus comprises more than 70 viruses circulating on virtually all continents except Antarctica. Mosquito-borne flaviviruses, such as yellow fever virus, serotypes of dengue virus 1-4, Japanese encephalitis virus and West Nile virus cause endemic and epidemic diseases with a high mortality rate around the world.

[0003] Os flavivirus são codificados por um genoma de RNA senso positivo, de fita simples e com aproximadamente 11 kb de comprimento. O genoma é um quadro de leitura aberto único que codifica 10 proteínas virais que são clivadas co- e pós-traducionalmente a partir da poliproteína, das proteínas estruturais do capsídeo (C) , da membrana (M) e do envelope (E) e das proteínas não estruturais (NS) 1, 2A, 2B, 3, 4A, 4B e 5. A poliproteína é flanqueada por regiões não codificantes 5' e 3' (Lindenbach & Rice, 2003) [0004] As sindromes que causam infecção humana por flavivirus variam de infecções assintomáticas a doenças graves e às vezes fatais, incluindo manifestações hemorrágicas de infecção grave por virus da febre amarela e dengue e encefalite causada por infecção com o virus da encefalite japonesa e o virus do Nilo Ocidental (Barrett & Higgs, 2007; Barrett & Monath, 2003; Gubler, 2004) . Flaviviruses are encoded by a single-stranded, single-stranded sense genome RNA of approximately 11 kb in length. The genome is a single open reading frame coding for 10 viral proteins that are cleaved co- and post-translationally from the polyprotein, the capsid (C), membrane (M) and envelope (E) structural proteins and non-structural proteins (NS) 1, 2A, 2B, 3A, 4B and 5. The polyprotein is flanked by 5 'and 3' non-coding regions (Lindenbach & Rice, 2003) Syndromes that cause human infection with flaviviruses range from asymptomatic infections to serious and sometimes fatal diseases, including haemorrhagic manifestations of severe yellow fever and dengue virus infection and encephalitis caused by infection with Japanese encephalitis virus and virus (Barrett & Higgs, 2007; Barrett & Monath, 2003; Gubler, 2004).

[0005] A febre amarela é endémica em diversas regiões da América do Sul e da África, e é espalhada pelo Aedes aegypti , o mosquito que também carrega o virus da dengue e Zika. Os primeiros sintomas - febre, náuseas, vómitos e dor muscular - são razoavelmente leves e geralmente duram apenas de 3 a 4 dias. Cerca de 15% dos acometidos pela doença, no entanto, apresentam uma segunda fase mais tóxica 24h após a recuperação dos sintomas iniciais. Os pacientes sofrem dor abdominal severa, tornam-se ictéricos e pode ocorrer sangramento da boca, nariz, olhos ou estômago. Cerca de metade das pessoas que entram na fase tóxica morrem no período de 7 a 10 dias.  Yellow fever is endemic in several regions of South America and Africa and is spread by the Aedes aegypti, the mosquito that also carries the dengue virus and Zika. Early symptoms - fever, nausea, vomiting and muscle pain - are fairly mild and usually last only 3 to 4 days. About 15% of those affected by the disease, however, present a second most toxic phase 24h after recovery from the initial symptoms. Patients experience severe abdominal pain, become jaundiced, and bleeding from the mouth, nose, eyes, or stomach may occur. About half of people who enter the toxic phase die within 7 to 10 days.

[0006] O pedido de patente internacional PCT/US2008/076806, revela composições e métodos de tratar um hospedeiro infectado com um vírus da família Flaviviridae, e que utilizou também uma biblioteca LOPAC para chegar a um determinado composto (veja abaixo) . Entretanto, o composto não é o mesmo e nem se assemelha ao da presente invenção. Nesse pedido foi utilizado um análogo de clemizol, com a fórmula que segue: International patent application PCT / US2008 / 076806 discloses compositions and methods of treating a host infected with a Flaviviridae family virus, and which also used an LOPAC library to reach a particular compound (see below). However, the compound is not the same and does not resemble that of the present invention. In this application, a clemizole analogue was used, having the formula:

Figure imgf000004_0001
Figure imgf000004_0001

[0007] Já a presente invenção relata a triagem de uma biblioteca de compostos farmacologicamente ativos, contendo diversos fármacos experimentais e de uso clinico, para a descoberta de inibidores da infecção de virus da febre amarela. O trabalho utilizou a estratégia de High Content Screening (HCS), ou Triagem de Alto Conteúdo. Esta tecnologia permite que coleções de compostos químicos, tanto de origem sintética quanto natural, sejam rapidamente tríadas contra o vírus em um ambiente fisiológico, com o vírus infectando células humanas. The present invention relates to the screening of a library of pharmacologically active compounds containing various experimental and clinically useful drugs for the detection of inhibitors of yellow fever virus infection. The work utilized the High Content Screening (HCS) or High Content Screening strategy. This technology allows collections of chemical compounds, both of synthetic and natural origin, to be rapidly triadic against the virus in a physiological environment, with the virus infecting human cells.

[0008] Nenhum documento do estado da técnica descreve a composição farmacêutica da presente invenção compreendendo o composto de fórmula (I) aqui definido e um veículo farmaceuticamente aceitável para o tratamento de infecção causada por vírus da família Flaviviridae e nem seu uso para a produção de um medicamento para o tratamento de infecção causada por vírus da família Flaviviridae.  No prior art document describes the pharmaceutical composition of the present invention comprising the compound of formula (I) defined herein and a pharmaceutically acceptable carrier for the treatment of infection caused by Flaviviridae family viruses and nor their use for the production of a medicament for the treatment of infection caused by viruses of the Flaviviridae family.

SUMÁRIO DA INVENÇÃO  SUMMARY OF THE INVENTION

[0009] A vacinação é um importante modo de prevenção da febre amarela. Entretanto, há grupos de risco que não podem se beneficiar de tal prevenção, como pessoas com mais de 60 anos, bebés com menos de 9 meses, gestantes ou portadores de imunodeficiência . Além disso, já houve epidemia da doença em Angola e, aparentemente, há riscos da doença se espalhar pela Ásia, especificamente países de alta taxa populacional como China e índia. Há muitos trabalhadores chineses em Angola e, é provável que não haja vacina suficiente para todos caso o vírus atinja estes países de alto índice populacional. Neste sentido, é importante um tratamento da doença para que haja aumento na taxa de sobreviventes. No momento não há um tratamento antiviral específico para a febre amarela, somente cuidados para tratar desidratação, insuficiência hepática e renal e febre. Este fato demonstra a importância do presente pedido de patente. [0009] Vaccination is an important way of preventing yellow fever. However, there are risk groups that can not benefit from such prevention, such as people over 60, babies under 9 months, pregnant women or people with immunodeficiency. In addition, there has already been an epidemic of the disease in Angola and, apparently, there are risks of the disease spread throughout Asia, specifically high population rate such as China and India. There are many Chinese workers in Angola, and there is probably not enough vaccine for everyone if the virus reaches these high-index countries. In this sense, it is important to treat the disease so that there is an increase in the rate of survivors. At the moment there is no specific antiviral treatment for yellow fever, only care to treat dehydration, liver and kidney failure and fever. This fact demonstrates the importance of this patent application.

[0010] Em um aspecto, a presente invenção se refere a uma composição farmacêutica para o tratamento de infecção causada por vírus da família Flaviviridae compreendendo o composto definido pela fórmula (I)  In one aspect, the present invention relates to a pharmaceutical composition for the treatment of infection caused by Flaviviridae family viruses comprising the compound defined by formula (I)

Figure imgf000005_0001
Figure imgf000005_0001

ou um sal farmaceuticamente aceitável do mesmo, e um veículo farmaceuticamente aceitável. Em uma concretização, o composto definido pela fórmula (I) pode ser utilizado na concentração de 12,5 mM a 25 mM. Em uma concretização adicional, o composto definido pela fórmula (I) é utilizado preferencialmente na concentração de 12,5 mM. or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In one embodiment, the compound defined by formula (I) can be used in the concentration of 12.5 mM to 25 mM. In a further embodiment, the compound defined by formula (I) is preferably used at the concentration of 12.5 mM.

[0011] Em outro aspecto, a invenção se refere ao uso da composição como definida anteriormente para a produção de um medicamento para o tratamento de infecção causada por vírus da família Flaviviridae. Em uma concretização adicional, os vírus da família Flaviviridae incluem vírus selecionados do grupo que consiste de: vírus do Nilo Ocidental, vírus da dengue, vírus Zika, vírus da encefalite transmitido por carrapato, vírus da encefalite de St. Louis, hepatite C e vírus da febre amarela. Em uma concretização ainda adicional, a infecção causada por vírus da família Flaviviridae é ocasionada pelo vírus da febre amarela. In another aspect, the invention relates to the use of the composition as defined above for the manufacture of a medicament for the treatment of infection caused by viruses of the Flaviviridae family. In a further embodiment, viruses of the Flaviviridae family include viruses selected from the group consisting of: West Nile virus, dengue virus, Zika virus, encephalitis virus transmitted by tick, St. Louis encephalitis virus, hepatitis C and yellow fever virus. In a still further embodiment, the virus infection caused by the Flaviviridae family is caused by the yellow fever virus.

BREVE DESCRIÇÃO DAS FIGURAS  BRIEF DESCRIPTION OF THE DRAWINGS

[0012] O objetivo da invenção, juntamente com vantagens adicionais da mesma, poderá ser melhor entendido mediante referência às figuras em anexo e às seguintes descrições :  The object of the invention together with further advantages thereof may be best understood by reference to the accompanying figures and the following descriptions:

[0013] Figura 1 - Imagens representativas do High Content Analysis de células Huh7 (azul) infectadas com YFV- YFP (amarelo) coradas com DAPI 72 h após a infecção. As imagens mostram o processo de análise automatizada de imagem: A) dados brutos; B) segmentação de núcleos de células hospedeiras; C) segmentação do citoplasma de células hospedeiras; D) intensidade YFP; E) Células infectadas (verde) .  Figure 1 - High Content Analysis representative images of Huh7 (blue) cells infected with YFV-YFP (yellow) stained with DAPI 72 h after infection. The images show the process of automated image analysis: A) raw data; B) segmentation of host cell nuclei; C) segmentation of host cell cytoplasm; D) YFP intensity; E) Infected cells (green).

[0014] Figura 2 - Triagem da biblioteca LOPAC1280 contra YFV-YFP. Correlação entre taxa celular (eixo Y) e atividade normalizada (eixo X) . Controle negativo representado em pontos vermelhos [(-) DMSO] ; pontos azuis indicam poços tratados com 6,25 ng/mL de Interferon alfa 2A [controle positivo - (+) EC100]; pontos verdes indicam poços infectados com mock [controle positivo - (+) MOCK] ; o ponto laranja representa a amostra selecionada para triagem secundária [hit] - atividade normalizada de mais de 70% e taxa celular próxima a 0,8.  Figure 2 - Screening of LOPAC1280 library against YFV-YFP. Correlation between cellular rate (Y axis) and normalized activity (X axis). Negative control represented by red dots [(-) DMSO]; blue spots indicate wells treated with 6.25 ng / mL of Interferon alfa 2A [positive control - (+) EC100]; green dots indicate wells infected with mock [positive control - (+) MOCK]; the orange dot represents the sample selected for secondary screening [hit] - normalized activity of more than 70% and cell rate close to 0.8.

[0015] Figura 3 - Imagens representativas obtidas do Operetta High Content System (objetiva de 20x WD - coloração de DNA com DAPI) mostrando os controles, o composto de referência Interferon alpha 2A (a 6.25 ng/mL) - ECiocv controle negativo (DMSO) e controle positivo (infectado com mock) , além de diversas concentrações do composto HA155 - LOPAC- 886. Figure 3 - Representative images obtained from the Operetta High Content System (objective of 20x WD - DNA staining with DAPI) showing the controls, the reference compound Interferon alpha 2A (at 6.25 ng / mL) - ECiocv control negative (DMSO) and positive (mock-infected) control, in addition to various concentrations of compound HA155-LOPAC-886.

[0016] Figura 4 - Atividade antiviral do composto HA155 - LOPAC-886 YFV-YFP infectando célula hospedeira Huh7. Eixo esquerdo-Y: valores da atividade normalizada (pontos e curvas pretos); Eixo Direito-Y: valores de taxa celular normalizada (quadrados e curvas vermelhos); Eixo X: Log de composto / concentração de amostra. Os valores médios (pontos e quadrados) e o desvio padrão (barras) são mostrados. Dados de duas experiências independentes.  Figure 4 - Antiviral activity of compound HA155-LOPAC-886 YFV-YFP infecting host cell Huh7. Left-Y axis: normalized activity values (black points and curves); Right-Y axis: normalized cell rate values (squares and red curves); X axis: Compound log / sample concentration. The mean values (points and squares) and the standard deviation (bars) are shown. Data from two independent experiments.

DESCRIÇÃO DETALHADA DA INVENÇÃO  DETAILED DESCRIPTION OF THE INVENTION

[0017] Embora a presente invenção possa ser suscetível a diferentes concretizações, é mostrada nos desenhos e na seguinte discussão detalhada, uma concretização preferida com o entendimento de que a presente descrição deve ser considerada uma exemplificação dos princípios da invenção e não pretende limitar a presente invenção ao que foi ilustrado e descrito aqui.  While the present invention may be susceptible to different embodiments, it is shown in the drawings and in the following detailed discussion, a preferred embodiment with the understanding that the present disclosure is to be considered an exemplification of the principles of the invention and is not intended to limit the present invention. invention which has been illustrated and described herein.

Composição farmacêutica para o tratamento de infecção causada por vírus da família Flaviviridae  Pharmaceutical composition for the treatment of infection caused by viruses of the family Flaviviridae

[0018] Em uma primeira concretização, a presente invenção se refere a uma composição farmacêutica para o tratamento de infecção causada por vírus da família Flaviviridae compreendendo o composto como definido pela fórmula (I)

Figure imgf000008_0001
ou um sal farmaceuticamente aceitável do mesmo, e um veiculo farmaceuticamente aceitável. Em outra concretização, o composto definido pela fórmula (I) pode ser utilizado na concentração de 12,5 a 25 mM. Ainda em outra concretização, o composto definido pela fórmula (I) é utilizado preferencialmente na concentração de 12,5 mM. In a first embodiment, the present invention relates to a pharmaceutical composition for the treatment of infection caused by Flaviviridae family viruses comprising the compound as defined by formula (I)
Figure imgf000008_0001
or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In another embodiment, the compound defined by formula (I) may be used in the concentration of 12.5 to 25 mM. In yet another embodiment, the compound defined by formula (I) is preferably used at the concentration of 12.5 mM.

Descrição do composto HA155 - LOPAC-886  Description of compound HA155-LOPAC-886

[0019] O composto definido por fórmula I  The compound defined by formula I

Figure imgf000008_0002
Figure imgf000008_0002

apresenta como sinónimos ácido ( Z ) -4- ( ( 4 ( 3- ( 4-Fluorobenzil ) - 2, 4-dioxo-l, 3-tiazolan-5-iliden) - metil ) fenoxi ) metil ) benzenoboronico, B— [ 4— [ [ 4— [ ( Z )— [ 3— [ (4- fluorofenil ) metil ] -2 , 4-dioxo-5- tiazolidinilideno ] metil ] fenoxi ] metil ] fenil ] -boronico . (Z) -4 - ((4- (3- (4-Fluorobenzyl) -2,4-dioxo-1,3-thiazolan-5-ylidene) methyl) phenoxy) methyl) benzeneboronic acid, B- [ 4 - [[4 - [(Z) - [3 - [(4-fluorophenyl) methyl] -2,4-dioxo-5-thiazolidinylidene] methyl] phenoxy] methyl] phenyl] boronic acid.

Apresenta também como fórmula empírica (Notação de Hill) C24H19BFN05S, peso molecular de 463,29, IC50 de 5,7 nM. It also presents as empirical formula (Hill Notation) C24H19BFN05S, molecular weight of 463.29, IC50 of 5.7 nM.

[0020] O composto de fórmula (I) é um inibidor de autotaxina IV. A autotaxina converte a lisofosfatidilcolina em ácido lisofosfatídico (LPA) , que pode mediar mudanças na proliferação celular, angiogênese e secreção de citocinas. HA-155 é um composto à base de ácido boronico que inibe a autotaxina (IC50 = 5,7 nM) por ligação seletiva à sua treonina catalítica (Hausmann J et al, 2011 e Albers, H. M. H. G. et al, 2011) . Demonstrou-se bloquear de forma dose- dependente a secreção de LPA induzida por trombina em plaquetas (Fulkerson Z et al, 2011) . The compound of formula (I) is an inhibitor of autotaxin IV. Autotaxin converts lysophosphatidylcholine to lysophosphatidylic acid (LPA), which can mediate changes in cell proliferation, angiogenesis and cytokine secretion. HA-155 is a boronic acid-based compound which inhibits autotaxin (IC 50 = 5.7 nM) by selective binding to its catalytic threonine (Hausmann J et al, 2011 and Albers, HMHG et al, 2011). Dose-dependent blockade of thrombin-induced LPA secretion in platelets has been demonstrated (Fulkerson Z et al, 2011).

[0021] A autotaxina (ATX) , uma fosfodiesterase segregada, hidrolisa a abundante fosfolipidio lisofosfatidilcolina (LPC) para produzir ácido lisofosfatidico (LPA) . O eixo de sinalização ATX-LPA foi relatado como envolvido na fibrose, inflamação e progressão do tumor, tornando ATX um alvo de drogas atraente. In vitro, HA155 - LOPAC-886 foi identificado como um inibidor à base de ácido borônico de ATX com base na estrutura cristalina de ATX em complexo com HA155. Além disso, as sínteses e atividades do HA155 - LOPAC-886 podem ser explicadas por dados estruturais. A fim de compreender melhor a diferença de atividade, realizaram-se experimentos de acoplamento molecular. O acoplamento molecular indicou uma forma de ligação notável para um dos isômeros, que diferia da forma de ligação original de HA155 - LOPAC-886 para ATX. Além disso, o aumento mediado pela trombina no LPA derivado de plaquetas foi completamente atenuado de maneira dose dependente por HA155 - LOPAC-886. HA155 - LOPAC-886 poderia inibir a autotaxina por ligação seletiva à sua treonina catalítica. O HA155 - LOPAC-886 mostrou bloquear de forma dose-dependente a secreção de LPA induzida por trombina nas plaquetas (Albers, H. M. H. G. et al, 2011 e Fulkerson Z et al, 2011) .  Autotaxin (ATX), a secreted phosphodiesterase, hydrolyzes the abundant phospholipid lysophosphatidylcholine (LPC) to produce lysophosphatidic acid (LPA). The ATX-LPA signaling axis has been reported as involved in fibrosis, inflammation and tumor progression, making ATX an attractive drug target. In vitro, HA155-LOPAC-886 was identified as a boronic acid-based inhibitor of ATX based on the crystal structure of ATX in complex with HA155. In addition, the syntheses and activities of HA155-LOPAC-886 can be explained by structural data. In order to better understand the difference in activity, molecular coupling experiments were performed. Molecular coupling indicated a remarkable binding form for one of the isomers, which differed from the original binding form of HA155-LOPAC-886 to ATX. In addition, the thrombin-mediated increase in platelet-derived LPA was completely attenuated in a dose dependent manner by HA155-LOPAC-886. HA155 - LOPAC-886 could inhibit autotaxin by selective binding to its catalytic threonine. HA155-LOPAC-886 has been shown to dose-dependently block platelet-mediated thrombin-induced LPA secretion (Albers, H. M. H. et al., 2011 and Fulkerson Z et al, 2011).

[0022] Conforme usado na presente invenção, o emprego do termo " farmaceuticamente aceitável" significa, essencialmente, ser não-tóxico ao indivíduo ao qual o material farmaceuticamente aceitável é administrado. As used in the present invention, the use of the term "pharmaceutically acceptable" essentially means being nontoxic to the subject to which the Pharmaceutically acceptable material is administered.

[0023] Ainda conforme usado na presente invenção, o emprego do termo "veiculo farmaceuticamente aceitável" significa um sólido não-tóxico, inerte, excipiente liquido semi-sólido, diluente, formulação auxiliar de qualquer tipo, tal como solução salina, exceto água. Alguns exemplos dos materiais que podem servir como veículos farmaceuticamente aceitáveis são açúcares, tais como lactose, glicose e sacarose, os amidos, tais como amido de milho e o amido de batata, a celulose e os seus derivados, tais como a carboximetilcelulose de sódio, a etilcelulose e o acetato de celulose, ciclodextrina; óleos, tais como óleo de amendoim, óleo de semente de algodão, óleo de girassol, óleo de gergelim, óleo de oliva, óleo de milho e óleo de semente de soja; glicóis, tais como propilenoglicol , polióleos, tais como glicerinaglicol , sorbitol, manitol e de polietileno; ésteres, tais como o laurato etílico, oleato etílico, ágar; agentes tamponantes, tais como o hidróxido de alumínio e hidróxido de magnésio; ácido algínico; salina isotônica, solução de Ringer; soluções tampões de álcool etílico e fosfato, emulsão oleosa em água contendo mycobacteria morta pela ação do calor ou componentes de sua parede celular (adjuvante completo de Freund) , DMSO, assim como outras substâncias não tóxicas compatíveis usadas em formulações farmacêuticas .  As used in the present invention, the use of the term "pharmaceutically acceptable carrier" means a nontoxic, inert solid, semisolid liquid excipient, diluent, auxiliary formulation of any type, such as saline, except water. Some examples of the materials that may serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, cyclodextrin; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols, such as glycerinaglycol, sorbitol, mannitol and polyethylene; esters, such as ethyl laurate, ethyl oleate, agar; buffering agents, such as aluminum hydroxide and magnesium hydroxide; alginic acid; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, oily emulsion in water containing mycobacteria killed by the action of heat or components of its cell wall (Freund's complete adjuvant), DMSO, as well as other compatible non-toxic substances used in pharmaceutical formulations.

[0024] Ainda, "vírus da família Flaviviridae" é utilizado neste pedido de patente como sinónimo de "flavivírus" e " flavivirose" como sinónimo de "infecção causada por vírus da família Flaviviridae".  Further, "Flaviviridae family virus" is used in this application as a synonym for "flavivirus" and "flavivirose" as synonymous with "infection caused by Flaviviridae family viruses".

Uso da composição para a produção de um medicamento para o tratamento de infecção causada por vírus da família Flaviviridae Use of the composition for the production of a medicament for the treatment of infection caused by the family virus Flaviviridae

[0025] Em uma segunda concretização, a presente invenção se refere ao uso da composição como definida anteriormente para a produção de um medicamento para o tratamento de infecção causada por vírus da família Flaviviridae. Em outra concretização, a infecção causada por vírus da família Flaviviridae incluem vírus selecionados do grupo que consiste de: vírus do Nilo Ocidental, vírus da dengue, vírus Zika, vírus da encefalite transmitido por carrapato, vírus da encefalite de St. Louis, hepatite C e vírus da febre amarela. Em outra concretização, a infecção causada por vírus da família Flaviviridae é ocasionada pelo vírus da febre amarela.  In a second embodiment, the present invention relates to the use of the composition as defined above for the manufacture of a medicament for the treatment of infection caused by Flaviviridae family viruses. In another embodiment, the virus infection of the Flaviviridae family includes viruses selected from the group consisting of: West Nile virus, dengue virus, Zika virus, tick-borne encephalitis virus, St. Louis encephalitis virus, hepatitis C virus and yellow fever viruses. In another embodiment, the virus infection caused by the Flaviviridae family is caused by the yellow fever virus.

EXEMPLOS  EXAMPLES

Compostos  Compounds

[0026] O composto de referência Interferon alpha 2A foi obtido de Sigma-Aldrich . A biblioteca LOPAC1280 contendo 1280 amostras foi adquirida da Sigma-Aldrich.  The reference compound Interferon alpha 2A was obtained from Sigma-Aldrich. The LOPAC1280 library containing 1280 samples was purchased from Sigma-Aldrich.

Células  Cells

[0027] Huh7 A linha celular Huh7 foi derivada de um carcinoma hepato celular humano. As células formam uma monocamada contígua que pode ser mantida por várias semanas. Este estoque foi adquirido na Coleção Japonesa de Bioressources de Pesquisa (JCRB) . Esta linhagem celular foi mantida em meio DMEM F12 (Sigma-Aldrich) , suplementado com 10% de FBS (Sigma Aldrich) , 100 U/ml de penicilina (GIBCO) e 100 pg/ml de estreptomicina (GIBCO) , doravante descrito como "DMEM F12".  Huh7 The Huh7 cell line was derived from a human hepatic cell carcinoma. The cells form a contiguous monolayer that can be maintained for several weeks. This inventory was purchased from the Japanese Bioressources Research Collection (JCRB). This cell line was maintained in DMEM F12 medium (Sigma-Aldrich), supplemented with 10% FBS (Sigma Aldrich), 100 U / ml penicillin (GIBCO) and 100æg / ml streptomycin (GIBCO), hereinafter described as " DMEM F12 ".

[0028] C6/36: A linhagem celular C6/36 foi derivada de larvas incubadas de Aedes albopictus. Células C6/36 exibem morfologia epitelial aderente. Este estoque foi cedido pelo Prof. Dr . Amilcar Tanuri, da Universidade Federal do Rio de Janeiro. Esta linhagem celular foi mantida em meio L15 ( Sigma-Aldrich) , suplementado com 10% de FBS[0028] C6 / 36: The C6 / 36 cell line was derived from incubated larvae of Aedes albopictus. C6 / 36 cells exhibit adherent epithelial morphology. This inventory was provided by Prof. Dr. Amilcar Tanuri, from the Federal University of Rio de Janeiro. This cell line was maintained in L15 medium (Sigma-Aldrich), supplemented with 10% FBS

(Sigma Aldrich) , 100 U/ml de penicilina (GIBCO) e 100 pg/ml de estreptomicina (GIBCO), doravante descrito como "L15". (Sigma Aldrich), 100 U / ml penicillin (GIBCO) and 100 pg / ml streptomycin (GIBCO), hereinafter described as "L15".

[0029] Vírus da Febre Amarela : vacina contra o vírus da febre amarela atenuado (YFV) 17D que expressa a proteína repórter fluorescente amarela (YFP) (YFV-YFP) foi doada por Laura Gil (Centro de Pesquisa Aggeu Magalhães, Fiocruz, Pernambuco, Brasil) . Os vírus da febre amarela foram obtidos a partir do sobrenadante de culturas de tecido C6/36 infectadas com o vírus . As culturas infectadas foram mantidas em meio L15 e congeladas em alíquotas de 250 pL . Preparação da solução composta  Yellow fever virus: YDV-YD virus (YFV) 17D virus expressing the yellow fluorescent reporter protein (YFP) (YFV-YFP) was donated by Laura Gil (Aggeu Magalhães Research Center, Fiocruz, Pernambuco , Brazil) . Yellow fever viruses were obtained from the supernatant of C6 / 36 tissue cultures infected with the virus. Infected cultures were maintained in L15 medium and frozen in 250 pL aliquots. Preparation of the compound solution

[0030] A biblioteca de amostras foi enviada em placas de 96 poços (25 pL/poço) a uma concentração estoque de 10 mM em 100% de sulfóxido de dimetila (DMSO) . As amostras foram transferidas manualmente para placas estoques de polipropileno de 384 poços (placas Mãe) (Greiner Bio-One) . Estas placas foram utilizadas para preparar novas placas em menor concentração. As amostras foram transferidas manualmente para novas placas de polipropileno de 384 poços (10 pL/poço) a uma concentração final de 1 mM em 100% de DMSO. Antes de executar o ensaio, placas intermediárias foram preparadas. As amostras foram diluídas na solução de tampão salina (PBS IX) até uma concentração final de 600 pM de composto e 6% de DMSO. Os hits selecionados foram coletados da placa mãe de 384 poços.  The sample library was sent into 96-well plates (25 μl / well) at a stock concentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Samples were transferred manually to 384 well polypropylene stock plates (Mother plates) (Greiner Bio-One). These plates were used to prepare new plates in a lower concentration. Samples were transferred manually to new 384-well polypropylene plates (10 μl / well) at a final concentration of 1 mM in 100% DMSO. Prior to performing the assay, intermediate plates were prepared. Samples were diluted in the buffered saline solution (PBS IX) to a final concentration of 600 pM compound and 6% DMSO. The selected hits were collected from the 384-well motherboard.

Ensaios de atividade antiviral  Antiviral activity assays

[0031] HCS permite a avaliação de amostras com atividade antiviral contra células Huh7 infectadas. Na triagem primária, 10 pL de cada amostra foram transferidas manualmente de placas intermediárias (Greiner Bio-One) para placas de ensaio de poliestireno preto de 384 poços (Greiner Bio-One) por meio de uma pipeta multicanal que produz uma concentração final de 10 mM com 1,0% de DMSO (diluição de 100 vezes) . Em seguida, as infecções foram realizadas com 3.000 células Huh7/poço co-plaqueadas com MOI 2,5 de YFP- YFP, todas ressuspendidas em DMEM F12 e dispensadas com a ajuda de uma pipeta multicanal. As placas foram incubadas durante 72 h a 37 °C / 5% de C02 sob atmosfera umidifiçada. Todas as placas continham o seguinte conjunto de poços controle: células Huh7 não infectadas, células infectadas tratadas com 6,25 ng/mL de Interferon alfa 2A e células infectadas tratadas com veiculo (1% DMSO, v/v) . O ensaio primário foi realizado em duplicata (isto é, dois experimentos independentes) . Na seleção confirmatória, as amostras selecionadas (ver abaixo) foram testadas em dose- resposta. O ensaio foi realizado da mesma forma que o primário. Os compostos foram diluídos em série por um fator de 2 (ou seja, em diluições seriadas de 2 vezes) em DMSO puro em 10 pontos de diluição em uma placa separada de polipropileno de 384 poços (Greiner BioOne) usando uma pipeta manual de 16 canais equipada com pontas descartáveis (ThermoScientific) . Para todas as amostras testadas, a concentração mais alta começou a 100 mM, exceto para Interferon alfa 2A, que começou a 6,25 ng/mL. Dez microlitros de solução composta foram transferidos para placas de ensaio, produzindo uma concentração final de 1% de DMSO (v/v) . Os controles descritos na seleção primária foram utilizados para este ensaio de confirmação. Os ensaios de confirmação foram realizados em duplicata (isto é, dois experimentos independentes) . HCS allows the evaluation of samples having antiviral activity against infected Huh7 cells. At 10 μl of each sample were manually transferred from Greiner Bio-One plates to 384-well black polystyrene assay plates (Greiner Bio-One) by means of a multichannel pipette which yields a final concentration of 10 mM with 1.0% DMSO (100 fold dilution). Infections were then performed with 3,000 Huh7 / well cells co-plated with MOI 2.5 of YFP-YFP, all resuspended in F12 DMEM and dispensed with the aid of a multichannel pipette. The plates were incubated for 72 h at 37øC / 5% CO 2 under a humidified atmosphere. All plates contained the following set of control wells: uninfected Huh7 cells, infected cells treated with 6.25 ng / ml Interferon alfa 2A and infected cells treated with vehicle (1% DMSO, v / v). The primary assay was performed in duplicate (i.e., two independent experiments). At confirmatory selection, the selected samples (see below) were tested in dose-response. The assay was performed in the same manner as the primer. Compounds were serially diluted by a factor of 2 (i.e., serial 2-fold dilutions) into neat DMSO at 10 dilution points in a separate 384-well polypropylene plate (Greiner BioOne) using a 16-channel manual pipette equipped with disposable tips (ThermoScientific). For all samples tested, the highest concentration started at 100 mM, except for Interferon alfa 2A, which started at 6.25 ng / mL. Ten microliters of composite solution was transferred to assay plates, yielding a final concentration of 1% DMSO (v / v). The controls described in the primary selection were used for this confirmatory assay. The confirmatory assays were performed in duplicate (ie two experiments).

[0032] No estágio final do ensaio, para as seleções primária e confirmatória, as placas foram fixadas com 4% de paraformaldeido (PFA) , coradas com DAPI ( Sigma-Aldrich) por 30 minutos no escuro à temperatura ambiente e quatro imagens de cada poço foram adquiridas no High Content Imaging System Operetta (PerkinElmer) com ampliação de 20X. As imagens foram analisadas pelo software de análise de alto conteúdo (HCA) Harmony (PerkinElmer) para identificação, segmentação e quantificação do núcleo da célula hospedeira, citoplasma e parasitas intracelulares (Figura 1) . O HCA forneceu como dados de saida para todas as imagens de um poço o número total de células, número total de células infectadas e intensidade de YFP de células infectadas. A taxa de infectadas para o número total de células foi então calculada e definida como a Taxa de Infecção (IR) . Os dados brutos para valores de IR foram normalizados para 1% de células infectadas tratadas com DMSO (controle negativo) e células não infectadas (controle positivo) para determinar a atividade antiviral normalizada, de acordo com a seguinte fórmula :  In the final stage of the assay, for the primary and confirmatory selections, the plates were fixed with 4% paraformaldehyde (PFA), stained with DAPI (Sigma-Aldrich) for 30 minutes in the dark at room temperature and four images of each well were purchased from the High Content Imaging System Operetta (PerkinElmer) with 20X magnification. The images were analyzed by Harmony's high content analysis software (HCA) (PerkinElmer) for identification, segmentation and quantification of the host cell nucleus, cytoplasm and intracellular parasites (Figure 1). The HCA provided as output data for all images from one well the total number of cells, total number of infected cells and YFP intensity of infected cells. The infected rate for the total number of cells was then calculated and defined as the Infection Rate (IR). The crude data for IR values were normalized to 1% of infected cells treated with DMSO (negative control) and uninfected cells (positive control) to determine the standardized antiviral activity, according to the following formula:

Atividade Norrralizacfe ( A) = [1- (ftv. H¾ - A/. P¾) / (ftv. H¾ - A/. P¾>) ] x 100Activity Norrralizacfe (A) = [1- (ft H¾-A / P¾) / (ft H¾-A / P¾ > )] x 100

Onde: Av. IRN: taxa de infecção média de poços de controle negativo. Where: Av. IR N : mean infection rate of negative control wells.

Av. IRp: taxa de infecção média de poços de controle positivo .  Av. IRp: mean infection rate of positive control wells.

Av. IRT: taxa de infecção média de poços do composto teste (em uma dada concentração) . Av. IR T : mean infection rate of test compound wells (at a given concentration).

[0033] O controle do ensaio de qualidade foi medido pelo fator Z'; este fator estatístico foi calculado para cada placa, considerando a média e o desvio padrão dos valores da taxa de infecção (IR) dos controles positivos (mock - infectados) e negativos (DMSO puro tratado) - de acordo com a seguinte fórmula (ii) : Control of the quality assay was measured by factor Z '; This statistical factor was calculated for each plate, considering the mean and standard deviation of the positive (mock - infected) and negative (pure treated DMSO) infection rate (IR) values - according to the following formula (ii):

(ii) Z' = 1 (Sdv.IRp + Sdv.n¾) / | (An.P¾> - An.P¾) |] (ii) Z '= 1 (Sdv.IRp + Sdv.n¾) / | (An.P¾ > - An.P¾) |]

Onde: Sdv.IRp : desvio padrão da taxa de infecção do controle positivo  Where: Sdv.IRp: standard deviation of the infection rate of the positive control

Sdv.IRN: desvio padrão da taxa de infecção do controle negativo Sdv.IR N : standard deviation of infection rate from negative control

Av.IRp: média da taxa de infecção do controle positivo AV.IRN: média da taxa de infecção do controle negativo Av.IRp: mean of infection rate of positive control AV.IR N : mean of infection rate of negative control

[0034] As placas que apresentaram fator Z'³ 0,5 foram consideradas aprovadas, pois indica uma separação satisfatória entre os controles de ensaio negativo e positivo .  Plaques showing Z'³ 0.5 factor were considered approved because it indicates a satisfactory separation between the negative and positive assay controls.

[0035] As leituras de análise de triagem primária foram processadas usando o Microsoft Excel e o Tibco Sporfire. Em relação aos dados de triagem secundária, os valores de atividade normalizados foram processados com o software Graphpad Prism, versão 6, para geração de ajuste de curva não-linear de dose-resposta sigmoidal (inclinação variável) e determinação de valores EC50 por interpolação. Para o propósito deste estudo, EC50 foi definido como a concentração do composto correspondente a 50% de atividade normalizada após 72 h de incubação do composto. A potência refere-se ao EC50 - quanto mais potente o composto, menor é seu EC50 - enquanto a eficácia se relaciona com a atividade máxima observada de um composto (em %) - quanto mais eficaz o composto, mais próxima é a sua atividade máxima de 100%. O mesmo racional é usado para a definição CC50: a concentração do composto, que reduz a taxa celular em 50%, quando comparada à média de controle negativo. O índice de seletividade (SI) foi calculado com base na taxa entre o valor composto EC50 e seu CC50 (SI = EC50/CC50) , sempre que a análise de dados dos compostos não gera valores de CC50 (porque não era tóxico nas concentrações testadas) a mais alta concentração testada é usada para estimar o SI (SI = Max [] testado / EC50) . Primary screening screens were processed using Microsoft Excel and Tibco Sporfire. Regarding the secondary screening data, the normalized activity values were processed with Graphpad Prism software, version 6, for generating a non-linear sigmoidal dose-response curve (variable slope) adjustment and EC50 values determination by interpolation. For the purpose of this study, EC 50 was defined as the concentration of the compound corresponding to 50% normalized activity after 72 h incubation of the compound. Power refers to the EC50 - the more potent the compound, the lower its EC50 - while the efficacy is related to the maximum observed activity of a compound (in%) - the more effective the compound, the closer its maximum activity of 100%. The same rationale is used for the CC50 definition: the compound concentration, which reduces the cell rate by 50%, when compared to the mean of negative control. The index of selectivity (SI) was calculated based on the rate between the EC50 compound value and its CC50 (SI = EC50 / CC50), where data analysis of the compounds did not generate CC50 values (because it was non-toxic at the concentrations tested). high concentration tested is used to estimate SI (SI = Max [] tested / EC 50 ).

Exemplo 1 :  Example 1:

[0036] A biblioteca LOPAC1280 foi rastreada a uma diluição de 100 vezes, e as placas de ensaio foram consideradas aprovadas com fator Z'³ 0,5; o fator Z' médio do ensaio foi de 0,84 ± 0,06. Os valores de taxa celular de todas as amostras testadas na biblioteca revelaram que apenas 9, 61% apresentaram toxicidade celular hospedeira superior a 50% na concentração testada (Figura 2) . Além disso, os hits primários foram selecionados como as melhores vinte e oito amostras mais ativas produzindo uma toxicidade celular hospedeira £ 20% e atividade normalizada igual ou aproximadamente 70%, à medida que as imagens individuais dos poços testados foram investigadas e confirmou a redução da taxa de infecção. A seleção de amostras na triagem primária resultou em uma taxa de hit global de 2,18% (Figura 2) . Os resultados estimulam o processo com os ensaios posteriores, uma vez que a concentração testada para os hits na triagem primária é considerada baixa (10 mM) . Foram observados diferentes perfis de toxicidade de células hospedeiras e antivirais a partir de amostras hit e controles (Figura 3) . O rastreio secundário foi realizado utilizando uma abordagem de dose-resposta de 10 pontos com 72 h de exposição da amostra para determinar a atividade antiviral de hits selecionados, tais como: valor EC50, atividade máxima (MA) e citotoxicidade contra células hospedeiras, como mostrado pelo valor CC50. A seletividade das amostras é mostrada pelo índice de seletividade (SI) (Tabela 1) . Um critério de confirmação de hit foi estabelecido da seguinte forma: pelo menos um ponto de atividade normalizada igual ou superior a 70% no gráfico de ajuste de curva gerado e um valor de índice de seletividade igual ou superior a 1,0 (Tabela 1 e Figura 4) . Apenas vinte e seis dos vinte e oito hits preencheram este critério na triagem confirmatória (92,85% do hit de confirmação) e três amostras apresentaram resultados prósperos, uma delas o composto utilizado na composição do presente pedido, o LOPAC-886 (EC50: 2,51 mM; MA: 99,48%) . Em amostras de 10 mM, o composto LOPAC-886 apresentou taxa celular de 0,96 e atividade normalizada de 97,05%. Os outros hits confirmados apresentaram EC50 moderado a alto. The LOPAC1280 library was screened at a 100-fold dilution, and the assay plates were considered approved with factor Z'³0.5; the mean Z factor of the assay was 0.84 ± 0.06. The cell rate values of all samples tested in the library revealed that only 9.61% had host cell toxicity greater than 50% at the concentration tested (Figure 2). In addition, the primary hits were selected as the best twenty-eight more active samples yielding a host cell toxicity of 20% and normalized activity equal to or approximately 70% as the individual images of the wells tested were confirmed and confirmed the reduction of rate of infection. Selection of samples at primary screening resulted in a global hit rate of 2.18% (Figure 2). The results stimulate the process with subsequent trials, since the concentration tested for hits on primary screening is considered low (10 mM). Different toxicity profiles of host and antiviral cells were observed from hit samples and controls (Figure 3). Secondary screening was performed using a 10-point dose-response approach with 72 h of sample exposure to determine the antiviral activity of selected hits, such as: EC 50 value, maximal activity (MA) and cytotoxicity against host cells as shown by the value CC50. The selectivity of the samples is shown by the selectivity index (SI) (Table 1). A hit confirmation criterion was established as follows: at least one normalized activity point equal to or greater than 70% in the generated curve fit graph and a selectivity index value equal to or greater than 1.0 (Table 1 and Figure 4). Only twenty-six of the twenty-eight hits fulfilled this criterion in confirmatory triage (92.85% of confirmatory hit) and three samples showed successful results, one of which was the compound used in the composition of the present application, LOPAC-886 (EC50: 2.51 mM, MA: 99.48%). In 10 mM samples, LOPAC-886 showed a cell rate of 0.96 and normalized activity of 97.05%. The other confirmed hits showed moderate to high EC50.

Tabela 1. Dados de triagem de confirmação: valor EC50 (em pg/mL) , valor CC50 (em pg/mL) , atividade máxima (em %) e índice de seletividade (SI) .  Table 1. Confirmation screening data: EC50 value (in pg / mL), CC50 value (in pg / mL), maximal activity (in%) and selectivity index (SI).

ECso CC50 Atividade  ECso CC50 Activity

índice de index of

Amostra (pM ou (pM ou Máxima Sample (pM or (pM or Maximum

seletividade ng/mL) ng/mL) (%)  selectivity ng / mL) ng / mL) (%)

Interferon  Interferon

0.39 ng/mL ND 100.2 > 253.9 a 2A  0.39 ng / mL ND 100.2> 253.9 to 2A

LOPAC-886 2 . 5 pM 59.4 pM 99.5 23.6  LOPAC-886 2. 5 pM 59.4 pM 99.5 23.6

Dados médios de duas repetições. índice de seletividade (SI) : taxa do valor CC50 pelo valor EC50 (CC50/EC50) . Sempre que o CC50 não é gerado por ajuste de curva, a concentração mais alta testada é usada para estimar o SI (Max [] testado/ECso) . Sempre que o EC50 não é gerado por ajuste de curva, a menor concentração testada é usada para estimar o SI (CC50/Min [] testado) . A atividade máxima foi obtida a partir do ponto de valor mais alto pela curva de regressão não-linear. Average data of two replicates. selectivity index (SI): rate of CC 50 value by EC 50 value (CC 50 / EC 50 ). Whenever the CC 50 is not generated by curve fitting, the highest concentration tested is used to estimate the SI (Max [] tested / ECso). Whenever the EC 50 is not generated by curve fitting, the lowest concentration tested is used to estimate the SI (CC 50 / Min [] tested). The maximum activity was obtained from the value by the nonlinear regression curve.

Referências References

[0037] Albers, H. M. H. G. et al . Structure-Based Design of Novel Boronic Acid-Based Inhibitors of Autotaxin. J. Med. Chem 54, 4619-4626 (2011) .  [0037] Albers, H.M.H.G. et al. Structure-Based Design of Novel Boronic Acid-Based Inhibitors of Autotaxin. J. Med. Chem. 54, 4619-4626 (2011).

[0038] Barrett A. D., Higgs S. Yellow fever: A disease that has yet to be conquered. Ann. Rev. Entomol. 52:209-229 (2007) .  [0038] Barrett A.D., Higgs S. Yellow fever: A disease that has yet to be conquered. Ann. Rev. Entomol. 52: 209-229 (2007).

[0039] Barrett A. D., Monath T.P. Epidemiology and ecology of yellow fever vírus. Adv. Vírus Res. 61:291-315 (2003) .  [0039] Barrett A.D., Monath T.P. Epidemiology and ecology of yellow fever virus. Adv. Virus Res. 61: 291-315 (2003).

[0040] Fulkerson, Z. et al . Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells *. (2011) . doi : 10.1074/jbc.Ml 11.276725  Fulkerson, Z. et al. Binding of Autotaxin to Integrins Localizes Lysophosphatidic Acid Production to Platelets and Mammalian Cells *. (2011). doi: 10.1074 / jbc.m1 11.276725

[0041] Gubler D.J. The changing epidemiology of yellow fever and dengue, 1900 to 2003: Full circle? Comp . Immunol. Microbiol. Infect. Dis. 27:319-330 (2004).  [0041] Gubler D.J. The changing epidemiology of yellow fever and dengue, 1900 to 2003: Full circle? Comp. Immunol. Microbiol. Infect. Dis. 27: 319-330 (2004).

[0042] Hausmann, J. et al . Structural basis for substrate discrimination and integrin binding by autotaxin. Nat. Struct. Mol . Blol. 18, 198-204 (2011) .  Hausmann, J. et al. Structural basis for substrate discrimination and integrin binding by autotaxin. Nat. Struct. Mol. Blol. 18, 198-204 (2011).

[0043] Lindenbach B.D., Rice C.M. Molecular biology of flaviviruses . Adv. Vírus Res. 59:23-61 (2003) .  [0043] Lindenbach B.D., Rice C.M. Molecular biology of flaviviruses. Adv. Virus Res. 59: 23-61 (2003).

Claims

RE INVIDICAÇÕES RE INVIDITIONS 1. Composição farmacêutica para o tratamento de infecção causada por vírus da família Flaviviridae caracterizada pelo fato de que compreende o composto como definido pela fórmula (I)  A pharmaceutical composition for the treatment of infection caused by viruses of the Flaviviridae family characterized in that it comprises the compound as defined by formula (I)
Figure imgf000019_0001
ou um sal farmaceuticamente aceitável do mesmo, e um veículo farmaceuticamente aceitável.
Figure imgf000019_0001
or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. 2. Composição farmacêutica, de acordo com a reivindicação 1, caracterizada pelo fato de que o composto definido pela fórmula (I) pode ser utilizado na concentração de 12,5 a 25 mM .  A pharmaceutical composition according to claim 1, characterized in that the compound defined by formula (I) can be used in the concentration of 12.5 to 25 mM. 3. Composição farmacêutica, de acordo com a reivindicação 2, caracterizada pelo fato de que o composto definido pela fórmula (I) é utilizado preferencialmente na concentração de 12,5 mM.  A pharmaceutical composition according to claim 2, characterized in that the compound defined by formula (I) is preferably used in the concentration of 12.5 mM. 4. Uso da composição como definida em qualquer uma das reivindicações 1 a 3 caracterizado pelo fato de ser na produção de um medicamento para o tratamento de infecção causada por vírus da família Flaviviridae.  Use of the composition as defined in any one of claims 1 to 3, characterized in that it is in the manufacture of a medicament for the treatment of infection caused by viruses of the Flaviviridae family. 5. Uso, de acordo com a reivindicação 4, caracterizado pelo fato de que a infecção causada por vírus da família Flaviviridae incluem vírus selecionados do grupo que consiste de: vírus do Nilo Ocidental, vírus da dengue, vírus Zika, vírus da encefalite transmitido por carrapato, vírus da encefalite de St. Louis, hepatite C e virus da febre amarela . Use according to claim 4, characterized in that the virus infection caused by the Flaviviridae family includes viruses selected from the group consisting of: West Nile virus, dengue virus, Zika virus, encephalitis virus transmitted by tick, virus of St. Louis encephalitis, hepatitis C and yellow fever virus. 6. Uso, de acordo com a reivindicação 5, caracterizado pelo fato de que a infecção causada por virus da família Flaviviridae é ocasionada pelo vírus da febre amarela.  Use according to claim 5, characterized in that the virus infection caused by the Flaviviridae family is caused by the yellow fever virus.
PCT/BR2018/050489 2017-12-29 2018-12-28 Pharmaceutical composition for treating infection caused by viruses from the flaviviridae family and use of the composition Ceased WO2019126860A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BRBR1020170286231 2017-12-29
BR102017028623-1A BR102017028623B1 (en) 2017-12-29 PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF INFECTION CAUSED BY VIRUSES OF THE FLAVIVIRIDAE FAMILY, AND USE OF THE COMPOSITION

Publications (1)

Publication Number Publication Date
WO2019126860A1 true WO2019126860A1 (en) 2019-07-04

Family

ID=67062764

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/BR2018/050489 Ceased WO2019126860A1 (en) 2017-12-29 2018-12-28 Pharmaceutical composition for treating infection caused by viruses from the flaviviridae family and use of the composition

Country Status (1)

Country Link
WO (1) WO2019126860A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012024620A2 (en) * 2010-08-20 2012-02-23 Amira Pharmaceuticals, Inc. Autotaxin inhibitors and uses thereof
EP2203168B1 (en) * 2007-09-18 2014-07-16 Stanford University Compositions for treating a flaviviridae family viral infection

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2203168B1 (en) * 2007-09-18 2014-07-16 Stanford University Compositions for treating a flaviviridae family viral infection
WO2012024620A2 (en) * 2010-08-20 2012-02-23 Amira Pharmaceuticals, Inc. Autotaxin inhibitors and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALBERS, H.M.H.G. ET AL.: "Structure-Based Design of Novel Boronic Acid-Based Inhibitors of Autotaxin", J. MED. CHEM., vol. 54, 26 May 2011 (2011-05-26), pages 4619 - 4626, XP055078298, doi:10.1021/jm200310q *
NITSCHE, C. ET AL.: "Peptide-Boronic Acid Inhibitors of Flaviviral Proteases: Medicinal Chemistry and Structural Biology", J. MED. CHEM., vol. 60, no. 1, 2017, pages 511 - 516, XP055622746 *

Also Published As

Publication number Publication date
BR102017028623A2 (en) 2019-07-16

Similar Documents

Publication Publication Date Title
Butts et al. A repurposing approach identifies off-patent drugs with fungicidal cryptococcal activity, a common structural chemotype, and pharmacological properties relevant to the treatment of cryptococcosis
Chen et al. Cinnamic acid inhibits Zika virus by inhibiting RdRp activity
De Moraes et al. In vitro synergistic interaction between amide piplartine and antimicrobial peptide dermaseptin against Schistosoma mansoni schistosomula and adult worms
Li et al. Methylene blue is a potent and broad-spectrum inhibitor against Zika virus in vitro and in vivo
KR20120049852A (en) Treatment and prevention of dengue virus infections
EP2349257B1 (en) Use of paf receptor for treating infections caused by flaviviridae
TW202143957A (en) Composition for preventing or treating sars coronavirus 2 infection disease
Yang et al. In vitro and in vivo antiviral activity of Maqian (Zanthoxylum myriacanthum var. pubescens) essential oil and its major constituents against strains of influenza virus
JP2023534077A (en) HERBAL COMPOSITION, METHOD FOR PRODUCING THE SAME, AND METHOD FOR PREVENTION OR TREATMENT OF VIRUS INFECTION BY ADMINISTRING THE SAME
Mir et al. Management of thrombocytopenia and flu-like symptoms in dengue patients with herbal water of Euphorbia hirta
CN110755436A (en) Medicine for preventing Zika virus infection and application of lycorine in preparation of medicine for preventing Zika virus infection
JP2023537229A (en) Interaction of SARS-CoV-2 protein with host cell molecular and cellular machinery and formulations for treating COVID-19
Chiu et al. Aquatic birnavirus capsid protein, VP3, induces apoptosis via the Bad-mediated mitochondria pathway in fish and mouse cells
CN117320748A (en) Inhibition of copy of COVID-19 by COVID-19 entry inhibitors
WO2019126860A1 (en) Pharmaceutical composition for treating infection caused by viruses from the flaviviridae family and use of the composition
BR102017028623B1 (en) PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF INFECTION CAUSED BY VIRUSES OF THE FLAVIVIRIDAE FAMILY, AND USE OF THE COMPOSITION
TWI605822B (en) Uses of an avocado extract, avocadenol b, and (2r,4r)-1,2,4-trihydroxyheptadec-16-yne, and health food containing the avocado extract
CN114748481B (en) Application of trorostat hippurate in the preparation of drugs against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection
CN114558022B (en) Application of brincidofovir in the preparation of anti-pseudorabies virus drugs
TW202227065A (en) Use of thioimidazolidinone drugs in the treatment of covid-19 disease
Zhou et al. The in vitro and in vivo antiviral effects of aloperine against Zika virus infection
CN114668763A (en) Application of etravirine in the preparation of drugs against tick-borne encephalitis virus, West Nile virus, yellow fever virus and chikungunya virus infection
Schieffelin Treatment of arenavirus infections
Chen et al. Tolvaptan, an FDA-approved drug, inhibits Zika virus infection both in vitro and in vivo.
Aktar et al. Recent Advances in Dengue, Viral Hepatitis, and HIV Infections and Related Management Strategies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18893816

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18893816

Country of ref document: EP

Kind code of ref document: A1