WO2013059896A1 - Use of pyranonaphthoquinone as an anti-viral agent; pharmaceutical composition containing pyranonaphthoquinones; medicament containing pyranonaphthoquinones for treating infections by dengue virus - Google Patents
Use of pyranonaphthoquinone as an anti-viral agent; pharmaceutical composition containing pyranonaphthoquinones; medicament containing pyranonaphthoquinones for treating infections by dengue virus Download PDFInfo
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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
- A61K31/33—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
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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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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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
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- 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
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- 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
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- 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 pharmaceutical composition described herein is used for the preparation of a medicament for the treatment of infections in human and non-human mammalian animals caused by viruses, the latter being of the flaviviridae family, preferably dengue virus and its different serotypes.
- This drug presents the pyranonaphthoquinones, already described above, may also have their derivatives or isomers or their salts.
- VERO cells were placed in contact with dengue virus stocks, in a ratio of one infectious viral particle to 10. cells for one hour for adsorption and entry of viral particles into cells. After this time the cells were washed with saline and culture medium containing 100 ⁇ of each compound and 200 ⁇ of Ribavirin as positive control. These cultures were incubated at 37 ° C for 72 h. After this time an aliquot of the cell free medium (supernatant) was collected and processed for RNA extraction, followed by reverse transcription and Real-Time PCR reaction using dengue-specific primers and probe type 2.
Abstract
The invention describes the use of pyranonaphthoquinones, the derivatives, isomers and salts thereof as anti-viral agents for inhibiting viral ATPase enzymes. Moreover, the invention also includes a pharmaceutical composition containing the pyranonaphthoquinones described.
Description
USO DAS PIRANONAFTOQUINONA COMO ANTIVIRAL; COMPOSIÇÃO FARMACÊUTICA CONTENDO AS PIRANONAFTOQUINONAS; MEDICAMENTO CONTENDO AS PIRANONAFTOQUINONAS PARA TRATAMENTO DE INFECÇÕES CAUSADAS POR VÍRUS DA DENGUE Use of pyranonaphthoquinone as an antiviral; PHARMACEUTICAL COMPOSITION CONTAINING PYRANONAFTOKINONES; MEDICINAL PRODUCT CONTAINING PYRANONAFTOKINONES FOR TREATMENT OF DENGUE VIRUS INFECTIONS
Campo da Invenção Field of the Invention
A presente invenção refere-se ao uso de compostos piranonaftoquinônicos incluídos nas famílias de compostos das quinonas, seus sais, suas formas esteriosoméricas e suas misturas racêmicas como um agente antiviral. The present invention relates to the use of pyranonaphthoquinonic compounds included in the families of quinone compounds, their salts, their steriosomeric forms and their racemic mixtures as an antiviral agent.
A invenção destina-se a área médica por tratar-se de uma composição farmacêutica contento os compostos piranonaftoquinônicos e o uso desta composição no tratamento de inflamações causadas por agentes virais em animais mamíferos humanos e não humanos. The invention is intended for the medical field as it is a pharmaceutical composition containing the pyranonaphthoquinonic compounds and the use of this composition in the treatment of inflammation caused by viral agents in human and non-human mammalian animals.
Antecedentes da Invenção Background of the Invention
As piranonaftoquinonas, um grupo de moléculas incluídas na família das quinonas, podem ser de origem natural ou sintética com múltiplas propriedades biológicas. Na natureza suas fontes mais frequentes são plantas superiores, artrópodes, fungos, liquens, bactérias, algas e vírus, conforme descrito por Pyranonaphthoquinones, a group of molecules included in the quinone family, may be of natural or synthetic origin with multiple biological properties. In nature their most frequent sources are higher plants, arthropods, fungi, lichens, bacteria, algae and viruses, as described by
Thompson, R. U.(Naturally Occuring Quinones IV: Recents Advances; Champman & Hall, Londres, 1997), com funções biológicas múltiplas nos mais diversos ciclos metabólicos destes organismos {Barreiro, E. J; da Silva, J. E. M.; Fraga, C. A. M.; Noções Básicas do Metabolismo dos Fármacos; Quím. Nova 1996, 19, 641). Um grande número de quinonas naturais possui aplicações biológicas práticas, algumas chegaram à produção industrial, como por exemplo, as vitaminas do tipo K, mitomicinas e antraciclinas, entre outras conforme descrito (M. N. da Silva, V. F. Ferreira, M. C. B. V. de Souza; Um panorama atual da química e da farmacologia de naftoquinonas, com ênfase na β-lapachona e derivados; Quim. Nova 2003, 26, 407). Thompson, R. U. (Naturally Occuring Quinones IV: Recents Advances; Champman & Hall, London, 1997), with multiple biological functions in the various metabolic cycles of these organisms {Barreiro, E. J; da Silva, J. E. M .; Fraga, C.A. M .; Basics of Drug Metabolism; Chem. Nova 1996, 19, 641). A large number of natural quinones have practical biological applications, some have reached industrial production, such as K-type vitamins, mitomycins and anthracyclines, among others as described (MN da Silva, VF Ferreira, MCBV de Souza; chemistry and pharmacology of naphthoquinones, with emphasis on β-lapachone and derivatives; Quim Nova 2003, 26, 407).
Algumas quinonas, como as do grupo da β-lapachona, são bons agentes bacterianos e antifúngicos como são relatadas (Guiraud, P.; Steiman, R.; Campos
Takaki, G.M.; Seigle-Murandi, E.; Simeon B.M.; Comparison of antibacterial and Antifungal Activities of Lapachol and β-Lapachone; Planta Medica 1994, 60, 373). Além disso, estudos comprovam que além de serem agentes bacterianos e antifúngicos, atuam no processo da prevenção da esquistossomose, por inibirem o processo de penetração das cercarias do Schistosoma mansoni em camundongos, como visto (Pinto, A. V.; Gilbert, B.; Pinto, M. C; ln Vitro and In Vivo Evaluation of the toxocity of 1 ,4-Naphthoquinone and 1 ,2-Naphthoquinone Derivatives against Trypanosoma cruzi; Ann. Trop. Med. Parasitei. 1978, 72, 523). Some quinones, such as those in the β-lapachone group, are good bacterial and antifungal agents as reported (Guiraud, P .; Steiman, R .; Campos. Takaki, GM; Seigle-Murandi, E .; Simeon BM; Comparison of antibacterial and Antifungal Activities of Lapachol and β-Lapachone; Medical Plant 1994, 60, 373). Moreover, studies prove that besides being bacterial and antifungal agents, they act in the process of prevention of schistosomiasis, by inhibiting the penetration process of Schistosoma mansoni cercariae in mice, as seen (Pinto, AV; Gilbert, B .; Pinto, M. C. Vitro and In Vivo Evaluation of the toxicity of 1,4-Naphthoquinone and 1,2-Naphthoquinone Derivatives against Trypanosoma cruzi (Ann. Trop. Med. Parasitei. 1978, 72, 523).
A ação das quinonas sobre os vírus está relacionada com a inibição das enzimas da família das polimerases, mais preciso das DNA polimerases, e também com a inibição da enzima transcriptase reversa. A ação sobre esta última enzima foi demonstrada sobre os vírus da mieloblastose aviária (AMV) e da leucemia murínica de Raucher(RLV). A ação inibitória da enzima DNA polimerase foi específica, pois comparada com outros inibidores, as quinonas apresentaram característica distintas e ineditismo em comparação com outros como relatado (Schuerch, A. R; Wehrli, W.; β-Lapachone, an Inhibitor of Oncornavirus Reverse Transcriptase and Eukariotic DNA Polymerase-a: Inhibitory Effect, Thiol Dependence and Specificity; Eur. J. Biochem. 1978, 84, 197) ou também relatado por outro grupo de pesquisadores (Chau, Y. P.; Shiah, S. G.; Don, M. J.; M. L. Kuo; Involvement of Hydrogen Peroxide in Topoisomerase Inhibitor beta-Lapachone-Induced Apoptosis andThe action of quinones on viruses is related to inhibition of polymerase family enzymes, more precise DNA polymerases, and also to inhibition of reverse transcriptase enzyme. The action on the latter enzyme has been demonstrated on avian myeloblastosis (AMV) and Raucher murine leukemia (RLV) viruses. The inhibitory action of the DNA polymerase enzyme was specific, as compared to other inhibitors, quinones had distinct characteristics and novelty compared to others as reported (Schuerch, A. R; Wehrli, W .; β-Lapachone, an Inhibitor of Oncornavirus Reverse. Transcriptase and Eukariotic DNA Polymerase-a: Inhibitory Effect, Thiol Dependence and Specificity; Eur. J. Biochem. 1978, 84, 197) or also reported by another group of researchers (Chau, YP; Shiah, SG; Don, MJ; ML Kuo; Involvement of Hydrogen Peroxide in Topoisomerase Inhibitor beta-Lapachone-Induced Apoptosis and
Differentiation in Human Leukemia Celis, Free Radical Biol. Med. 1998, 24, 660). Differentiation in Human Leukemia Celis, Free Radical Biol. Med. 1998, 24, 660).
Outros estudos demonstram que mesmo em baixas concentrações essas substâncias foram capazes de induzirem à morte de células cancerosas da próstata humana, com características de processo apoptótico. Este novo modo de atuação biodinâmica vem tornar esta quinona um grande potencial como droga de valor para a quimioterapia de câncer, particularmente no câncer de próstata (Li, J. C; Wang, C; Pardee B. A.; Induction of Apoptosis by β-Lapachone in Human Prostate Câncer Celis; Câncer Res. 1995, 55, 3712). Other studies show that even at low concentrations these substances were able to induce the death of human prostate cancer cells, with characteristics of apoptotic process. This new mode of biodynamic action makes this quinone a great potential as a valuable drug for cancer chemotherapy, particularly in prostate cancer (Li, J. C; Wang, C; Pardee BA; Induction of Apoptosis by β-Lapachone in Human Prostate Cancer Celis; Cancer Res. 1995, 55, 3712).
Muitas patentes foram concedidas para o uso das lapachonas como quimioterápicos, porém, até o presente momento nenhuma patente apresentou o uso das lapachonas como agentes antivirais do vírus da dengue. A patente internacional WO0977797 menciona um grupo de moléculas da família das lapachonas que são
utilizadas para tratamento do câncer de próstata em mamíferos. Tais moléculas induzem, de forma seletiva, a morte das células cancerosas do epitélio da próstata de mamíferos. Outro pedido internacional WO96033988 diz respeito ao uso de lapachonas e seus análogos sozinhos ou com quimioterapia ou radioterapia para indução da morte programada de células cancerosas. Principal fator para essa induzir a morte nas células cancerosas, está no fato dessas lapachonas e seus análogos, quando associados com um tratamento quimioterápico ou radioterápico, são inibidores irreversíveis da enzima topoisomerase freando o processo de replicação celular. Many patents have been granted for the use of lapachones as chemotherapeutic agents, but so far no patent has presented the use of lapachones as dengue virus antiviral agents. International patent WO0977797 mentions a group of lapachone family molecules that are used to treat prostate cancer in mammals. Such molecules selectively induce the death of cancer cells in mammalian prostate epithelium. Another international application WO96033988 relates to the use of lapachones and their analogs alone or with chemotherapy or radiotherapy to induce programmed cancer cell death. A major factor in inducing cancer cell death is the fact that these lapachones and their analogues, when associated with chemotherapy or radiotherapy, are irreversible inhibitors of the topoisomerase enzyme that hampers the process of cell replication.
Descrição das Figuras Description of the Figures
Figura 1 -Curva dose-resposta da replicação do vírus da Dengue (sorotipo 2) na presença do composto LVM138. A produção virai para cada concentração do composto utilizada foi medida por PCR quantitativo e apresentada como porcentagem de inibição em relação ao controle virai sem a adição do composto. Para o cálculo do IC50% foi utilizada a regressão não linear de Hill. Figure 1 - Dose response curve of dengue virus replication (serotype 2) in the presence of compound LVM138. Viral production for each compound concentration used was measured by quantitative PCR and presented as a percentage of inhibition relative to the viral control without the addition of compound. For the calculation of the IC50%, Hill's nonlinear regression was used.
Figura 2- Curva dose-resposta da replicação do vírus da Dengue (sorotipo 2) na presença do composto LVM137. A produção virai para cada concentração do composto utilizada foi medida por PCR quantitativo e apresentada como porcentagem de inibição em relação ao controle virai sem a adição do composto. Para o cálculo do IC50% foi utilizada a regressão não linear de Hill. Figure 2- Dose response curve of dengue virus replication (serotype 2) in the presence of compound LVM137. Viral production for each compound concentration used was measured by quantitative PCR and presented as a percentage of inhibition relative to the viral control without the addition of compound. For the calculation of the IC50%, Hill's nonlinear regression was used.
Sumário da Invenção Summary of the Invention
O principal objeto dessa invenção trata-se do uso das piranonaftoquinonas de fórmula geral I, II e III, assim como seus derivados, seus isômeros e seus sais como um agente antiviral, The main object of this invention is the use of pyranonaphthoquinones of general formula I, II and III, as well as their derivatives, isomers and salts thereof as an antiviral agent.
O último objeto desta invenção trata-se de uma composição farmacêutica que contêm as piranonaftoquinonas, de fórmula geral I, II e III, assim como seus derivados, seus isômeros e seus sais. The last object of this invention is a pharmaceutical composition containing the pyranonaphthoquinones of general formula I, II and III, as well as their derivatives, isomers and salts thereof.
O último objeto desta invenção trata-se de um medicamento para tratamento das infecções causadas por vírus, em animais mamíferos humanos e não humanos. The last object of this invention is a medicament for treating virus infections in human and non-human mammalian animals.
Descrição Detalhada da Invenção Detailed Description of the Invention
O principal objeto desta invenção é o uso das piranonaftoquinonas de fórmula geral I, II e III abaixo, assim como seus derivados, seus isômeros e sais, como agente contra infecções causadas por vírus em animais mamíferos humanos e não humanos. The main object of this invention is the use of the pyranonaphthoquinones of formula I, II and III below, as well as their derivatives, isomers and salts thereof, as an agent against infections caused by viruses in human and non-human mammalian animals.
Os radicais RI, R2, R3, R4, R5 e R6 das fórmulas moleculares acima, podem apresentar como substituintes grupos funcionais conjugados, ou independentes, alquilícos variados, dos tipos lineares, cíclicos, arílicos simples. Ainda podem apresentar grupos funcionalizados como amínicos, guanidínicos, sulfâmico, hidroxílicos, ésteres, éteres, tioésteres, tioéteres, halogênios, sendo preferencialmente, os grupos substituintes apresentados na tabela abaixo.
The radicals R1, R2, R3, R4, R5 and R6 of the above molecular formulas may be substituted, or independently, varied alkyl conjugated functional groups of the linear, cyclic, simple aryl types. They may still have functionalized groups such as amines, guanidines, sulfamic, hydroxyls, esters, ethers, thioesters, thioethers, halogens, preferably the substituent groups shown in the table below.
As piranonaftoquinonas desta invenção são usadas como agentes contra infecções causadas por vírus em animais mamíferos humanos e não humanos. Para fins dessa invenção, as piranonaftoquinonas são agentes contra infecções causadas por qualquer tipo de vírus, porém, sendo preferencialmente infecções causadas por vírus da família fl viviridae. Sendo as piranonaftoquinonas agentes contra infecções causadas por vírus da Hepatite A, hepatite B, hepatite C, contra infecções causadas por vírus da febre amarela, vírus do Oeste do Nilo e outros causadores de encefalite, vírus da diarréia bovina, vírus da febre suína. Porém, para fins desta invenção, as piranonaftoquinonas são agentes contra infecções causadas preferencialmente por vírus da dengue e seus diferentes sorotipos. The pyranonaphthoquinones of this invention are used as agents against virus infections in human and non-human mammalian animals. For the purposes of this invention, pyranonaphthoquinones are agents against infections caused by any type of virus, but are preferably infections caused by viruses of the family viviridae. Pyranonaphthoquinones are agents against infections caused by Hepatitis A virus, hepatitis B, hepatitis C virus, infections caused by yellow fever virus, West Nile virus and other encephalitis virus, bovine diarrhea virus, swine fever virus. However, for the purposes of this invention, pyranonaphthoquinones are agents against infections caused preferably by dengue viruses and their different serotypes.
As piranonaftoquinonas, já descritas anteriormente, de fórmula geral I, II e III, assim como seus derivados, seus isômeros e sais são usadas para preparação de uma composição farmacêutica destinada para o tratamento de infecções causadas por vírus da família flaviviridae, principalmente, para infecções causadas por vírus da dengue e suas isoformas.
A eficácia no tratamento das piranonaftoquinonas já descrita está no fato de assumirem uma ação inibitória sobre as enzimas ATPase dos vírus, principalmente sobre a enzima NS3 dos vírus da família flaviviridade. The previously described pyranonaphthoquinones of general formula I, II and III, as well as their derivatives, isomers and salts thereof are used for the preparation of a pharmaceutical composition intended for the treatment of infections caused by flaviviridae viruses, mainly for infections caused by dengue viruses and their isoforms. The efficacy in the treatment of pyranonaphthoquinones already described lies in the fact that they assume an inhibitory action on ATPase virus enzymes, especially on the flavivirity family virus NS3 enzyme.
Uma composição farmacêutica pode conter as piranonaftoquinonas de fórmula geral (I), (II), (III), assim como seus derivados, seus isômeros e sais. Além dessas piranonaftoquinonas, a composição farmacêutica apresenta substâncias inativas, como corantes, dispersantes, edulcorantes, emolientes, antioxidantes, conservantes, estabilizadores de ph, flavorizantes e outros conhecidos por um técnico na arte. A pharmaceutical composition may contain the pyranonaphthoquinones of formula (I), (II), (III) as well as their derivatives, isomers and salts. In addition to these pyranonaphthoquinones, the pharmaceutical composition features inactive substances such as colorants, dispersants, sweeteners, emollients, antioxidants, preservatives, ph stabilizers, flavorants and others known to one skilled in the art.
As substâncias ativas nessa composição são as piranonaftoquinonas de fórmula geral I, II e III já descritas anteriormente, assim como seus derivados, seus isômeros e sais. Tais substâncias devem apresentar uma quantidade farmaceuticamente aceitável, que para fins desta invenção compreende uma faixa 5 a ΙΟΟμΜ, sendo preferencialmente 12 a 50μΜ. The active substances in this composition are the pyranonaphthoquinones of formula I, II and III already described above, as well as their derivatives, isomers and salts. Such substances should have a pharmaceutically acceptable amount, which for the purposes of this invention comprises a range 5 to ΜμΜ, preferably 12 to 50μΜ.
A composição farmacêutica aqui descrita é utilizada para a preparação de um medicamento utilizado no tratamento de infecções em animais mamíferos humanos e não humanos, causadas por vírus, sendo este da família flaviviridae, sendo preferencialmente o vírus da dengue e seus diferentes sorotipos. Esse medicamento apresenta as piranonaftoquinonas, já descritas anteriormente, pode ainda apresentar seus derivados ou seus isômeros ou seus sais. The pharmaceutical composition described herein is used for the preparation of a medicament for the treatment of infections in human and non-human mammalian animals caused by viruses, the latter being of the flaviviridae family, preferably dengue virus and its different serotypes. This drug presents the pyranonaphthoquinones, already described above, may also have their derivatives or isomers or their salts.
O medicamento desta invenção pode estar na forma de cápsula, comprimido de liberação lenta, comprimidos de liberação controlada, pastilhas, xaropes, formas farmacêuticas encapsuladas em sistemas de nanopartículas ou micropartículas, formas farmacêuticas injetáveis. The medicament of this invention may be in capsule form, slow release tablet, controlled release tablets, tablets, syrups, dosage forms encapsulated in nanoparticle or microparticle systems, injectable dosage forms.
Os seguintes exemplos experimentais servem para ilustrar a presente invenção sem, contudo, limitar o escopo da invenção apresentada. As nomenclaturas dadas nos experimentos as pirononaftoquinonas, possuem finalidade ilustrativa, todas estão compreendidas dentro das fórmulas gerais de pirononaftoquinonas, que são objetos da patente, assim como seus radicais.
The following experimental examples serve to illustrate the present invention without, however, limiting the scope of the present invention. The nomenclatures given in the experiments, pyronaphthoquinones, are for illustrative purposes, all of which are comprised within the general formulas of pyrononephtoquinones, which are the subject of the patent, as well as their radicals.
Exemplo 1) Ensaios Inibidores do Vírus da Dengue Example 1) Dengue Virus Inhibitor Assays
Para realização dos testes das substâncias I a III frente ao vírus da dengue, comparativos com a Ribavirina, se utilizou um método de triagem onde células VERO eram colocadas em contato com estoques do vírus da dengue, em uma proporção de uma partícula virai infecciosa para 10 células por uma hora para a adsorção e entrada das partículas virais nas células. Após este tempo as células eram lavadas com solução salina e meio de cultura contendo 100 μΜ de cada um dos compostos e 200 μΜ de Ribavirina como controle positivo. Estas culturas eram incubadas a 37 °C por 72 h. Após este tempo uma alíquota do meio livre de células (sobrenadante) era coletada e processado para extração de RNA, seguido de transcrição reversa e reação de PCR em Tempo-Real utilizando iniciadores e sonda específicos para dengue do tipo 2. Aqueles compostos que mostravam inibição de pelo menos 10X na quantidade de RNA virai (progénie virai liberada no sobrenadante) em relação ao controle sem droga, seguiam para um ensaio de dose-resposta. Para este ensaio o procedimento era equivalente ao descrito acima, sendo que eram adicionadas diferentes concentrações dos compostos. Os resultados deste ensaio serviram para calcular a maior e menor porcentagem de inibição para cada composto. Desta forma, para as naftoquinonas do tipo II, LVM0137 e LVM0138 obteve-se, respectivamente 99 > 99,99% (nas concentrações de 25 e 12,5 μΜ, respectivamente).
Exemplo2) Ensaios de replicação viral. For the testing of dengue virus substances I to III compared to Ribavirin, a screening method was used where VERO cells were placed in contact with dengue virus stocks, in a ratio of one infectious viral particle to 10. cells for one hour for adsorption and entry of viral particles into cells. After this time the cells were washed with saline and culture medium containing 100 μΜ of each compound and 200 μΜ of Ribavirin as positive control. These cultures were incubated at 37 ° C for 72 h. After this time an aliquot of the cell free medium (supernatant) was collected and processed for RNA extraction, followed by reverse transcription and Real-Time PCR reaction using dengue-specific primers and probe type 2. Those compounds that showed inhibition of at least 10X in the amount of viral RNA (viral progeny released in the supernatant) relative to the drug-free control, followed for a dose response assay. For this assay the procedure was equivalent to that described above, with different concentrations of compounds being added. The results of this assay served to calculate the highest and lowest inhibition percentages for each compound. Thus, for type II naphthoquinones, LVM0137 and LVM0138, 99> 99.99% (25 and 12.5 μΜ, respectively) were obtained respectively. Example 2) Viral Replication Assays.
Afim de confirmar o resultado do exemplo 1 , outros dois ensaios de medida da replicação virai foram realizados (TCID50 % e Ensaio de redução de plaque virai). No ensaio de TCID50 %, células VERO eram colocadas em contato com estoques do vírus da dengue. Após este tempo as células eram lavadas e meio de cultura contendo 50 μΜ de cada um dos compostos e 200 μΜ de Ribavirina era adicionado como controle positivo do ensaio. Estas culturas eram incubadas a 37 °C por 72 h. Após este tempo o sobrenadante era coletado e utilizado para infecção de nova cultura de células VERO em sextuplicata. O efeito citopático virai era observado diariamente e anotados os poços que apresentavam 50 % de efeito em relação ao controle sem vírus. Estes dados eram utilizados para o cálculo do título virai conforme descrito por Reed e Munch. Observou- se inibição de mais de 99 % do título virai para ambos os compostos, conforme descrito (tabela abaixo). O ensaio de redução de plaque virai é realizado a partir da infecção de monocamadas de células Vero em Placas de 6 poços, com cerca de 50 unidades infecciosas virais. Após a etapa de adsorção as monocamadas são lavadas e é acrescentado meio semi-sólido acrescido de diferentes concentrações dos compostos. As células são cultivadas por sete dias. Após este período as células são fixadas e coradas com o corante cristal violeta. As plaques virais são contadas manualmente. Calcula-se a porcentagem de inibição em relação ao controle de vírus. Para o composto LVM0137 houve inibição de 50 % do número de plaques na concentração de 6,2 μΜ e para o composto LVM0138 houve inibição de mais de 50 % na concentração de 3,1 μΜ, conforme demonstrado na tabela abaixo.
In order to confirm the result of Example 1, two other viral replication measurement assays were performed (TCID 50% and Viral Plaque Reduction Assay). In the 50% TCID assay, VERO cells were placed in contact with dengue virus stocks. After this time the cells were washed and culture medium containing 50 μΜ of each compound and 200 μΜ of Ribavirin was added as a positive assay control. These cultures were incubated at 37 ° C for 72 h. After this time the supernatant was collected and used for infection of new VERO cell culture in sextuplicate. The viral cytopathic effect was observed daily and the wells that showed 50% effect compared to the control without virus were noted. These data were used for the calculation of viral titer as described by Reed and Munch. More than 99% inhibition of viral titer was observed for both compounds as described (table below). The viral plaque reduction assay is performed from vero cell monolayer infection in 6-well plates with about 50 viral infectious units. After the adsorption step the monolayers are washed and semi-solid medium added with different concentrations of the compounds is added. The cells are cultured for seven days. After this period the cells are fixed and stained with the crystal violet dye. Viral plaques are counted manually. The inhibition percentage relative to virus control is calculated. Compound LVM0137 inhibited 50% of the number of plaques at a concentration of 6.2 μΜ and compound LVM0138 inhibited more than 50% at a concentration of 3.1 μΜ, as shown in the table below.
Exemplo 3 ) teste de inibição das enzimas ATPases Example 3) ATPase Enzyme Inhibition Test
Para a realização da triagem dos compostos contra a atividade ATPásica, foi utilizado o ensaio colorimétrico de Fiske, C. H.; Subbarow, Y. ; "The Nature of the inorganic phosphate in voluntary muscle"; Science 1927, 65, 401-403, que mede a extensão de hidrólise do ATP para ADP, à partir da quantidade de Pi (fosfato inorgânico) liberada na reação. A triagem dos compostos foi realizada utilizando 0,6 μΜ de NS3 íntegra em reações contendo 40 mM de Tris-HCl (pH 7,5), 5 mM de DTT, 100 mM de KCl, 5 mM deFor the screening of compounds against ATPase activity, the colorimetric assay of Fiske, C. H .; Subbarow, Y.; "The Nature of the inorganic phosphate in voluntary muscle"; Science 1927, 65, 401-403, which measures the extent of hydrolysis from ATP to ADP from the amount of Pi (inorganic phosphate) released in the reaction. Compound screening was performed using 0.6 μ of intact NS3 in reactions containing 40 mM Tris-HCl (pH 7.5), 5 mM DTT, 100 mM KCl, 5 mM
MgC e 1 mM de ATP. A enzima foi pré-incubada com os compostos por 10 minutos a 30 °C, e a atividade cinética foi iniciada pela adição de 1 mM de ATP seguido de incubação a 30 °C por 15 minutos. As densidades óticas das amostras foram medidas a 660 nm no EspectraMax M2e (Molecular Devices) . Os parâmetros cinéticos de velocidade inicial (Vi), foram calculados por regressões linear e/ou polinomial de 3a ordem utilizando-se o programa SigmaPlot versão 10.0 e Excel 2007, respectivamente, sendo os valores de Vi convertidos em atividade relativa (%). Os compostos que apresentaram a média da atividade relativa <90% foram convertidos para percentual de inibição e selecionados para a testagem in vivo. MgC and 1 mM ATP. The enzyme was preincubated with the compounds for 10 minutes at 30 ° C, and kinetic activity was initiated by the addition of 1 mM ATP followed by incubation at 30 ° C for 15 minutes. The optical densities of the samples were measured at 660 nm on the SpectraMax M2e (Molecular Devices). The kinetic parameters of initial velocity (V i) were calculated by linear regression and / or polynomial 3 the order using the SigmaPlot version 10.0 and Excel 2007, respectively, and the Vi values converted into relative activity (%). Compounds with a mean relative activity <90% were converted to inhibition percentage and selected for in vivo testing.
Foram testados as piranonaftoquinonas abaixo contra a atividade ATPase da NS3 íntegra do DENV-2. Nessa triagem inicial foram identificados 14 compostos capazes de inibir a atividade The following pyranonaphthoquinones were tested against ATPase activity of DENV-2 intact NS3. In this initial screening 14 compounds were identified capable of inhibiting the activity
ATPásica da NS3 íntegra, sendo o composto 2TIOPMeB (LVM 142) o que apresentou maior efeito inibitório.
Whole NS3 ATPase, with the compound 2TIOPMeB (LVM 142) showing the greatest inhibitory effect.
Claims
REIVINDICAÇÕES
1) Uso das piranonaftoquinonas de fórmula geral I, II e III, assim como seus derivados, seus isômeros, seus sais são caracterizados por serem agentes contra infecções causadas por vírus em animais mamíferos humanos e não humanos. 1) Use of the pyranonaphthoquinones of general formula I, II and III, as well as their derivatives, their isomers, their salts, are characterized as being agents against infections caused by viruses in human and non-human mammalian animals.
2) Uso das piranonafitoquinonas de acordo com a reivindicação 1 , caracterizados por apresentarem como substituintes nos radicais R1 , R2, R3, R4, R5 e R6 grupos alquílicos variados, dos tipos lineares, cíclicos, arílicos simples. Ainda podem apresentar grupos funcionalizados como amínicos, guanidínicos, sulfâmico, hidroxílicos, ésteres, éteres, tioésteres, tioéteres e halogênios. Use of the pyranonaphoquinones according to claim 1, characterized in that they have as substituents on the radicals R 1, R 2, R 3, R 4, R 5 and R 6 varied alkyl groups of the linear, cyclic and simple aryl types. They may still have functionalized groups such as amines, guanidines, sulfamic, hydroxyls, esters, ethers, thioesters, thioethers and halogens.
3) Uso das piranonaftoquinonas de acordo com a reivindicação 1 , caracterizados pelos radicais R1 , R2, R3, R4, R5 e R6, serem preferencialmente Use of the pyranonaphthoquinones according to claim 1, characterized in that the radicals R1, R2, R3, R4, R5 and R6 are preferably
4) Composição farmacêutica caracterizada por conter as piranonaftoquinonas de fórmula geral (I), (II), (III), assim como seus derivados, seus isômeros e sais, destinada para o tratamento de infecções causadas por vírus da família flaviviridae, principalmente, para infecções causadas por vírus da dengue e suas isoformas. 4. Pharmaceutical composition characterized in that it contains the pyranonaphthoquinones of general formula (I), (II), (III), as well as their derivatives, isomers and salts, intended for the treatment of infections caused by flaviviridae viruses, mainly for infections caused by dengue virus and its isoforms.
5) Composição farmacêutica de acordo com a reivindicação 4, caracterizada por conter além das piranonaftoquinonas, outras substâncias inativas como corantes, dispersantes, edulcorantes, emolientes, antioxidantes, conservantes, estabilizadores de ph, flavorizantes e outros conhecidos por um técnico na arte. Pharmaceutical composition according to Claim 4, characterized in that it contains, in addition to pyranonaphthoquinones, other inactive substances such as colorants, dispersants, sweeteners, emollients, antioxidants, preservatives, ph stabilizers, flavorants and others known to one skilled in the art.
6) Composição farmacêutica de acordo com a reivindicação 4, caracterizada por conter uma quantidade farmaceuticamente aceitável das piranonaftoquinonas entre 5 a Ι ΟΟμΜ, sendo preferencialmente 12 a 50μΜ. Pharmaceutical composition according to Claim 4, characterized in that it contains a pharmaceutically acceptable amount of the pyranonaphthoquinones from 5 to Μ ΟΟμΜ, preferably from 12 to 50μΜ.
7) Medicamento que contem as piranonaftoquinonas de fórmula geral (I), (II), (III) caracterizado por ser usado no tratamento de infecções em animais mamíferos humanos e não humanos, causados por vírus, sendo este da família flaviviridae, sendo preferencialmente o vírus da dengue e seus diferentes sorotipos. (7) A medicinal product containing pyranonaphthoquinones of general formula (I), (II), (III) which is used for the treatment of infections in human and non-human mammalian animals caused by viruses, the latter being of the flaviviridae family, preferably dengue virus and its different serotypes.
8) Medicamento de acordo com a reivindicação 7, caracterizado por estar nas seguintes fórmulas farmacêuticas cápsula, comprimido de liberação lenta, comprimidos de liberação controlada, pastilhas, xaropes, formas farmacêuticas encapsuladas em sistemas de nanopartículas ou micropartículas, formas farmacêuticas injetáveis.
Medicament according to claim 7, characterized in that the following pharmaceutical formulas include capsule, slow release tablet, controlled release tablets, tablets, syrups, pharmaceutical forms encapsulated in nanoparticle or microparticle systems, injectable pharmaceutical forms.
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| BRPI1106509-5 | 2011-10-25 | ||
| BRPI1106509-5A BRPI1106509A2 (en) | 2011-10-25 | 2011-10-25 | USE OF PYRONOPHATOQUINONE AS ANTIVIRAL; PHARMACEUTICAL COMPOSITION CONTAINING PYRANONAFTOKINONES; MEDICINAL PRODUCT CONTAINING PYRANONAFTOKINONES FOR TREATMENT OF DENGUE VIRUS INFECTIONS |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994004145A1 (en) * | 1992-08-21 | 1994-03-03 | Dana Farber Cancer Institute | Treatment of human viral infections |
| US5763625A (en) * | 1995-04-25 | 1998-06-09 | Wisconsin Alumni Research Foundation | Synthesis and use of β-lapachone analogs |
| US5824700A (en) * | 1996-02-20 | 1998-10-20 | Wisconsin Alumni Research Foundation | Ortho-quinone derivatives novel synthesis therefor and their use in the inhibition of neoplastic cell growth |
| WO2000061142A1 (en) * | 1999-04-14 | 2000-10-19 | Dana-Farber Cancer Institute, Inc. | Method and composition for the treatment of cancer |
-
2011
- 2011-10-25 BR BRPI1106509-5A patent/BRPI1106509A2/en not_active Application Discontinuation
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2012
- 2012-10-24 WO PCT/BR2012/000397 patent/WO2013059896A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994004145A1 (en) * | 1992-08-21 | 1994-03-03 | Dana Farber Cancer Institute | Treatment of human viral infections |
| US5763625A (en) * | 1995-04-25 | 1998-06-09 | Wisconsin Alumni Research Foundation | Synthesis and use of β-lapachone analogs |
| US5824700A (en) * | 1996-02-20 | 1998-10-20 | Wisconsin Alumni Research Foundation | Ortho-quinone derivatives novel synthesis therefor and their use in the inhibition of neoplastic cell growth |
| WO2000061142A1 (en) * | 1999-04-14 | 2000-10-19 | Dana-Farber Cancer Institute, Inc. | Method and composition for the treatment of cancer |
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