WO2011130817A1 - Pharmaceutical formulation and method for reducing hepatotoxicity - Google Patents

Pharmaceutical formulation and method for reducing hepatotoxicity Download PDF

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Publication number
WO2011130817A1
WO2011130817A1 PCT/BR2011/000129 BR2011000129W WO2011130817A1 WO 2011130817 A1 WO2011130817 A1 WO 2011130817A1 BR 2011000129 W BR2011000129 W BR 2011000129W WO 2011130817 A1 WO2011130817 A1 WO 2011130817A1
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Prior art keywords
resveratrol
treatment
trans
tuberculosis
stilbene
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PCT/BR2011/000129
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French (fr)
Portuguese (pt)
Inventor
André Arigony An SOUTO
Diógenes Santiago SANTOS
Luiz Augusto Basso
Maria Martha Campos
Natália Fontana NICOLETTI
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União Brasileira De Educação E Assistência - Mantenedora Da Puc Rs
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Publication of WO2011130817A1 publication Critical patent/WO2011130817A1/en

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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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis

Definitions

  • the present invention describes a pharmaceutical formulation and a method for preventing and / or reducing hepatotoxicity associated with the treatment of tuberculosis by shortening treatment time and avoiding resistant strains of Mycobacterium tuberculosis.
  • the pharmaceutical formulation of the present invention comprises resveratrol, a tuberculostatic compound and a pharmaceutically acceptable carrier.
  • the present invention is in the fields of chemistry, pharmacy and medicine.
  • TB Active tuberculosis
  • the first-line treatment indicated for all forms of pulmonary and extrapulmonary TB is two months of rifampin (RIF), pyrazinamide (PZ) and isoniazid (INH), followed by 4 months of RIF and INH ( HR).
  • RIF has bactericidal activity against M. tuberculosis by inhibiting bacterial DNA-dependent RNA polymerase.
  • INH is a bacterial catalase oxidase (KatG) activated prodrug and actively reduces bacilli growth by inhibiting mycolic acid biosynthesis, which is an essential component of the M. tuberculosis cell wall.
  • PZA is activated by bacterial pyrazinamidase, which occurs only under acidic conditions (pH 5.5).
  • the active metabolite is pyrazinoic acid which inhibits fatty acid synthesis in M. tuberculosis. This drug is used within the first two months of treatment to reduce the duration of therapy and is not used alone.
  • hepatotoxicity The most common adverse effects related to tuberculostatic use are: hepatotoxicity, various skin manifestations and neurological and gastrointestinal disorders, with hepatotoxicity being the most common. serious of these effects.
  • Drugs used in TB treatment regimens interact with each other and with other drugs, which increases the risk of toxicity.
  • Anti-TB drugs undergo metabolization and the incidence of hepatotoxicity during treatment with multiple drug use ranges from 2% to 28%.
  • Adverse reactions caused by antituberculosis drugs contribute significantly to nonadherence to treatment, which decreases their effectiveness and increases the emergence of drug resistance.
  • Hepatotoxicity induced by anti-TB drugs is associated with significant morbidity and mortality and may decrease treatment effectiveness. Asymptomatic elevations of transaminases are common during treatment, but hepatotoxicity can be fatal when not diagnosed early and when therapy is not stopped early.
  • the present invention is a pharmaceutical formulation for preventing hepatotoxicity in the treatment of tuberculosis by shortening treatment time and avoiding resistant strains of Mycobacterium tuberculosis and the use of such pharmaceutical formulation.
  • PI 0514026-9 A2 discloses a combination comprising at least one optionally esterified or salified omega-3 fatty acid, at least one statin, coenzyme Q10, resveratrol, at least one polyonasol, pantethine, selenium and zinc. This combination is favored with a synergistic effect and is useful in treating forms of disease due to insulin resistance and cardiovascular disease.
  • the present invention differs from the document cited in that it uses resveratrol to prevent hepatotoxicity caused by drugs used in the treatment of tuberculosis.
  • US 2009/0163580 A1 describes putative treatment and prevention formulations and processes for aging (anti-aging composition) and for diseases or conditions of all diseases reactive oxygen-dependent species, such as Alzheimer's disease, Parkinson's disease, diabetes mellitus, cardiovascular disease, cancer, hepatitis and estrogen deficiency-related diseases, including osteoporosis and breast cancer, and to improve the athletic performance of humans, including resveratrol and two or more of the following additional functionalities or ingredients: formulation slow release of resveratrol, pterostilbene; quercetin, fisetin and naringenin.
  • resveratrol two or more of the following additional functionalities or ingredients: formulation slow release of resveratrol, pterostilbene; quercetin, fisetin and naringenin.
  • the present invention differs from that document in that it describes a resveratrol-based formulation to prevent hepatotoxicity caused by the treatment of tuberculosis, thus resulting in faster treatment and thus avoiding the emergence of resistant strains of Mycobacterium tuberculosis.
  • CN101259107 describes a resveratrol formulation for the treatment of hepatic fibrosis when generated by chronic hepatitis.
  • the present invention differs from that document in preventing hepatotoxic effects during treatment with tuberulostatic agents.
  • WO200908901 1-A2 describe the use of resveratrol as a possible tuberculostatic agent.
  • the present invention differs from these documents in that it utilizes a pharmaceutical formulation for prevention of hepatotoxicity in the treatment of tuberculosis.
  • ICC administration 4 caused a significant increase in the release of transaminases, lactate dehydrogenase, ⁇ -glutamine transpeptidase, creatinine kinase, total bilirubin, urea and serum uric acid.
  • Liver injury was more severe in animals receiving oral UCC than those exposed to CCI 4 (sc).
  • THE Resveratrol treatment was able to attenuate liver damage induced by acute CCI4 poisoning and showed great curative effect against lipid peroxidine and bypassed variable enzyme serum as well as maintained glutathione status for control.
  • Resveratrol treatment decreased CCI- 4 induced liver damage.
  • resveratrol has the potential to have healing effects against liver damage. However, this work does not suggest that this effect is also present when administering agents useful in the treatment of tuberculosis.
  • Figure 1 depicts the reversal of aspartate aminotransferase activity.
  • AST after treatment with resveratrol (RSV) isoniazid (INH) + rifampicin (RIF), where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
  • Figure 2 shows the reversal of alanine aminotransferase (ALT) activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, where: where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
  • Figure 3 shows the reversal of myeloperoxidase (MPO) activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, where: where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
  • MPO myeloperoxidase
  • Figure 4 shows the maintenance of catalase activity levels after resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, where where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
  • Figure 5 shows mouse liver sections in saline (A) and RSV (B) groups showing normal liver histological architecture. The group treated with INH + RIF (C) drugs presented moderate steatosis, while the group treated with RSV had normal liver histology (D).
  • the present invention is directed to a pharmaceutical formulation useful in the treatment of tuberculosis and which exhibits reduced hepatotoxicity as compared to existing formulations.
  • This formulation has the advantage of shortening treatment time and also prevents the emergence of resistant strains of Mycobacterium tuberculosis.
  • An object of the present invention is a pharmaceutical formulation comprising:
  • a further object of the present invention is a process for reducing hepatotoxicity associated with the administration of at least one compound useful in the treatment of tuberculosis comprising the step of administering a composition comprising:
  • the pharmaceutical formulation of the present invention is a formulation useful in the treatment of tuberculosis and has reduced side effects compared to existing formulations, in addition to shortening treatment time by increasing the dose of tuberculosis-indicated drugs and preventing the emergence of resistant strains.
  • Mycobacterium tuberculosis This formulation comprises:
  • Examples of compounds useful in the treatment of tuberculosis include, without limitation, ethambutol, rifampicin, pyrazinamide, isoniazid, streptomycin, ciprofloxacin, morfloxacin, corticosteroids, and combinations thereof.
  • the most common adverse effects related to the use of these agents are hepatotoxicity, various skin manifestations and neurological and gastrointestinal disorders, with hepatotoxicity being the most serious of these effects.
  • Drugs used in TB treatment regimens interact with each other and with other drugs, which increases the risk of toxicity.
  • Antituberculosis drugs undergo metabolization and the incidence of hepatotoxicity during treatment with multiple drug use ranges from 2% to 28%.
  • resveratrol and preferably frans-resveratrol
  • hepatotoxicity reduction process of the invention may be applied to resveratrol derivatives, such as their methylated and / or acetylated derivatives.
  • methylated derivatives include trans-3,5-dimethoxy-4'-hydroxy stilbene, trans-3,5,4'-trimethoxy stilbene and trans-3,5-hydroxy-4'-methoxy stilbene, as examples
  • Acetylated derivatives include trans-3,5-diacetyl-4'-hydroxy stilbene, trans-3,5,4'-triacetyl stilbene and trans-3,5-hydroxy-4'-acetyl stilbene.
  • the ratio of resveratrol: compound useful in the treatment of tuberculosis ranges from 0.4: 1 to 2: 1.
  • compositions means any composition with an active principle, for prophylactic, palliative and / or curative purposes, and a pharmaceutically acceptable carrier / carrier, said composition acting upon administration. oral, topical, parenteral, enteral and / or intrathecal.
  • the process for reducing hepatotoxicity associated with the administration of at least one compound useful in the treatment of tuberculosis is a process comprising the step of administering a composition comprising: a) resveratrol;
  • the mammal is man.
  • the compounds of the present invention may be administered orally as tablets, capsules (each includes sustained release or scheduled release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. They can also be given by infusion and subcutaneous or intramuscular injection, all using doses known to those skilled in the art in the pharmaceutical field. They may be administered alone, but generally are administered with a pharmaceutically acceptable carrier, selected based on the route of administration chosen and the standard of pharmaceutical practice.
  • the dosage regimen for the compounds of the present invention will, of course, vary according to known factors such as: pharmacodynamic characteristics of a specific agent and modality and route of administration, race, age, sex, health, medical condition and receptor weight, nature and extent of symptoms, type of concurrent treatment, frequency of treatment, route of administration, hepatic and renal function of the patient / user and the desired effect.
  • oral solid forms preferably have, in addition to the active ingredient, a pharmaceutically acceptable carrier comprising one or more solvents such as lactose, dextrose, sucrose, cellulose, cornstarch or potato starch; one or more lubricants such as silica, powder, stearic acid, magnesium or calcium stearate, or polyethylene glycol, one or more bonding agents (binders) such as starches, mucilage, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; one or more disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sugary agents; one or more wetting agents such as lecithin, polysorbate, lauryl sulfate and non-toxic pharmacologically inactive substances generally used in pharmaceutical formulations and widely known to those skilled in the art.
  • solvents such as lactose, dextrose, sucrose, cellulose, cornstarch or potato starch
  • compositions of the present invention may be manufactured in known ways, for example, by mixing, granulating, tableting, sugar coating, or film coating processes.
  • Syrups may have pharmaceutically acceptable carriers, such as sucrose or sucrose with glycerin and / or manita (mannitol) and / or sorbitol.
  • Suspensions and emulsions may have a pharmaceutically acceptable carrier, such as a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.
  • Intramuscular compounds may have, in addition to the active ingredient, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, propylene glycol and a suitable amount of lidocaine hydrochloride.
  • a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, propylene glycol and a suitable amount of lidocaine hydrochloride.
  • oral forms are preferred.
  • the animals were kept, until they reached the desired weight, in shelves equipped with temperature controlled air inlet and outlet filters (22 ⁇ 1 ° C) and 12h light-dark cycle (lights on at 7h; lights off at 7 pm).
  • the animals were kept in rodent cages, filled with pine shavings (changed 3 times a week), in a number of 6 animals per cage.
  • the animals received pelleted feed and filtered water ad libitum. No experimental procedure was performed in the space intended to maintain the animals in order to avoid the production of any kind of behavioral stress.
  • the laboratory temperature was maintained at 22 ⁇ 1 ° C. No analgesic methods were used, as they could interfere with the parameters to be evaluated.
  • the cervical dislocation method was used - a highly recommended method that provides the necessary speed, efficacy and ease for animals weighing less than 100 g.
  • Blood collection was performed by puncture of the abdominal aorta (400 to 500 ⁇ of blood) and subsequently the animals' liver was collected for the analyzes described below. It is noteworthy that the same animals were used for blood and liver tests in order to reduce the total number of animals in the study.
  • 4 groups of 6 animals totaling an N of 24 animals. The groups were divided into 1) saline group; 2) RSV group; 3) RSV + INH + RIF group; 4) INH + RIF group.
  • mice Hepatotoxicity was induced by previously described methodology (Chowdhury A, Santra A, Bhattacharjee K, Ghatak S, Saha DR, Dhali GK. Mitochondrial oxidative stress and permeability transition in isoniazid and rifampicin induced liver injury in mice. 45 (1): 117-26).
  • the animals received a combination of 25-50 mg / kg INH and 50-100 mg / kg RIF orally once daily for three consecutive days.
  • Control animals received vehicle (1% DMSO in saline; 10 ml / kg, V.O.) at the same time intervals.
  • the animals were euthanized 24 h after the last dose of antituberculosis agents. Immediately, the blood and liver of the mice were collected for biochemical analysis.
  • FIG. 1 demonstrates the reversal of AST activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, pointing to the hepatoprotective effect of resveratrol, a statistically significant effect (p ⁇ 0.0001).
  • liver A portion of the liver ( ⁇ 350 mg) was used to determine MPO activity according to the literature (Souza DG, Cassali GD, Poole S, Teixeira MM. Effects of inhibition of PDE4 and TNF-alpha on local and remote lesions following ischaemia and reperfusion injury Br J Pharmacol 2001; 134 (5): 985-94).
  • the liver was removed and homogenized at 5% in EDTA / NaCl buffer (pH 4.7) and centrifuged at 10,000 rpm for 15 min at 4 ° C.
  • MPO myeloperoxidase
  • Example 7 Histological evaluation for liver injury determination

Abstract

The present invention describes a pharmaceutical composition and a method for preventing and/or reducing hepatotoxicity associated with a treatment for tuberculosis, reducing the duration of the treatment and avoiding the creation of resistant Mycobacterium tuberculosis strains. The pharmaceutical formulation according to the present invention contains resveratrol, a tuberculostatic agent, and a pharmaceutically acceptable carrier. The present invention pertains to the fields of chemistry, pharmacy and medicine.

Description

Relatório Descritivo de Patente de Invenção  Patent Invention Descriptive Report
FORMULAÇÃO FARMACÊUTICA E PROCESSO PARA REDUZIR PHARMACEUTICAL FORMULATION AND PROCESS TO REDUCE
HEPATOTOXI CIDADE Campo da Invenção HEPATOTOXI CITY Field of the Invention
A presente invenção descreve uma formulação farmacêutica e um processo para evitar e/ou reduzir a hepatoxicidade associada ao tratamento da tuberculose diminuindo o tempo de tratamento e evitando cepas resistentes da Mycobacterium tuberculosis. A formulação farmacêutica da presente invenção compreende resveratrol, um composto tuberculostático e um veículo farmaceuticamente aceitável. A presente invenção se situa nos campos da química, farmácia e medicina.  The present invention describes a pharmaceutical formulation and a method for preventing and / or reducing hepatotoxicity associated with the treatment of tuberculosis by shortening treatment time and avoiding resistant strains of Mycobacterium tuberculosis. The pharmaceutical formulation of the present invention comprises resveratrol, a tuberculostatic compound and a pharmaceutically acceptable carrier. The present invention is in the fields of chemistry, pharmacy and medicine.
Antecedentes da Invenção Background of the Invention
A Tuberculose (TB) ativa é tratada com múltiplas drogas. O tratamento de primeira linha indicado para os casos de todas as formas de TB pulmonar e extrapulmonar consiste no uso de dois meses de rifampicina (RIF), pirazinamida (PZ) e isoniazida (INH), seguido de 4 meses de uso RIF e INH (RH). A RIF possui atividade bactericida contra M. tuberculosis pela inibição da RNA polimerase dependente do DNA bacteriano. A INH é um pró-fármaco ativado pela catalase-oxidase bacteriana (KatG) e reduz ativamente o crescimento dos bacilos pela inibição da biossíntese de ácidos micólicos, que é um componente essencial da parede celular do M. tuberculosis. Um outro pró- fármaco, a PZA, é ativado pela pirazinamidase bacteriana, o que ocorre apenas em condições ácidas (pH 5,5). O metabólito ativo é o ácido pirazinóico que inibe a síntese de ácidos graxos no M. tuberculosis. Esta droga é utilizada nos dois meses iniciais de tratamento, a fim de reduzir a duração da terapia, não sendo utilizada de forma isolada.  Active tuberculosis (TB) is treated with multiple drugs. The first-line treatment indicated for all forms of pulmonary and extrapulmonary TB is two months of rifampin (RIF), pyrazinamide (PZ) and isoniazid (INH), followed by 4 months of RIF and INH ( HR). RIF has bactericidal activity against M. tuberculosis by inhibiting bacterial DNA-dependent RNA polymerase. INH is a bacterial catalase oxidase (KatG) activated prodrug and actively reduces bacilli growth by inhibiting mycolic acid biosynthesis, which is an essential component of the M. tuberculosis cell wall. Another prodrug, PZA, is activated by bacterial pyrazinamidase, which occurs only under acidic conditions (pH 5.5). The active metabolite is pyrazinoic acid which inhibits fatty acid synthesis in M. tuberculosis. This drug is used within the first two months of treatment to reduce the duration of therapy and is not used alone.
Os efeitos adversos mais frequentes relacionados ao uso de tuberculostáticos são: hepatoxicidade, manifestações cutâneas variadas e distúrbios neurológicos e gastrointestinais, sendo a hepatotoxicidade o mais sério destes efeitos. As drogas usadas nos esquemas de tratamento de TB apresentam interações entre si e com outras drogas, o que aumenta o risco de toxicidade. Os fármacos anti-TB sofrem metabolização e a incidência de hepatotoxicidade durante o tratamento com a utilização múltiplas drogas varia entre 2% e 28%. As reações adversas causadas pelas drogas antituberculose contribuem significativamente para a não aderência ao tratamento, o que diminui sua efetividade e aumenta o surgimento de resistência às drogas. A hepatotoxicidade induzida por drogas anti-TB está associada com morbimortalidade significativa e pode diminuir a efetividade do tratamento. Elevações assintomáticas das transaminases são comuns durante o tratamento, mas a hepatotoxicidade pode ser fatal quando não diagnosticada precocemente e quando a terapia não é interrompida a tempo. The most common adverse effects related to tuberculostatic use are: hepatotoxicity, various skin manifestations and neurological and gastrointestinal disorders, with hepatotoxicity being the most common. serious of these effects. Drugs used in TB treatment regimens interact with each other and with other drugs, which increases the risk of toxicity. Anti-TB drugs undergo metabolization and the incidence of hepatotoxicity during treatment with multiple drug use ranges from 2% to 28%. Adverse reactions caused by antituberculosis drugs contribute significantly to nonadherence to treatment, which decreases their effectiveness and increases the emergence of drug resistance. Hepatotoxicity induced by anti-TB drugs is associated with significant morbidity and mortality and may decrease treatment effectiveness. Asymptomatic elevations of transaminases are common during treatment, but hepatotoxicity can be fatal when not diagnosed early and when therapy is not stopped early.
A presente invenção consiste em uma formulação farmacêutica para evitar a hepatoxicidade no tratamento da tuberculose diminuindo o tempo de tratamento e evitando cepas resistentes da Mycobacterium tuberculosis e o uso dessa formulação farmacêutica.  The present invention is a pharmaceutical formulation for preventing hepatotoxicity in the treatment of tuberculosis by shortening treatment time and avoiding resistant strains of Mycobacterium tuberculosis and the use of such pharmaceutical formulation.
No âmbito patentário, foram localizados alguns documentos relevantes que serão descritos a seguir.  In the patent area, some relevant documents were found, which will be described below.
O documento PI 0514026-9 A2 descreve uma combinação compreendendo pelo menos um ácido graxo ômega-3, opcionalmente esterificado ou salificado, pelo menos uma estatina, coenzima Q10, resveratrol, pelo menos um policonasol, pantetina, selênio e zinco. Essa combinação é favorecida com um efeito sinergístico e é útil no tratamento de formas de doença devido à resistência a insulina e em doenças cardiovasculares. A presente invenção difere do documento citado pelo fato de utilizar o resveratrol para evitar a hepatotoxicidade causada por medicamentos utilizados no tratamento da tuberculose.  PI 0514026-9 A2 discloses a combination comprising at least one optionally esterified or salified omega-3 fatty acid, at least one statin, coenzyme Q10, resveratrol, at least one polyonasol, pantethine, selenium and zinc. This combination is favored with a synergistic effect and is useful in treating forms of disease due to insulin resistance and cardiovascular disease. The present invention differs from the document cited in that it uses resveratrol to prevent hepatotoxicity caused by drugs used in the treatment of tuberculosis.
O documento US 2009/0163580 A1 descreve formulações e processos de tratamento e prevenção putativos para envelhecimento (composição anti- envelhecimento) e para doenças ou condições de todas as doenças espécies dependente reativas de oxigénio, como a doença de Alzheimer, doença de Parkinson, diabetes mellitus, doenças cardiovasculares, câncer, hepatite e doenças associadas à deficiência de estrogênio, incluindo osteoporose e câncer de mama e para melhorar o desempenho atlético dos seres humanos, incluindo resveratrol e dois ou mais das seguintes funcionalidades ou ingredientes ativos adicionais: formulação de lenta liberação de resveratrol, pterostilbeno; quercetina, fisetina e naringenina. A presente invenção difere desse documento por descrever uma formulação baseada em resveratrol para evitar a hepatotoxicidade causada pelo tratamento da tuberculose, resultando assim em um tratamento mais rápido e evitando-se, assim, o surgimento de cepas resistentes de Mycobacterium tuberculosis. US 2009/0163580 A1 describes putative treatment and prevention formulations and processes for aging (anti-aging composition) and for diseases or conditions of all diseases reactive oxygen-dependent species, such as Alzheimer's disease, Parkinson's disease, diabetes mellitus, cardiovascular disease, cancer, hepatitis and estrogen deficiency-related diseases, including osteoporosis and breast cancer, and to improve the athletic performance of humans, including resveratrol and two or more of the following additional functionalities or ingredients: formulation slow release of resveratrol, pterostilbene; quercetin, fisetin and naringenin. The present invention differs from that document in that it describes a resveratrol-based formulation to prevent hepatotoxicity caused by the treatment of tuberculosis, thus resulting in faster treatment and thus avoiding the emergence of resistant strains of Mycobacterium tuberculosis.
O documento CN101259107 descreve uma formulação com resveratrol para o tratamento de fibroses hepáticas quando geradas por hepatites crónicas. A presente invenção difere desse documento por prevenir efeitos hepatotóxicos durante o tratamento com agentes tuberulostáticos.  CN101259107 describes a resveratrol formulation for the treatment of hepatic fibrosis when generated by chronic hepatitis. The present invention differs from that document in preventing hepatotoxic effects during treatment with tuberulostatic agents.
Os documentos US2007212395-A1 , US2006205679-A1, US2004029871 The documents US2007212395-A1, US2006205679-A1, US2004029871
(A), WO200908901 1-A2, WO2007149865-A2, WO200908901 1-A2 descrevem o uso do resveratrol como possível agente tuberculostático. A presente invenção difere desses documentos por utilizar uma formulação farmacêutica para prevenção da hepatoxicidade no tratamento da tuberculose. (A), WO200908901 1-A2, WO2007149865-A2, WO200908901 1-A2 describe the use of resveratrol as a possible tuberculostatic agent. The present invention differs from these documents in that it utilizes a pharmaceutical formulation for prevention of hepatotoxicity in the treatment of tuberculosis.
O artigo intitulado "Resveratrol ameliorates carbon tetrachloride-induced acute liver injury in mice" de Fan, Guijuan et al. National Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Peop. Rep. China. Environmental Toxicology and Pharmacology (2009), 28(3), 350-356. Publisher: Elsevier B.V., CODEN: ETOPFR ISSN: 1382-6689, descreve a avaliação do potencial hepatoprotetor do resveratrol (30 mg / kg, via oral) em ratos após 2 rotas diferentes (por via oral e sc) de exposição ao tetracloreto de carbono (CCU, 1 ,0 mL / kg). A administração de CCI4 causou um aumento significativo na liberação de transaminases, desidrogenase lactato, transpeptidase γ-glutamina, quinase de creatinina, bilirrubina total, uréia e ácido úrico no soro. Lesão hepática foi mais grave nos animais que receberam CCU por via oral do que aqueles que foram expostos a CCI4 por via (sc). O tratamenro com resveratrol foi capaz de atenuar os danos hepáticos induzidos por intoxicação aguda de CCI4 e mostrou grande efeito curativo contra peroxidina lipídica e desviou de soro enzimático variáveis, bem como manteve o status de glutationa para controle. Tratamento do resveratrol diminuiu danos induzidos por CCI4 no fígado. Aparentemente, o resveratrol tem potencial para exercer efeitos curativos contra lesões no fígado. No entanto, esse trabalho não sugere que esse efeito esteja também presente quando da administração de agentes úteis no tratamento da tuberculose. The article entitled "Resveratrol ameliorates carbon tetrachloride-induced acute liver injury in mice" by Fan, Guijuan et al. National Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Peop. China Rep. Environmental Toxicology and Pharmacology (2009), 28 (3), 350-356. Publisher: Elsevier BV, CODEN: ETOPFR ISSN: 1382-6689 describes the evaluation of resveratrol hepatoprotective potential (30 mg / kg orally) in rats after 2 different routes (orally and sc) of carbon tetrachloride exposure (CCU, 1.0 mL / kg). ICC administration 4 caused a significant increase in the release of transaminases, lactate dehydrogenase, γ-glutamine transpeptidase, creatinine kinase, total bilirubin, urea and serum uric acid. Liver injury was more severe in animals receiving oral UCC than those exposed to CCI 4 (sc). THE Resveratrol treatment was able to attenuate liver damage induced by acute CCI4 poisoning and showed great curative effect against lipid peroxidine and bypassed variable enzyme serum as well as maintained glutathione status for control. Resveratrol treatment decreased CCI- 4 induced liver damage. Apparently resveratrol has the potential to have healing effects against liver damage. However, this work does not suggest that this effect is also present when administering agents useful in the treatment of tuberculosis.
Do que se depreende da literatura pesquisada, não foram encontrados documentos antecipando ou sugerindo os ensinamentos da presente invenção, de forma que a solução aqui proposta possui novidade e atividade inventiva frente ao estado da técnica.  From what can be inferred from the researched literature, no documents were found anticipating or suggesting the teachings of the present invention, so that the solution proposed here has novelty and inventive activity in relation to the state of the art.
Breve Descrição das Figuras Brief Description of the Figures
A Figura 1 descreve a reversão da atividade aspartato aminotransferase Figure 1 depicts the reversal of aspartate aminotransferase activity.
(AST) após o tratamento com resveratrol (RSV) isoniazida (INH)+ rifampicina (RIF), onde: 1- Salina; 2- Resveratrol (RSV); 3- Isoniazida (INH) + Rifampicina (RIF) e 4- Resveratrol (RSV) + Isoniazida (INH) + Rifampicina (RIF). (AST) after treatment with resveratrol (RSV) isoniazid (INH) + rifampicin (RIF), where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
A Figura 2 mostra a reversão da atividade da alanina aminotransferase (ALT) após o tratamento com resveratrol (RSV)+ isoniazida (INH) + rifampicina (RIF), onde: onde: 1- Salina; 2- Resveratrol (RSV); 3- Isoniazida (INH) + Rifampicina (RIF) e 4- Resveratrol (RSV) + Isoniazida (INH) + Rifampicina (RIF).  Figure 2 shows the reversal of alanine aminotransferase (ALT) activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, where: where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
A Figura 3 mostra a reversão da atividade mieloperoxidase (MPO) após o tratamento com resveratrol (RSV) + isoniazida (INH) + rifampicina (RIF), onde: onde: 1- Salina; 2- Resveratrol (RSV); 3- Isoniazida (INH) + Rifampicina (RIF) e 4- Resveratrol (RSV) + Isoniazida (INH) + Rifampicina (RIF).  Figure 3 shows the reversal of myeloperoxidase (MPO) activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, where: where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF).
A Figura 4 mostra a manutenção dos níveis de atividade da catalase após o tratamento com resveratrol (RSV) + isoniazida (INH) + rifampicina (RIF), onde onde: 1- Salina; 2- Resveratrol (RSV); 3- Isoniazida (INH) + Rifampicina (RIF) e 4- Resveratrol (RSV) + Isoniazida (INH) + Rifampicina (RIF). A figura 5 mostra secções de fígado dos camundongos nos grupos salina (A) e RSV (B) apresentaram arquitetura histológica hepática normal. O grupo tratado com as drogas INH + RIF (C) apresentou esteatose moderada, enquanto o grupo pré-tratado com RSV apresentou histologia hepática normal (D). Figure 4 shows the maintenance of catalase activity levels after resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, where where: 1- Saline; 2- Resveratrol (RSV); 3- Isoniazid (INH) + Rifampicin (RIF) and 4- Resveratrol (RSV) + Isoniazid (INH) + Rifampicin (RIF). Figure 5 shows mouse liver sections in saline (A) and RSV (B) groups showing normal liver histological architecture. The group treated with INH + RIF (C) drugs presented moderate steatosis, while the group treated with RSV had normal liver histology (D).
Sumário da Invenção Summary of the Invention
Em um aspecto, a presente invenção tem como objeto uma formulação farmacêutica útil no tratamento da tuberculose e que apresenta reduzida hepatotoxicidade quando comparada às formulações já existentes. Essa formulação tem como vantagem a diminuição do tempo de tratamento e também evita o surgimento de cepas resistentes da Mycobacterium tuberculosis.  In one aspect, the present invention is directed to a pharmaceutical formulation useful in the treatment of tuberculosis and which exhibits reduced hepatotoxicity as compared to existing formulations. This formulation has the advantage of shortening treatment time and also prevents the emergence of resistant strains of Mycobacterium tuberculosis.
É um objeto da presente invenção uma formulação farmacêutica que compreende:  An object of the present invention is a pharmaceutical formulation comprising:
a) resveratrol;  a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável.  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier.
É um adicional objeto da presente invenção uma formulação farmacêutica útil na manufatura de um medicamentoto útil no tratamento da tuberculose.  It is a further object of the present invention a pharmaceutical formulation useful in the manufacture of a medicament useful in the treatment of tuberculosis.
É um objeto adicional da presente invenção um processo para reduzir a hepatotoxicidade associada à administração de pelo menos um composto útil no tratamento da tuberculose compreendendo a etapa de administrar uma composição compreendendo:  A further object of the present invention is a process for reducing hepatotoxicity associated with the administration of at least one compound useful in the treatment of tuberculosis comprising the step of administering a composition comprising:
a) resveratrol;  a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável;  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier;
a um mamífero necessitando de tratamento. Estes e outros objetos da invenção serão imediatamente valorizados pelos versados na arte e pelas empresas com interesses no segmento, e serão descritos em detalhes suficientes para sua reprodução na descrição a seguir. Descrição Detalhada da Invenção to a mammal in need of treatment. These and other objects of the invention will be immediately appreciated by those skilled in the art and companies having an interest in the segment, and will be described in sufficient detail for reproduction in the following description. Detailed Description of the Invention
Os exemplos aqui mostrados têm o intuito somente de exemplificar uma das inúmeras maneiras de se realizar a invenção, contudo sem limitar, o escopo da mesma.  The examples shown herein are intended solely to exemplify one of the numerous ways of carrying out the invention, but without limiting the scope thereof.
Formulação Farmacêutica Pharmaceutical Formulation
A formulação farmacêutica da presente invenção é uma formulação útil no tratamento da tuberculose e apresenta reduzidos efeitos colaterais quando comparada com as formulações existentes, além de diminuir o tempo de tratamento através do aumento da dose dos fármacos indicados para tuberculose e evitar o surgimento de cepas resistentes da Mycobacterium tuberculosis. Essa formulação compreende:  The pharmaceutical formulation of the present invention is a formulation useful in the treatment of tuberculosis and has reduced side effects compared to existing formulations, in addition to shortening treatment time by increasing the dose of tuberculosis-indicated drugs and preventing the emergence of resistant strains. Mycobacterium tuberculosis. This formulation comprises:
a) resveratrol;  a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável.  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier.
Exemplos de compostos úteis no tratamento da tuberculose incluem, sem limitações, etambutol, rifampicina, pirazinamida, isoniazida, streptomicina, ciprofloxacina, morfloxacina, corticosteróides, e combinações dos mesmos. Os efeitos adversos mais frequentes relacionados ao uso desses agentes são: hepatotoxicidade, manifestações cutâneas variadas e distúrbios neurológicos e gastrointestinais, sendo a hepatotoxicidade o mais sério destes efeitos. As drogas usadas nos esquemas de tratamento de TB apresentam interações entre si e com outras drogas, o que aumenta o risco de toxicidade. Os fármacos antituberculose sofrem metabolização e a incidência de hepatotoxicidade durante o tratamento com a utilização múltiplas drogas varia entre 2% e 28%. As reações adversas causadas pelas drogas antituberculose contribuem significativamente para a não aderência ao tratamento, o que diminui sua efetividade e aumenta o surgimento de resistência às drogas. Embora, de acordo com a presente invenção, o uso de resveratrol, e preferivelmente de frans-resveratrol, seja preferencial na preparação da composição da invenção, os técnicos no assunto também irão identificar que o processo de redução da hepatotoxicidade da invenção pode ser aplicado aos derivados de resveratrol, como seus derivados metilados e/ou acetilados. Exemplos de derivados metilados incluem trans-3,5-dimetoxi-4'-hidroxi- estilbeno, trans-3,5,4'-trimetoxi-estilbeno e trans-3,5-hidroxi-4'-metoxi-estilbeno, enquanto exemplos de derivados acetilados incluem trans-3,5-diacetil-4'- hidroxi-estilbeno, trans-3,5,4'-triacetil-estilbeno e trans-3,5-hidroxi-4'-acetil- estilbeno. Examples of compounds useful in the treatment of tuberculosis include, without limitation, ethambutol, rifampicin, pyrazinamide, isoniazid, streptomycin, ciprofloxacin, morfloxacin, corticosteroids, and combinations thereof. The most common adverse effects related to the use of these agents are hepatotoxicity, various skin manifestations and neurological and gastrointestinal disorders, with hepatotoxicity being the most serious of these effects. Drugs used in TB treatment regimens interact with each other and with other drugs, which increases the risk of toxicity. Antituberculosis drugs undergo metabolization and the incidence of hepatotoxicity during treatment with multiple drug use ranges from 2% to 28%. Adverse reactions caused by antituberculosis drugs contribute significantly to nonadherence to treatment, which decreases their effectiveness and increases the emergence of drug resistance. Although according to the present invention, the use of resveratrol, and preferably frans-resveratrol, is preferred in the preparation of the composition of the invention, those skilled in the art will also identify that the hepatotoxicity reduction process of the invention may be applied to resveratrol derivatives, such as their methylated and / or acetylated derivatives. Examples of methylated derivatives include trans-3,5-dimethoxy-4'-hydroxy stilbene, trans-3,5,4'-trimethoxy stilbene and trans-3,5-hydroxy-4'-methoxy stilbene, as examples Acetylated derivatives include trans-3,5-diacetyl-4'-hydroxy stilbene, trans-3,5,4'-triacetyl stilbene and trans-3,5-hydroxy-4'-acetyl stilbene.
Preferencialmente, a proporção de resveratrol: composto útil no tratamento da tuberculose varia entre 0,4:1 a 2:1.  Preferably, the ratio of resveratrol: compound useful in the treatment of tuberculosis ranges from 0.4: 1 to 2: 1.
Para os efeitos da presente invenção, por "composições farmacêuticas" entende-se toda e qualquer composição com um princípio ativo, com finalidade profilática, paliativa e/ou objetivos curativos, e um veículo/transportador farmaceuticamente aceitável, a dita composição agindo através da administração oral, tópica, parenteral, enteral e/ou intratecal.  For the purposes of the present invention, "pharmaceutical compositions" means any composition with an active principle, for prophylactic, palliative and / or curative purposes, and a pharmaceutically acceptable carrier / carrier, said composition acting upon administration. oral, topical, parenteral, enteral and / or intrathecal.
Processo de Redução da Hepatotoxicidade Hepatotoxicity Reduction Process
O processo para reduzir a hepatotoxicidade associada à administração de pelo menos um composto útil no tratamento da tuberculose é um processo que compreende a etapa de administrar uma composição compreendendo: a) resveratrol;  The process for reducing hepatotoxicity associated with the administration of at least one compound useful in the treatment of tuberculosis is a process comprising the step of administering a composition comprising: a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável;  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier;
a um mamífero necessitando de tratamento. to a mammal in need of treatment.
Preferencialmente, o mamífero é o homem.  Preferably, the mammal is man.
Os compostos da presente invenção podem ser administrados em dose oral, como comprimidos, cápsulas (cada um inclui formulações de liberação sustentada ou liberação programada), pílulas, pós, grânulos, elixires, tinturas, suspensões, xaropes e emulsões. Eles também podem ser administrados por infusão e injeção subcutânea ou intramuscular, todos eles usando doses conhecidas para técnicos no assunto na área farmacêutica. Eles podem ser administrados isoladamente, mas em geral, são administrados com um veículo farmaceuticamente aceitável, selecionados com base na via de administração escolhida e no padrão da prática farmacêutica. The compounds of the present invention may be administered orally as tablets, capsules (each includes sustained release or scheduled release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. They can also be given by infusion and subcutaneous or intramuscular injection, all using doses known to those skilled in the art in the pharmaceutical field. They may be administered alone, but generally are administered with a pharmaceutically acceptable carrier, selected based on the route of administration chosen and the standard of pharmaceutical practice.
O regime de dose para os compostos da presente invenção irão variar, é claro, de acordo com os fatores conhecidos, tais como: características farmacodinâmicas de um agente específico e modalidade e via de administração, raça, idade, sexo, saúde, condição médica e peso do receptor, natureza e extensão dos sintomas, tipo de tratamento simultâneo, frequência de tratamento, via de administração, função hepática e renal do paciente/usuário e do efeito almejado. Por exemplo, formas sólidas orais, preferencialmente possuem, além do princípio ativo, um veículo farmaceuticamente aceitável compreendendo um ou mais solventes, tais como, lactose, dextrose, sacarose, celulose, amido de milho ou fécula de batata; um ou mais lubrificantes, como sílica, pó, ácido esteárico, estearato de magnésio ou cálcio, ou de polietileno glicol, um ou mais agentes de ligação (aglutinantes), tais como, amidos, mucilagem, gelatina, metilcelulose, carboximetilcelulose ou polivinilpirrolidona; um ou mais agentes desagregadores, tais como, amido, ácido algínico, alginatos ou glicolato de amido sódico; misturas efervescentes; corantes; agentes açucarados; um ou mais agentes umectantes, como a lecitina, polissorbato, lauril sulfato e substâncias farmacologicamente inativas e não-tóxicas geralmente utilizadas em formulações farmacêuticas e amplamente conhecidas pelos técnicos da área.  The dosage regimen for the compounds of the present invention will, of course, vary according to known factors such as: pharmacodynamic characteristics of a specific agent and modality and route of administration, race, age, sex, health, medical condition and receptor weight, nature and extent of symptoms, type of concurrent treatment, frequency of treatment, route of administration, hepatic and renal function of the patient / user and the desired effect. For example, oral solid forms preferably have, in addition to the active ingredient, a pharmaceutically acceptable carrier comprising one or more solvents such as lactose, dextrose, sucrose, cellulose, cornstarch or potato starch; one or more lubricants such as silica, powder, stearic acid, magnesium or calcium stearate, or polyethylene glycol, one or more bonding agents (binders) such as starches, mucilage, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; one or more disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sugary agents; one or more wetting agents such as lecithin, polysorbate, lauryl sulfate and non-toxic pharmacologically inactive substances generally used in pharmaceutical formulations and widely known to those skilled in the art.
As composições da presente invenção podem ser fabricadas de maneiras conhecida, por exemplo, por meio de mistura, granulação, prensagem de comprimido, revestimento de açúcar, ou processos de revestimento por filme. Xaropes podem ter veículos farmaceuticamente aceitáveis, tais como, sacarose ou sacarose com glicerina e/ou manita (manitol) e/ou sorbitol. Suspensões e emulsões podem ter veículo farmaceuticamente aceitável, como por exemplo, uma goma natural, ágar, alginato de sódio, pectina, metilcelulose, carboximetilcelulose ou álcool polivinílico. As formas destinadas a injeções intramusculares podem ter, além do princípio ativo, veículo farmaceuticamente aceitável, tais como, água estéril, azeite, oleato de etila, propilenoglicol e uma quantidade adequada de cloridrato de lidocaína. Na presente invenção, as formas orais são as preferenciais. The compositions of the present invention may be manufactured in known ways, for example, by mixing, granulating, tableting, sugar coating, or film coating processes. Syrups may have pharmaceutically acceptable carriers, such as sucrose or sucrose with glycerin and / or manita (mannitol) and / or sorbitol. Suspensions and emulsions may have a pharmaceutically acceptable carrier, such as a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. Forms intended for injections Intramuscular compounds may have, in addition to the active ingredient, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, propylene glycol and a suitable amount of lidocaine hydrochloride. In the present invention, oral forms are preferred.
A seguir a presente invenção será ilustrada pelo exemplo abaixo, que não deve ser considerado como limitante da técnica.  In the following the present invention will be illustrated by the example below, which is not to be construed as limiting the art.
Realização preferencial  Preferred Achievement
Animais: Para a realização dos experimentos foram utilizados camundongos BALB/c machos, de aproximadamente 4 semanas, pesando entre 25 e 30g. Etapas: Animals: The experiments used male BALB / c mice, approximately 4 weeks old, weighing between 25 and 30g. Phases:
Os animais foram mantidos, até atingirem o peso desejado, em estantes equipadas com filtros de entrada e saída de ar, com temperatura controlada (22 ± 1 °C) e ciclo claro-escuro de 12 h (luzes acesas às 7 h; luzes apagadas às 19 h). Os animais foram mantidos em gaiolas apropriadas para roedores, preenchidas com maravalha de pinus (trocadas 3 vezes por semana), em número de 6 animais por gaiola. Os animais receberam ração peletizada e água filtrada ad libitum. Nenhum procedimento experimental foi realizado no espaço destinado à manutenção dos animais, a fim de evitar a produção de qualquer tipo de estresse comportamental.  The animals were kept, until they reached the desired weight, in shelves equipped with temperature controlled air inlet and outlet filters (22 ± 1 ° C) and 12h light-dark cycle (lights on at 7h; lights off at 7 pm). The animals were kept in rodent cages, filled with pine shavings (changed 3 times a week), in a number of 6 animals per cage. The animals received pelleted feed and filtered water ad libitum. No experimental procedure was performed in the space intended to maintain the animals in order to avoid the production of any kind of behavioral stress.
Durante os procedimentos experimentais, a temperatura do laboratório foi mantida em 22 ± 1 °C. Não foram utilizados métodos analgésicos, pois os mesmos poderiam interferir nos parâmetros a serem avaliados. Para eutanásia, foi utilizado o método de deslocamento cervical - método altamente recomendado, que apresenta a rapidez, a eficácia e a facilidade necessárias, para animais com menos de 100 g. A coleta de sangue foi realizada por punção da aorta abdominal (400 a 500 μΙ de sangue) e, posteriormente, o fígado dos animais foi coletado para as análises descritas abaixo. Destaca-se que os mesmos animais foram utilizados para as análises sanguíneas e hepáticas, a fim de reduzir o número total de animais no estudo. Para a realização dos experimentos foram utilizados 4 grupos de 6 animais, totalizando um N de 24 animais. Os grupos foram divididos em 1) grupo salina; 2) grupo RSV; 3) grupo RSV+INH+RIF; 4) grupo INH+RIF. During the experimental procedures, the laboratory temperature was maintained at 22 ± 1 ° C. No analgesic methods were used, as they could interfere with the parameters to be evaluated. For euthanasia, the cervical dislocation method was used - a highly recommended method that provides the necessary speed, efficacy and ease for animals weighing less than 100 g. Blood collection was performed by puncture of the abdominal aorta (400 to 500 μΙ of blood) and subsequently the animals' liver was collected for the analyzes described below. It is noteworthy that the same animals were used for blood and liver tests in order to reduce the total number of animals in the study. For the experiments were used 4 groups of 6 animals, totaling an N of 24 animals. The groups were divided into 1) saline group; 2) RSV group; 3) RSV + INH + RIF group; 4) INH + RIF group.
Exemplo 1 - Indução de hepatotoxicidade  Example 1 - Hepatotoxicity Induction
Para a indução de hepatotoxicidade utilizou-se metodologia descrita previamente (Chowdhury A, Santra A, Bhattacharjee K, Ghatak S, Saha DR, Dhali GK. Mitochondrial oxidative stress and permeability transition in isoniazid and rifampicin induced liver injury in mice. J Hepatol 2006;45(1):117-26). Os animais receberam uma combinação de 25-50 mg/kg de INH e 50-100 mg/Kg de RIF, por via oral, uma vez ao dia, durante três dias consecutivos. Os animais controle receberam veículo (DMSO 1 % em solução salina; 10 ml/kg, V.O.), nos mesmos intervalos de tempo. Os animais foram submetidos à eutanásia 24 h após a administração da última dose dos agentes antituberculose. Imediatamente, foram coletados o sangue e o fígado dos camundongos para as análises bioquímicas.  Hepatotoxicity was induced by previously described methodology (Chowdhury A, Santra A, Bhattacharjee K, Ghatak S, Saha DR, Dhali GK. Mitochondrial oxidative stress and permeability transition in isoniazid and rifampicin induced liver injury in mice. 45 (1): 117-26). The animals received a combination of 25-50 mg / kg INH and 50-100 mg / kg RIF orally once daily for three consecutive days. Control animals received vehicle (1% DMSO in saline; 10 ml / kg, V.O.) at the same time intervals. The animals were euthanized 24 h after the last dose of antituberculosis agents. Immediately, the blood and liver of the mice were collected for biochemical analysis.
Exemplo 2 - Tratamento com resveratrol (RSV) e agentes antituberculose Diferentes grupos de animais foram tratados com RSV (50-100 mg/kg), por via oral, 30 minutos antes da indução da hepatotoxicidade e mais duas vez ao dia, de 6 em 6 horas, até o 3o dia após a primeira aplicação de INH + RIF (sempre no mesmo horário). Animais controle foram tratados com veículo (DMSO 1 % em solução salina; 10 ml/kg, V.O), nos mesmos períodos de tempo; um quarto grupo de animais foi tratado apenas com RSV (100 mg/kg), seguindo os mesmos intervalos de tempo já descritos. As doses e intervalos de administração do RSV foram selecionados com base em estudos prévios da literatura. Os animais foram submetidos à eutanásia 24 h após a administração da última dose dos agentes antituberculose. Imediatamente, foram coletados o sangue e o fígado dos camundongos para as análises bioquímicas. Example 2 - Treatment with resveratrol (RSV) and antituberculosis agents Different groups of animals were treated with RSV (50-100 mg / kg) orally 30 minutes before hepatotoxicity induction and twice a day every 6 6 hours up to 3 days after the first application of INH + RIF (always at the same time). Control animals were treated with vehicle (1% DMSO in saline; 10 ml / kg, VO) at the same time periods; A fourth group of animals were treated with RSV alone (100 mg / kg), following the same time intervals already described. The doses and intervals of administration of RSV were selected based on previous studies in the literature. The animals were euthanized 24 h after the last dose of antituberculosis agents. Immediately, the blood and liver of the mice were collected for biochemical analysis.
Exemplo 3 - Determinação de AST (as parta to aminotransferase) Example 3 - Determination of AST (as part to aminotransferase)
Foram coletados cerca de 400 a 500 μΙ de sangue total através da punção da aorta abdominal. Em seguida, as amostras foram centrifugadas por 20 min, a 10000 rpm e o soro foi separado para posterior determinação da função hepática, através da dosagem dos níveis séricos de AST em modo cinético, utilizando kits disponíveis comercialmente, de acordo com as recomendações do fabricante (Labtest, Brasil). A absorbância foi medida em 490 nm e os resultados expressos em U/ml. A figura 1 demonstra a reversão da atividade da AST após o tratamento com resveratrol (RSV) + isoniazida (INH) + rifampicina (RIF), apontando o efeito hepatoprotetor do resveratrol, um efeito estatisticamente significativo (p<0,0001). About 400 to 500 μΙ of whole blood were collected by puncture of the abdominal aorta. Then the samples were centrifuged for 20 min at 10,000 rpm and the serum was separated for further determination of liver function by measuring serum AST levels in kinetic mode using commercially available kits according to manufacturer's recommendations (Labtest, Brazil). Absorbance was measured at 490 nm and results expressed in U / ml. Figure 1 demonstrates the reversal of AST activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, pointing to the hepatoprotective effect of resveratrol, a statistically significant effect (p <0.0001).
Exemplo 4 - Determinação da ALT (alanina aminotranferase) Example 4 - Determination of ALT (Alanine Aminotranferase)
Foram coletados cerca de 400 a 500 μΙ de sangue total através da punção da aorta abdominal. Em seguida, as amostras foram centrifugadas por 20 min, a 10000 rpm e o soro foi separado para posterior determinação da função hepática, através da dosagem dos níveis séricos de ALT em modo cinético, utilizando kits disponíveis comercialmente, de acordo com as recomendações do fabricante (Labtest, Brasil). A absorbância foi medida em 490 nm e os resultados expressos em U/ml. A figura 2 demonstra de forma significativa (p<0,0001), a reversão da atividade da ALT após o tratamento com resveratrol (RSV) + isoniazida (INH) + rifampicina (RIF), indicado o efeito hepatoprotetor do resveratrol.  About 400 to 500 μΙ of whole blood were collected by puncture of the abdominal aorta. The samples were then centrifuged for 20 min at 10,000 rpm and the serum was separated for later liver function determination by measuring serum kinetic ALT levels using commercially available kits according to the manufacturer's recommendations. (Labtest, Brazil). Absorbance was measured at 490 nm and results expressed in U / ml. Figure 2 significantly demonstrates (p <0.0001) the reversal of ALT activity following resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, indicating the resveratrol hepatoprotective effect.
Exemplo 5 - Determinação da atividade da mieloperoxidase (MPO)  Example 5 - Determination of Myeloperoxidase (MPO) Activity
Uma porção do fígado (± 350 mg) foi utilizada para a determinação da atividade da MPO, segundo a literatura (Souza DG, Cassali GD, Poole S, Teixeira MM. Effects of inhibition of PDE4 and TNF-alpha on local and remote injuries following ischaemia and reperfusion injury. Br J Pharmacol 2001 ;134(5):985-94). O fígado foi removido e homogeneizado a 5 % em tampão EDTA/ NaCI (pH 4.7) e centrifugado a 10.000 rpm por 15 min, a 4°C. Após seguidas etapas de lavagem, o sobrenadante foi utilizado na reação enzimática para detecção da MPO, realizada na presença de tetrametilbenzidina (TMB, 1 ,6 mM), NaPO 80 mM e peróxido de hidrogénio (H2O2, 0,3 mM). A absorbância foi medida em 595 nm e os resultados expressos em densidade óptica (DO) por mg de tecido. A MPO é um indicativo indireto da migração de neutrófilos, parâmetro utilizado como indicador de estresse oxidativo e resposta inflamatória. A figura 3 demonstra a reversão da atividade da mieloperoxidase (MPO) após o tratamento com resveratrol (RSV) + isoniazida (INH) + rifampiciha (RIF). O efeito inibitório observado para o resveratrol foi estatisticamente significativo (p<0,0001). A portion of the liver (± 350 mg) was used to determine MPO activity according to the literature (Souza DG, Cassali GD, Poole S, Teixeira MM. Effects of inhibition of PDE4 and TNF-alpha on local and remote lesions following ischaemia and reperfusion injury Br J Pharmacol 2001; 134 (5): 985-94). The liver was removed and homogenized at 5% in EDTA / NaCl buffer (pH 4.7) and centrifuged at 10,000 rpm for 15 min at 4 ° C. Following washing steps, the supernatant was used in the enzymatic reaction to detect MPO, performed in the presence of tetramethylbenzidine (TMB, 1.6 mM), 80 mM NaPO and hydrogen peroxide (0.3 mM H2O2). Absorbance was measured at 595 nm and results expressed as optical density (OD) per mg of tissue. MPO is an indirect indicator of neutrophil migration, a parameter used as an indicator of oxidative stress and inflammatory response. Figure 3 demonstrates the reversal of myeloperoxidase (MPO) activity following resveratrol (RSV) + isoniazid (INH) + rifampiciha (RIF) treatment. The inhibitory effect observed for resveratrol was statistically significant (p <0.0001).
Exemplo 6 - Determinação da atividade da catalase Example 6 - Determination of Catalase Activity
Uma porção do fígado (± 100 mg) foi utilizada para a determinação da atividade da catalase, segundo a literatura (Aebi, H. Catalase in vitro. Methods Enzymol.1984; 105: 121 -126). Para tal, a degradação do H2O2 foi monitorada a 240 nm, a cada 30 s, durante 2 min. O coeficiente de extinção de H2O2 utilizado para determinar a atividade da catalase foi igual a 43.6 M"1.cm"1. Os resultados foram expressos em mmol/min/mg de proteína. A determinação da atividade da catalase, enzima peroxissomal detoxificante responsável pela manutenção dos baixos níveis de peróxido de hidrogénio (H2O2) nas células, é um parâmetro utilizado como indicador de estresse oxidativo. A figura 4 demonstra a manutenção dos níveis de atividade da catalase após o tratamento com resveratrol (RSV) + isoniazida (INH) + rifampicina (RIF), um efeito estatisticamente significante (P < 0,01). A portion of the liver (± 100 mg) was used for the determination of catalase activity according to the literature (Aebi, H. Catalase in vitro. Methods Enzymol.1984; 105: 121-126). To this end, H 2 O 2 degradation was monitored at 240 nm every 30 s for 2 min. The H 2 O 2 extinction coefficient used to determine catalase activity was 43.6 M "1 .cm " 1 . Results were expressed as mmol / min / mg protein. The determination of catalase activity, a detoxifying peroxisomal enzyme responsible for maintaining low levels of hydrogen peroxide (H 2 O 2 ) in cells, is a parameter used as an indicator of oxidative stress. Figure 4 shows the maintenance of catalase activity levels after resveratrol (RSV) + isoniazid (INH) + rifampicin (RIF) treatment, a statistically significant effect (P <0.01).
Exemplo 7 - Avaliação histológica para a determinação de lesão hepática Example 7 - Histological evaluation for liver injury determination
Para a avaliação histológica, uma porção do fígado foi fixada em formaldeído (10 %), durante 24 h. Posteriormente, os tecidos foram processados em parafina e secções seriadas de 5 μιτι foram coradas com hematoxilina e eosina. Os cortes foram analisados quanto à presença de esteatose e células necróticas. As imagens apresentadas na Figura 5 demonstram uma redução marcante da esteatose causada pela associação de INH e RIF, em camundongos tratados com resveratrol. For histological evaluation, a portion of the liver was fixed in formaldehyde (10%) for 24 h. Subsequently, tissues were processed in paraffin and serial 5 μιτι sections stained with hematoxylin and eosin. The sections were analyzed for the presence of steatosis and necrotic cells. The images shown in Figure 5 demonstrate a marked reduction in steatosis caused by the association of INH and RIF in resveratrol-treated mice.
Os versados na arte valorizarão os conhecimentos aqui apresentados e poderão reproduzir a invenção nas modalidades apresentadas e em outros variantes, abrangidos no escopo das reivindicações anexas.  Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments within the scope of the appended claims.

Claims

Reivindicações  Claims
FORMULAÇÃO FARMACÊUTICA E PROCESSO PARA REDUZIR PHARMACEUTICAL FORMULATION AND PROCESS TO REDUCE
HEPATOTOXICIDADE 1. Formulação farmacêutica caracterizada por compreender: HEPATOTOXICITY 1. Pharmaceutical formulation comprising:
a) resveratrol;  a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável.  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier.
2. Formulação, conforme reivindicação 1, caracterizada pelo resveratrol ser escolhido do grupo que compreende trans-resveratrol, trans-3,5-dimetoxi-4'- hidroxi-estilbeno, trans-3,5,4'-trimetoxi-estilbeno, trans-3,5-hidroxi-4'-metoxi- estilbeno, trans-3,5-diacetil-4'-hidroxi-estilbeno, trans-3,5,4'-triacetil-estilbeno, trans-3,5-hidroxi-4'-acetil-estilbeno e mistura dos mesmos.  Formulation according to Claim 1, characterized in that resveratrol is selected from the group comprising trans-resveratrol, trans-3,5-dimethoxy-4'-hydroxy stilbene, trans-3,5,4'-trimethoxy stilbene, trans -3,5-hydroxy-4'-methoxy-stilbene, trans-3,5-diacetyl-4'-hydroxy-stilbene, trans-3,5,4'-triacetyl-stilbene, trans-3,5-hydroxybutyl 4'-acetyl stilbene and mixture thereof.
3. Formulação, conforme reivindicação 2, caracterizada pelo resveratrol ser trans-resveratrol.  Formulation according to claim 2, characterized in that resveratrol is trans-resveratrol.
4. Formulação, conforme reivindicação 1 , caracterizada pelo composto útil no tratamento da tuberculose ser escolhido do grupo que compreende etambutol, rifampicina, pirazinamida, isoniazida, streptomicina, ciprofloxacina, morfloxacina, corticosteróides, e combinações dos mesmos.  Formulation according to Claim 1, characterized in that the compound useful in the treatment of tuberculosis is chosen from the group comprising ethambutol, rifampicin, pyrazinamide, isoniazid, streptomycin, ciprofloxacin, morphfloxacin, corticosteroids, and combinations thereof.
5. Formulação, conforme reivindicação 2, caracterizada pelo composto ser isoniazida e rifampicina.  Formulation according to claim 2, characterized in that the compound is isoniazid and rifampicin.
6. Formulação, conforme reivindicação 1 , caracterizada pela proporção resveratroixomposto útil no tratamento da tuberculose estar compreendida na faixa que vai de 0,4:1 a 2:1.  Formulation according to claim 1, characterized in that the resveratroboxin ratio useful in the treatment of tuberculosis ranges from 0.4: 1 to 2: 1.
7. Formulação farmacêutica compreendendo:  A pharmaceutical formulation comprising:
a) resveratrol;  a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável;  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier;
caracterizada por ser utilizada no preparo de um medicamento útil no tratamento da tuberculose. characterized in that it is used in the preparation of a medicament useful in the treatment of tuberculosis.
8. Formulação, conforme reivindicação 7, caracterizada pelo resveratrol ser escolhido do grupo que compreende trans-resveratrol, trans-3,5-dimetoxi-4'- hidroxi-estilbeno, trans-3,5,4'-trimetoxi-estilbeno, trans-3,5-hidroxi-4'-metoxi- estilbeno, trans-3,5-diacetil-4'-hidroxi-estilbeno, trans-3,5,4'-triacetil-estilbeno, trans-3,5-hidroxi-4'-acetil-estilbeno e mistura dos mesmos. Formulation according to Claim 7, characterized in that resveratrol is selected from the group comprising trans-resveratrol, trans-3,5-dimethoxy-4'-hydroxy stilbene, trans-3,5,4'-trimethoxy stilbene, trans -3,5-hydroxy-4'-methoxy-stilbene, trans-3,5-diacetyl-4'-hydroxy-stilbene, trans-3,5,4'-triacetyl-stilbene, trans-3,5-hydroxybutyl 4'-acetyl stilbene and mixture thereof.
9. Formulação, conforme reivindicação 8, caracterizada pelo resveratrol ser trans-resveratrol.  Formulation according to claim 8, characterized in that resveratrol is trans-resveratrol.
10. Formulação, conforme reivindicação 7, caracterizada pelo composto útil no tratamento da tuberculose ser escolhido do grupo que compreende etambutol, rifampicina, pirazinamida, isoniazida, streptomicina, ciprofloxacina, morfloxacina, corticosteróides, e combinações dos mesmos.  Formulation according to Claim 7, characterized in that the compound useful in the treatment of tuberculosis is selected from the group comprising ethambutol, rifampicin, pyrazinamide, isoniazid, streptomycin, ciprofloxacin, morphfloxacin, corticosteroids, and combinations thereof.
11. Formulação, conforme reivindicação 8, caracterizada pelo composto ser isoniazida e rifampicina.  Formulation according to claim 8, characterized in that the compound is isoniazid and rifampicin.
12. Formulação, conforme reivindicação 7, caracterizada pela proporção resveratrol:composto útil no tratamento da tuberculose estar compreendida na faixa que vai de 0,4:1 a 2:1.  Formulation according to claim 7, characterized in that the resveratrol: compound useful in the treatment of tuberculosis ranges from 0.4: 1 to 2: 1.
13. Processo para reduzir a hepatotoxicidade associada à administração de pelo menos um composto útil no tratamento da tuberculose caracterizado por compreender a etapa de administrar uma formulação farmacêutica compreendendo:  A process for reducing hepatotoxicity associated with the administration of at least one compound useful in the treatment of tuberculosis comprising the step of administering a pharmaceutical formulation comprising:
a) resveratrol;  a) resveratrol;
b) pelo menos um composto útil no tratamento da tuberculose; e c) um veículo farmaceuticamente aceitável;  b) at least one compound useful in the treatment of tuberculosis; and c) a pharmaceutically acceptable carrier;
a um mamífero necessitando de tratamento. to a mammal in need of treatment.
14. Processo, conforme reivindicação 13, caracterizado pela formulação farmacêutica ser conforme descrita nas reivindicações de 1 a 6.  Process according to claim 13, characterized in that the pharmaceutical formulation is as described in claims 1 to 6.
15. Processo, conforme reivindicação 13, caracterizado pelo mamífero ser o homem.  Process according to claim 13, characterized in that the mammal is male.
PCT/BR2011/000129 2010-04-22 2011-04-25 Pharmaceutical formulation and method for reducing hepatotoxicity WO2011130817A1 (en)

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CN107951870A (en) * 2016-10-14 2018-04-24 复旦大学 A kind of pharmaceutical composition of the suppression drug-resistant staphylococcus aureus containing resveratrol
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