Influence of silymarin administration on hepatic glutathione-conjugating enzyme system in rats treated with antitubercular drugs

OBJECTIVE: This study evaluated the influence of simultaneous administration of silymarin (SIL), a hepatoprotective and antioxidant agent, on the status of glutathione (GSH) and its metabolising enzymes in the liver tissue of rats treated with antitubercular drugs, i.e. isoniazid (INH), rifampicin (RIF) and pyrazinamide (PYR). METHODS: Male Wistar albino rats (n = 24) were randomly divided into four groups. Group I received saline as they served as controls. Group II rats were administered antitubercular drugs (INH 25 mg/kg + RIF 50 mg/kg + PYR 140 mg/kg orally) daily for 45 days. Group III animals were treated with SIL (50 mg/kg orally) simultaneously with the antitubercular drugs for the same period. Group IV animals were treated with SIL alone. The status of GSH, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-s-transferase (GST) in liver tissue was evaluated at the end of the study. RESULTS: Administration of antitubercular drugs caused a significant decrease (p < 0.001) in the status of GPx, GST and GR and of non-enzymic (GSH) antioxidants in liver tissue when compared with saline-treated control rats. Simultaneous treatment of SIL with antitubercular drugs completely prevented decreases in the levels of all the above parameters. Treatment with SIL alone enhanced the activities of GST (p < 0.001) and GPx (p < 0.05) and did not alter glutathione levels compared with control. CONCLUSION: A fall in the status of glutathione and its conjugating enzymes upon administration of antitubercular drugs denotes an impairment of the antioxidant defence mechanism. Simultaneous administration of SIL afforded complete protection of the liver against this abnormality, an effect that could have been due to the strong antioxidant properties of SIL.     

Hepatoprotective activity of chitosan against isoniazid and rifampicin-induced toxicity in experimental rats

Tuberculosis is a dangerous disease and its death toll is increasing year by year. Intake of isoniazid and rifampicin, the most common antitubercular drugs, lead to fatal hepatotoxic condition. We have studied the protective effect of chitosan supplementation against the hepatotoxicity induced by antitubercular drugs with respect to the changes in the levels of protein, albumin-globulin ratio, urea and bilirubin in the serum and diagnostic marker enzymes (alanine amino transferase, aspartate amino transferase, acid phosphatase and alkaline phosphatase), protein, glycoprotein conjugates (hexose, hexosamine and sialic acid), lipid peroxidation and reduced glutathione in the liver tissue of normal and experimental groups of rats. Co-administration of chitosan was found to significantly prevent the antitubercular drugs-induced alterations in the levels of diagnostic marker enzymes, bilirubin and albumin/globulin ratio in experimental groups of rats. Isoniazid and rifampicin-induced lipid peroxidation was also found to be prevented by the administration of chitosan. Further, chitosan administration increased the levels of urea and protein (in serum and liver) in experimental groups compared to hepatotoxicity-induced group of rats. Levels of glycoconjugates were also maintained to near normal level by chitosan co-administration. From the results obtained, it can be concluded that chitosan is beneficial against antitubercualr drugs-induced hepatoxicity.     

水飞蓟宾胶囊预防抗结核药物肝损害的临床观察

[摘要]目的:探讨水飞蓟宾胶囊在结核病治疗中预防药物肝损害的价值。方法:我院2007年7月~2009年1月期间住院治疗112例肺结核患者,治疗组(47例)在采取抗结核方案基础上应用水飞蓟宾胶囊（70mg,口服,tid）护肝,对照组(65例)未应用水飞蓟宾胶囊,采取抗结核方案与治疗组相同,观察两组肝功能损害的情况。结果:抗结核治疗期间治疗组发生肝功异常比例明显低于对照组,两组比较有统计学差异(P<0.05)。治疗组发生肝功能异常停用抗结核药物给予保肝治疗,肝功能恢复正常平均时间低于对照组,两组比较有统计学差异（P<0.05）。结论:结核病患者在常规抗结核治疗的同时应用水飞蓟宾胶囊可以降低肝损害的发生率,发生肝损害的程度也可减轻。     

Synthesis, characterization and in-vitro antitubercular activity of isoniazid-gelatin conjugate

Objectives A novel and simple method to synthesize antitubercular‐protein conjugate by solid phase synthesis was developed employing a carboxypolystyrene resin. The aim was to covalently bind a drug with antitubercular activity, isoniazid, to a biomacromolecule, gelatin, widely used in the pharmaceutical, cosmetic and food industry. Methods Calorimetric and ¹H NMR analyses were performed to verify the bond formation between the antitubercular drug and gelatin. After absorption isoniazid delivers toxic metabolites and so an oxidation test with tert‐butyl hydroperoxide was performed to assess the amount of toxic metabolites released from the prodrug (gelatin linked to isoniazid), compared with isoniazid itself. Key findings Spectrophotometric analysis revealed that the protein derivative was an excellent isoniazid prodrug since there was a 40% reduction in release of toxic metabolites (isonicotinic acid) by the prodrug. The results clearly showed that antitubercular moieties, covalently linked to a natural polymer, allowed the introduction of peculiar features for specific pharmaceutical applications into the macromolecule. In addition, antitubercular activity of the new polymer was determined by Middlebrook 7H11 medium against Mycobacterium tuberculosis complex. Conclusions The new isoniazid‐gelatin conjugate showed significant antitubercular activity and for this reason should be useful as an efficacious tool in the treatment of tuberculosis.     

Spray-dried fucoidan microparticles for pulmonary delivery of antitubercular drugs

Pulmonary tuberculosis accounts for 80% of cases and the delivery of antitubercular drugs into the lungs allows targeting the infected organ and, possibly, reducing systemic drug toxicity. This work aimed at using fucoidan as matrix of inhalable microparticles that associate two first-line antitubercular drugs, for an application in pulmonary tuberculosis therapy. Fucoidan is composed of fucose and sulphated sugar residues, moieties described as being recognised by surface receptors of alveolar macrophages, which host mycobacteria. Inhalable fucoidan microparticles loaded with antitubercular drugs were successfully produced with high association efficiencies of either isoniazid (95%) or rifabutin (81%). The microparticles evidenced no cytotoxicity on lung epithelial cells (A549). However, rifabutin-loaded microparticles showed a certain degree of toxicity on macrophage-like cells (THP-1) at the highest tested concentration (1?mg/mL). Furthermore, microparticles showed favourable aerodynamic properties for deep lung delivery (MMAD 2.0–3.8?µm) and, thus, show potential for an application as inhalable tuberculosis therapy.     

Inhibition of siderophore biosynthesis in Mycobacterium tuberculosis with nucleoside bisubstrate analogues: structure-activity relationships of the nucleobase domain of 5'-O-[N-(salicyl)sulfamoyl]adenosine

5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) is a prototype for a new class of antitubercular agents that inhibit the aryl acid adenylating enzyme (AAAE) known as MbtA involved in biosynthesis of the mycobactins. Herein, we report the structure-based design, synthesis, biochemical, and biological evaluation of a comprehensive and systematic series of analogues, exploring the structure-activity relationship of the purine nucleobase domain of Sal-AMS. Significantly, 2-phenyl-Sal-AMS derivative 26 exhibited exceptionally potent antitubercular activity with an MIC99 under iron-deficient conditions of 0.049 microM while the N-6-cyclopropyl-Sal-AMS 16 led to improved potency and to a 64-enhancement in activity under iron-deficient conditions relative to iron-replete conditions, a phenotype concordant with the designed mechanism of action. The most potent MbtA inhibitors disclosed here display in vitro antitubercular activity superior to most current first line TB drugs, and these compounds are also expected to be useful against a wide range of pathogens that require aryl-capped siderphores for virulence.     

Protective potential [correction of potencial] of Euphorbia hirta against cytotoxicity induced in hepatocytes and a HepG2 cell line

Medicinal plants play a key role in human health care. Frustration over the side effects of allopathic drugs has driven the medical world to take asylum in the plant kingdom for the treatment of various ailments. Euphorbia hirta belonging to the family of Euphorbiacae has been reported to possess antibacterial, antiviral, and anticancer activity. The aim of the present study was to investigate the protective effect of E. hirta against antitubercular drug-induced cytotoxicity in freshly isolated hepatocytes. The extent of cytotoxicity of the plant extracts was also analyzed using human liver derived HepG2 cell line by estimating the viability of cells (MTT assay). The alcoholic plant extract normalized the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), triacylglycerol (TAG), cholesterol, total protein, albumin, total and direct bilirubin, which were altered due to antitubercular drug intoxication. A dose-dependent increase in percent viability was observed when antitubercular drug exposed HepG2 cells were treated with different concentrations of plant extracts (125, 250, 500 and 1000 microg/mL) which were compared with a standard hepatoprotective drug silymarin. The highest percentage viability of HepG2 was observed at a concentration of 1000 microg/mL. The results suggest that E. hirta exerts protection against antitubercular drug-induced cytotoxicity in this vitro model system.     

Effect of chitosan supplementation on antitubercular drugs-induced hepatotoxicity in rats

We have studied the protective effect of chitosan on isoniazid- and rifampicin-induced hepatotoxicity with respect to the changes in the levels of diagnostic marker enzymes (in serum), lipid components and lipid peroxidation (in serum and liver). The oral administration of antitubercular drugs caused a significant elevation in the levels of diagnostic marker enzymes and cholesterol, triglycerides, free fatty acids and lipid peroxidation in serum and liver of experimental rats. There was a slight decline in the level of phospholipids in liver tissue also observed. Co-administration of chitosan significantly prevented the antitubercular drugs-induced elevation in the levels of serum diagnostic marker enzymes (alanine amino transferase, aspartate amino transferase, lactate dehydrogenase, acid phosphatase and alkaline phosphatase) in experimental groups of rats. It exerted a significant antilipidemic effect against isoniazid- and rifampicin-induced hepatitis by maintaining the levels cholesterol, triglycerides, free fatty acids and phospholipids in serum and liver at near normalcy. A tendency to prevent the isoniazid- and rifampicin-induced lipid peroxidation was also observed. The results of the present study indicated that the hepatoprotective effect of chitosan might be ascribable to its antilipidemic effect and/or antioxidant property.     

Identification of 1-[4-Benzyloxyphenyl)-but-3-enyl]-1H-azoles as New Class of Antitubercular and Antimicrobial Agents

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结核分枝杆菌泛酸合成酶抑制剂的实验研究

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Colloidal carriers: a rising tool for therapy of tuberculosis

Tuberculosis (TB) is the second most deadly infectious disease, caused mainly by M. tuberculosis in humans, usually affecting the lungs; it also attacks other parts of the body. The design of novel antibiotics attempts to overcome drug resistance, to shorten the treatment course, and to reduce drug interactions with antiretroviral therapies. Overcoming technological drawbacks of these therapeutic agents as well as improving the effectiveness of the drugs by targeting the infection reservoirs remain the central aims of pharmaceutical technology. In this framework, colloidal carriers appear as one of the most promising approaches for the development of more effective and compliant medicines by releasing the drugs slowly over prolonged time periods and reducing the current costs of treatment. Due to unique physicochemical properties (ultrasmall and controllable size, large surface area to mass ratio, high reactivity, and functionalizable structure) of colloidal carriers, they can facilitate the administration of antitubercular drugs, thereby overcoming some of the limitations in traditional antitubercular therapeutics. In recent years, encapsulation of antitubercular drugs in colloidal carrier systems is emerging as an innovative and promising alternative with enhanced therapeutic effectiveness and reduced undesirable side effects of the encapsulated drugs. The present review aims to describe the current conventional as well as combination drug therapy with special consideration towards the emerging role of novel colloidal carriers designed and targeted against TB. Colloidal carriers employing drugs alone or in combination targeted towards the site of action could lead to reduction in duration of conventional treatment, higher patient fulfillment, and prevention of antitubercular drug resistance or toxicity.     

Synthesis and antitubercular activity of N-(2-naphthyl)glycine hydrazide analogues

N-(2-Naphthyl)glycine hydrazide analogues were synthesized and tested for possible in vitro antitubercular activity. N-(2-Naphthyl)alanine hydrazide (3), N-methyl-N-(2-naphthyl)glycine hydrazide (5), N-(6-methoxy-2-naphthyl)glycine hydrazide (7), and 3-(2-naphthylamino)butyric acid hydrazide (23) showed potent inhibitory action against Mycobacterium tuberculosis H37Rv in Youman's medium at concentrations ranging from 0.5 to 10.0 micrograms/mL. These compounds showed significant inhibitory action against isonicotinic acid hydrazide and streptomycin-resistant strains of M. tuberculosis. N-(6-Quinolyl)glycine hydrazide (18) and 3-(2-quinolylamino)butyric acid hydrazide (24), which are bioisosteres of compounds 1 and 23, showed loss of antitubercular activity at low concentrations.     

Pharmacokinetic and pharmacodynamic behaviour of antitubercular drugs encapsulated in alginate nanoparticles at two doses

This study was designed to evaluate the pharmacokinetics and tissue distribution of free and alginate-encapsulated antitubercular drugs in mice at different doses. Alginate nanoparticles encapsulating isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) were prepared by controlled cation-induced gelification of alginate. The formulation was orally administered to mice at two dose levels (D1 and D2). A comparison was made in mice receiving free drugs at equivalent doses. Drugs were analysed by high performance liquid chromatography (HPLC). The average size of alginate nanoparticles was found to be 235.5+/-0.0 nm with a polydispersity index of 0.44; drug encapsulation was 70-90% for INH and PZA, 80-90% for RIF and 88-95% for EMB. In the free drug groups, plasma levels of RIF and INH were higher and PZA and EMB levels were lower in the D1 group (per body surface area of mice) compared with the D2 group (recommended human dose). The plasma drug levels of all drugs were higher in the D1 encapsulated group compared with D2, resulting in higher values of area under the plasma drug concentration-time curve (AUC(0-infinity)). The relative bioavailabilities of all drugs encapsulated in alginate nanoparticles were significantly higher compared with free drugs. Drug levels were maintained at or above the minimum inhibitory concentration (MIC(90)) until Day 15 in organs after administration of encapsulated drugs, whilst free drugs stayed at or above the MIC(90) up to Day 1 only irrespective of dose. The levels of drugs in various organs remained above the MIC at both doses for equal periods, demonstrating their equiefficiency. Alginate nanoparticles hold great potential in reducing dosing frequency of antitubercular drugs.     

Structural analysis of nanocapsules by nuclear magnetic resonance

An oral formulation based on poly (DL-lactide-co-glycolide) (PLG) microparticles was developed for delivery of antituberculous drugs. PLG entrapped antitubercular drugs when administered orally, were found to release the drugs in a sustained manner. This formulation was found to be stable in the acidic environment of gastric fluid whereas, in the intestinal fluid the drug release was obtained up to 20 days as indicated by in vitro studies. Pharmacokinetic analysis of the data revealed changes in C(max); AUC(o-alpha); t(1/2) (a) and t(1/2) (e) when drugs were given entrapped in PLG microparticles. Higher peak concentration, area under the concentration time curve and delayed elimination rate of entrapped drugs indicated the potential of PLG for effective treatment of tuberculosis. Further, work is being carried out to evaluate the chemotherapeutic efficacy of the antitubercular drugs encapsulated in PLG microspheres.     

Oxidative activation of thiacetazone by the Mycobacterium tuberculosis flavin monooxygenase EtaA and human FMO1 and FMO3

Thiacetazone (TAZ) and ethionamide (ETA) are, respectively, thiourea- and thioamide-containing second line antitubercular prodrugs for which there is an extensive clinical history of cross-resistance in Mycobacterium tuberculosis. EtaA, a recently identified flavin-containing monooxygenase (FMO), is responsible for the oxidative activation of ETA in M. tuberculosis. We report here that EtaA also oxidizes TAZ and identify a sulfinic acid and a carbodiimide as the isolable metabolites. Both of these metabolites are derived from an initial sulfenic acid intermediate. Oxidation of TAZ by EtaA at basic pH favors formation of the carbodiimide, whereas neutral or acidic conditions favor formation of the sulfinic acid. The same metabolites are formed from TAZ by human FMO1 and FMO3. The sulfenic acid and carbodiimide metabolites, but not the sulfinic acid product, readily react with glutathione, the first to regenerate the parent drug and the second to give a glutathione adduct. These reactions may contribute to the antitubercular activity and/or toxicity of TAZ.     

Shikimate kinase: a potential target for development of novel antitubercular agents

Tuberculosis (TB) remains the leading cause of mortality due to a bacterial pathogen, Mycobacterium tuberculosis. However, no new classes of drugs for TB have been developed in the past 30 years. Therefore there is an urgent need to develop faster acting and effective new antitubercular agents, preferably belonging to new structural classes, to better combat TB, including MDR-TB, to shorten the duration of current treatment to improve patient compliance, and to provide effective treatment of latent tuberculosis infection. The enzymes in the shikimate pathway are potential targets for development of a new generation of antitubercular drugs. The shikimate pathway has been shown by disruption of aroK gene to be essential for the Mycobacterium tuberculosis. The shikimate kinase (SK) catalyses the phosphorylation of the 3-hydroxyl group of shikimic acid (shikimate) using ATP as a co-substrate. SK belongs to family of nucleoside monophosphate (NMP) kinases. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices. The shikimate kinases are composed of three domains: Core domain, Lid domain and Shikimate-binding domain. The Lid and Shikimate-binding domains are responsible for large conformational changes during catalysis. More recently, the precise interactions between SK and substrate have been elucidated, showing the binding of shikimate with three charged residues conserved among the SK sequences. The elucidation of interactions between MtSK and their substrates is crucial for the development of a new generation of drugs against tuberculosis through rational drug design.     

Current status and future development of antitubercular chemotherapy

Tuberculosis (TB), which kills more people than any other infectious disease, was declared a global emergency by the World Health Organization in 1993. The emergence of new Mycobacterium tuberculosis strains that are resistant to some or all current antitubercular drugs seriously hampers the control of the disease. Up to 50 million people may be infected with drug-resistant TB, with resistance being caused by inconsistent or partial treatment when patients do not comply with long-term chemotherapy. Resistance is often a corollary to HIV infection. Besides being more fatal, drug-resistant TB is more difficult and more expensive to treat. In addition to this human cost, TB also represents a significant economic burden for developing countries. Therefore, new approaches to the treatment of TB are needed. During the last few years, important efforts have been made in order to elucidate the molecular mechanism of action of antitubercular drugs and understand the genetic basis of acquired drug resistance in M. tuberculosis. The identification of novel targets requires the characterisation of biochemical pathways specific to mycobacteria. Many unique metabolic processes occur during the biosynthesis of cell wall components, including arabinogalactan and mycolic acids. In this review, the mode of action of first- and second-line agents, as well as the potentiality of some promising drugs that are still at an early stage of development will be described. Finally, some of the attractive targets offered by the mycobacterial cell wall for the rational design of new antitubercular drugs for a future and more effective control of the disease will be examined.     

Current status and future development of antitubercular chemotherapy

Tuberculosis (TB), which kills more people than any other infectious disease, was declared a global emergency by the World Health Organization in 1993. The emergence of new Mycobacterium tuberculosis strains that are resistant to some or all current antitubercular drugs seriously hampers the control of the disease. Up to 50 million people may be infected with drug-resistant TB, with resistance being caused by inconsistent or partial treatment when patients do not comply with long-term chemotherapy. Resistance is often a corollary to HIV infection. Besides being more fatal, drug-resistant TB is more difficult and more expensive to treat. In addition to this human cost, TB also represents a significant economic burden for developing countries. Therefore, new approaches to the treatment of TB are needed. During the last few years, important efforts have been made in order to elucidate the molecular mechanism of action of antitubercular drugs and understand the genetic basis of acquired drug resistance in M. tuberculosis. The identification of novel targets requires the characterisation of biochemical pathways specific to mycobacteria. Many unique metabolic processes occur during the biosynthesis of cell wall components, including arabinogalactan and mycolic acids. In this review, the mode of action of first- and second-line agents, as well as the potentiality of some promising drugs that are still at an early stage of development will be described. Finally, some of the attractive targets offered by the mycobacterial cell wall for the rational design of new antitubercular drugs for a future and more effective control of the disease will be examined.     

Plasma pooling methodology as a faster and cheaper tool to evaluate bioequivalence of rifampicin component of FDCs of antitubercular drugs

Rifampicin is one of the most important first line drugs used in fixed dose combinations (FDCs) of antitubercular drugs. The chances of reduced bioavailability of rifampicin from FDCs necessitated its evaluation against standard formulations of individual drugs in bioequivalence studies. This study was undertaken to evaluate the importance of plasma pooling methodology as a rapid and cheaper tool to evaluate bioequivalence of rifampicin component of FDCs of antitubercular drugs. Plasma samples of volunteers obtained from bioequivalence studies were pooled according to volunteer and time-wise pooling. Area under the plasma concentration versus time curves (AUCs) of rifampicin was reduced in the pooled plasma samples when compared to the individual samples. However, the ratio of AUCs of FDCs to that of separate formulations was found to be comparable for both the individual samples and pooled samples data. Concentration-time profiles of rifampicin obtained after time-wise pooling were subjected to non-compartmental analysis for determination of pharmacokinetic parameters. Whereas bioequivalence estimates were determined using individual AUC values obtained from volunteer-wise pooling. Results indicated the possibility of using plasma pooling methodology in bioequivalence estimation of rifampicin to simplify and accelerate the registration process.