A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm^?1 shift from 1306 cm^?1 in aqueous solution to 1314 cm^?1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding ability. In non-hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm^?1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm^?1; in water, a strong hydrogen-bonding solvent, 1306 cm^?1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm^?1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

A novel synergistic galactomannan-based unit dosage form for sustained release of acarbose

In the current study, the potential of a novel combination of a galactomannan with acarbose (100 mg) was evaluated for attaining a desired hypoglycaemic effect over a prolonged period of time. Three major antidiabetic galactomannans viz., fenugreek gum, boswellia gum, and locust bean gum were selected in order to achieve a synergistic effect in the treatment alongwith retardation in drug release. In vitro studies indicated that batches containing various proportions of fenugreek gum (AF40-60) were able to control drug release for a longer duration of approximately 10–12 h. In contrast, the matrices prepared using boswellia and locust bean gum were able to sustain the release for relatively shorter durations. Drug release mainly followed first-order release kinetics owing to the highly soluble nature of the drug. In vivo study depicted a significant reduction (p < 0.001) in the postprandial blood glucose and triglyceride levels in the diabetic rats on treatment with formulation AF40. Thus, the developed system provides a better control of the postprandial glycaemic levels and it also obviates the need of conventional multiple dosing of acarbose. Furthermore, it also reduces the occurrence of side effects like diarrhea and loss of appetite.     

A new rapid colourimetric method for testing Mycobacterium tuberculosis susceptibility to isoniazid and rifampicin: a crystal violet decolourisation assay

The aim of this study was to investigate the performance of a new and accurate method for the detection of isoniazid (INH) and rifampicin (RIF) resistance among Mycobacterium tuberculosis isolates using a crystal violet decolourisation assay (CVDA). Fifty-five M. tuberculosis isolates obtained from culture stocks stored at -80ºC were tested. After bacterial inoculation, the samples were incubated at 37ºC for seven days and 100 µL of CV (25 mg/L stock solution) was then added to the control and sample tubes. The tubes were incubated for an additional 24-48 h. CV (blue/purple) was decolourised in the presence of bacterial growth; thus, if CV lost its colour in a sample containing a drug, the tested isolate was reported as resistant. The sensitivity, specificity, positive predictive value, negative predictive value and agreement for INH were 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively, and 88.8%, 100%, 100%, 94.8% and 96.3%, respectively, for RIF. The results were obtained within eight-nine days. This study shows that CVDA is an effective method to detect M. tuberculosis resistance to INH and RIF in developing countries. This method is rapid, simple and inexpensive. Nonetheless, further studies are necessary before routine laboratory implementation.     

结核分枝杆菌异烟肼耐药性的分子遗传和生物化学机理

枝菌酸是结核分枝杆菌细胞壁组分之一。异烟肼作用的主要靶标是参与枝菌酸生物合成的蛋白质。临床分离耐药株多数在KatG蛋白发生点突变,该蛋白也是INH作用靶标之一。但是,现有数据还不能解释INH耐药和INH杀灭细菌的全部机理。我们研究结核分枝杆菌耐药机理应该高起点,综合利用生物信息学、基因芯片、蛋白质组学、结构生物学等技术进行研究。     

Isonicotinoylhydrazone analogs of isoniazid: relationship between superoxide scavenging and tuberculostatic activities

Superoxide scavenging activity (SSA) of recently synthesized isonicotinoylhydrazones, analogs of the clinically used anti-tuberculosis drug isoniazid (INH), was investigated using xanthine/xanthine oxidase system to generate the superoxide anion. The isonicotinoylhydrazones exhibited well expressed SSA, whereas INH did not show any SSA. All of the isonicotinoylhydrazones had a tuberculostatic activity when tested with the standard strain of Mycobacterium tuberculosis H₃₇R_v and some of them had a higher tuberculostatic activity than INH. A lower acute toxicity was also observed compared to INH. Moreover, a correlation was observed between LD₅₀ and SSA for the isonicotinoylhydrazones studied. An explanation is suggested for the higher tuberculostatic activity and lower acute toxicity of some of the isonicotinoylhydrazones as compared to that of INH. A new route to less toxic derivatives of INH with potential tuberculostatic activity is proposed.     

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.     

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.     

Lack of liver injury in Wistar rats treated with the combination of isoniazid and rifampicin

Isoniazid (INH) can cause serious idiosyncratic liver injury. An animal model would greatly facilitate mechanistic studies, but it is essential that the mechanism in the model be similar to the liver injury that can occur in humans. We attempted to replicate a previous study in which Wistar rats treated with INH and rifampicin (RMP) developed liver injury, which was promising because of its delayed onset similar to the liver injury that can occur in humans. Wistar rats were treated with either a high dose of INH (150 mg/kg/day) or a combination of INH and RMP (75 mg/kg/day and 50 mg/kg/day, respectively) for up to 4 weeks. However, we did not observe any liver injury or evidence of an inflammatory infiltrate as had been reported; rather, we observed an increase in CTLA4-positive cells in the cervical lymph nodes as well as a decrease in serum CXCL1 and MCP-1. In short, we were unable to reproduce a previously reported model of delayed onset INH-induced liver injury in Wistar rats.     

Controlled Release Hydrophilic Matrix Tablet Formulations of Isoniazid: Design and In Vitro Studies

The aim of the present investigation was to develop oral controlled release matrix tablet formulations of isoniazid using hydroxypropyl methylcellulose (HPMC) as a hydrophilic release retardant polymer and to study the influence of various formulation factors like proportion of the polymer, polymer viscosity grade, compression force, and release media on the in vitro release characteristics of the drug. The formulations were developed using wet granulation technology. The in vitro release studies were performed using US Pharmacopoeia type 1 apparatus (basket method) in 900 ml of pH 7.4 phosphate buffer at 100 rpm. The release kinetics was analyzed using Korsmeyer–Peppas model. The release profiles were also analyzed using statistical method (one-way analysis of variance) and f ₂ metric values. The release profiles found to follow Higuchi’s square root kinetics model irrespective of the polymer ratio and the viscosity grade used. The results in the present investigation confirm that the release rate of the drug from the HPMC matrices is highly influenced by the drug/HPMC ratio and viscosity grade of the HPMC. Also, the effect of compression force and release media was found to be significant on the release profiles of isoniazid from HPMC matrix tablets. The release mechanism was found to be anomalous non-Fickian diffusion in all the cases. In the present investigation, a series of controlled release formulations of isoniazid were developed with different release rates and duration so that these formulations could further be assessed from the in vivo bioavailability studies. The formulations were found to be stable and reproducible.     

Synthesis of new verapamil analogues and their evaluation in combination with rifampicin against Mycobacterium tuberculosis and molecular docking studies in the binding site of efflux protein Rv1258c

New verapamil analogues were synthesized and their inhibitory activities against Mycobacterium tuberculosis H37Rv determined in vitro alone and in combination with rifampicin (RIF). Some analogues showed comparable activity to verapamil and exhibited better synergies with RIF. Molecular docking studies of the binding sites of Rv1258c, a M. tuberculosis efflux protein previously implicated in intrinsic resistance to RIF, suggested a potential rationale for the superior synergistic interactions observed with some analogues.     

Simultaneous determination of isoniazid and p‐aminosalicylic acid by capillary electrophoresis using chemiluminescence detection

It was found that isoniazid (ISO) or p‐aminosalicylic acid (PAS) could enhance the chemiluminescence (CL) emission from Cu (II)‐luminol‐hydrogen peroxide system, and the increased chemiluminescence signals were proportional to their concentrations, respectively. Based on this phenomenon, a chemiluminescence method coupled to capillary electrophoresis (CE) was established for simultaneous determination of ISO and PAS. The CE conditions including running buffer and running voltage were investigated in detail. The effects of the pH of H₂O₂ solution and the concentrations of luminol, H₂O₂ and Cu (II) on the CL signal were also investigated carefully. Under the optimized conditions, the analysis could be accomplished within 10 min, with the limits of detection of 0.3 µg mL^–1 for ISO and 1.1 µg mL^–1 for PAS, corresponding to 7.2 and 26.4 pg per injection (24 nL), respectively. Finally, the method was validated by determining the two analytes in pharmaceutical preparation and spiked human serum samples. The results of pharmaceutical tablet analysis were in good agreement with the labeled amounts. The recoveries for ISO and PAS in human serum were in the range of 92–104% and 90–113%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

相对定量分析异烟肼、链霉素单耐药与多耐药结核分枝杆菌临床分离株的蛋白质组差异

【目的】探讨异烟肼(isoniazid,INH)、链霉素(streptomycin,SM)单耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)与INH/SM多耐药MTB蛋白质组差异。【方法】应用i TRAQ结合Nano LC-MS/MS定量蛋白质组学技术,分析临床分离INH、SM或INH/SM耐药MTB与H37Rv标准株间均表达差异蛋白;并以INH/SM耐药MTB与H37Rv比值为对照,相对定量分析单耐药与多耐药MTB蛋白表达差异倍数;运用DAVID 6.7分析差异蛋白生物功能;STITCH 5.0分析差异蛋白与INH和SM相互作用。【结果】与H37Rv标准株比较,58个蛋白在INH、SM耐药与INH/SM耐药MTB间均有表达差异,共同差异蛋白生物功能主要为氧化还原酶活性和转移酶活性;主要参与丙酸代谢信号通路。共同差异蛋白中,与INH/SM耐药MTB比较,Rv2986c和Rv1908c在INH、SM耐药MTB均表达上调1.25倍;Rv3133c和Rv0577则均表达下调0.7倍;生物信息学预测发现以上4种蛋白可直接或间接与INH、SM进行相互作用。【结论】INH、SM单耐药和INH/SM多耐药MTB蛋白表达谱有较大差异,蛋白Rv2986c、Rv1908c、Rv3133c和Rv0577表达水平及相互作用可能与INH和SM耐药有关。     

A novel method for selective isotope labeling of bacterially expressed proteins

Summary A novel method for isotope labeling in selected amino acids is presented for use with the T7 RNA polymerase system. The protocol is illustrated with the DNA-binding domain from the E2 protein of bovine papillomavirus, BPV-1. On addition of rifampicin, protein expression occurs exclusively from the gene controlled by the T7 promoter. Since the bacteria are now dedicated to the production of E2 protein, labeling with specific amino acids is efficiently performed. For example, 10 mg/l of ¹⁵N-labeled phenylalanine is shown to be sufficient for incorporation of the label, without scrambling, and without the use of an auxotrophic strain.     

Detection of mutations associated with resistance to rifampicin and isoniazid in Mycobacterium tuberculosis by polymerase chain reaction-ligase detection reaction

Mycobacterium tuberculosis （MTB） infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB （MDR-TB）, defined as being resistant to at least rifampicin （RMP） and isoniazid （1NH）,     

Biochemical studies with bi-resistant mutants (ethambutol plus streptomycin and isoniazid plus streptomycin) ofMycobacterium smegmatis ATCC 607

Biochemical characteristics of bi-resistant mutants (resistant to ethambutol plus streptomycin or isoniazid plus streptomycin) of mycobacteria isolated by replica plating fromMycobacterium smegmatis ATCC were compared with those of the drug-susceptible strains. Reduced incorporation of [¹⁴C]uracil, [³H]lysine and [¹⁴C]acetate into RNA, protein and phospholipids respectively was seen in the resistant mutants. Total phosphorlipids were enhanced in ethambutol plus streptomycin resistant mutant and decreased in isoniazid plus streptomycin resistant mutant. There were similar changes in levels of individual phospholipids. The resistant mutants revealed an accumulation of phospholipids in the cell wall, and a marked decrease of phospholipids in the cell membrane in comparison to the susceptible strain. Several qualitative alterations in the polypeptide profile (with respect to number and molecular weight) of the crude protein extract and of different subcellular compartments were seen in the resistant mutants.     

A novel design of bioartificial kidneys with improved cell performance and haemocompatibility

Treatment with bioartificial kidneys had beneficial effects in animal experiments and improved survival of critically ill patients with acute kidney injury in a Phase II clinical trial. However, a Phase II b clinical trial failed. This and other results suggested various problems with the current design of bioartificial kidneys. We propose a novel design to improve various properties of device, including haemocompatibility and cell performance. An important feature of the novel design is confinement of the blood to the lumina of the hollow fibre membranes. This avoids exposure of the blood to the non‐haemocompatible outer surfaces of hollow fibre membranes, which usually occurs in bioartificial kidneys. We use these outer surfaces as substrate for cell growth. Our results show that commercial hollow fibre membranes can be directly applied in the bioreactor when human primary renal proximal tubular cells are grown in this configuration, and no coatings are required for the formation of robust and functional renal epithelia. Furthermore, we demonstrate that the bioreactor unit produces significant amounts of interleukins. This result helps to understand the immunomodulatory effects of bioartificial kidneys, which have been observed previously. The novel bioartificial kidney design outlined here and the results obtained would be expected to improve the safety and performance of bioartificial kidneys and to contribute to a better understanding of their effects.     

Spectrofluorimetric determination of eptifibatide in human plasma and dosage form

A spectrofluorimetric method for the determination of eptifibatide is presented based on its native fluorescence. The type of solvent and the wavelength of maximum excitation and emission were carefully selected to optimize the experimental conditions. Under the specified experimental conditions, the linearities obtained between the emission intensity and the corresponding concentrations of eptifibatide were in the range 0.1–2.5 μg/ml for the calibration curve constructed for direct determination of eptifibatide in dosage form and 0.05–2.2 μg/ml for the calibration curve constructed in spiked human plasma with a good correlation coefficient (r > 0.99). The lower limit of quantification for the calibration curve constructed in human plasma was 0.05 μg/ml. Recovery results for eptifibatide in spiked plasma samples and in dosage form, represented as mean ± % RSD, were 95.17 ± 1.94 and 100.29 ± 1.33 respectively. The suggested procedures were validated according to the International Conference on Harmonization (ICH) guidelines for the direct determination of eptifibatide in its pure form and dosage form and United States Food and Drug Administration (US FDA) Guidance for Industry, Bioanalytical Method Validation for the assay of eptifibatide in human plasma.     

摄入异烟肼对家蚕幼虫体内谷胱甘肽氧化还原循环和谷胱甘肽S-转移酶活性的影响

为了建立家蚕Bombyx mori的药物筛选和毒性评价模型, 以剂量为2 000 mg/kg的抗结核模药异烟肼饲喂家蚕5龄第3天幼虫后检测其中肠和脂肪体的抗氧化解毒相关代谢的变化。结果表明： 雌蚕中肠组织中, 总谷胱甘肽(GSH+2GSSG)、 还原型谷胱甘肽(reduced glutathione, GSH)和氧化型谷胱甘肽(oxidized glutathione, GSSG)含量均呈现迅速上升再缓慢下降趋势; 谷胱甘肽S 转移酶(glutathione S-transferase, GST)活性升高到较大值后逐渐降低; GSH/GSSG的比值下降表明, 在72 min后中肠组织向氧化态转移。脂肪体组织中, 总谷胱甘肽、 GSH和GSSG含量变化均呈现迅速下降再迅速上升的趋势; GST活性达到最大值后逐渐降低后趋于平稳; GSH/GSSG比值升高表明, 在72 min后脂肪体组织向还原态转移。无论雌蚕还是雄蚕, 总谷胱甘肽、 GSH和GSSG含量以及GST活性均是脂肪体高于中肠。雌蚕的总谷胱甘肽含量、 GSH和GSSG含量高于雄蚕, 但雄蚕的GST活性高于雌性。结果说明, 摄入异烟肼引起了家蚕幼虫体内谷胱甘肽氧化还原状态的改变和酶活性的变化, 在这个过程中脂肪体起主要解毒代谢作用。     

A new fluorescence probe for sofosbuvir analysis in dosage form and spiked human plasma

A simple, rapid, and low-cost technique was developed to allow reliable analysis of the anti-hepatitis C drug sofosbuvir in bulk, tablet form, and spiked human plasma. This method depends on the ability of sofosbuvir to quench the fluorescence of the newly synthesized 2-amino-3-cyano-4,6-dimethylpyridine (reagent 3 ). Elemental analysis and spectral data were used to validate the structure of the synthesized reagent. The newly synthesized reagent exhibited a satisfactory level of fluorescence emission at 365 nm after excitation at 247 nm. All experimental variables that might affect the quenching process were analyzed and optimized. Linearity, range, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) were all validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The concentration range was shown to be linear between 0.1 and 1.5 μg/mL. The technique was effectively utilized for sofosbuvir analysis in both its tablet dosage form and spiked human plasma, with mean percentage recoveries of 100.13 ± 0.35 and 94.26 ± 1.69, respectively.