Effects of four Indian medicinal herbs on Isoniazid-, Rifampicin- and Pyrazinamide-induced hepatic injury and immunosuppression in guinea pigs

AIM: To evaluate and compare the hepatoprotective and immunomodulatory effects of Curcuma longa (CL), Ocimum sanctum (OS), Tinospora cordifolia (TC) and Zizyphus mauritiana (ZM) on liver injury and immunosuppression induced by Isoniazid (INH), Rifampicin (RIF) and Pyrazinamide (PZA). METHODS: Duncan Hartley guinea pigs, weighing 700-1050 g, were treated orally with 50 mg/kg of INH, 100 mg/kg of RIF and 300 mg/Kg of PZA for 21-d. 200 mg/kg (bw) of each herb crude extract was administered to the herb control group and 2-h previous to INH + RIF + PZA (AKT) doses to the Herb + AKT groups. Serum alanine aminotransferase (ALT), aspertate aminotransferase (AST) bilirubin and Alkaline Phosphatase (ALP) were assessed on d 0 and 21 in all the groups. Phagocytic % (P%), Phagocytic Index (PI) and Chemotactic Index (CI) were also measured as immunologic parameters. Histological analysis was carried out to assess injury to the liver. RESULTS: The AKT treated control group showed hepatotoxicity as judged by elevated serum AST 5-fold, AST/ALT ratio 4-fold, ALP 2-fold and hepatological changes, such as focal necrosis, portal triaditis and steatosis. Immune function was suppressed as judged by decreased P% (51.67 ?1.68 vs 40.61 ?1.28, P < 0.01), PI (2.0725±0.05 vs 0.61±0.05, P < 0.001) and CI (1.8525±0.04 vs 0.695±0.07, P < 0.001). All four herb treated groups showed normal liver histology, enzyme levels and increased P%, while PI and CI were enhanced in the TC and ZM treated groups, respectively. CL + AKT, TC + AKT and ZM + AKT showed nearly normal histology with minimal inflammation and microvesicular steatosis, while OS + AKT showed partial protection. Hepatotoxicity was prevented by restricting the rise of AST by 2-fold in CL + AKT and TC + AKT groups and by 3-fold in OS + AKT and ZM + AKT groups, AST/ALT by 2-fold and ALP to normal levels in all four groups. All four herb + AKT groups showed normal to enhanced neutrophil function. CONCLUSION: All four herbs showed hepatoprotective potential and prevented immunosuppression. CL and TC showed the highest hepatoprotective activity, while TC and ZM showed strong immunostimulatory activity.

Application of Near Infrared Diffuse Reflectance Spectroscopy with Radial Basis Function Neural Network to Determination of Rifampincin Isoniazid and Pyrazinamide Tablets

Partial least squares（PLS）,back-propagation neural network（BPNN）and radial basis function neural network（RBFNN）were respectively used for estalishing quantative analysis models with near infrared（NIR）diffuse reflectance spectra for determining the contents of rifampincin（RMP）,isoniazid（INH）and pyrazinamide（PZA）in rifampicin isoniazid and pyrazinamide tablets.Savitzky-Golay smoothing,first derivative,second derivative,fast Fourier transform（FFT）and standard normal variate（SNV）transformation methods were applied to pretreating raw NIR diffuse reflectance spectra.The raw and pretreated spectra were divided into several regions,depending on the average spectrum and RSD spectrum.Principal component analysis（PCA）method was used for analyzing the raw and pretreated spectra in different regions in order to reduce the dimensions of input data.The optimum spectral regions and the models＇ parameters were chosen by comparing the root mean square error of cross-validation（RMSECV）values which were obtained by leave-one-out cross-validation method.The RMSECV values of the RBFNN models for determining the contents of RMP,INH and PZA were 0.00288,0.00226 and 0.00341,respectively.Using these models for predicting the contents of INH,RMP and PZA in prediction set,the RMSEP values were 0.00266,0.00227 and 0.00411,respectively.These results are better than those obtained from PLS models and BPNN models.With additional advantages of fast calculation speed and less dependence on the initial conditions,RBFNN is a suitable tool to model complex systems.

Organolanthanide Compounds Containing Methanesulfonate and Pyrazinamide Ligand in Styrene Polymerization

The synthesis of organolanthanide compounds identified as LnCp^＊（MS）2PzA, Ln = Sm, Tb, Yb （MS = methanesulfonate, Cp^＊ = pentamethylcyclopentadienyl, and PzA = pyrazinamide）, by the reaction of coordination compounds Ln（MS）3（PzA）4 with NaCp in THF was reported. The complexes were formulated according to elemental analyses, complexometric titration with EDTA （%Ln）, and ^1H NMR. IR spectroscopy revealed that PzA coordinates with lanthanide （Ⅲ） ions and methanesulfonate coordinates via oxygen atoms in a non-equivalent manner. In preliminary catalytic studies, these compounds were active in styrene polymerization that used MAO as a cocatalyst with an activity of 12.3 kg PS molSm^-1h^-1. Differential scanning calorimetry （DSC） of polystyrene showed that the polymer was mainly atactic.

The Influence of Pyrazinamide Monoresistance on Treatment Outcomes in Tuberculosis Patients from Southern China

This study aimed to explore the influence of pyrazinamide (PZA) monoresistance on the treatment outcome of otherwise drug susceptible tuberculosis (TB). A cohort of 194 TB patients that were infected with strains susceptible to isoniazid (INH), rifampin (RIF) and ethambutol (EMB) were included in a retrospective study at the Guangzhou Chest Hospital. We reported 148 (76.3%) PZA- susceptible TB cases and 46 (23.7%) PZA-monoresistance TB cases identified by the BACTEC MGIT 960 system. All patients were treated with the standard 6 months WHO recommended regimen, which included 2 months of INH + RIF + EMB + PZA in the intensive-phase, and the subsequent 4 months of INH + RIF during continuation-phase. Bacterial burden in the lungs was estimated using sputum smear acid-fast bacillary count while the lung lesions and cavitations were examined by X-ray at the end of first 2 months of chemotherapy. After intensive-phase treatment, there were 164 (84.5%) cases of smear-negative conversion and 151 (77.9%) cases of total or partial lesion elimination. The rates of smear-negative conversion (78.3%) and lesion elimination (39.1%) of the PZA-monoresistant patients were similar with the PZA-sensitive group (P > 0.05). However, lung cavitation was more likely to be resolved in PZA-sensitive patients than in the PZA-patients (X2 = 9.623, P = 0.002). The smear-negative conversion rates were 95.9% for the PZA-sensitive patients and 87.0% for the PZA-monoresistant patients after 6 months of treatment (X2 = 3.461, P = 0.063). Together, our data suggest that PZA-monoresistance contributes to the delay of resolution of the lung cavitations in the Southern China population without affecting the sputum conversion and lesion elimination rates.

High Resolution Melting Curve Analysis for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis Clinical Isolates

Background: Pyrazinamide (PZA) is one of the most important drugs for tuberculosis (TB) treatment, however, its susceptibility is not routinely tested. High-resolution melting (HRM) curve analysis has been widely used for many applications. In this study, HRM assay was developed and evaluated for the detection of PZA resistance in Mycobacterium tuberculosis clinical isolates. Methods: Ninety five M. tuberculosis clinical isolates with different susceptibility patterns to anti-TB drugs were used to evaluate this assay. Isolates were phenotypically (Bactec MGIT 960) and genotypically (HRM and pncA gene sequencing) analysed for PZA resistance. Results: Bactec MGIT 960 analysis revealed that 29 of the 95 M. tuberculosis isolates were PZA resistant. In comparison to the Bactec MGIT 960, HRM showed a sensitivity of 47.7% and specificity of 74.6%, and the overall agreement between the two methods was 68.4%. Based on DNA sequencing, a correlation of 0.67 (significant at p-value pncA mutations was observed. PZA resistance was strongly associated with multi-drug resistant (MDR)-TB as it was shown in 79.3% of the MDR isolates included in the study. Conclusion: HRM is simple and useful for screening clinical M. tuberculosis isolates for PZA resistance, however, further modifications to improve its performance are required.

Performance of the microscopic observation drug susceptibility assay in pyrazinamide susceptibility testing for Mycobacterium tuberculosis

Background Drug susceptibility assay is very important in tuberculosis therapy. Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis （M. tuberculosis） is difficult and time consuming by conventional methods. In this study, we aimed to evaluate the performance of the microscopic observation drug susceptibility （MODS） assay in the detection of pyrazinamide resistance in M. tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen （L J） proportion method. Methods M. tuberculosis clinical isolates （n=132） were tested by the MODS and the Wayne assay： the results were compared with those obtained by the LJ proportion method. Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods. Results Compared to the LJ results, the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively. Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant （3 tests）, in 1 of the 4 strains （LJ only）, in 42 of 48 strains （at least I test）, but no mutations in 1 strain sensitive according to the MODS assay only. The MODS assay, Wayne assay and LJ proportion method provided results in a median time of 6, 7 and 26 days respectively. Conclusions MODS assay offers a rapid, simple and reliable method for the detection of pyrazinamide resistance in M. tuberculosis and is an optimal alternative method in resource limited countries.

Calcitriol enhances pyrazinamide treatment of murine tuberculosis

Background: Tuberculosis is a leading cause of morbidity and mortality in humans worldwide. There is an urgent need for new and effective drugs to treat tuberculosis and shorten the duration of tuberculosis therapy. 1, 25-dihydroxy vitamin D3 (1,25 (OH)2D3) has been reported to have a synergistic effect with pyrazinamide (PZA) in killing tubercle bacilli in vitro. The addition of 1,25 (OH)2D3 to standard tuberculosis treatment should benefit patients if the adjunctive drug has a synergistic effect in vivo. Thus, in this study, calcitriol (bioactive 1,25 (OH)2D3) was administered to mice undergoing treatment for Mycobacterium tuberculosis (M.tb) infection with PZA, a first-line anti-tuberculosis drug, to determine whether vitamin D3 enhances the therapeutic effect. Methods: C57BL/6 female mice were infected with the M.tb H37Rv strain through aerosol exposure. Calcitriol and PZA, either alone or in combination, were orally administered to the M.tb infected mice. The effect of calcitriol on PZA activity was determined by evaluating the bacterial burden and analyzing the histopathological lesions in the lungs and spleen. To investigate the expression of inflammatory cytokines and anti-microbial peptide genes, we determined the transcriptional levels of interferon-γ(IFN-γ), interleukin-4 (IL-4), mouse β-defensin-2 (mBD2), and cathelicidin LL-37 through real-time quantitative polymerase chain reaction. The protein levels of IFN-γ were detected by enzyme-linked immunosorbent assay. Differences between groups were analyzed with independent samples t-test or one-way analysis of variance. Results: Calcitriol alone had little effect on tuberculosis infection, whereas PZA, compared with saline control treatment, decreased the bacterial burden (spleens: PZA vs. saline, 4.82 ± 0.22 vs. 5.22 ± 0.40 Log10 colony-forming units [CFU]/gram, t = 2.13, P < 0.05;lungs: PZA vs. saline, 5.55 ± 0.15 vs. 6.83 ± 0.46 Log10 CFU/gram, t = 6.56, P < 0.01) and pathological lesions in the lungs. Simultaneous administration of calcitriol with PZA, compared with PZA alone, decreased the bacterial load (spleen: calcitriol + PZA vs. PZA, 4.37 ± 0.13 vs. 4.82 ± 0.22 Log10 CFU/gram, t = 4.36, P < 0.01;lung: calcitriol + PZA vs. PZA, 5.03 ± 0.32 vs. 5.55 ± 0.15 Log10 CFU/gram, t = 3.58, P < 0.01) and attenuated the lung lesions (gross pathological score: calcitriol + PZA vs. PZA, 3.25 ± 0.50 vs. 2.50 ± 0.58, t = 1.96, P < 0.05;affected area of total lung area: calcitriol + PZA vs. PZA, 30.75%± 6.50% vs. 21.55%± 2.99%, t = 2.66, P < 0.05). Further studies demonstrated calcitriol significantly increased the expression of anti-inflammatory cytokine IL-4 but suppressed production of the pro-inflammatory cytokine IFN-γ(IL-4: calcitriol vs. saline, 5.69 ± 0.50 vs. 2.80 ± 0.56 fold of control, t= 6.74, P < 0.01;IFN-γ: calcitriol vs. saline, 1.36 ± 0.11 vs. 4.13 ± 0.83 fold of control, t= 5.77, P < 0.01). In addition, calcitriol alone or in combination with PZA significantly enhanced the transcriptional level of anti-microbial peptides (cathelicidin LL-37: calcitriol vs. saline, 10.59 ± 1.03 vs. 2.80 ± 0.90 fold of control, t = 9.85, P < 0.01;mBD2: calcitriol vs. saline, 7.92 ± 0.62 vs. 1.79 ± 0.45 fold of control, t = 13.82, P < 0.01), whereas PZA exerted a negative effect on anti-microbial peptide gene expression. Conclusions: Calcitriol as adjunctive treatment can result in beneficial treatment outcomes in M.tb infection by suppressing the inflammatory response and up-regulating the expression of anti-microbial peptides. These results indicate the feasibility of using calcitriol adjunctively with standard chemotherapy for the treatment of M.tb infection.

Evaluation of antihepatotoxic potential of Solanum xanthocarpum fruit extract against antitubercular drugs induced hepatopathy in experimental rodents

Objective:To assess the hepatoprotective effect of Solanum xanthocarpum(S. xanthocarpum) fruit extract against antitubercular drug-induced liver toxicity in experimental animals.Methods:Ethanolic(50%) fruit extract ofS. xanthocarpum(100, 200 and 400 mg/kg bw) was administered daily for 35 days in experimental animals. Liver toxicity was induced by combination of three antitubercular drugs [isoniazid(I) 7.5 mg/kg, rifampicin(R) 10 mg/kg and pyrazinamide(P) 35 mg/kg] given orally as suspension for 35 days in rats. The hepatoprotective activity was assessed using various biochemical parameters like aspartate aminotransferase(AST), alanine aminotransferase(ALT), alkaline phosphatise(ALP), total bilirubin(TBL), albumin(ALB), total protein(TP), lactate dehydroginase(LDH), and serum cholesterol(CHL). Meanwhile,in vivoantioxidant activities as lipid peroxidation(LPO), reduced glutathione(GSH), superoxide dismutase(SOD) and catalase(CAT) were measured in rat liver homogenate. The biochemical observations were supplemented by histopathological examination.Results:The results demonstrated that treatment withS.xanthocarpumsignificantly(P<0.05-P<0.001) and dose-dependently prevented drug induced increase in serum levels of hepatic enzymes. Furthermore,S. xanthocarpumsignificantly(up toP<0.001) reduced the LPO in the liver tissue and restored activities of defence antioxidant enzymes GSH, SOD and CAT towards normal levels. Histopathology of the liver tissue showed that S. xanthocarpumattenuated the hepatocellular necrosis and led to reduction in inflammatory cells infiltration.Conclusions:The results of this study strongly indicate the protective effect of S. xanthocarpumagainst liver injury which may be attributed to its hepatoprotective activity, and thereby scientifically support its traditional use.

Anti-Tuberculosis Drug Induced Liver Injury and Ursodeoxycholic Acid

Hepatotoxicity induced by standard anti-tuberculosis drugs (isoniazid, rifampicin, pyrazinamide) can result in significant morbidity and, rarely, even mortality. This major adverse side-effect of anti-tuberculosis treatment has a negative impact on patient adherence and patient outcomes as well as on tuberculosis control. Early recognition and prompt withdrawal of the offending drugs are the most critical interventions in the management of anti-tuberculosis drug-induced liver injury. No drug or herbal extract has been shown until recently to prevent or reverse anti-tuberculosis drug-induced hepatotoxicity. Ursodeoxycholic acid is the only FDA approved drug for the treatment of primary biliary cholangitis and has also been successfully used in various cholestatic liver diseases. Although still experimental, recent controlled clinical studies suggested that oral administration of ursodeoxycholic acid may prevent the onset of anti-tuberculosis drug-induced liver injury and accelerate the recovery of liver injury. These clinical data are supported by experimental models of anti-tuberculosis drug-induced hepatotoxicity. There is an urgent need for further randomized clinical trials to document the promising hepatoprotective properties of ursodeoxycholic acid.

Clinical and experimental research in antituberculosis drug-induced hepatotoxicity:a review

Drug-induced liver injury is the common adverse effect seen in patients receiving antituberculosis drugs （ATDs）. There are several risk factors associated with the development of hepatotoxicity in such patients. Though there have been appreciable efforts taken by carrying out studies investigating the efficacy of several natural and synthetic compounds in minimising this effect, the only choice available for clinicians is withdrawal of drugs. This review would give a precise idea of ATD-induced hepatotoxicity its underlying mechanisms and alternative therapies for the same.

Anti-hepatotoxic and antioxidant influence of Ipomoea carnea against anti-tubercular drugs induced acute hepatopathy in experimental rodents

Objective:To assess the hepatoprotective effct of Ipomoea carnea(I.carrnea)extract against antitubercular drug-induced liver toxicity in experimental animals.Methods:I.carnea extracts(125,250 and 500 mg/kg,p.o.body weight)were administered daily for 35 d in experimental animals.Liver toxicity was induced by combination of three antitubercular drugs(isoniazid 7.5 mg/kg,rifampicin 10 mg/kg and pyrazinamide 35 mg/kg)given orally as suspension for 35 d in rats.Treatment groups received I.carnea extracts along with antitubercular drugs.The hepatoprotective activity was assessed using various biochemical parameters like aspartate aminotransferase,alanine aminotransferase,alkaline phosphatise and total bilirubin.Meanwhile,in-vivo antioxidant activities as lipid peroxidation,reduced glutathione,superoxide dismutase and catalase were measured in rat liver homogenate along with ATPase and G-6-Pase.The biochemical observations were supplemented by histopathological examination.Results:Obtained results demonstrated that treatment with I.carnea extracts significantly(P<0.05-P<0.001)and dose-dependently prevented drug induced increase in serum levels of hepatic enzymes.Furthermore,I.carnea extracts significantly(up to P<0.001)reduced the lipid peroxidation in the liver tissue and restored activities of defence antioxidant enzymes,reduced glutathione,superoxide dismutase and catalase towards normal levels.Histopathology of the liver tissue showed that I.carnea extracts attenuated the hepatocellular necrosis,massive fatty changes and led to reduction in inflammatory cells infiltration.Conclusions:The results of this study strongly indicate the protective effect of I.carnea extracts against liver injury,which may be attributed to its hepatoprotective activity,and there by scientifically support its traditional use.