Quality analysis of some drugs against tuberculosis an in vitro study / Монголын эм зүй, эм судлал

Introduction@#According to the statistical data of 2019, 10 million people in 202 countries have been registered as tuberculosis cases, 1.6 million people has died around the world. In 2019, 4,089 new tuberculosis cases were determined, of which 265 died in Mongolia.@*Purpose@#The purpose of this study is analyze quality of some drugs used in the treatment of certain groups of tuberculosis in accordance with international standards.@*Materials and methods@#Isoniazid 300 mg, Rifampicin 150 mg, Pyrazinamide 400 mg and Ethambutol 400 mg, which are commonly used in Mongolia, were analyzed according to the pharmacopoeia methods of United States, Chinese and British.@*Results@#Appearance - Complies with the requirements of the certificate for sensory examination. Identification - It was determined by an infrared spectrophotometry. Average weight - Isoniazid 547.5 mg, Rifampicin 277.6 mg, Pyrazinamide 640.8 mg, Etambutol 399.6 mg. DisintegrationIsoniazid decomposes in 2.5 minutes, Pyrazinamide decomposes in 4.6 minutes, Etambutol decomposes in 8.5 minutes. Friability – Isoniazid - 0.4%, Pyrazinamide 10.4%, Hardness – Isoniazid 0.4%, Pyrazinamide is - 49.7 N, Dissolution – Isoniazid not less than - 95.7%, Rifampicin - 78.7%, Pyrazinamide - 95.7%, Assay - Etambutol is - 399.6 mg, Rifampicin is 139.94 mg, Isoniazid is - 293.6 mg Pyrazinamide - 492.5 mg complied with the standard requirements. @*Conclusion@#Analysis of tuberculosis drugs quality by pharmacopoeia methods including its appearance, identification, average weight, friability, hardness, disintegration, dissolution and assay, the isoniazid 300 mg tablets are complied with the United States pharmacopoeia, Rifampicin 150 mg tablets met British pharmacopoeia, and Ethambutol 400 mg tablets are complied with Chinese pharmacopoeia. Pyrazinamide 400 mg tablets did not complies the friability requirements of the United States Pharmacopoeia and also satisfied with the other requirements.

Pyrazinamide-isoniazid hybrid: synthesis optimisation, characterisation, and antituberculous activity / Híbrido pirazinamida-isoniacida: optimización de síntesis, caracterización y actividad antituberculosa / Híbrido pirazinamida-isoniazida: otimização da síntese, caracterização e atividade antituberculose

Abstract Over time, the effective resistance mechanisms to various first- and second-line drugs against the disease of tuberculosis make its treatment extremely difficult. This work presents a new approach to synthesizing a hybrid of antituberculosis medications: isoniazid (INH) and pyrazinamide (PZA). The synthesis was performed using ultrasound-assisted synthesis to obtain an overall yield of 70%, minimizing the reaction time from 7 to 1 h. The evaluation of the biological activity of the hybrid (compound 2) was tested using the tetrazolium microplate assay (TEMA), showing inhibition in the growth of Mycobacterium tuberculosis H37Rv at a concentration of 0.025 mM at pH 6.0 and 6.7.

Resumen Debido a los grandes mecanismos de resistencia a lo largo del tiempo de diversos fármacos de primera y segunda línea contra la enfermedad de la tuberculosis, el tratamiento sigue dificultándose. Este trabajo presenta un nuevo enfoque para sintetizar un híbrido de fármacos antituberculosos: isoniazida (INH) y pirazinamida (PZA). La síntesis fue asistida por ultrasonido con el fin de obtener un rendimiento global del 70%, minimizando el tiempo de reacción de 7 a ' h. La evaluación de la actividad biológica del híbrido (compuesto 2) se probó usando el ensayo de microplaca de tetrazolio (TEMA), que mostró una inhibición en el crecimiento de Mycobacterium tuberculosis H37Rv a una concentración de 0,025 mM a pH 6,0 y 6,7.

Resumo Devido aos grandes mecanismos de resistência ao longo do tempo a diversos fármacos de primeira e segunda linha contra a tuberculose, o que torna seu tratamento extremamente difícil. Este trabalho apresenta uma nova abordagem para sintetizar um híbrido de fármacos antituberculose: isoniazida (INH) e pirazinamida (PZA) A síntese foi realizada utilizando a síntese assistida por ultrassom de forma a obter um rendimento global de 70%, minimizando o tempo de reação de 7 h para ' h. A avaliação da atividade biológica do híbrido (composto 2) foi testada utilizando o ensaio de microplaca de tetrazólio (TEMA), mostrando uma inibição no crescimento de Mycobacterium tuberculosis H37Rv na concentração de 0,025 mM em pH 6,0 e 6,7.

Analysis of pyrazinamide resistance of multidrug-resistant Mycobacterium tuberculosis in Henan Province and its relationship with the mutation of drug resistance-related gene pncA / 中华传染病杂志

Objective:To evaluate the performance of the sequencing method of pncA mutations in detecting pyrazinamide (PZA)resistance in multidrug-resistant tuberculosis (MDR-TB) patients in Henan Province. Methods:Sputum samples of 152 MDR-TB patients were collected from 10 drug-resistance surveillance areas in Henan Province from January to December 2018. Questionnaire survey was conducted to collect the information, such as age, gender, treatment history and sputum culture results. The questionnaire and strain samples were sent to Henan Provincial Center for Disease Control and Prevention for further study. PZA susceptibility test was performed using the liquid medium for Mycobacterium tuberculosis. The mutations of pncA were detected by polymerase chain reaction (PCR)and sequencing, then compared with the sequence of standard strain H37Rv. The association between PZA resistant phenotype and treatment outcomes was also investigated. Chi square test and independent sample t test were used for statistical analysis. Results:Among 152 MDR-TB isolates, 105 showed phenotypically PZA resistant. The proportion of PZA resistance in the isolates with isoniazid, rifampicin, ethambutol and streptomycin resistance, pre-extensively drug-resistant and extensively drug-resistant (XDR) were 80.39%(82/102), 81.13%(43/53) and 92.59%(25/27), respectively. One hundred and two isolates had mutations in the pncA gene. Based on the results of the phenotypic drug sensitivity test, the sensitivity of pncA gene mutation detection for PZA resistance was 89.52%(95% confidence interval ( CI) 81.64%-94.39%), and the specificity was 89.36%(95% CI 76.10%-96.01%). These MDR-TB isolates harbored 100 different mutation patterns in the pncA gene, including 80 point mutations and 20 indel mutations, and 13 isolates harbored multiple mutations. Seven strains had mutation in the promoter of pncA, including -7G insertion, -11T to C and -12A to G. The relative expression levels of pncA mRNA in the three groups were 0.21±0.05, 0.31±0.08 and 0.33±0.03, respectively, which were all lower than that of H37Rv(1.00). The differences were statistically significant ( t=4.57, 2.43 and 3.65, respectively, all P<0.05). The difference of the sputum negative conversion rates between patients with PZA-resistant isolates and those with PZA susceptible isolates was statistically significant at different time periods after treatment ( χ2=10.01, P=0.02). The negative conversion rate of PZA-resistant patients at the end of six months of treatment was 1.08%(1/93), and that of PZA-sensitive patients was 7.14%(3/42). Conclusions:The PZA resistance in MDR-TB isolates is associated with pncA mutations, which are scatered and diversified. The sputum negative conversion time of PZA-resistant patients is prolonged.

Effects of Rat Intestinal Flora on the Pharmacokinetic Parameters of Pyrazinamide and Its Active Metabolite Py- razinoic Acid / 中国药房

OBJECTIVE：To study the effects of rat intestinal flora on the pharmacokinetic parameters of pyrazinamide and its active metabolite pyrazinoic acid. METHODS ：Totally 16 SD rats were randomly divided into trial group and control group ，with 8 rats in each group. Trial group was given mixed antibiotics （streptomycin sulfate+neomycin sulfate ）intragastrically to construct pseudoaseptic rat model. After modeling ，both groups were given pyrazinamide intragastrically （150 mg/kg）. Before and 0.167， 0.333，0.667，1，1.5，2，3，4，6，9 h after administration ，0.1 mL blood sample was collected from orbital venous plexus ，and 0.3 mL blood sample was collected from orbital venous plexus 12，24 h after administration. Using phenacetin as internal standard ， LC-MS/MS method was adopted to determine the plasma concentration of pyrazinamide and pyrazinoic acid. The determination was performed on Agilent ZORBAX SB-Aq column with mobile phase consisted of 0.2％ formic acid （containing 8 mmol/L ammonium acetate）-methanol（gradient elution ）at the flow rate of 1 mL/min. The column temperature was set at 30 ℃，and sample size was 10 μL. The ion source was ESI and the temperature of ion source was 500 ℃. The collision gas was nitrogen and the pressure was 10 psi. The temperature of mass transfer interface was 100 ℃. The mass spectrum monitoring mode was multi reaction monitoring ， and the collection mode was positive ion mode. The monitoring transition ion-pairs were m/z 124.0→79.0（pyrazinamide），m/z 125.1→79.1（pyrazinic acid ）and m/z 180.0→110.2（internal standard ）. The de-clustering potential and collision voltage were 55， 26 and 85 V，24，23 and 28 V，respectively. The pharmacokinetic parameters were calculated and compared by using DAS 2.1.1 software. RESULTS ：The linear ranges of pyrazinamide and pyrazinoic acid were 25-5 000 ng/mL（r＝0.997 6）and 100-12 500 ng/mL（r＝0.999 0）. The lower limits of quantification were 25 and 100 ng/mL，respectively. Intra-batch and inter-batch accuracy were 92.93％-100.50％，and RSDs of intra-batch and inter-batch precision and matrix effect tests were all lower than or equal to 8.42％（n＝6 or n＝3）. Compared with control group ，tmax of pyrazinamide in trial group was prolonged significantly （P＜0.01）； there was no statistical significance in other pharmacokinetic parameters between 2 groups（P＞0.05）. CONCLUSIONS ：The absorption of single dose pyrazinamide is delayed with the change of intestinal flora in rats.

Características clínicas de la hepatotoxicidad asociada a la pirazinamida en pacientes de un hospital de Lima, Perú / Clinical characteristics of pyrazinamide-associated hepatotoxicity in patients at a hospital in Lima, Peru

RESUMEN Con el objetivo de determinar las características de la enfermedad hepática inducida por el medicamento (DILI) se realizó un estudio de pacientes adultos con diagnóstico de tuberculosis y esquema de tratamiento antituberculoso con pirazinamida. El análisis de causa efecto de la DILI fue mediante el proceso de reexposición. Se encontraron 10 pacientes con DILI asociada a pirazinamida, la mediana de edad y de estancia hospitalaria fue de 40,5 años (rango 22-76) y 41 días (rango 11-130), respectivamente. La mediana de presentación del evento fue de 14 días (rango 3-46), 4 pacientes presentaron ictericia, 5 tuvieron patrón hepatocelular, 3 mixtas y 2 colestásicos. La presentación de la DILI fue leve en 6 casos (60%) y moderados en 3 (30%). En conclusión, la DILI asociada a la pirazinamida requiere estancia hospitalaria prolongada, se presenta con ictericia en un poco más de un tercio de los casos siendo el patrón predominante el hepatocelular.

ABSTRACT In order to determine the characteristics of drug-induced liver injury (DILI), adult patients diagnosed with tuberculosis and with an anti-tuberculosis treatment scheme including pyrazinamide were studied. The re-exposure process was used for the cause-effect analysis of the DILI. A total of 10 patients were found with pyrazinamide-associated DILI; the median age and hospital stay were 40.5 years (from 22 to 76 years) and 41 days (from 11 to 130 days), respectively. The median time in which the events appeared was 14 days (from 3 to 46 days); jaundice was observed in 4 patients and radiological patterns such as hepatocellular, mixed and cholestatic were found in 5, 3 and 2 patients, respectively. Mild presentation of DILI was observed in 6 cases (60%) and moderate in 3 (30%). In conclusion, pyrazinamide-associated DILI required prolonged hospital stay, presented jaundice in little more than a third of the cases, and radiologically, the hepatocellular pattern predominated.

Calcitriol enhances pyrazinamide treatment of murine tuberculosis / 中华医学杂志(英文版)

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.

Pyrazinamide-Induced Urticaria and Angioedema: a Case Report / 대한내과학회지

Pyrazinamide (PZA) is an anti-tuberculosis drug and an essential component of the standard four-drug regimen for tuberculosis. Here, we report a case of immediate angioedema secondary to PZA administration intended for pulmonary tuberculosis treatment. A previously healthy 48-year-old woman was diagnosed with pulmonary tuberculosis and tuberculous lymphadenitis. Thirty minutes after taking the first dose of isoniazid, rifampicin, pyrazinamide, and ethambutol, the patient developed facial edema, generalized rash, and dizziness. An oral provocation test was performed on the four drugs, and 1,000 mg pyrazinamide showed a positive result characterized by 50 minutes of urticaria, angioedema, and hypotension. As the prevalence of tuberculosis increases, prescriptions for anti-tuberculosis drugs may increase as well. Clinicians should be aware of the possibility of immediate hypersensitivity as well as delayed hypersensitivity to anti-tuberculosis drugs.

In vitro cytotoxicity of HRZ (isoniazid + rifampin + pyrazinamide) /transforming growth factor β1 siRNA nanoliposomes and the underlying mechanism / 中国感染与化疗杂志

Objective To study the in vitro cytotoxicity of HRZ (isoniazid + rifampin + pyrazinamide) / transforming growth factor (TGF) β1 siRNA nanoliposomes on human macrophages and the underlying mechanism. Methods Self-made nanoliposomes were used to study with the cultured human macrophages in vitro. MTT assay was used to detect cell proliferation. Flow cytometry was used to analyze apoptosis and cell cycle. Electron microscopy was used to observe autophagy. RT-PCR and Western blot were employed to analyze the silenced expression of target gene TGF-β1. Results HRZ/TGF-β1 siRNA nanoliposomes (triple liposome) inhibited macrophage proliferation within certain range of concentration, and cell cycle was captured in G2 phase. The HRZ / TGF-β1 SiRNA nanoliposomes could significantly inhibit the expression of target gene TGF-β1 in human macrophages. Conclusions The self-made triple liposome has evident effect in silencing the target gene. It is a promising biomaterial, which meets the required specifications in terms of cytotoxicity.

Research progress of association between gene mutation and pyrazinamide resistance in Mycobacterium tuberculosis / 中国人兽共患病学报

Pyrazinamide (PZA) is an important anti-tuberculosis drug especially for treating multidrug-resistant tuberculo sis (MDR-TB).In recent years,the incidence of Mycobacterium tuberculosis' resistance to pyrazinamide has increased.At present,the mechanism of drug resistance has not been clearly elucidated.In this review,the association between gene mutation and pyrazinamide resistance in Mycobacterium tuberculosis will be summarized.

Establishment of a rat model of hyperuricemia associated with uric acid excretion disorder / 中国比较医学杂志

Objective To explore a reliable method to establish a rat model of hyperuricemia associated with abnormal uric acid excretion, and to lay the foundation for the study of pathogenesis of uric acid excretion disorder and the optimization of the treatment plan.Methods The models were established respectively by potasium oxonate(300 mg/kg) with pyrazinamide (300 mg/kg) or ethambutol(250 mg/kg).Continuous dosing for 1, 3 and 5 weeks, to determine the content of uric acid in rat blood, urine, and stool, the function of liver and kidney was detected and pathological examination was performed.Results The blood uric acid in the potasium oxonate and ethambutol group was increased first and then decreased, while in the potasium oxonate and pyrazinamide group were increased steadily and the excretion of uric acid in urine was stable during the continuous administration.The two methods showed no harmful effect on the liver and kidney function.Conclusions A stable rat model of hyperuricemia associated with uric acid excretion disorder can be effectively established by potassium oxonate and pyrazinamide, exhibiting similar manifestations of clinical hyperuricemia and uric acid excretion disorder.

Rapid detection on resistance of pyrazinamide for Mycobacterium tuberculosis by microplate allochroic silica gel assay / 国际检验医学杂志

Objective To establish a method of judge the pyrazinamide(PZA) susceptibility for Mycobacterium tuberculosis(MTB) with 24-hole micro-liquid culture silica gel color plate,and evaluate the clinical application value of this method.Methods According to the result of MGIT960,to detect PZA drug susceptibility for 30 MTB clinical isolates of whose PZA susceptibility were known with silica gel color plate.The effects of different pH value,different inoculation concentrations on the results were observed,and the optimum detection conditions were discussed.Finally,the 98 MTB clinical isolated of whose PZA susceptibility were unknown were simultaneously detected by gel color plate and MGIT960,the sensitivity,specificity and accuracy were judged by PZA drug sensitivity.Results The best pH was 5.8-5.9 and the best concentration was 2.5×10-1 mg/mL in gel color plate.The best results were read after 7-14 d.The sensitivity,specificity and accuracy were 95.50%,96.30%,and 98.21% respectively just at PZA critical concentration was 100 μg/mg.The sensitivity,specificity and accuracy were 90.90%,92.59%,and 91.84% respectively just at PZA critical concentration was 200 μg/mg.Conclusion The PZA susceptibility for MTB with 24-hole micro-liquid culture silica gel color plate is accurate,rapid,low-cost and simple.

Analysis on Pyrazinamide Drug Susceptibility of Mycobacterium Tuberculosis Clinical Strains / 现代检验医学杂志

Objective To research the pyrazinamide drug susceptibility of Mycohacterium tuberculosis,and provide reference for clinical medication and prevention.Methods Bactec MGIT 960 system was used to test the resistance of isoniazid,rifam picin,ethambutol,streptomycin and pyrazinamide for 153 strains of Mycobacterium tuberculosis.Results Of 153 Mycobacterium tuberculosis,34 were resistant to PZA,and the resistant rate was 22.2 ％.There was no PZA single drug resistance.A mong patients with and without INH resistance,RFP resistance,EMB resistance and Sm resistance,the proportions of PZA resistance were respectively 40.5 ％ (34/84) vs 0％(0/69),47.5％(29/61) vs 5.4％(5/92),75％(6/8) vs 19.3％(28/145),53.1％ (26/49) vs 7.7％ (8/104).Among the 54 multidrug-resistant (MDR) strains 53.7％ were resistant to PZA,which was significantly higher than 5.1 ％ (5/99) among the nonMDR-TB strains (x2 =47.854,P＜0.05).In multivariate logistic analysis,resistance to Sm (OR=0.270,95％CI:0.091～0.802) and MDR-TB (OR=0.281,95％CI:0.087～0.911) were risk factors to PZA resistance.Conclusion The PZA resistance rate among MDR-TB isolates was high.PZA resistance would be associated with SM resistance and MDR-TB.The drug susceptibility test for PZA is very important to MDR-TB patients.

Relapse for patients with primary smear-positive pulmonary tuberculosis and pyrazinamide-resistance / 实用医学杂志

Objective To explore the relapse rate of the patients with primary smear-positive pulmonary tu-berculosis and pyrazinamide-resistance. Methods Retrospective analysis was made on the relapse for 150 patients with primary smear-positive pulmonary tuberculosis , who had been diagnosed and completed treatment in Guangzhou Chest Hospital from January 2012 to August 2013 , and had followed up two years. According to the re-sults of drug susceptibility test before treatment, they were divided into pyrazinamide-sensitive (114 cases) and pyrazinamide-resistant (36 cases) groups. Results (1)By the end of the treatment, the recovery rates in the sensi-tive group and resistant group were 98.25%and 88.89%respectively (P=0.044). The rate of the lesions absorption was 99.12%and 94.44%respectively (P=0.143). The rate of the cavity shrinking was 89.01% and 70.37% re-spectively (P = 0.039). The rate of the relapse was 3.57% and 6.25% respectively (P = 0.867) within 2 years fol-low-up in the sensitive group and the resistant group. Conclusions PZA has certain effects on the patients with primary smear-positive pulmonary tuberculosis. Those who are tolerant would have lower incidence for cavity shrink-ing. But the relapse rate for two-year follow-up showed there were not significant differences in two groups.

An in vitro experimental study on the physical and elution properties of PMMA bone cement loaded with rifampicin, iso-niazid, pyrazinamide and moxifloxacin / 中华骨科杂志

Objective To investigate the feasibility of Antituberculotic?loaded bone cement (ATLBC) prepared by mix?ing the anti?TB drugs Rifampicin (RFP), Isoniazid (INH), Pyrazinamid (PZA), Moxifloxacin (MFX) with Palacos R PMMA bone cement in Total Joint Arthroplasty treatment for Joint Tuberculosis. Methods Forty grams of Palacos R bone cement powder without antibiotics was mixed with 1 or 2 grams of RFP, INH, PZA and MFX respectively. According to ISO 5833:2002 stan?dard, 8 groups of ATLBC standard test specimen were prepared as experiment group and Palacos R PMMA bone cement with?out antibiotics was prepared as control group. Physical properties (such as the average dough time, curing time, maximum tem?perature), mechanical strength (such as the compressive strength, the bending resistance strength, the modulus of elasticity) and the concentrations of eluant drug in different time points of ATLBC were detected. Results In RFP (1 g), RFP (2 g), INH (1 g) and INH (2 g) group, the average dough time and curing time were longer than those in control group, which exceeded the standard scope of ISO, while the average maximum temperature was significantly lower than that in control group. The INH ( 1 g) group and INH (2 g) group hardened after mixing for 14 days. The RFP (1 g) group and RFP (2 g) group hardened after mixing for 30 days. Twenty minutes after mixing and hardening, the compressive strength, bending resistance strength and modulus of elastic?ity were significantly lower than the specified values of ISO standard. The physical properties and mechanical strength in PZA ( 1 g) group, PZA (2 g) group, MFX (1 g) group, MFX (2 g) group and control group were in accordance with the specified values of ISO standard, and they hardened after 20 minutes. In RFP (1 g) group, RFP (2 g) group, INH (1 g) group, INH (2 g) group, PZA (1 g) group, PZA (2 g) group, MFX (1 g) group and MFX (2 g) group, the concentration of eluant could maintain for 3 days, 7 days, 90 days, 90 days, 45 days, 60 days, 60 days and 60 days respectively. Conclusion RFP and INH mixing with Palacos R PMMA bone cement can hinder the aggregation of bone cement so they are unsuitable for preparing ATLBC. PZA and MFX mixing with Palacos R PMMA bone cement do not affect the physical properties of bone cement, with excellent mechanical strength and elu?tion properties. Because the minimal inhibitory concentration of PZA is higher and its antimicrobial activity maintains shorter time, while MFX maintains longer time in antimicrobial activity, it's more suitable for the preparation of ATLBC.

The correlation study between PZA and anti-tuberculosis drug-induced liver injury / 实用医学杂志

Objective To study the correlation of PAZ with anti-tuberculosis treatment regimen and drug-induced liver injury in tuberculosis patients with HBV-DNA positive in order to provide an optimized treatment regimen. Methods from Jan 2013 to Dec 2014, 199 pulmonary tuberculosis with HBV-DNA positive patients and 103 pulmonary tuberculosis patients without HBV in our hospital were collected. They were assigned as follows:122 cases were anti tuberculosis treatment with antiviral therapy,64 cases were A(HRZE),58 cases were B (HRE). 77 cases were anti tuberculosis treatment but not antiviral therapy , 41 cases were C (HRZE), 36 cases were D(HRE) and 103 patients without HBV were E (HRZE, the contrast group). We had observed the liver injury for 2 months after the treatment. Results 1.Incidence of liver injury was 34.38% in group A , higher than the cases in group B(20.69%,P > 0.05). 2.Incidence of liver injury in group C was apparently higher than in group D (73.17% vs. 30.56%,P 0.05)4.Incidence of liver injury in group A was lower than group C (34.38% vs. 73.17%,P 0.05). Conclusion Although anti tuberculosis treatment combined with antiviral therapy can be partially reduce the incidence of liver injury and relieve the severity of liver injury in tuberculosis patients infected with HBV , but PZA toxicity to hepatocytes is a major risk factor for liver injury , and we need to change the treatment plan to reduce the occurrence of liver injury.

Determinação simultânea de Rifampicina, Isoniazida, Pirazinamida e Etambutol em comprimidos com doses fixas combinadas / Method for simultaneously determining the Rifampin, Isoniazid, Pyrazinamide and Ethambutol in the combined fixed-dose tablets

Os comprimidos utilizados no tratamento da tuberculose possuem quatro fármacos associados, isoniazida, pirazinamida, etambutol e rifampicina, e são distribuídos gratuitamente pelo Sistema Único de Saúde. Os métodos analíticos oficiais para analisar este medicamento estão especificados na Farmacopeia Americana 36a edição e na Farmacopeia Internacional 4a edição. Porém, estes compêndios oficiais não possuem monografias para análise simultânea dos quatro fármacos. O objetivo deste estudo foi desenvolver uma metodologia para determinar simultaneamente os princípios ativos em comprimidos dose fixa combinada, utilizando-se cromatografia a líquido de alta eficiência com detector de ultravioleta-visível, pois é de grande importância para o controle da qualidade do medicamento. O método desenvolvido utilizou coluna cromatográfica C18 (250 x 4,6) mm e 5 μm, fase móvel constituída de fase aquosa (85 % tampão formiato de amônio 0,3 mol/L pH 5, 15 % metanol e 0,005 mol/L de Cu2+ ou 250 mg/L de CuSO4.5H2O) e fase orgânica (metanol, 0,1 % de trietilamina e 0,2 % de ácido fórmico). O fluxo foi de 1,0 mL/min e comprimento de onda de 265 nm para isoniazida, pirazinamida e o etambutol e de 335 nm para rifampicina. Este método apresentou desvio padrão relativo inferior a 2,0 % na precisão e linearidade para os quatro fármacos estudados.

Tablets containing isoniazid, pyrazinamide, ethambutol and rifampicin are used for tuberculosis treatment, and theyare freely distributed by the Brazilian National Health System. The official analytical methods for testing those substances in fixed-dose combined tablet are described in the United States Pharmacopeia and theInternational Pharmacopoeia. None of these official compendiums refers to the methodologies forconducting the simultaneous analysis of these four drugs. This study aimedatdeveloping an analytical methodology to determine simultaneously allof four drugs in tablets for tuberculosis treatment, bymeans ofhigh performance liquid chromatography with ultraviolet-visible detector. The developed method used a chromatographic column with octadecylsilane stationary phase (250 mm x 4.6 mm x 5 μm particle size). The mobile phase was aqueous (85 % ammonium formate buffer pH 5, 15 % methanol and 250 mg CuSO4.5H2O), and organic phase (methanol, 0.1 % triethylamine and 0.2 % formic acid). The flow was 1.0 mL/min, at a wavelength of 265 nm or, when the equipment allowed, a wavelength of 265 nm for isoniazid, pyrazinamide and ethambutol and 335 nm for rifampicin. The developed methodology showed satisfactory results regarding the precision parameter, with relative standard deviation lower than 2.0 % for the studied drugs.

Molecular characteristics of pyrazinamide resistance in Mycobacterium tuberculosis strains / 中华微生物学和免疫学杂志

Objective To investigate the correlation between the mutation of pncA gene and the susceptibility to pyrazinamide ( PZA) in Mycobacterium tuberculosis complex ( MTBC) strains and to analyze the mutation of panD and rpsA genes in wild type isolates without pncA gene mutation.Methods The sus-ceptibilities of 108 MTBC strains to first-line drugs including PZA were detected by using the MGIT 960 TB system.PCR was performed to amplify the 16S rDNA and pncA, panD and rpsA genes.The PCR products were analyzed by DNA sequencing analysis .Results Among the 78 multidrug-resistant MTBC strains , 47 isolates (60%) were resistant to PZA.Four out of 30 (13%) strains that were sensitive to ethambutol , iso-niazid, rifampicin and streptomycin (EIRS) were resistant to PZA.The drug-resistant MTBC strains showed higher resistance rate to PZA than that of the EIRS sensitive strains .There were 49 ( 96%) PZA-resistant isolates and 4 (7%) PZA-sensitive isolates occurred pncA gene mutation.Most of the pncA gene mutations in the genomes of PZA-resistant strains were base substitution mutation , especially the His57Asp substitu-tion.The pncA gene mutations centralized in the regions of 160-169, 203-289, 309-396 and 413-467.Seven novel mutation sites of pncA gene were observed including T175C, C188A, G insertion at 68, AGC insertion at 235, C insertion at 339, CC insertion at 392 and GT deletion at 395.The mutation sites founded in the genomes of PZA-sensitive strains were different from those of the PZA-resistant strains .No mutation of the pncA gene and the upstream regulatory sequence was found in two PZA-resistant strains , NJ44 and NJ108 . The sequence analysis of panD and rpsA gene showed that the NJ 108 strain had panD gene mutation at G419A, but no mutation was detected in the NJ 44 strain.Conclusion The multidrug-resistant MTBC strains showed higher resistance rate to PZA .The pncA gene mutation was common in PZA-resistant MTB strains and the panD gene mutation was also worthy of attention .

Resistência primária e adquirida à pirazinamida em pacientes com tuberculose pulmonar atendidos em um hospital de referência no Recife / Primary and acquired pyrazinamide resistance in patients with pulmonary tuberculosis treated at a referral hospital in the city of Recife, Brazil

OBJETIVO: Verificar a resistência primária e adquirida à pirazinamida em cepas de Mycobacterium tuberculosis provenientes de amostras de escarro de pacientes com tuberculose pulmonar. MÉTODOS: Estudo prospectivo e descritivo realizado no período entre abril e novembro de 2011 em um hospital de referência para o tratamento de tuberculose em Recife (PE). Culturas, testes de sensibilidade a fármacos e testes da pirazinamidase foram realizados em um laboratório particular na mesma cidade. RESULTADOS: Dos 71 pacientes incluídos no estudo, 37 eram virgens de tratamento e 34 eram casos de retratamento. Desses, 0 (0,0%) e 14 (41,2%), respectivamente, apresentaram cepas resistentes à pirazinamida. Desses 14 isolados, 10 (90,9%) apresentaram resultados negativos no teste da pirazinamidase. Dos 60 isolados que apresentaram resultados positivos para o teste da pirazinamidase, 56 (93,3%) eram sensíveis à pirazinamida. CONCLUSÕES: A elevada frequência de cepas resistentes à pirazinamida em pacientes em retratamento da tuberculose destaca a necessidade da realização de testes de sensibilidade à pirazinamida antes de se escolher um novo esquema de tratamento.

OBJECTIVE: To determine primary and acquired resistance to pyrazinamide in Mycobacterium tuberculosis strains isolated in sputum samples from patients with pulmonary tuberculosis. METHODS: This was a prospective, descriptive study conducted between April and November of 2011 at a referral hospital for tuberculosis in the city of Recife, Brazil. Cultures, drug sensitivity tests, and tests of pyrazinamidase activity were conducted in a private laboratory in Recife. RESULTS: Of the 71 patients included in the study, 37 were treatment-naïve and 34 represented cases of retreatment. Pyrazinamide-resistant strains were isolated in 14 (41.2%) of the 34 patients who had previously been treated for tuberculosis and in none of the 37 treatment-naïve patients. Of the 14 isolates, 10 (90.9%) tested negative for pyrazinamidase activity. A total of 60 isolates tested positive for pyrazinamidase activity. Of those, 56 (93.3%) were found to be sensitive to pyrazinamide. CONCLUSIONS: The high frequency of pyrazinamide-resistant strains (41.2%) in patients previously treated for tuberculosis highlights the need for drug susceptibility testing prior to the adoption of a new treatment regimen.

Activity of 5-chloro-pyrazinamide in mice infected with Mycobacterium tuberculosis or Mycobacterium bovis.

Background & objectives: Pyrazinamide is an essential component of first line anti-tuberculosis regimen as well as most of the second line regimens. This drug has a unique sterilizing activity against Mycobacterium tuberculosis. Its unique role in tuberculosis treatment has lead to the search and development of its structural analogues. One such analogue is 5-chloro-pyrazinamide (5-Cl-PZA) that has been tested under in vitro conditions against M. tuberculosis. The present study was designed with an aim to assess the activity of 5-Cl-PZA, alone and in combination with first-line drugs, against murine tuberculosis. Methods: The minimum inhibitory concentration (MIC) of 5-Cl-PZA in Middlebrook 7H9 broth (neutral pH) and the inhibitory titre of serum from mice that received a 300 mg/kg oral dose of 5-Cl-PZA 30 min before cardiac puncture were determined. To test the tolerability of orally administered 5-Cl-PZA, uninfected mice received doses up to 300 mg/kg for 2 wk. Four weeks after low-dose aerosol infection either with M. tuberculosis or M. bovis, mice were treated 5 days/wk with 5-Cl-PZA, at doses ranging from 37.5 to 150 mg/kg, either alone or in combination with isoniazid and rifampicin. Antimicrobial activity was assessed by colony-forming unit counts in lungs after 4 and 8 wk of treatment. Results: The MIC of 5-Cl-PZA against M. tuberculosis was between 12.5 and 25 μg/ml and the serum inhibitory titre was 1:4. Under the same experimental conditions, the MIC of pyrazinamide was >100 μg/ml and mouse serum had no inhibitory activity after a 300 mg/kg dose; 5-Cl-PZA was well tolerated in uninfected and infected mice up to 300 and 150 mg/kg, respectively. While PZA alone and in combination exhibited its usual antimicrobial activity in mice infected with M. tuberculosis and no activity in mice infected with M. bovis, 5-Cl-PZA exhibited antimicrobial activity neither in mice infected with M. tuberculosis nor in mice infected with M. bovis. Interpretation & conclusion: Our findings showed that 5-Cl-PZA at doses up to 150 mg/kg was not active in chronic murine TB model. Further studies need to be done to understand the mechanism and mode of inactivation in murine model of tuberculosis.

Blood Levels of Pyrazinamide in Children at Doses Administered Under the Revised National Tuberculosis Control Program.

Objectives: To evaluate the blood levels, pharma-cokinetics and pharmacodynamic indices of pyrazinamide (PZA) in children suffering from tuberculosis, at doses administered under the weight band system of Revised National Tuberculosis Control Program of India (RNTCP) of India. Design: Prospective, open-label, non-randomized single-dose study. Setting: 20 children in the age group 5-12 years attending outpatient tuberculosis clinic of a tertiary hospital. Outcome Measures: Blood levels of pyrazinamide after single dose administration, as per the weight band system of RNTCP. Results: Group I (n=7) included children who received pyrazinamide within the recommended 30-35 mg/kg dose (mean 31.9+0.8 mg/kg) and Group II (n=13) included those who received a dose lower than 30 -35 mg/kg (mean 28.1±0.3 mg/ kg). The Cmax (95% CI of difference 2.2, 13.2; P=0.008) and AUC (95% CI of difference 28.6, 208.1; P=0.01) were significantly lower in Group II. The duration of time for which the concentration was maintained above 25 μg ml-1 was 4-8 h in Group I and 3-5.5 h in Group II (95% CI of difference 0.1, 2.0; P=0.03). The half life, elimination rate constant, clearance and volume of distribution were comparable in the two groups. The ratios of Cmax and AUC to MIC (25 μg ml-1) in children were lower than that recommended for PZA in adults. Conclusions: Lower blood concentrations are being attained in children receiving PZA doses under the existing weight band system of RNTCP of India. The weight bands may need to be revised and dose recommendations be based on pharmacokinetic and efficacy data in children.