O controle de impurezas do tipo N-Nitrosaminas em medicamentos e insumos farmacêutico ativos / The control of N-Nitrosamine impurities in drugs and active pharmaceutical ingredients

As N-nitrosaminas são compostos orgânicos caracterizados pela ligação de um grupo nitroso a um grupo amina e potencial carcinogênico em exposições por longos períodos. Recentemente, a presença de N-nitrosaminas foi identificada em insumos farmacêuticos ativos (IFAs) e/ou medicamentos como valsartana (anti-hipertensivo), nizatidina, ranitidina (antiulcerosos) e metformina (antidiabético), acarretando no recolhimento de diversos lotes de produtos disponíveis no mercado. Desde então, ações de controle têm sido implementadas pelas agências reguladoras, visando à proteção da saúde do paciente. O objetivo deste trabalho foi elaborar uma revisão narrativa sobre N-nitrosaminas em medicamentos, abordando mecanismos de formação, recomendações regulatórias e aspectos toxicológicos e analíticos de monitoramento. Para isto, foram utilizadas como fontes de consulta bases de dados de órgãos oficiais de saúde, nacionais e internacionais, dentre outras. Verificou-se que a causa mais discutida para a ocorrência de N-nitrosaminas em medicamentos é o emprego de agentes nitrosantes durante a síntese de fármacos. Sendo assim, a principal estratégia de controle é baseada no gerenciamento de risco quanto à formação e toxicidade, para que melhorias nos processos produtivos possibilitem a mitigação destas impurezas. As análises laboratoriais confirmatórias de identificação e quantificação de N-nitrosaminas envolvem técnicas de elevada sensibilidade, destacando-se a cromatografia gasosa ou líquida acoplada à espectroscopia de massas.

Nitrosamines are organic compounds characterized by the chemical bonding of a nitroso group to an amine group, and carcinogenic potential in long-term exposure. Recently, the presence of nitrosamines was identified in active pharmaceutical ingredients (APIs) and/or finished pharmaceutical products (FPPs) such as valsartan (antihypertensive), nizatidine, ranitidine (antiulcer) and metformin (antidiabetic), resulting in the recall of several batches of marketed drugs. Since then, regulatory agencies implemented the control of nitrosamines in drugs, aiming to protect the patient's health. This work conducted a narrative review of nitrosamines in drugs, the mechanisms of formation, regulatory recommendations, and toxicological and analytical aspects of monitoring. For this review, national and international databases of official health agencies were used as reference sources, among others. It was found that the most discussed cause for the occurrence of nitrosamines in drugs is the use of nitrosating agents during the synthesis of APIs. Therefore, the main control strategy is based on risk management regarding formation and toxicity, and the implementation of adjustments in manufacturing processes that enable the mitigation of these impurities. Confirmatory analysis for the identification and quantification of nitrosamines requires highly sensitive techniques, as gas or liquid chromatography coupled with mass spectroscopy.

Efficacy and safety of secukinumab in patients with psoriatic arthritis: A meta-analysis of different dosing regimens

The appropriate dosing regimens of secukinumab for psoriatic arthritis (PsA) are not well defined. We performed a meta-analysis to evaluate the efficacy and safety of different dosing regimens of secukinumab in the treatment of PsA. A systematic search was conducted using major electronic databases to identify relevant randomized controlled trials (RCTs) comparing secukinumab 300 mg versus secukinumab 150 mg in patients with PsA. Meta-analysis was performed using Review Manager software (version 5.3). Six studies with a total of 1141 patients were included. At week 24, secukinumab 300 mg was associated with a higher American College of Rheumatology 20% response (ACR 20), ACR 50, PASI 75 response rate, and dactylitis resolution rate than secukinumab 150 mg, especially in the anti-TNF-IR subgroup. At week 52, secukinumab 300 mg was associated with a higher psoriasis area and severity index (PASI) 75 and PASI 90 response rate than secukinumab 150 mg. There was no significant difference between secukinumab 300 mg and secukinumab 150 mg in the risk of any adverse events (AEs) and serious AEs at either week 24 or week 52. Secukinumab 300 mg was significantly more effective than 150 mg, especially for patients with PsA who have failed TNF therapy, and it was well tolerated.

Methylation in the promoter regions of WT1, NKX6-1 and DBC1 genes in cervical cancer tissues of Uygur women in Xinjiang

Abstract This study aimed to explore: 1) DNA methylation in the promoter regions of Wilms tumor gene 1 (WT1), NK6 transcription factor related locus 1 gene (NKX6-1) and Deleted in bladder cancer 1 (DBC1) gene in cervical cancer tissues of Uygur women in Xinjiang, and 2) the correlation of gene methylation with the infection of HPV16/18 viruses. We detected HPV16/18 infection in 43 normal cervical tissues, 30 cervical intraepithelial neoplasia lesions (CIN) and 48 cervical cancer tissues with polymerase chain reaction (PCR) method. Methylation in the promoter regions of the WT1, NKX6-1 and DBC1 genes in the above-mentioned tissues was measured by methylation-specific PCR (MSP) and cloning sequencing. The expression level of these three genes was measured by real-time PCR (qPCR) in 10 methylation-positive cervical cancer tissues and 10 methylation-negative normal cervical tissues. We found that the infection of HPV16 in normal cervical tissues, CIN and cervical cancer tissues was 14.0, 36.7 and 66.7%, respectively. The infection of HPV18 was 0, 6.7 and 10.4%, respectively. The methylation rates of WT1, NKX6-1 and DBC1 genes were 7.0, 11.6 and 23.3% in normal cervical tissues, 36.7, 46.7 and 30.0% in CIN tissues, and 89.6, 77.1 and 85.4% in cervical cancer tissues. Furthermore, WT1, NKX6-1 and DBC1 genes were hypermethylated in the high-grade squamous intraepithelial lesion (CIN2, CIN3) and in the cervical cancer tissues with infection of HPV16/18 (both P< 0.05). The expression of WT1, NKX6-1 and DBC1 was significantly lower in the methylation-positive cervical cancer tissues than in methylation-negative normal cervical tissues. Our findings indicated that methylation in the promoter regions of WT1, NKX6-1 and DBC1 is correlated with cervical cancer tumorigenesis in Uygur women. The infection of HPV16/18 might be correlated with methylation in these genes. Gene inactivation caused by methylation might be related to the incidence and development of cervical cancer.

A highly sensitive method for the detection of Chrysanthemum virus B

Background: Chrysanthemum plants are subject to serious viral diseases. The viruses cause severe losses of the quantity and quality of chrysanthemum. The most problematic pathogen of chrysanthemum is typically considered Chrysanthemum virus B (CVB). Thus, a method for the simultaneous detection of CVB is needed. Results: We used gene-specific primers, which were derived from the coat protein gene region of the virus, for reverse transcription to obtain cDNA. Nested amplification polymerase chain reaction (PCR) was employed to detect the viral gene. This method was sensitive enough to detect the virus at up to 10-9 dilution of the cDNA. Conclusion: A highly specific and sensitive nested PCR-based assay has been described for detecting CVB. This new method is highly specific and sensitive for the detection of CVB, which is known to infect chrysanthemum plants in the fields. Further, this protocol has an advantage over traditional methods as it is more cost-effective. This assay is ideal for an early stage diagnosis of the disease.

Amelioration of mitochondrial dysfunction in heart failure through S-sulfhydration of Ca2+/calmodulin-dependent protein kinaseⅡ / 中国药理学与毒理学杂志

OBJECTIVE To determine the functional role of hydrogen sulfide (H2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca2 +/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) using wild type and CSE knockout mouse models. METHODS Continuous subcutaneous injection isoprenaline (7.5 mg·kg-1·d-1), once a day for 4 weeks to induce heart failure in Male C57BL/6 (6-8 weeks old) mice and CSE-/- mice. 150 μmol·L-1 H2O2 was used to induce oxidative stress in H9c2 cells. Echocardiograph was used to detect cardiac parameters. H&E stain and Masson stain was to observation histopathological changes. Western blot was used to detect protein expression and activity. The siRNA was used to silence protein expression. HPLC was used to detect H2S level. Biotin assay was used to detect the level of S- sulfhydration protein. RESULTS Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H2S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKⅡ phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKⅡ activity was found to be elevated in CSE-/- mice as compared to wild type animals and the phosphorylation status of CaMKⅡ appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKII leading to reduced activity of this protein however, in CSE-/- mice S-sulfhydration was abolished following SPRC treatment. CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKⅡ is presented. SPRC mediated S-sulfhydration of CaMKII was found to inhibit CaMKⅡ activity and to preserve cardiovascular homeostasis.

Amelioration of mitochondrial dysfunction in heart failure through S-sulfhydration of Ca2+/calmodulin-dependent protein kinaseⅡ / 中国药理学与毒理学杂志

OBJECTIVE To determine the functional role of hydrogen sulfide (H2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca2 +/calmodulin-dependent protein kinaseⅡ (CaMKⅡ) using wild type and CSE knockout mouse models. METHODS Continuous subcutaneous injection isoprenaline (7.5 mg·kg-1 per day), once a day for 4 weeks to induce heart failure in male C57BL/6 (6-8 weeks old) mice and CSE-/- mice. 150 μmol·L-1 H2O2 was used to induce oxidative stress in H9c2 cells. Echocardiograph was used to detect cardiac parameters. H&E stain and Masson stain was to observation histopathological changes. Western blot was used to detect protein expression and activity. The siRNA was used to silence protein expression. HPLC was used to detect H2S level. Biotin assay was used to detect the level of S-sulfhydration protein. RESULTS Treatment with S-propyl-L-cysteine (SPRC) or sodium hydrosulfide (NaHS), modulators of blood H2S levels, attenuated the development of heart failure in animals, reduced lipid peroxidation, and preserved mitochondrial function. The inhibition CaMKⅡ phosphorylation by SPRC and NaHS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds. Interestingly, CaMKⅡ activity was found to be elevated in CSE-/- mice as compared to wild type animals and the phosphorylation status of CaMK Ⅱ appeared to relate to the severity of heart failure. Importantly, in wild type mice SPRC was found to promote S-sulfhydration of CaMKⅡ leading to reduced activity of this protein however, in CSE-/- mice S-sulfhydration was abolished following SPRC treatment. CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of CaMKⅡ is presented. SPRC mediated S-sulfhydration of CaMKⅡ was found to inhibit CaMKⅡ activity and to preserve cardiovascular homeostasis.

Over-expression of Mycobacterium neoaurum 3-ketosteroid-∆ 1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3, 17-dione to androst-1, 4-diene-3, 17-dione

Background: 3-Ketosteroid-∆¹-dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen asa new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd n) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The Km and Vmax values of purified KSDD were 8.91 µM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADDfromADefficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.

Effects of Sleeve Gastrectomy Surgery with Modified Jejunoileal Bypass on Body Weight, Food Intake and Metabolic Hormone Levels of Rats / 华中科技大学学报（医学）（英德文版）

This study examined the effects of a combined surgery of sleeve gastrectomy (SG) and modified jejunoileal bypass (JIB) on the body weight,food intake,and the plasma levels of active glucagon-like peptide-1 (GLP-1) and total ghrelin of rats.Rats were divided into 3 groups in terms of different surgical protocol:SG-JIB (n=12),SG (n=12),JIB (n=12) and sham surgery groups (n=10).In SG-JIB group,rats was subjected to sleeve gastrectomy and end to side anastomosis of part of the jejunum (25 cm from the ligament of Treitz) to the ileum 25 cm proximal to the cecum.The body weight and food intake were evaluated during 10 consecutive weeks postoperatively.The levels of active GLP-1 and total ghrelin in the plasma of the rats were measured by ELISA assay.The results showed that the SG-JIB treated rats relative to SG- or JIB-treated ones produced a sustained reduction in food intake and weight gain.The level of active GLP-1 was elevated and total ghrelin level decreased in SG-JIB-treated rats as compared with SG- or JIB-treated ones.It was concluded that SG-JIB could efficiently reduce the body weight and food intake,alter obesity-related hormone levels of the rats,indicating that SG-JIB may be potentially used for the treatment of obesity.

Clinical analysis of 266 cases with hand foot and mouth disease in Shenyang area 2009 / 中国小儿急救医学

Objective To summarize the prevention and treatment of hand food and mouth disease　(HFMD),especially the severe cases.Methods We analyzed the data including gender,age,time of onset,place of residence,contact history,clinical manifestation,accessory examinations and clinical outcome of 266　HFMD cases hospitalized in our department from Apr to Oct 2009,and identified the clinical and epidemiologic features associated with HFMD.Results Age group ranged from 4 months to 11 years with 87.6％(233/266) of the cases≤5 years.Most cases were resident in rural or suburban area,the number of which　was 3 times as much as urban.Neurological complications were more common,especially in the cases with increases of white blood cell and neutrophil number in peripheral blood.There were 40 cases with etiological examination results.Twelve cases were infected with enterovirus 71 ( EV71 ) and the others were infected with Coxsackievirus A16.Twelve cases with EV71 infection were complicated with meningoencephalitis,encephalitis,encephalomyelitis,one of whom was brain stem encephalitis.Conclusion The high-risk group of HFMD are the children less than 5 years.The suburban and rural are the key areas of prevention and control.The neurological complications are common,especially in the case with EV71 infection and significant increases of white blood cell and neutrophil number in peripheral blood.

A general evaluation indexing system for hospital administrators / 中华医院管理杂志

A general evaluation indexing system is constructed based on hospital specifics, in HR evaluation theory, on job orientation, HR competence, and by means of expert consultation and level of analysis. Four tier-1 indexes were earmarked as knowledge, basic competence, management capability, and research capability; 32 tier-2 indexes include education background, foreign language command, computer capability, legal knowledge, and professional knowledge, each having different weight. This indexing system enables hospitals to screen their management staff objectively, reliably and efficiently. Moreover, it serves as a reference not only for their recruitment, training, screening and promotion, but also for their personal development.