Determination of procarbazine in human plasma by liquid chromatography with electrospray ionization mass spectrometry

Procarbazine is a cytotoxic chemotherapeutic agent used in the treatment of lymphomas and brain tumors. Its pharmacokinetic behavior remains poorly understood even though more than 30 years have elapsed since the drug was approved for clinical use. To characterize the pharmacokinetics of procarbazine in brain cancer patients during a phase I trial, a method for determining the drug in human plasma by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) was developed and thoroughly validated. Plasma samples were prepared for analysis by precipitating proteins with trichloroacetic acid and washing the protein-free supernatant with methyl tert-butyl ether to remove excess acid. The solution was separated on a Luna C-18 analytical column using methanol-25 mM ammonium acetate buffer, pH 5.1 (22:78, v/v) as the mobile phase at 1.0 ml/min. A single-quadrupole mass spectrometer with an electrospray interface was operated in the selected-ion monitoring mode to detect the [M+H](+) ions at m/z 222.2 for procarbazine and at m/z 192.1 for the internal standard (3-dimethylamino-2-methylpropiophenone). Procarbazine and the internal standard eluted as sharp, symmetrical peaks with retention times (mean+/-S.D.) of 6.3+/-0.1 and 9.9+/-0.3 min, respectively. Calibration curves of procarbazine hydrochloride in human plasma at concentrations ranging from 0.5 to 50 ng/ml exhibited excellent linearity. The mean absolute recovery of the drug from plasma was 102.9+/-1.0%. Using a sample volume of 150 microl, procarbazine was determined at the 0.5 ng/ml (1.9 nM) lower limit of quantitation with a mean accuracy of 105.2% and an interday precision of 3.60% R.S.D. on 11 different days over 5 weeks. During this same time interval, the between-day accuracy for determining quality control solutions of the drug in plasma at concentrations of 2.0, 15 and 40 ng/ml ranged from 97.5 to 98.2% (mean+/-S.D., 97.9+/-0.4%) and the precision was 3.8-6.2% (mean+/-S.D., 5.1+/-1.2%). Stability characteristics of the drug were thoroughly evaluated to establish appropriate conditions to process, store and prepare clinical specimens for chromatographic analysis without inducing significant chemical degradation. The sensitivity achieved with this assay permitted the plasma concentration-time profile of the parent drug to be accurately defined following oral administration of standard doses to brain cancer patients.     

Aurora-A高表达对食管癌细胞侵袭和 MMP-9表达的影响

目的:建立Aurora-A高表达的稳定细胞系,并探讨Aurora-A高表达对食管癌细胞侵袭和MMP-9蛋白表达与活性的影响。方法:利用脂质体法稳定转染Aurora-A表达质粒,通过Westernblot和RT-PCR鉴定Aurora-A高表达的稳定细胞系。利用细胞体外侵袭实验观察细胞的侵袭能力,并通过Western blot、ELISA和明胶酶谱的方法观察MMP-9的蛋白表达水平和活性。结果:建立了Aurora-A高表达的稳定细胞系,且Aurora-A高表达能增加食管癌细胞的体外侵袭能力,并提高了MMP-9的蛋白表达水平和活性。结论:初步阐明了Aurora-A促进食管癌细胞侵袭的作用以及分子机制,为Aurora-A可能成为治疗食管癌的有效靶点提供了依据。     

A new sentinel surveillance system for severe influenza in England shows a shift in age distribution of hospitalised cases in the post-pandemic period

Background

The World Health Organization and European Centre for Disease Prevention and Control have highlighted the importance of establishing systems to monitor severe influenza. Following the H1N1 (2009) influenza pandemic, a sentinel network of 23 Trusts, the UK Severe Influenza Surveillance System (USISS), was established to monitor hospitalisations due to confirmed seasonal influenza in England. This article presents the results of the first season of operation of USISS in 2010/11.

Methodology/Principal Findings

A case was defined as a person hospitalised with confirmed influenza of any type. Weekly aggregate numbers of hospitalised influenza cases, broken down by flu type and level of care, were submitted by participating Trusts. Cases in 2010/11 were compared to cases during the 2009 pandemic in hospitals with available surveillance data for both time periods (n = 19). An unexpected resurgence in seasonal A/H1N1 (2009) influenza activity in England was observed in December 2010 with reports of severe disease. Reported cases over the period of 4 October 2010 to 13 February 2011 were mostly due to influenza A/H1N1 (2009). One thousand and seventy-one cases of influenza A/H1N1 (2009) occurred over this period compared to 409 at the same Trusts over the 2009/10 pandemic period (1 April 2009 to 6 January 2010). Median age of influenza A/H1N1 (2009) cases in 2010/11 was 35 years, compared with 20 years during the pandemic (p = <0.0001).

Conclusions/Significance

The Health Protection Agency successfully established a sentinel surveillance system for severe influenza in 2010/11, detecting a rise in influenza cases mirroring other surveillance indicators. The data indicate an upward shift in the age-distribution of influenza A/H1N1 (2009) during the 2010/11 influenza season as compared to the 2009/10 pandemic. Systems to enable the ongoing surveillance of severe influenza will be a key component in understanding and responding to the evolving epidemiology of influenza in the post-pandemic era.