1. Immunization No Response HIV Drug Concentration Of Three Components Of Triptolide In Plasma And Its Relationship With Abnormal Immune Activation 2. High Performance Liquid Chromatography Determination Of Serum, Cerebrospinal Fluid And Pancreatic Juice

BACKGROUNDSince the application of highly active antiretroviral therapy (HAART) in the 1990 s, the breakthrough progress in the treatment of acquired immune deficiency syndrome (AIDS) has been made. HAART can transform human immunodeficiency virus (HIV)-infected patients outcome by adequately suppressing HIV-1 replication and in turn leads to sustained CD4+T cells recovery in most patients.However, approximately 20% of patients do not achieve adequate CD4+T cell reconstitution despite prolonged virologic suppression. These patients are often called immunologic non-responders (INRs), which significantly increases the morbidity and mortality of opportunistic infection, tumor, cardiovascular disease, liver disease, etc. So, Finding an effective therapy that can reverse immune nonresponse solves the unmet need.Among kinds of influence factors, immune activation represents the main driving force of immune reconstruction failure. Tripterygium wilfordii Hook F (TwHF), a traditional Chinese medication, has a history of hundreds of years. It has been found to have anti-inflammatory, anticancer and potent immunosuppressive properties and been used widely in clinic for the treatment of autoimmune diseases, rheumatoid arthritis, dermatosis, etc. It is called "glucocorticoid of traditional Chinese medicine" as a result of perfect effects. Pharmacological characteristics in vivo and vitro about several monomers were studied, and triptolide, tripterine, triptonide, demethylzeylasteral, triptolidenol was deemed to account for the immunosuppression and anti-inflammatory. The effect of reversing immune nonresponse of TwHF was preliminarily confirmed in a pilot study about INRs in department of infectious diseases, Peking Union Medical College Hospital.Unfortunately, the preparations of TwHF in the market, like tripterygium tablets, tripterygium glycosides tablets and tripterygii hypoglauci, have dozens of monomer compositions. The studies about the monomers of tripterygium that can reverse immune reconstruction failure of INRs and their mechanism of action have not been reported, neither the drug concentration and pharmacokinetics of these monomers in healthy volunteers and HIV-infected patients. Our studies will try to find answers of these three questions. So, the relativity between tripterine, triptolide, triptolidenol and CD4+T cells, immune activation biomarkers CD38 and HLA-DR was studied.OBJECTIVESAn ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of tripterine, triptolide and triptolidenol in plasma of healthy volunteers (n=1) and INRs (n=19) respectively. Then, pharmacokinetics of the three ones in healthy volunteers were studied using non-compartmental model of Winnonlin 1.5. The relativity between drug concentration of the three ones and the percentage of CD4+T cells growth, the growth value of CD4+T cells and the change value of immune activation biomarkers CD38(%) and HLA-DR(%) was analyzed using SPSS 13.0. Which would be helpful to find effective monomers and their mechanism. The basic studies and drug developments were expected to improve on the basis of our study. Above all, our study would be meaningful to HIV-infected patients.METHODSAt first, an UPLC-MS/MS method for the determination drug concentration of tripterine, triptolide and triptolidenol was developed and validated. The content of tripterine, triptolide and triptolidenol in the preparations of tripterygium glycosides tablets was determinated.Blood samples were collected at 0,0.5,1,2,3,4,6,8,12,24 h after healthy volunteers (n=1) received tripterygium glycosides tablets 20 mg as a single dose extravascular administration. The drug concentration of tripterine, triptolide and triptolidenol was determinated by developed UPLC-MS/MS method. The pharmacokinetics of the three ones were analyzed using non-compartmental model of Winnonlin 1.5.All INRs received tripterygium glycosides tablets 10 or 20 mg, tid, as extravascular administration. Blood samples were collected just before the next dose. The drug concentration of tripterine, triptolide and triptolidenol were determinated by developed UPLC-MS/MS method. The relativity between drug concentration of the three ones and the percentage of CD4+T cells growth, the growth value of CD4+T cells and the change value of immune activation biomarkers CD38(%) and HLA-DR(%) was analyzed using SPSS 13.0.RESULTSAn UPLC-MS/MS method using electronic spray ion with positive ionization ions (ESI+) monitored in the multiple reaction monitoring (MRM) mode was developed and validated for the determination of tripterine, and another UPLC-MS/MS method using ESI+ monitored in the MRM mode was developed and validated for simultaneously determination of triptolide and triptolidenol.The mean value of tripterine, triptolide and triptolidenol in the preparations of tripterygium glycosides tablets was 22.941(15.163-31.755) μg/tablet,7.649(3.598-14.495) ng/tablet and 1423.475(817.895-2233.433) ng/tablet respectively.Healthy volunteers received tripterygium glycosides tablets 20 mg as a single dose extravascular administration. Non-compartmental analysis was used to analyze the pharmacokinetic parameters. The maximum concentration of tripterine in plasma (Cmax) was 0.52 ng·mL-1 and time to Cmax (Tmax) was 3.0 h, and the elimination half-life (t1/2) in plasma was 16.76 h. Unfortunately, the drug concentration of triptolide and triptolidenol was lower than the lowest limit of quantification (LLOQ). The pharmacokinetic parameters of triptolide and triptolidenol failed to be gained.All INRs received tripterygium glycosides tablets at a dosage of 10 or 20 mg, tid, as extravascular administration, the drug concentration of tripterine was (0.28-4.01) ng-mL"1. As for drug concentration of triptolide and triptolidenol, the same problem happened as above, the drug concentration of the two ones was lower than LLOQ.All dates about drug concentration, the percentage of CD4+T cells growth, the growth value of CD4+T cells and the change value of immune activation biomarkers CD38(%) and HLA-DR(%) were showed normal distribution characteristics using normality test with Shapiro-Wilk test, the value of p>0.05. There was no relativity between the drug concentration and the percentage of CD4+T cells growth, the growth value of CD4+T cells, the change value of CD38(%) and HLA-DR(%) as the correlation coefficient was 0.072、-0.211、0.310、0.424, respectively with Pearson correlation analysis and the value of p was 0.7988、0.445、0.261、0.116, respectively with hypothesis test. Then, two groups were divided according to different dosage (20 mg and 10 mg). The drug concentration of tripterine, the percentage of CD4+T cells growth, the growth value of CD4+T cells, the change value of CD38(%) and HLA-DR(%) of the two groups was not showed remarkable difference in statistics with Independent-Sample Test. Another two groups were divided according to the percentage of CD4+T cells change value. The drug concentration of the two groups was not showed remarkable difference in statistics with Independent-Sample Test.CONCLUSIONThere is no relativity between drug concentration of tripterine and CD4+T cells, immune activation biomarkers in INRs with Pearson correlation analysis, The drug concentration, the percentage of CD4+T cells growth, the growth value of CD4+T cells, the change value of CD38(%) and HLA-DR(%) of the two groups divided according to different dosage was not showed remarkable difference in statistics with Independent-Sample Test, which may be affected by confounding factors or small sample size. So, whether tripterine can reverse immune reconstruction failure or not was not conformed. The drug concentration of triptolide and triptolidenol was lower than LLOQ, the relativity between drug concentration of triptolide, triptolidenol and immune activation biomarkers in INRs can not be analyzed.BACKGROUNDGram-positive bacterial is responsible for community and hospital infection, staphylococcus aureus, enterococcus, streptococcus are most common pathogens. Bacteremia or sepsis and intracranial infection with a poor prognosis is caused when G+ bacterial breaks out into bloodstream and brain tissue. In recent years, with the wide application of antibiotics, bacterial resistance is increasingly serious. The sensitivity of antimicrobial agents sensitive to G+ bacterial decreased gradually. Linezolid is characterized with broad spectrum and powerful resistance to G+ bacteria activity, strong lipid solubility, the high rate of drug penetration into tissue and devoid of renal side-effects.So far, the studies about the pharmacokinetics and penetration rates of linezolid in pancreatic tissues have not be reported in the domestic and overseas. At the same time, studies about the pharmacokinetics and pharmacodynamics of linezolid in patients with severe infection and multiple drug resistant have not be reported in the domestic. The aim of this study was to develop and validate a new chromatographic method to determinate linezolid concentration in human serum, cerebrospinal fluid and pancreatic juice. The pharmacokinetics, pharmacodynamics and penetrability of linezolid in pancreatic tissue of Chinese patients with severe infection and multidrug resistance would be studied further, which can provide reference for clinical rational use of the drug.OBJECTIVETo develop a high-performance liquid chromatographic (HPLC) method for determination of linezolid in human serum, cerebrospinal fluid (CSF) and pancreatic juice.METHODSWith risperidone as internal standard, samples were extracted by methyl tert-butyl ether:n-propanol=90:10 (v/v) after added 0.1 mol·L-1 NaOH, then separated on a Shima-pack CLC-ODS-C18 (150 mm X 6.0 mm,5 um) column with mobile phase consisted of methanol:acetonitrile:phosphate buffer solution (0.02 mol·L-1 KH2PO4, pH value was adjusted to about 3.5 by H3PO4)= 20:16:64 (v/v/v) with a flow rate of 1.0 mL·min-1 at 35℃. The detection wavelength was set at 254 nm.RESULTSFor serum, the linear range of linezolid was from 0.25 to 40.0 mg·L-1 (r=0.999), the limit of quantitation was 0.25 mg·L-1, for the low, middle and high concentrations (0.8, 8.0,25.0 mg·L-1) of check samples:the intra-run RSDs were 1.64%-2.38%(n=5) and inter-run RSDs were 4.53%-6.34% (n=5), the mean method recoveries were 93.781%-97.393%(n=5), the mean extraction recoveries were 72.861%-77.539%(n=5). For CSF, the linear range of linezolid was from 0.1 to 20.0 mg·L-1 (r=0.999), the limit of quantitation was 0.1 mg·L-1, for the low, middle and high concentrations (0.2,2.0,15.0 mg·L-1) of check samples:the intra-run RSDs were 3.84%-4.83%(n=5) and inter-run RSDs were 6.04%-8.16%(n=5), the mean method recoveries were 106.910%-110.971% (n=5), the mean extraction recoveries were 73.226%-80.603%(n=5). For pancreatic juice, the linear range of linezolid was from 0.1 to 20.0 mg·L-1 (r=0.999), the limit of quantitation was 0.1 mg·L-1, for the low, middle and high concentrations (0.2,2.0,15.0 mg·L-1) of check samples:the intra-run RSDs were 2.96%-5.30%(n=5) and inter-run RSDs were 4.68%-6.40%(n=5), the mean method recoveries were 97.178%-105.072% (n=5),the mean extraction recoveries were 73.333%-76.010%(n=5). The mean extraction recoveries of IS were more than 87.00%.CONCLUSIONThe method is sensitive, simple and accuracy, and it can be used for clinical study, especially used in the patient with drug combination.