Suberoylanilide hydroxamic acid (SAHA) attenuates memory impairment in the offspring of rats exposed to sevoflurane anesthesia.

BACKGROUND: SAHA was reported to enhance the expression of miR-129-5p, which was predicted to bind to 3' UTR of CASP-6, a gene playing crucial roles in the pathogenesis of memory impairment. Whether SAHA/miR-129-5p/CASP-6 is involved in the pathogenesis of prenatal exposure to sevoflurane remains to be explored. METHODS: Morris water maze test was performed to evaluate the functional parameters of learning and memory. Quantitative real-time qPCR was carried out to analyze the expression of miRNAs and CASP-6 mRNA under different conditions. RESULTS: Sevoflurane exposure of pregnant rats and SAHA treatment of the offspring had no effect on the blood gases, litter size, survival rate and weight. SAHA administration remarkably reversed the learning and memory impairment in prenatal rats caused by sevoflurane exposure. Mechanistically, the abnormal expression of miR-129-5p and CASP-6 in the offspring of pregnant rats exposed to sevoflurane was effectively restored by SAHA treatment. The luciferase activity of CASP-6 vector was effectively inhibited by miR-129-5p in primary neuron cells of rats. Moreover, the expression of CASP-6 mRNA and protein was significantly suppressed by miR-129-5p and SAHA treatment in a dose-dependent manner. CONCLUSION: Our work demonstrated that the administration of SAHA suppressed the expression of CASP-6 via modulating the expression of miR-129-5p, and SAHA may rescue the apoptosis of neurons caused by exposure to sevoflurane. The underlying mechanism might be the ability of SAHA to relieve learning and memory impairment in the offspring of the pregnant rats exposed to sevoflurane.

[Evaluation on birth defects surveillance system in four counties of Shanxi province, China].

OBJECTIVE: To evaluate the reliability of the birth defects surveillance system in four counties with high prevalence of birth defects (Pingding, Xiyang, Taigu and Zezhou counties) in Shanxi province, China. METHODS: One township was selected from each county as study site. The health workers chosen from township or village level were trained to visit families on the outcomes of each pregnancy who gave birth during year 2003 in the study site. The number of births and cases collected in the study were compared with that from the surveillance system. The number of births reported by surveillance system in four counties was also compared with the data from the local government. The criteria of evaluation were: 1) number of the missing report of births should < or = 5%, 2) the number of missing report on major external birth defects cases should < or = 10%. Researchers from the Peking University were responsible for examining the quality of surveillance in some terminal units of surveillance system. RESULTS: The numbers of births reported in the study and from the surveillance system for four-township were 1043 and 997, respectively. 46 births were missing and the rate of misreporting for births was 4.4%. The numbers of birth defects cases reported in the study and from the surveillance system were 30 and 29, respectively. 1 case of birth defect as missed, and rate of misreporting for birth defects cases was 3.3%. The total number of births reported from surveillance was similar to that in the study in four counties, with a difference of 1.2%. Birth registry data was rather readable and special health workers responsible for surveillance work were present in all the terminal units of the surveillance system. CONCLUSION: The misreporting of births and cases existed in the birth defects surveillance system of the four counties in Shanxi province, but were lower than the allowable criteria. The surveillance units had better registration, reporting and administration of births and birth defect cases. Hence, the quality of the data from the surveillance system in these four counties was reliable.

Pharmacokinetics of sifuvirtide, a novel anti-HIV-1 peptide, in monkeys and its inhibitory concentration in vitro.

AIM: To study the pharmacokinetics of sifuvirtide, a novel anti-human immunodeficiency virus (HIV) peptide, in monkeys and to compare the inhibitory concentrations of sifuvirtide and enfuvirtide on HIV-1-infected-cell fusion. METHODS: Monkeys received 1.2 mg/kg iv or sc of sifuvirtide. An on-line solid-phase extraction procedure combined with liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS) was established and applied to determine the concentration of sifuvirtide in monkey plasma. A four-(127)I iodinated peptide was used as an internal standard. Fifty percent inhibitory concentration (IC(50)) of sifuvirtide on cell fusion was determined by co-cultivation assay. RESULTS: The assay was validated with good precision and accuracy. The calibration curve for sifuvirtide in plasma was linear over a range of 4.88-5000 microg/L, with correlation coefficients above 0.9923. After iv or sc administration, the observed peak concentrations of sifuvirtide were 10 626+/-2886 microg/L and 528+/-191 microg/L, and the terminal elimination half-lives (T(1/2)) were 6.3+/-0.9 h and 5.5+/-1.0 h, respectively. After sc, T(max) was 0.25-2 h, and the absolute bioavailability was 49%+/-13%. Sifuvirtide inhibited the syncytium formation between HIV-1 chronically infected cells and uninfected cells with an IC(50) of 0.33 microg/L. CONCLUSION: An on-line SPE-LC/MS/MS approach was established for peptide pharmacokinetic studies. Sifuvirtide was rapidly absorbed subcutaneously into the blood circulation. The T(1/2) of sifuvirtide was remarkably longer than that of its analog, enfuvirtide, reported in healthy monkeys and it conferred a long-term plasma concentration level which was higher than its IC(50) in vitro.

Pharmacokinetics of His-tag recombinant human endostatin in Rhesus monkeys.

AIM: To study the pharmacokinetics and accumulation of an Escherichia coli expressed His-tag fused recombinant human endostatin (rh-endostatin) in Rhesus monkeys. METHODS: Rh-endostatin was iv or sc injected in Rhesus monkeys, and the rh-endostatin concentration in serum samples was determined by an enzyme immunoassay (EIA) method. The serum drug concentration-time data were analyzed by compartmental analysis using the practical pharmacokinetic program 3p97. RESULTS: Following iv administration at a dose rate of 1.5, 4.5, and 13.5 mg/kg in rhesus monkeys, the concentration-time curves of rh-endostatin were best fitted to a three-compartment open model. AUC(0-infinity) linearly increased with dose, while Cls exhibited no significant difference among different dose groups. The terminal half-lives (lambda3) were 8+/-8, 3.1+/-1.4, and 20+/-14 h, respectively. After sc administration at a dose rate of 1.5 mg/kg, the concentration-time curve was best fitted to a two-compartment open model, with a terminal half-life (T(1/2beta)) of 8+/-3 h. Bioavailability following sc injection was approximately 70%+/-3%. After consecutive iv injection of rh-endostatin at a dose rate of 1.5 mg.kg(-1).d(-1) for 7 d, the AUC(0-24 h) substantially increased from 22+/-13 mg.h.L(-1) (d 1) to 50+/-29 mg.h.L(-1) (d 7), with an accumulation factor of 2.3+/-0.6 (P < 0.05). CONCLUSION: The pharmacokinetic behavior of rh-endostatin in Rhesus monkeys complies with linear kinetics within the examined dose range. It tends to be accumulated in bodies after repeated administration at a dose level of 1.5 mg.kg(-1).d(-1) for more than 7 consecutive days.

Detection and quantitation of PS20, a phosphorothioate oligodeoxynucleotides in monkey plasma.

AIM: To establish the method for quantitation of the phosphorothioate oligodeoxynucleotides (S-ODNs) in plasma. METHODS: Two solid-phase extraction columns combined with a strong anion-exchange column were utilized to remove proteins and lipids in plasma, and the salts were removed by a reverse-phase column followed by dialysis with a 2500 Da-cutoff membrane. The concentration of the tested S-ODNs, PS20, and its metabolites extracted from the plasma were determined by the method of non-gel sieving capillary electrophoresis (NGCE) with diode array detection in the presence of internal standard (IS). RESULTS: The method was with good base number specificity. Relative standard deviation % of both intra and inter assay were all less than 10 %, and the total mean recovery was about 91 %. The methodology was successfully used to determine the PK behavior of an anti-tumor antisense S-ODNs in monkeys and identify the metabolites with single base difference. CONCLUSION: The combined method of solid-phase extraction and NGCE could be used to study the pharmacokinetics of S-ODNs, and the main parameters of the methodology met the requirement of PK study.

Antisense candidates against protein kinase C-alpha designed based on phylogenesis and simulant structure of mRNA.

AIM: To optimize the antisense drug design by the combined method of phylogenetic analysis and secondary structure prediction and to get ideal candidates. METHODS: The phylogenetic analysis and the secondary structure simulation were performed by computer. Oligodeoxynucleotides (ODN) were designed against the full-conserved blocks with low local reaction free energy of protein kinase C (PKC)-alpha mRNA. The in vitro effects of ODN were evaluated by human A549 lung carcinoma cells and mouse B16-BL6 melanoma cells, the expression of target mRNA was detected by in situ hybridization and RT-PCR. The in vivo effects of ODN were also evaluated by models of A549 xenografts in nude mice and B16 melanoma in mice. RESULTS: Three ODN had significantly lower IC50 values than that of ISIS3521, the positive control, on A549 cells in vitro. Five ODN inhibited the growth of B16-BL6 cells with IC50 <100 nmol/L, while IC50 of ISIS3521 was >200 nmol/L. In situ hybridization and RT-PCR showed that the best candidate AP1261 inhibited the expression of PKC-alpha at mRNA level in a dose-dependent manner. AP1261 inhibited the growth of A549 and B16 tumors in vivo at 0.005-0.5 mg.kg(-1).d(-1). The inhibitory rate of AP1261 on A549 tumors was greater than that of ISIS3521 at the same dose. ISIS3521 did not affect the growth of B16 tumors. CONCLUSION: AP1261 may be of value as an antitumor agent or adjuvant and the combined method of phylogenetic analysis and secondary structure prediction is a potential helpful tool for antisense drug design.

Pharmacokinetics and partial thromboplastin time after intravenous recombinant hirudin variant-2 in rhesus monkeys.

AIM: To study the pharmacokinetics (PK) and changes of kaolin partial thromboplastin time (KPTT) following single or multiple (7 d) dosing of a novel recombinant hirudin variant-2 (rHV-2) via the route of iv bolus injection (50 % of the total dose) plus infusion (the remained 50 % of the dose) in rhesus monkeys. METHODS: A crossover design was applied to research the PK and KPTT profiles of rHV-2 after single (with total dose at 1, 3, and 6 mg/kg, respectively) and multiple dosing (3 mg/kg). An enzyme-linked immunosorbent assay (ELISA) method was utilized to determine the level of rHV-2 in plasma. RESULTS: The concentration profiles of rHV-2 during or after administration were dependent both on the loading dose and the infusion rate. Mean Cmax after bolus in three single dose groups were 2.90, 9.78, and 15.68 mg/L, respectively. Infusions at rate of 8.35, 25, and 50 g/kg/h in 1 h resulted in steady-state levels of 0.73-0.86, 1.94-2.04, and 5.41-5.59 mg/L, respectively. The plasma rHV-2 levels during or after administration among doses were significantly different at most of the time points. Area under concentration-time curve (AUC) increased linearly with dose but systemic clearances were similar among different groups. KPTT was significantly prolonged (compared with baseline) at all dose levels, and trended to increase with dose. CONCLUSION: Both the loading dose and the infusion rate are very important for controlling the rHV-2 level, and the data may be helpful for optimizing dosage-regimen in clinical trials.

[Metabolism, Distribution and Excretion of Recombinant Human Thrombopoietin in Mice]

The metabolism, distribution and excretion profiles of recombinant human thrombopoietin (rhTPO) in mice were studied by means of (125)I-labeled rhTPO ((125)I-rhTPO) combined with size exclusive high performance liquid chromatography (SHPLC) or trichloroacetic acid (TCA) precipitation analysis. (125)I-rhTPO was prepared by iodogen method. Purification was performed on Sephacryl S-200 HR gel. Radioactive-purity of (125)I-rhTPO identified by SHPLC was (96.9 +/- 1.5)% (n = 3). The proliferation effect of TPO dependent cell line (TD-3) and the increase of peripheral platelet counts in mouse by (125)I-rhTPO demonstrated that (125)I-labeled protein maintained the biological activities of TPO both in vitro and in vivo. SHPLC analysis of serum and urine samples taken after sc 1 micro g/mouse (345 kBq/mouse) of (125)I-rhTPO revealed that there were two lower molecular weight (125)I-degradation metabolites ((125)I-MI and (125)I-MII) other than parent molecule. (125)I-MI was mainly found in urine, and (125)I-MII was detected both in serum and in urine. The maximal concentration of (125)I-rhTPO was reached at 2 hours after injection. The terminal half-life was 10.8 hours, which was much longer than those of other peptides. TCA precipitable radioactivity in tissue showed that the radioactivity in bone marrow was rather high. The highest level was found in urinary system. Levels in adrenals, lymph nodes, and fat were near to that in serum. Lowest was found in brain. The main excretion route was urinary system and (98 +/- 5.6)% of (125)I-rhTPO was excreted within 72 hours after dosing.