AIM: To study concentration-time profiles and pharmacokinetics of recombinant human basic fibroblast growth factor (rhbFGF) following intravenous bolus injection plus infusion of the drug (20%:80%) at 10, 20 and 40 μg·kg-1 and compared with bolus injection in rhesus monkey. METHODS: The specificity, sensitivity, precision and accuracy of determination by enzyme labeled immunosorbent assay were satisfied. RESULTS: The endogenous level of bFGF was (144 ± 101) ng·L-1 in plasma. cmax of (33 ± 16), (56 ± 8) and (91 ± 28) μg·L-1, respectively, was observed immediately after injection. Levels maintained at 22-27, 37-44, 63-65 μg·L-1, respectively during infusion. Concentration rapidly decreased after the end of administration, with an average terminal t1/2 of 2.6-2.9 h. AUC(0-∞) increased proportional with dose, and systemic clearance Cls was closed among doses. CONCLUSION: In dosage range studied pharmacokinetic behavior appeared as linear kinetic. The parameters of the drug after bolus were similar to bolus plus infusion.

Objective To evaluate biodistribution and pharmacokinetics pattern of ~(131)I-labeled rch24which is the region-grafted (humanized) anti-carcinoembryonic antigen (CEA) monoclonal antibody in nude mice. Methods Nude mice bearing cancer xenografts received intravenous injections of ~(131)I- rch24, then blood, plasma, heart, liver, spleen, lung, kidney, tumor and other tissues were taken at different time point for determination the concentration of radioactivity and calculate the T/NT value. Nude mice were packeted randomly to four group of high, medium, low dose and continuous administration, blood drug concentration was detected by ELISA method at the different intervals. Then, draw the concentration-time curve and calculate the pharmacokinetics paramete. Results After administration, radioactivity of the tumour was significantly enhanced whereas radioactivity of normal tissues decreased gradually. For single administration, at the dose of low to medium, pharmacokinetics pattern was linearity -kinetics whereas for high dose group,pharmacokinetics paramete shown some behavior of non-linearity-kinetics. Conclusion Our results suggest that the ~(131)I-labeled region-grafted (humanized) anti-CEA monoclonal antibody rch24 exhibit a considerable targeting activity so as to ~(131)I radioisotopes can be concentrated specifically in tumor. The pharmacokinetics pattern of this medicine was different at different dose.

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC–ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin−1. Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39–400.00ngmL−1, the correlation coefficients (r2) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL−1. The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC–MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.

Adenovirus vectors are one of the most promising gene transfer systems. They are of great value for gene therapy because these vectors achieve temporal high-level transgene expression and high gene transfer efficiency. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. Perfusion cultivation of 293 cells is one of the most commonly used methods to produce adenovirus vectors and it is suitable for industrialized production specially. Experimental studies had been carried out to produce recombinant adenovirus containing the green fluorescent protein gene (Ad-GFP) by perfusion cultivation of HEK-293 N3S cells in a 5L stirring bioreactors. Perfusion rate was 1-2 volume/day. To infect the 293 N3S cells with Ad-GFP at the density of (2-4) x 10(6) cells/ ml. The time of collecting cells was 48 hours post infection. After three rounds of freeze/thaw and centrifugation, the crude viral lysates were stored at--80 degrees C until use. Then to get the Ad-GFP products by 2 x CsCl-gradient purification. The purity of the products was determined by the A260/A280 ratio and a high performance liquid chromatography (HPLC) assay. The infective titer was determined by a TCID50 assay. The culture term was 10-12 days. The infectious titer, the number of virus particle and the ratio of infectious titer to virus particle for the product were 1.0 x 10(11) IU/mL, 1.68 x 10(12) VP/mL and 6.0% IU/VP respectively. The A260/A280 ratio was 1.33, and the purity determined by HPLC was 99.2%. The cell specific productivity was around 1000 IU/cell. By perfusion cultivation of 293 N3S cells in a 5L stirring bioreactors, we established the production process for Ad-GFP, which paves a way to produce other recombinant adenovirus for gene therapy.

Objectives:To develop a method to quantitate puerarin in beagle plasma samples by on-line solid phase extraction column switching liquid chromatography tandem mass spectrometry,and to study the pharmacokinetics(PK) of puerarin preparationsa in beagles.Methods:The mobile phase of analytical column consisted of acetonitrile/methanol/0.1% formic acid(25/25/50) at a flow-rate of 0.2 ml/min.The analytical column was Grace Vydac C8 column.The mobile phase of SPE extraction column consisted of acetonitrile/0.1% formic acid(5/95) at a flow-rate of 2.0 ml/min.SPE extraction column was packed with Zorbax C18,with average particle size 50 μm.Puerarin was analyzed by a linear ion trap mass spectrometer,LTQ-MS,operating in the negative ion and selective reaction monitoring(SRM) acquisition mode.The SRM detection setup for the analytic peurarin was m/z 415.2→195.2.Results:Validation of the methodology demonstrated that the linear calibration curves covered the range of 0.39-400 ng/ml,the determinate coefficients(r2) were above 0.999.The lower limit of detection(LLOD) was 0.39 ng/ml.The intra-and inter-batch precisions were less than 7.6% and 6.4%,respectively.The accuracy of beagle plasma samples containing puerarin at the QC levels was-0.51%,1.20%,and-2.31%,respectively.The single run time of an individual sample was 6.5 min.Conclusion:The established on-line SPE-LC/MS/MS system needs no complicated pretreatment procedures of samples,the analytical speed is rapid,and the specificity,linearity,sensitivity,and precision of the methodology fulfil the requirements of PK study.The results showed that the developed method is feasible and reliable to be applied in preclinical or clinical PK studies of isoflavone drugs.

Objective To prepare a highly purified 125 I -pegylated thymosin α1 (PEG-TA1) with satisfied bioactivity for using in the distribution and excretion study in animals. Methods Tyr-PEG-TA1 was iodinated by means of Iodogen method. The ZipTip pipette tips were utilized to monitor the reaction process for the optimization of reaction conditions. A two-step gel filtration chromatography with Sephadex G50 followed by Sephadex G10 was applied to purify the labelled products. The bioactivity assay of PEG-TA1, Tyr-PEG-TA1 and 125 I -Tyr-PEG-TA1 were performed by an enzyme immunoassay (EIA) method. Results The optimum condition of labelling reaction was: shaking for 20 min at 25℃ in 0.1 mol/L phosphate buffer (0.1mol/L NaCl) with the pH value of 7.0. Slight but no significant difference of bioactivity among the PEG-TA1, Tyr-PEG-TA1 and the 125 I -Tyr-PEG-TA1 was observed. After the two-step purification process, a product of 125 I -Tyr-PEG-TA1 with radiochemical purity of 95% and specific activity of 39.4 Bq/ng was obtained. Conclusion A successful preparation of 125 I -Tyr-PEG-TA1 was achieved, and the radiochemical purity and the specific activity of the ~(125)I labeled molecules satisfied the requirements of non-clinical pharmacokinetic study.;

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-∞) linearly increased with dose, while Cl s exhibited no significant difference among different dose groups. The terminal half-lives (λ 3 ) 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 1/2β ) 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.

目的:使用新型DOTA类螯合剂p-SCN-BZ-DOTA对进口人鼠嵌合型抗CD20单抗美罗华进行90Y标记,确定最佳标记条件。方法:采用先螯合抗体再标记核素的方法制备放免复合物,在固定抗体、螯合剂、核素用量及缓冲液的前提下,采用正交设计考察标记反应中的3个关键因素pH值、温度、标记时间对其比活度的影响,确定最佳标记条件。结果:不同标记条件下,放免复合物的比活度波动在0.122~0.327 mCi/mg之间,统计分析后发现,最佳标记条件为:pH值7.5,4℃反应2 h。结论:使用新型螯合剂p-SCN-BZ-DOTA可成功制备90Y-抗CD20单抗放射免疫药物,其放化纯度可满足临床研究使用。

AIM: To study biodistributional profiles of recombinant antibreast cancer immunotherapeutic fusion protein HSP-MUC1 vaccine after subcutaneous in tumor-bearing mice. METHODS: HSP-MUC1 of variant tissue was detected by ^125I-HSP-MUC1 combined with trichloroacetic acid (TCA) precipitation or sizeexclusive high performance liquid chromatography (SHPLC). RESULTS: ^125I-HSP-MUC1 distributed widely. The highest

AIM: To study the metabolism, distribution, and excretion profiles of recombinant human glucagon-like peptide-1(7-36)[(rhGLP-1(7-36))] in Wistar rats. METHODS: The dose was set at 25 μg·kg^-1. ^125I-rhGLP-1(7-36) was prepared by iodogen method and determined by size exclusive HPLC (SHPLC) or trichloroacetic acid (TCA) precipitation analysis. RESULTS:SHPLC analysis after iv or sc administration of rhGLP-1(7-36)

AIM: To study the pharmacokinetic profiles and biodistribution of recombinant human parathyroid hormone (1-34) [rhPTH(1-34)] after administration in rats. METHODS: ^125I-rhPTH(1-34) was prepared by iodogen methods and purified by gel filtration. Immunoactivity of ^125I-labeled peptide was assayed by enzyme immunoassay (EIA). Concentrations of ^125I-rhPTH(1-34) in serum and tissues of rats were determined by the method of trichloroacetic acid (TCA) precipitation. Concentrations of

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.

目的:了解DNA疫苗在BALB/c小鼠体内的生物分布情况, 建立DNA疫苗体内生物分布情况研究方法.方法:乙肝核酸疫苗(H BV DNA)经32P 标记、分离、纯化、鉴定后,胫前肌注射给药,结合三氯乙酸(TCA)沉淀,研究肌注乙肝核酸疫苗后BALB/c小鼠体内的生物分布情况.结果:质粒DNA疫苗除注射局部肌肉分布较多外,其他一些重要组织也有分布, 其中腺体组织(肾上腺、胰腺、胸腺)中的浓度最高,其次为排泄物(尿、肠内粪),脑组织中浓度最低.各组织中可沉淀放射性按血浆浓度时间曲线下面积(AUC)从高到低排列依次为胸腺、肾上腺、膀胱、生殖腺、脂肪、肠内粪、胰腺、颌下腺、脾、小肠、眼球、肝、肠内容、肾、甲状腺、肺、淋巴结、对侧肌肉、心脏和脑.结论:32P标记DNA疫苗后各组织β计数结合三氯乙酸沉淀法研究核酸疫苗生物分布情况,方法灵敏、可靠,易于分析.

It is difficult to analyze protein drug in biological sample since the concentration of protein drug in it is very low,and lots of impurities with similar structure as protein drug will disturb the analysis. In this research, a method combining immobilized affinity chromatography with biological mass spectrometry to extract and characterize recombined protein drug from animal serum was developed. Protein drug was extracted and enriched from rhesus serum after i.v. 13.5 mg/kg rh-endostatin 2 min and 20 min with two affinity chromatography methods, immobilized immunoaffinity chromatography (IAC) and immobilized metal ion affinity chromatography (MAC). The mass information of protein drug, metabolites and drug-protein complexes in the gel was acquired by matriv-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of affinity gel directly. Protein drug in the affinity gel was digested with trypsin and the masses of generated peptides were measured with MALDI-TOF-MS. The data obtained from peptide mass fingerprinting (PMF) was searched in protein database, and collagen XVIII [Homo sapiens] (ID of SWISS-PROT/TrEMBL:Q8WX15), a prototype of endostatin in vivo was identified.

扼要介绍重组蛋白多肽类药物的临床药代动力学研究的内容、方法和特点.综述了重组内皮抑制素、重组糖基化血小板生成素、改构肿瘤坏死因子、重组糖基化尿激酶原、人源化单抗trastuzumab和重组人肿瘤坏死因子受体临床药代动力学研究的方法学、内容和结果.

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.

目的:在肿瘤患者中观察重组人肿瘤坏死因子α突变体rhTNF-αD11a的药代动力学.方法:在单次肌内注射160×104 U和240×104U及连续肌内注射160×104U,qd×7d后,用ELISA法测定其抗原浓度.结果:方法学检验表明,灵敏度、特异性、线性、线性范围、精密度和回收率均符合要求.肌注0.5 h后血清浓度显著高于内源水平(P≤0.05～0.001),24 h后多数患者恢复至药前水平.Cmax和AUC随剂量增高,平均Cmax分别为10.5和14.7 pg-eq·mL-1,AUC分别为50.1和106.1 pg-eq·h·mL-1,但无统计学差异.Tmax随剂量有延长趋势.连续注射7次后浓度明显低于首次注射(P＜0.05).蓄积因子(AUC第7次/AUC第1次)为0.65.结论:在研究剂量范围内单次肌内注射药代动力学近似线性,连续注射后药物浓度呈降低趋势.