COVID-19康复者恢复期血浆及COVID-19静注人免疫球蛋白的IgG亚类检测分析

目的:探讨COVID-19康复者恢复期血浆及COVID-19静注人免疫球蛋白(human intravenous immunoglobulin,IVIG)(COVID-19-IVIG)IgG及亚类的水平。方法:选择2020年2月至3月国药集团武汉血液制品有限公司采集的COVID-19康复者恢复期血浆及制备的COVID-...     

一种新的静注人免疫球蛋白制剂制备工艺的研究

目的初步探讨一种新的静注人免疫球蛋白的制备工艺对制品得率和主要质量指标的影响。方法以组分Ⅱ+Ⅲ经过溶解、辛酸沉淀、Capto-Q层析、Macrocap-Q层析、收集流穿液,经超滤、浓缩,制备静注人免疫球蛋白,检测蛋白液中Ig A、Ig M、白蛋白、PKA、ACA含量、纯度、收率,并与低温乙醇法比较。结果与低温乙醇法比较,辛酸沉淀法可显著降低制品中Ig A、Ig M、白蛋白、PKA含量,提高产品得率。结论采用辛酸沉淀工艺制备静注人免疫球蛋白可提高制品的质量和产量。     

静注人免疫球蛋白中IgA的3种去除方法效果比较

目的    探索滤板压滤法、复合膜法和离子交换层析法对静注人免疫球蛋白中残留IgA的去除效果。方法  对3种过滤方法进行多批次的规模化生产静注人免疫球蛋白中分子大小分布、蛋白回收率和IgA含量的考察,分析不同过滤方法的去除效果。结果  3种过滤方法平均IgA去除率分别为86.6%、92.7%和97.9%;平均蛋白回收率为81.6%、95.3%和97.1%。通过单因素方差分析,发现3种方法并无明显统计学差异。结论  3种过滤方式各有其优缺点,滤板压滤法应用范围管但去除量有限;复合膜法使用方便但成本较高;离子交换层析法分离效果好但审批手续繁琐,不同企业应选择适合自己的过滤方式。     

Properties and efficacy of a human immunoglobulin M preparation for intravenous administration

From Cohn fraction III a new immunoglobulin (Ig) preparation (Pentaglobin) was prepared. This preparation contains 72% IgG and is enriched in IgM (12%) and IgA (16%). Due to a treatment with beta-propiolactone it is suitable for intravenous application. This IgM-enriched immunoglobulin preparation is prominent in high antibody titers (passive hemagglutination) against gram-negative as well as gram-positive germs and shows significantly higher efficacy in mouse protection tests than intravenous standard IgG. The high IgM content of this preparation in particular is responsible for the binding of bacterial antigens. Thus a marked advancement in the treatment of bacterial infections is to be expected by this new intravenously tolerable immunoglobulin preparation.     

Use of omeprazole sulfone in a single plasma sample as a probe for CYP3A4

Objective The hydroxylation of omeprazole, measured as the ratio of omeprazole/5-hydroxyomeprazole in a plasma sample taken 3 h after an oral dose, is an established method to determine CYP2C19 activity, and the ratio of omeprazole AUC/omeprazole sulfone AUC has been used for assessing CYP3A4 activity. The aim of this study was to determine whether the latter ratio from a single 3-h sample can also be used for CYP3A4 phenotyping.Methods Plasma levels of omeprazole and omeprazole sulfone were analyzed by reversed-phase high-performance liquid chromatography in a blood sample drawn 3 h after intake of a single oral 20-mg dose of omeprazole by 22 healthy subjects and five patients with newly diagnosed epilepsy. The procedure was repeated on the 4th day of 200 mg of ketoconazole intake (10 subjects), after 3 weeks of 150–200 mg twice-daily carbamazepine (five patients), and on the 6th day of 4 mg twice-daily tolterodine (12 subjects). Five subjects also took 100 mg and 50 mg of ketoconazole for 3 days before concomitant intake with omeprazole.Results The mean log10(omeprazole/omeprazole sulfone) ratio was 0.18 3 h after intake of omeprazole alone. After concomitant intake of ketoconazole, the corresponding value was 1.38 (p<0.001); after intake of carbamazepine it was −0.42 (p<0.05); and after tolterodine it was 0.29 (not significant). In the five subjects taking increasing doses of ketoconazole, the ratio was 0.11, 0.79, 1.2, and 1.5 after 0, 50, 100, and 200 mg of ketoconazole, respectively. The correlation between the metabolic ratios from the AUC_(0-6h) and from the single 3-h samples was very good, with a correlation coefficient of 0.92 (p<0.001).Conclusions A single blood sample taken 3 h after intake of 20 mg of omeprazole can be reliably used to phenotype for both CYP2C19 and CYP3A4 activity.Financial support was obtained from the National Network for Drug Development, within the Foundation for Strategic Research, Sweden.     

重组人淋巴细胞功能相关抗原3-抗体融合蛋白质量控制方法与质量标准研究

研究建立重组人淋巴细胞功能相关抗原3-抗体融合蛋白(rhLFA3-IgG1)质量控制方法和质量标准。采用针对Jurkat细胞表面CD2分子的竞争结合试验测定融合蛋白的体外活性，以分子筛和离子交换色谱分别确定制品纯度，将制品还原、羧甲基化后再以胰蛋白酶裂解并经反相高效液相色谱进行肽图分析，采用ELISA法分别测定蛋白A与CHO宿主细胞蛋白残留量。建立了重组人淋巴细胞功能相关抗原3-抗体融合蛋白生物学活性、理化特性与残留杂质等质量控制方法和质量标准。所建立的质量控制方法和质量标准具有保证产品安全有效、质量可控的特点，可用于重组人淋巴细胞功能相关抗原3-抗体融合蛋白(rhLFA3-IgG1)的质量控制与产品检定。     

Beta-propiolactone as a sterilizing agent in the manufacture of intravenous immunoglobulin preparations

It was demonstrated with phi X174, phi e and x phages that the beta-propiolactone step, which eliminated the anticomplementary activity of the intravenous immunoglobulin (IgG) preparation. Intraglobin, inactivates more than 10(6) viruses/ml. This means additional safety with respect to those viral diseases for which specific and sensitive diagnostic methods are not available at present.     

Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-450IIE1

Human cytochrome P-450IIE1 has been implicated in the oxidation of a number of substrates, including protoxins and -carcinogens. To date, no drugs have been identified that are exclusive substrates for the protein and are applicable for use as noninvasive probes of the in vivo function of the enzyme in humans. Chlorzoxazone was found to be oxidized only to 6-hydroxychlorzoxazone in human liver microsomes. Results of steady-state kinetics are consistent with the view that only a single enzyme catalyzes the reaction. The microsomal reaction was strongly inhibited by rabbit anti-P-450IIE1 and, in a competitive manner, by known P-450IIE1 substrates. Rates of chlorzoxazone 6-hydroxylation in different human liver microsomal preparations were well correlated with levels of immunochemically measured P-450IIE1 and rates of (CH3)2NNO oxidation. Chlorzoxazone 6-hydroxylation was also found to be catalyzed by purified human liver P-450IIE1. These results provide strong evidence that P-450IIE1 is the primary catalyst of chlorzoxazone 6-hydroxylation in human liver. Rates of chlorzoxazone 6-hydroxylation vary considerably among human liver samples, and chlorzoxazone 6-hydroxylation may have potential use as a noninvasive probe in estimating the in vivo expression of human P-450IIE1 and its significance as a risk factor in the toxicity and carcinogenicity of a number of solvents, nitrosamines, and drugs.     

Use of a benzyloxy-substituted lactone cyclooxygenase-2 inhibitor as a selective fluorescent probe for CYP3A activity in primary cultured rat and human hepatocytes.

The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor DFB [3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one] is metabolized in human and rat liver microsomal incubations and hepatocytes to a fluorescent metabolite, DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one]. This process is CYP3A-mediated in both species, as demonstrated by incubations with recombinant CYP3A enzymes and experiments with inhibitory antibodies. Measurement of DFH fluorescence can be used as a rapid readout of CYP3A activity following microsomal or cultured hepatocyte incubations. In rat and human hepatocytes treated with prototypical inducers, the formation of DFH was linear for the first 30 min, with no secondary metabolism of DFH, such as phase II glucuronidation, observed at early time points. Using a panel of four prototypical inducers (phenobarbital, dexamethasone, phenytoin, and rifampicin), the correlation between testosterone 6beta-hydroxylation in cultured human hepatocytes and CYP3A enzyme level in cell lysate was confirmed. DFB debenzylation was then shown to correlate well with testosterone 6beta-hydroxylation in hepatocytes treated with these four inducers. Primary cultured rat and human hepatocyte induction assays were optimized for 24- and 96-well plates, respectively. Controls were established to evaluate whether test compounds demonstrate time-dependent CYP3A inhibition to avoid false negative results. Thus, the use of DFB, a fluorogenic CYP3A-selective probe substrate, affords a fast, efficient, and robust assay for the measurement of CYP3A induction in both rat and human cultured primary hepatocytes.     

比色法测定静注人乙肝免疫球蛋白中麦芽糖的含量

目的:建立一种快速简便检测静注人乙肝免疫球蛋白(HBIGIV)中麦芽糖的含量。方法用比色法测定静注人乙肝免疫球蛋白中麦芽糖的含量。结果麦芽糖含量的线性范围5mg﹒mL-1～40mg﹒mL-1,平均回收率(n=6)为99.4%,RSD为1.13%。结论本方法准确、简便,适用于测定静注人乙肝免疫球蛋白中麦芽糖含量的测定。     

Use of cafeine as a probe for rapid determination of cytochrome P-450 CYP1A2 activity in humans

目的：建立快速测定细胞色素Ｐ４５０ＣＹＰ１Ａ２酶活性的高压液相色谱方法．方法：取３００μＬ血浆样品，用β羟乙基茶碱作内标，经５ｍＬ氯仿／异丙醇（９∶１）萃取处理后，用００５％的乙酸、乙腈和甲醇作为基本流动相，采用梯度洗脱程序在ＯＤＳ柱上分离待测组分，紫外检测波长２８２ｎｍ．结果：无内源性物质干扰测定．次黄嘌呤、内标和咖啡因快速基线分离，三者的保留时间均小于１３分钟．次黄嘌呤和咖啡因的检测下限均为０１μｍｏｌ·Ｌ－１，线性范围分别为１－１００μｍｏｌ·Ｌ－１和１－２００μｍｏｌ·Ｌ－１，相关系数分别为０９９９９和０９９８７，变异系数分别小于６％和１０％．两者的平均相对回收率为９６％－１０８％．结论：本方法快速、灵敏，可用于人群ＣＹＰ１Ａ２酶活性研究．     

Use of 1-anilino-8-naphthalene sulfonate as a fluorescent probe in the investigation of drug interactions with human alpha-1-acid glycoprotein and serum albumin

We report a rapid method for the characterization of the human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) interactions with drugs. The binding of 1-anilino-8-naphthalene sulfonate (ANS) to AAG and HSA was measured by fluorescence spectroscopy. Fluorescence data indicated that ANS was bound tightly to at least one site on AAG, with an affinity constant of 1.35 x 10(6) M-1. The fluorescence of an ANS:AAG complex was quenched by the binding of various drugs. Fluorescence quenching of the HSA:ANS complex showed a single site with an affinity constant of 0.72 x 10(6) M-1. The interaction of AAG and HSA with ANS or other drugs was also studied by comparative equilibrium dialysis. [14C]Pipequaline was used as an AAG and HSA site marker. [14C]Pipequaline seems to share sites I (azapropazone) and II (diazepam and ibuprofen) of HSA. However, high concentrations of warfarin were unable to displace [14C]pipequaline. On the other hand, it was shown that palmitic acid decreased, whereas bilirubin increased the pipequaline binding.     

高效液相色谱法测定人乙肝免疫球蛋白中麦芽糖的含量

目的建立高效液相色谱法,测定静脉注射用人乙肝免疫球蛋白(HBIGIV)中麦芽糖的含量.方法采用氨基柱Kromasil 100-5NH2(4.6 mm×250 mm)及示差折光检测器,以0.004 mol·L-1硫酸溶液做流动相,柱温50℃,流速0.8 mL·min-1.结果麦芽糖含量的线性范围5～40 g·L-1,平均回收率(n=6)为99.4%,RSD为1.13%.结论本方法准确、简便,适用于测定人乙肝免疫球蛋白静脉注射液中麦芽糖含量的测定.     

比色法测定静注人乙肝免疫球蛋白中麦芽糖的含量

目的：建立一种快速简便检测静注入乙肝免疫球蛋白（HBIGIV）中麦芽糖的含量。方法用比色法测定静注入乙肝免疫球蛋白中麦芽糖的含量。结果麦芽糖含量的线性范围5mg．mL—1～40mg，mL—1，平均回收率（n=6）为99．4％，RSD为1．13％。结论本方法准确、简便，适用于测定静注入乙肝免疫球蛋白中麦芽糖含量的测定。     

Characterization of human OCT1-mediated transport of DAPI as a fluorescent probe substrate

The present study was conducted to assess the functional characteristics of human organic cation transporter 1 (hOCT1) for the transport of 4',6-diamidino-2-phenylindol (DAPI), a fluorescent compound that may be used as a probe substrate for rapid assays of its functionality. The specific uptake of DAPI by hOCT1 heterologously introduced into Madin-Darby canine kidney II cells by stable transfection was found to be, when assessed by DAPI-derived fluorescence intensity, rapid and saturable with a Michaelis constant of 8.94 μM, indicating that DAPI is a good substrate of hOCT1. The specific uptake of DAPI was insensitive to the membrane potential and extracellular pH, indicating a mode of operation different from that for typical cationic substrates such as tetraethylammonium (TEA), for which hOCT1 has been suggested to be driven by an inside-negative membrane potential and favor higher pH for optimal operation. However, many organic cations were found to inhibit the specific DAPI uptake with extents well correlated with those of inhibition of the specific uptake of [(14) C]TEA, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of hOCT1.     

定量ELISA法检测健康人血浆抗呼吸道合胞病毒IgG抗体的可行性研究

目的  确认定量ELISA法检测健康人血浆抗呼吸道合胞病毒（respiratory syncytial virus,RSV）IgG抗体的可行性。方法  采用定量ELISA法检测健康人血浆,对初检的抗-RSV阳性血浆（抗-RSV效价>11 U/ml）进行复检和中和试验。结果  检测3041份血浆,初检和复检的血浆抗-RSV阳性率分别为5.4%和4.7%。定量ELISA法的特异性和精密性均良好,抗-RSV阳性血浆的有效检出率为87.8%。结论  定量ELISA法可用于健康人血浆抗RSV IgG抗体的大规模筛检。     

Use of the triazolotriazine [3H]ZM 241385 as a radioligand at recombinant human A2B adenosine receptors

Radiolabeled ZM 241385 (4-(2-[7-amino-2- ?furyl??1,2,4?triazolo?2,3-a??1,3,5?triazin-5-ylaminoethyl)p henol), has previously been used as a high affinity radioligand for the labeling of A2A adenosine receptors in cell membranes. Another subtype, the A2B receptor, is the least well-defined subtype of adenosine receptors and lacks selective pharmacological probes. In the present study, we have used [3H]ZM 241385 as a radioligand to label recombinant human A2B adenosine receptors in HEK-293 cell membranes, that do not express A2A adenosine receptors, and found that the pharmacological profile is consistent with the SAR of A2B receptors. Saturable, specific binding (Kd 33.6 nM, Bmax 4.48 pmol/mg protein) that was best described by a one-site model was found, and specific binding was approximately 75% of total binding. [3H]ZM 241385 binding was displaceable by a large number of compounds known to interact with A2B receptors; thus, this method has promise as a tool in the search for agonists and antagonists selective for this subtype. Xanthine analogs, which are antagonists, proved to be the most potent displacers. The Ki of XAC, xanthine amine congener, was 12.3 nM, while CPX (8-cyclopentyl-1,3-dipropylxanthine) was less potent. The non-selective triazoloquinazoline antagonist CGS 15943 (Ki 16.4 nM), which is similar in structure to ZM 241385, was slightly less potent than XAC. The non-xanthine A2B-antagonist alloxazine displaced [3H]ZM 241385-binding with a Ki of 462 nM, similar to its affinity in functional assays. Adenosine derivatives known to activate this receptor subtype, such as NECA (5'-N-ethylcarboxamidoadenosine) and R-PIA (N6-phenylisopropyladenosine), were considerably less potent than the 8-substituted xanthines examined.     

用咖啡因作探药,快速测定人体细胞色素P—450CYP1A2酶的活性

AIM: To develop a rapid HPLC method for the determination of cytochrome P-450 CYP1A2 activity. METHODS: A 300-microL plasma was prepared by extraction with 5-mL chloroform/isopropanol (9:1), and beta-hydroxyletheophylline was added as internal standard (IS). Samples were separated on an ODS column by a gradient elution system, of which mobile phase consisted of 0.05% acetic acid, acetonitrile, and methanol. The compounds of interest were monitored at 282 nm by UV detector. RESULTS: No potential interfering peaks were found. Paraxanthine (17X), IS and caffeine (137X) were rapidly eluted with baseline resolution, and their retention time was less than 13 min. The detection limits of both 17X and 137X were 0.1 mumol.L-1. Linear relations ranged over 1-100 mumol.L-1 and 1-200 mumol.L-1 with correlation coefficient of 0.9999 and 0.9987, respectively, for 17X and 137X. The coefficients of variation were within 6% for 17X, and 10% for 137X. The average recoveries for both compounds were ranged from 96% to 108%. CONCLUSION: This method is sensitive and rapid, and can be used for population studies of CYP1A2.     

2,5-Diphenyloxazole as a probe for microsomal mono-oxygenation in human and rat liver

2,5-Diphenyloxazole (PPO) is metabolized to one major fluorescent product by human liver and rat liver microsomes. PPO metabolism by human-liver microsomes involves more than one cytochrome P-450 isozyme, termed low-affinity and high-affinity components. At a substrate concentration of 0.1 microM, 95% of activity is due to the high-affinity component whereas at 100 microM 69% of activity is due to the low-affinity component. Inhibition studies with metyrapone and alpha-naphthoflavone at 0.1 microM and 100 microM suggest that the high-affinity component may reflect a 3-methylcholanthrene-inducible form of cytochrome. Therefore studies at low substrate concentrations may be a useful tool for cytochrome P-450 studies in man. Rat liver microsomes show linear kinetics indicating the involvement of one major form of cytochrome P-450.