MB病毒灭活新鲜冰冻血浆中部分有效成分的探讨

目的 探讨经过病毒灭活的新鲜冰冻血浆中部分有效成分的质量变化.方法 采集50袋新鲜血浆,分成A、B两组,分别对其进行检测,内容包括PT,TT,APTT,Fg,TP、ALB及凝血因子FⅧ、FⅤ.结果 病毒灭活冰冻血浆中的TP、凝血因子FⅧ、FⅤ和Fg等的含量均有不同程度的降低.但大都在30%以内,在可接受的范围内.结论 采用MB光化学法病毒灭活血浆不影响临床应用,其利大于弊.     

蝮蛇抗栓酶对血浆凝块降解作用的体外实验研究

本文通过体外实验研究了蝮蛇抗栓酶对血浆凝块的降解作用,以尿激酶(UK)作对照,结果表明:蝮蛇抗酸酶与UK相似,能使血浆凝块析出液中纤维蛋白(原)降解产物(FDP)增加,同时使血浆凝块体积减小,且随剂量增加,FOP升高,凝块体积减小越显著。提示蝮蛇抗栓酶具有溶栓作用,且有剂量效应关系。     

亚甲蓝病毒灭活新鲜冰冻血浆制备冷沉淀的可行性研究

目的 评价分析亚甲蓝灭活后血浆制备冷沉淀的可行性.方法 通过对同源血浆亚甲蓝病毒灭活前后制备冷沉淀FⅧ和Fbg含量的比较得出结论.结果 用亚甲蓝病毒灭活新鲜冰冻血浆制备的冷沉淀,FⅧ和Fbg的含量均有明显降低.结论 用于制备冷沉淀的新鲜血浆不易进行亚甲蓝病毒灭活.     

In vitro evaluation of acetylcholinesterase reactivators as potential antidotes against tabun nerve agent poisonings

Searching for new potent acetylcholinesterase (AChE; E.C. 3.1.1.7) reactivators (oximes) is a very time-consuming process. At our department, we are able to synthesize more than 50 new AChE reactivators per year. Owing to this fact, we have to select promising reactivators using our in vitro method (potentiometric titration, pH 8 and temperature 25 degrees C; source of cholinesterases, rat brain homogenate; time of inhibition by nerve agents, 30 min; time of reactivation, 10 min) prior to in vivo experiments. For this purpose, we are using two-phase in vitro evaluation of reactivator potency. In the first phase, reactivation potency of all newly synthesized AChE reactivators is tested at two concentrations: 10(-3) M and 10(-5) M. Afterwards, all reactivators achieving reactivation potency over 15% (especially at the concentration 10(-5) M) are tested. The second phase consists of the measurement of the relationship between concentration of the oxime and its reactivation ability. In most cases, the reactivation bell-shaped curve is obtained. The most potent AChE reactivators are selected and provided for further experiments during our development process.     

In vitro selection and efficacy of topical skin protectants against the nerve agent VX

Against highly toxic chemicals that are quickly absorbed in the skin, topical formulations could adequately complement specific protective suits and equipments. In this work, we evaluated in vitro and compared the skin protection efficacy against the nerve agent VX of four different topical formulations: oil-in-water and water-in-oil emulsions, a perfluorinated-based cream and a hydrogel. Semi-permeable silicone membrane, pig-ear and human abdominal split-thickness skin samples mounted in diffusion cells were compared as in vitro permeation tests. The results showed that silicone membrane could be used instead of skin samples to screen for potentially effective formulations. However, the results indicated that due to potentially significant interactions between formulations and skin, relevant ranking of formulations according to their protective efficacy could require tests with skin samples. The main phase of emulsions, water or oil, was not found to be critical for skin protective efficacy against VX. Instead, specific film-forming ingredients such as perfluorinated-based polymers and silicones could significantly affect the skin protective efficacy of formulations. We showed that a hydrogel containing specific hydrophilic polymers was by far the most effective of the formulations evaluated against VX skin permeation in vitro.     

In vitro reactivation potency of novel symmetrical bis-pyridinium oximes for electric eel acetylcholinesterase inhibited by nerve agent sarin

This communication describes synthesis and in vitro evaluation of a series of novel bis-pyridinium oximes connected by bis-methoxymethyl benzene, 1,4-bis-methoxymethyl (cis)-but-2-ene and 1,4-bis-methoxymethyl but-2-yne linkers as reactivators of sarin inhibited acetylcholinesterase (AChE). The reactivation data of synthesized oximes were compared with those of 2-PAM and obidoxime. The efficacy of oximes such as 1,4-dimethoxy cis-but-2-ene bis-[4,4′-(hydroxyiminomethyl)-pyridinium] dichloride (3g), 1,4-dimethoxy benzene bis-[3,3′-(hydroxyimino-methyl) pyridinium] dichloride (3b) and 1,3-dimethoxy benzene bis-[3,3′-(hydroxy-iminomethyl) pyridinium] dichloride (3e) were found to be more than that of obidoxime in reactivating sarin inhibited AChE. The oxime 3g was able to reactivate 25% of AChE activity in comparison to 20% and 5% reactivation exhibited by 2-PAM and obidoxime respectively at a concentration of 10⁻⁴ M. The pKa of the oximes were determined and correlated with the reactivation potential.     

浅谈新鲜冰冻血浆融化后不同放置时间的临床应用意义

目的探讨新鲜冰冻血浆融化后24h内凝血因子的变化,为指导临床治疗提供理论依据。方法采用SYSMEX CA-1500型自动血凝分析仪,对20份血浆样品分别于融化后0、6、12、24h测定凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白(FIB)、凝血酶时间(TT)、凝血因子Ⅶ、Ⅷ、Ⅸ(FⅦ、FⅧ、FⅨ)活性水平。结果新鲜血浆融化后24h之内无明显改变的有PT、FIB、TT(P>0·5);其他指标在不同时间段各有明显的改变,同时显示FⅧ半衰期为12~24h。结论新鲜冰冻血浆融化后放置会有凝血因子活性衰减,为保证输血质量,应尽可能融化后立即输注。     

In vitro human plasma leucine(5)-enkephalin degradation is inhibited by a select number of drugs with the phenothiazine molecule in their chemical structure

A number of drugs with the phenothiazine molecule in their chemical structure inhibit in a dose-dependent manner human plasmatic aminopeptidase leucine(5)-enkephalin (LEU) metabolism. Half-life peptide degradation was significantly increased by thioridazine > fluphenazine > As-1397 [10-(alpha-diethylaminopropionyl)phenothiazine] >/= promethazine >/= chlorpromazine (final drug conc. 10(-4) M); t1/2 (+/- SD) 21.2 +/- 1.1, 19.6 +/- 1.0, 17.2 +/- 0.9, 17.1 +/- 1.0, and 17.1 +/- 1.1 min, respectively. Control and bacitracin (known aminopeptidase inhibitor) values were 11.8 +/- 1.0 and 31.3 +/- 1.7 min, respectively. These drugs significantly decreased (listed in the same order) LEU degradation initial velocity; Iv (+/- SD) 0.77 +/- 0.2, 0.82 +/- 0.2, 0.92 +/- 0.3, 0.93 +/- 0.2, 0.94 +/- 0.3 pg LEU/min, respectively. Control and bacitracin 1.10 +/- 0.3 and 0.20 +/- 0.1 pg LEU/min, respectively. Values represent results from 5 samples, each obtained by pooling 6 individual plasmas (4 male and 2 female; n = 30 healthy, drug-free volunteers). However, neither the phenothiazines ethopropazine, methotrimeprazine, prochlorperazine and trifluoperazine nor the various commonly used heterocyclic antipsychotics tested, e.g., molindone, loxapine, clozapine, haloperidol, sulpiride and thiothixene inhibited plasma LEU degradation kinetics. Our results failed to show correlations between chemical structure, antipsychotic properties and ability to inhibit plasmatic aminopeptidase LEU degradation. Whereas, presence of the phenothiazine molecule appears to be necessary for enzyme inhibition, only five out of nine substituted phenothiazines tested exhibited this effect. Furthermore, there was a lack of correlation between phenothiazines antipsychotic properties and their capacity to inhibit aminopeptidase activity, a property shown by promethazine (antihistaminic) and As-1397 (selective butyrylcholinesterase inhibitor) but lacking in prochlorperazine and trifluoperazine. Our results provide information which could lead to the rational design of agents capable to modulate the bioavailability of enkephalin and other endogenous aminopeptidase-degraded peptides believed to be involved in the etiology and/or pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.     

Studies on in vitro degradation of anhydroecgonine methyl ester (methylecgonidine) in human plasma

The presence of the cocaine pyrolysis product anhydroecgonine methyl ester (AEME, methylecgonidine) in plasma indicates the smoking of cocaine. The stability of this analyte in human plasma has not been studied. In the present investigation AEME and its hydrolysis product anhydroecgonine (AE, ecgonidine) were assayed in plasma and buffers using gas chromatography-mass spectrometry. It was found that 50% of AEME in human plasma was hydrolyzed to AE within 5 days at room temperature and within 13 days at 4 degrees C. Addition of esterase inhibitors such as sodium fluoride or echothiophate iodide reduced the hydrolysis significantly. Hydrolysis of AEME also occurred in buffers with pH values above 5, but the hydrolysis rate was significantly lower when compared with plasma and could be markedly increased by the addition of butyrylcholine esterase. The data suggest that AEME is degraded via two mechanisms: chemical hydrolysis at basic pH and enzymatic hydrolysis by butyrylcholine esterase. Therefore, proper storage conditions must be provided for the assay of AEME in plasma.     

病毒灭活血浆与新鲜冰冻血浆在血浆置换治疗中的效果分析

目的探讨血浆置换中应用病毒灭活血浆与新鲜冰冻血浆的效果差异。方法2012年1-6月洛阳市26例进行血浆置换患者分别采用新鲜冰冻血浆及病毒灭活血浆治疗,并对两组治疗前后的血凝、蛋白等指标进行比较。结果应用病毒灭活血浆组治疗后患者PT、APTT与治疗前比较差异有统计学意义（P〈0．05）,两组治疗前后清蛋白检测差异无统计学意义（P〉0．05）。结论大量应用血浆制品时,病毒灭活血浆安全性仍较高,但若患者有出血倾向则易选用新鲜冰冻血浆（FFP）做置换液,临床效果更佳。     

新鲜冰冻血浆融化后不同放置时间凝血因子的变化

目的探讨新鲜冰冻血浆融化后24h内凝血因子的变化,为指导临床治疗提供理论依据。方法采用SYSMEX CA-1500型自动血凝分析仪,对20份血浆样品分别于融化后0、6、12、24h测定凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白(FIB)、凝血酶时间(TT)、凝血因子Ⅶ、Ⅷ、Ⅸ(FⅦ、FⅧ、FⅨ)活性水平。结果新鲜血浆融化后24h之内无明显改变的有PT、FIB、TT(P>0.5);其他指标在不同时间段各有明显的改变,同时显示FⅧ半衰期为12～24h。结论新鲜冰冻血浆融化后放置会有凝血因子活性衰减,为保证输血质量,应尽可能融化后立即输注。     

更昔洛韦药物-树脂复合物的体外释药动力学研究

目的制备更昔洛韦药物-树脂复合物并对其体外释药动力学进行考察。方法考察更昔洛韦药物树脂在不同释放介质、不同离子浓度及不同温度下的释药动力学。结果确定更昔洛韦药物树脂体外释药控制在37℃下,以0．1mol·L^-1HCl为释放介质进行释放度考察,其体外释药行为符合Viswannathan方程。结论体外释药动力学研究表明,更昔洛韦药物树脂的释药行为与释放介质中的离子种类及温度有关,离子强度对释药行为的影响不大。     

新鲜冰冻血浆制备时间对冷沉淀凝血因子的质量影响

目的探讨不同制备时间新鲜冰冻血浆对冷沉淀凝血因子(简称冷沉淀)中Ⅷ因子含量(FⅧ)和纤维蛋白原(Fg)含量的影响,以期找到制备冷沉淀凝血因子原料血浆的最佳及最长的制备允许间隔时间。方法以200份的血液样本作为研究对象,分别于全血采集后6、8、13、18h内制备成新鲜冰冻血浆,并标识,将四组不同制备时间段的新鲜冰冻血浆分别制备成冷沉淀凝血因子,检测其FⅧ活性和Fg含量。结果四组不同制备时间段的新鲜冰冻血浆制备成冷沉淀凝血因子FⅧ含量活性随制备时间延长明显下降,组间比较差异有统计学意义(P<0.05),Fg含量比较差异无统计学意义(P>0.05);其中6h合格率为96%,8h合格率为94%,13h合格率为86%,均满足质控要求的75%受控要求,而18h内制备的新鲜冰冻血浆分离出来的冷沉淀凝血因子合格率只有54%,达不到冷沉淀凝血因子的质量要求,6、8、13h合格率与18h比较差异均有统计学意义(P<0.05)。结论为了保证冷沉淀凝血因子的质量及满足临床用血需求量,用于制备冷沉淀凝血因子的新鲜冰冻血浆最佳选择在采集后6~8h内,最长不宜超过13h分离并速冻的新鲜冰冻血浆作为原料浆。     

输注新鲜冰冻血浆对晚期肝癌患者的临床疗效观察

目的探讨输注新鲜冰冻血浆对晚期肝细胞癌患者的临床疗效。方法统计我院2013年1月至2015年1月的晚期肝细胞癌患者共125例,分为血浆组(92例)和对照组(33例)。对照组给予内科常规治疗和介入治疗,血浆组患者在进行内科常规治疗和介入治疗的同时对其进行血浆输注辅助治疗,血浆输注隔天1次,每次200~400mL,持续输注2周,检测2组患者治疗后肝功能、彩色多普勒超声检查腹腔积液深度等指标的变化,同时评估患者焦虑、疼痛评分。结果血浆组患者治疗14d后丙氨酸转氨酸、天冬氨酸转氨酶、总胆红素较对照组均降低(P<0.05),白蛋白、胆碱酯酶、凝血酶原活动度较对照组均明显升高(P<0.05)。Child-Pugh肝功能评级较对照组明显好转(P<0.05),同时患者焦虑程度和疼痛程度得到缓解。结论对晚期肝癌患者输注新鲜冰冻血浆可显著改善患者肝功能及临床体验。     

In vitro detoxification of cyclosarin (GF) by modified cyclodextrins

Developing potent detoxification strategies for prophylaxis and therapy against organophosphate (OP) intoxication still represents a challenging task. Clinical application of numerous investigated substances including enzymes and low molecular scavengers like metal ions or nucleophiles could not yet be realised due to profound disadvantages. Presenting a promising attempt, cyclodextrins (CDs) efficiently enhance the degradation of some organophosphorus compounds. The present study examined the in vitro GF degradation mediated by three CDs and a nucleophilic precursor performed by mass spectrometric detection with ammonia chemical ionisation. All four compounds caused a notable enhancement of GF detoxification that was synergistically accelerated in the case of 2-O-(3-carboxy-4-iodosobenzyl)-β-cyclodextrin (IBA-β-CD) with the alpha-nucleophile 2-iodosobenzoic acid (IBA) grafted on the secondary face of β-cyclodextrin (β-CD). In vitro toxicokinetic investigations of CD derivatives are needed to evaluate the effect of slow terminal elimination phase of the more toxic (-)-GF shown for two CD-derivatives underlining the necessity of detecting the complete kinetic course of inactivation. The observed effect of fast high affinity binding (20-30%) represents an additional therapeutic option of an extremely rapid reduction of GF concentration in vivo. Distinctive differences in the course of reaction are detected depending on β-CD-derivatives, allowing a first inference of possible mechanisms and relevance of attached substituents. However, further profound investigation needs to be done to evaluate the basis of a clinical application of substituted CDs as potential detoxification agents.     

In vitro evaluation of drug release kinetics from liposomes by fractional dialysis

A new dialysis method was designed with the purpose of studying drug release rate from liposomes. The liposomes were diluted directly in the continuous phase and dialysed against a small volume of buffer. The dialysate was changed at 10 min intervals and the amount of carboxyfluorescein released was calculated from a standard curve. To evaluate the new method, parallel measurements were recorded with the traditional carboxyfluorescein assay, which is based on direct measurements on liposomes containing a self-quenching concentration. The new method is called fractional dialysis, and is shown to have sensitivity similar to that of the carboxyfluorescein assay. In certain cases the fractional dialysis method gives a more accurate release profile. It is suggested that fractional dialysis can be a useful alternative to other techniques for recording the in vitro release characteristics of specific drugs from liposomes or other colloidal carriers.     

In vitro protection of plasma cholinesterases by metoclopramide from inhibition by mipafox

Metoclopramide (MCP) is a dopamine receptor antagonist and serotonin receptor agonist widely used as an antiemetic and gastric prokinetic drug. In addition MCP is a reversible inhibitor of cholinesterases from human central nervous system and blood. Metoclopramide may have a cholinesterase protective effect against inhibition by organophosphates. The purpose of the study was to quantify in vitro, by means of the IC(50) shift, the extent of MCP conferred protection, using mipafox (MPFX) as an inhibitor. Mipafox is a neuropathic organophosphate. Cholinesterase activities (with acetylthiocholine [ChE-A] and butyrylthiocholine [ChE-B] as substrates) in human plasma were measured photometrically in the presence of different MPFX concentrations and the IC(50) was calculated. Determinations were repeated in the presence of increasing MCP concentrations. It appears that the shift induced by the presence of MCP increases with the MCP concentration in a linear manner. In the presence of a clinically easily achievable plasma concentration of 1 micro M MCP, the IC(50) of MPFX for cholinesterase 'shifts' by a factor of ca. 3-6. The protective effect of MCP on cholinesterase could be of practical relevance in the treatment of organophosphate poisoning. We conclude that in vivo testing of MCP as an organophosphate protective agent is warranted.     

In vitro protection of plasma cholinesterases by metoclopramide from inhibition by paraoxon

Metoclopramide (MCP) is a dopamine receptor antagonist and serotonine receptor agonist widely used as an antiemetic and gastric prokinetic drug. In addition MCP is a reversible inhibitor of cholinesterases from human central nervous system and blood. MCP may have a cholinesterase protective effect against inhibition by organophosphates. The purpose of the study was to quantify "in vitro" by means of the IC(50)-shift the extent of MCP conferred protection, using paraoxon (POX) as an inhibitor. POX is a widely used organophospate responsible for a large number of accidental or suicidal exposures. Cholinesteratic activities (ChE) (with acetyl-thiocholine (A) and butyryl-thiocholine (B) as substrates) in human plasma were measured photometrically in the presence of different POX concentrations and IC(50) was calculated. Determinations were repeated in the presence of increasing MCP concentrations. It appears that the shift induced by the presence of MCP increases with the MCP concentration in a linear manner. In the presence of a clinically easily achievable plasma concentration of 1 micro M MCP the IC(50) of POX for ChE 'shifts' by a factor of approximately 2-3. The protective effect of metoclopramide on cholinesterases could be of practical relevance in the treatment of paraoxon poisoning. We conclude that in vivo testing of MCP as an organophosphate protective agent is warranted.