阿仑膦酸钠的定量分析方法综述

目的综述阿仑膦酸钠的定量分析方法。方法以国内外有代表性的论文为依据,对阿仑膦酸钠的各种分析方法进行了整理、归纳和总结。结果与结论为广大药师和药物分析者提供了便利,以便更好地进行该类药物的质量控制与研究。     

RP-HPLC法测定阿仑膦酸钠注射液含量

目的建立用于阿仑膦酸钠注射液含量的分析方法。方法采用岛津LC-10Avp高效液相色谱系统,HAMILTON PRP-1不锈钢柱（150mm×4.1mm,5μm）,以乙腈-甲醇-0.05mol.L-1柠檬酸盐/0.05mol.L-1磷酸盐缓冲液（用磷酸调pH8.0）（25∶5∶70）为流动相,流速为1.0mL.min-1,柱温为35℃,检测波长266nm。结果阿仑膦酸钠进样量在0.42~4.20μg范围内,方法的线性关系良好;方法的平均回收率为99.52%;RSD为0.58%。在该色谱条件下,阿仑膦酸钠注射液样品测定不受辅料干扰。结论本法简便、准确、灵敏度高,适用于阿仑膦酸钠注射液的常规检测,为该药的质量控制提供了可靠的分析手段。     

柱前衍生HPLC法测定阿仑膦酸钠骨化三醇片中阿仑膦酸钠的含量

目的建立阿仑膦酸钠骨化三醇片中阿仑膦酸钠的含量测定方法。方法用0.1%FMOC的乙腈溶液进行柱前衍生,色谱柱填料为苯乙烯-二乙烯苯共聚物（Hamilton PRP-1柱适用）;流动相为乙腈-甲醇-0.05mol/L磷酸氢二钠和0.05mol/L枸橼酸钠混合液（用磷酸调pH8.0）（20∶5∶75）;检测波长266nm。结果阿仑膦酸钠（以阿仑膦酸计）在5～15μg/ml的浓度范围内有良好的线性关系（r=0.99998,n=5）;平均回收率为100.8%,RSD=1.1%（n=9）。结论该方法稳定,准确度、精密度高,重复性好,可用于阿仑膦酸钠骨化三醇片中阿仑膦酸钠的含量测定。     

高效液相色谱法测定阿仑膦酸钠片有关物质

目的:建立高效液相色谱法检测阿仑膦酸钠片的有关物质.方法:通过柱前衍生反应,以苯乙烯基-二乙基苯共聚物为填充剂的色谱柱,采用梯度洗脱方法进行检测.结果:通过方法学验证此方法完全可用于检测阿仑膦酸钠片的有关物质.结论:本方法高效、准确、灵敏.     

阿仑膦酸钠与大豆异黄酮联合治疗骨质疏松30例

目的 比较阿仑膦酸钠与大豆异黄酮联合用药与单纯使用阿仑膦酸钠对骨质疏松的疗效.方法 将60例骨质疏松患者随机均分为两组,治疗组30例口服阿仑膦酸钠和大豆异黄酮片,对照组30例口服阿仑膦酸钠和安慰剂,用药6个月后对两组患者进行疼痛改善的临床疗效评定并检测骨密度.结果 两纽患者的疼痛程度均有改善,骨密度也明显升高,但治疗组骨密度提高更明显.结论 阿仑膦酸钠与大豆异黄酮联合使用对骨质疏松的治疗效果比阿仑膦酸钠单独使用更好.     

衍生化高效液相色谱-紫外检测法测定阿仑膦酸钠和电喷雾离子阱质谱法分析其衍生物

目的:以9-氯甲酸芴甲酯(FMOC-Cl)为柱前衍生化试剂,采用电喷雾离子阱质谱(ESI-IT-MS)技术对其衍生化物进行定性鉴别及高效液相色谱-紫外检测法测定阿仑膦酸钠片的含量。方法:阿仑膦酸钠与FMOC-Cl衍生化反应后,采用LinkSil C₁₈色谱柱(5.0 μm,250 mm×4.6 mm),乙腈-甲醇-0.4%乙二胺四乙酸(用氢氧化钠调pH 7.16)(25:5:70)为流动相,测定阿仑膦酸钠的含量,并用ESI-MS获得质谱图对其衍生化产物的主要碎片进行分析。结果:在负离子模式下,阿仑膦酸钠的衍生化产物主要碎片离子为m/z 470、426、408、366、344和326。阿仑膦酸钠质量浓度在1~40 mg·L^-1范围内线性关系良好,平均回收率为101.2%,最低检测限为0.2 mg·L^-1。结论:该HPLC-UV柱前衍生法样品处理简单,测定灵敏,准确度高,可以用来测定阿仑膦酸钠片剂含量。ESI-IT-MS分析为进一步对阿仑膦酸钠的体内代谢过程与结构修饰研究提供了理论指导和实验依据。     

伊班膦酸钠输注液和阿仑膦酸钠片治疗老年性骨质疏松症的疗效-成本分析

目的：探究分析伊班膦酸钠注射液和阿仑膦酸钠片治疗老年骨质疏松的疗效,并对其疗效-成本进行分析。方法：将我院自2013年12月～2014年12月期间收治的200例老年性骨质疏松症患者随机分为两组：伊班膦酸钠组和阿仑膦酸钠组,各100例。结果：治疗1个疗程后,伊班膦酸钠组患者腰椎骨密度较治疗前增加7.57%,阿仑膦酸钠组患者较治疗前增加5.39%,伊班膦酸钠注射液产生单位骨密度增高效果的成本为630.85元,阿仑膦酸钠片的成本为734.17元。结论：据药物经济学分析,阿仑膦酸钠片更适于治疗老年性骨质疏松症。     

阿仑膦酸钠肠溶片和普通片的人体尿药排泄动力学比较

目的:建立人尿液中阿仑膦酸钠的芴甲氧羰酰氯(Fmoc-Cl)衍生化HPLC-荧光检测法,用于阿仑膦酸钠肠溶片和普通片的人体药代动力学比较研究。方法:20名男性健康志愿者分别给药70 mg阿仑膦酸钠肠溶片或普通片,采集0～24 h内不同时间段的尿液样本,帕米膦酸钠内标法测定受试者尿液阿仑膦酸钠浓度并计算排泄量,DAS 2.1软件计算尿液排泄动力学参数。结果:阿仑膦酸钠浓度在1～500 ng.mL-1范围响应与浓度呈良好的线性关系(r>0.990 0)。批内及批间精密度和准确度均符合要求。阿仑膦酸钠普通片和肠溶片的尿药累积排泄量分别是(466±261)和(306±234)μg,最大排泄速率分别是(190±118)和(86±57)μg.h-1。结论:建立的测定法专属耐用灵敏度适宜,阿仑膦酸钠肠溶片与普通片剂相比,药代动力学行为发生明显改变。     

高效液相色谱法测定阿仑膦酸钠的尿药浓度

目的建立阿仑膦酸钠尿药浓度的高效液相色谱-荧光检测法。方法采用Shiseido Capcell Pak C18(150 mm×4.6 mm,5μm)色谱柱;梯度洗脱,流动相0.01 mol.L-1磷酸氢二钠(A)-乙腈(B);流速为1.0 mL.min-1;柱温35℃;内标物:帕米膦酸钠;荧光检测:激发波长为260 nm,发射波长为310 nm。结果阿仑膦酸钠峰与内标物峰面积比值在20～1 000μg.L-1呈良好的线性关系(r=0.996 1)。方法准确度与日内及日间RSD均符合要求,定量下限为20μg.L-1,提取回收率稳定,成功应用于4名男性健康受试者体内阿仑膦酸钠尿药浓度的测定。结论该法为临床研究阿仑膦酸钠体内尿药浓度提供了方法。     

阿仑膦酸钠片不良反应26例分析

目的 探讨阿仑膦酸钠片所致不良反应的类型与特点.方法 对这一年来我院老年病区应用阿仑膦酸钠片出现的不良反应,进行类型归纳和分析.结果 阿仑膦酸钠片的不良反应临床表现大致有4类,以胃肠道反应最为常见.结论 阿仑膦酸钠片耐受性良好,不良反应低,是一种安全可靠的药品.     

Tetrodotoxin alleviates acute heroin withdrawal syndrome: a multicentre, randomized, double-blind, placebo-controlled study

1. Tetrodotoxin (TTX) is a powerful sodium channel blocker extracted from the puffer fish. The efficacy and safety of TTX as monotherapy for the treatment of acute heroin withdrawal syndrome were evaluated in the present study. This 7-day, multicentre, randomized, double-blind, placebo-controlled study was carried out between December 2008 and October 2009. In total, 216 patients who met the Diagnostic and Statistical Manual of Mental Disorders IV diagnosis of heroin addiction were recruited. After providing written informed consent, subjects were randomly assigned to double-blind treatment in one of the following groups: 5 μg TTX group (group 1), 10 μg TTX group (group 2) or the placebo group (group 3). 2. Evidence suggests that both 5 and 10 μg TTX significantly reduced withdrawal symptoms by day 3 compared with placebo, and there was no significant difference in the incidence of adverse events in the three groups. 3. In conclusion, this clinical trial shows that TTX (5 and 10 μg given t.i.d.) is effective in alleviating opiate withdrawal symptoms with few side-effects.     

极化型与去极化型心脏停搏液对离体大鼠心肌细胞胞质游离钙离子含量的影响

目的观察极化型心脏停搏液(TTX组)与传统的去极化型停搏液(St.Thomas-2号液,STH-2)对急性分离的大鼠心肌细胞胞质静息游离钙离子含量的影响。方法45只健康Wistar大鼠,随机分为正常对照组、STH-2组和TTX组。采用急性酶学分离的方法获取成熟大鼠心室肌细胞,持续灌流给药,利用离子成像技术,观察不同配方的心脏停搏液对于急性分离的大鼠心肌细胞胞质游离钙离子浓度的影响。结果①STH-2组和TTX组细胞质内游离钙离子含量均较灌流前后升高(P<0.05);②STH-2组恢复正常台式液灌流后15min与灌流前比较差异有统计学意义,而TTX组灌流前后的细胞内钙离子含量差异无统计学意义。结论与传统的STH-2号液相比,采用加有钠离子通道选择性的拮抗剂TTX的极化型心脏停搏液可以减少心肌细胞在停搏液灌流过程中的胞内钙离子含量,减轻钙超载,因而具有较好的心肌细胞保护作用。     

Transfer profile of intramuscularly administered tetrodotoxin to non-toxic cultured specimens of the pufferfish Takifugu rubripes

Tetrodotoxin (TTX) was intramuscularly administered to non-toxic cultured specimens of the pufferfish Takifugu rubripes to investigate TTX transfer/accumulation profiles in the pufferfish body. In two groups of test fish administered either 50 MU/individual of TTX standard (purified TTX; PTTX) or crude extract of toxic pufferfish ovary (crude TTX; CTTX), TTX rapidly transferred from the muscle via the blood to other organs. The toxin transfer profiles differed between groups, however, from 4 to 72 h. In the PTTX group, little TTX was retained in the liver, and most (>96%) of the toxin remaining in the body transferred/accumulated in the skin after 12 h, whereas in the CTTX group, a considerable amount of toxin (15%-23% of the administered toxin or 28%-58% of the remaining toxin) was transferred/retained in the liver for up to 24 h, despite the fact that 89% of the remaining toxin transferred/accumulated in the skin at the end of rearing period (168 h). The total amount of toxin remaining in the entire body at 1-4 h was approximately 60% of the administered toxin in both groups, which decreased at 8-12 h, and then increased again to approximately 60%-80% at 24-168 h. Immunohistochemical observation revealed that the toxin accumulated in the skin was localized at the basal cells of the epidermal layer.     

河豚毒素对缺血再灌注大鼠心肌细胞凋亡及相关蛋白表达的影响

目的研究河豚毒素（TTX）心脏停搏液对缺血再灌注大鼠心肌细胞凋亡及凋亡相关蛋白表达的影响。方法取Wistar大鼠24只,建立Langendorff-Neely离体心脏灌注模型,随机分为3组（n=8）：基础组、STH-2组、TTX组。用TUNEL法检测心肌细胞凋亡指数,免疫组化测定凋亡相关蛋白Bcl-2、Bax、p53的表达,Westernblot检测Bcl-2蛋白表达。结果STH-2组凋亡指数（AI）（11.20±0.79）%高于TTX组（6.92±1.10）%和基础组（1.34±0.23）%（P〈0.01）。TTX组Bcl-2的PEI为（22.63±1.10）%高于STH-2组（13.51±0.67）%和基础组（2.52±0.58）%（P〈0.01）;STH-2组Bax的PEI为（8.04±0.57）%高于TTX组（6.05±0.46）%和基础组（2.14±0.57）%（P〈0.01）。TTX组Bcl-2/Bax为3.78±0.41高于STH-2组1.75±0.15（P〈0.01）。TTX组Bcl-2的相对蛋白含量为（86.1±0.98）%高于STH-2组（53.2±1.76）%和基础组（23.7±2.61）%（P〈0.01）。p53蛋白在各组均未见明显阳性表达。结论与STH-2比较,TTX能减少缺血再灌注大鼠心肌细胞凋亡数,其抗凋亡的机制可能与上调Bcl-2表达,下调Bax表达,提高Bcl-2/Bax比值有关。     

Human ScFv that block sodium ion channel activity of tetrodotoxin

Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C₁₁H₁₇N₃O₈) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.     

Toxin-neutralizing effect and activity-quality relationship for mice tetrodotoxin-specific polyclonal antibodies

The polyclonal antibodies specific for tetrodotoxin (TTX) were prepared from mice and their capacity of neutralizing TTX was investigated so as to explore the possibility of developing TTX antitoxin. Haptenic TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH) chemically to form artificial antigen TTX-TTH. BALB/c mice were immunized with TTX-TTH and ascites were induced by intraperitoneal administration of Freund's adjuvant. Twenty strains of TTX-specific ascites antibody with apparent affinity varying from 10(-4) to 10(-7)M were obtained. KM mice were challenged with lethal doses (1LD = 14.0 microg/kg, i.p.) of TTX neutralized by antibodies to evaluate the power of antitoxin. The potential of TTX-neutralizing of the antibodies was approved by the increase in survival animal challenged by lethal doses of TTX pre-incubated in vitro or neutralized in vivo with TTX specific antibodies. The highest protection was observed with all animals survived challenge of 1.5 x LD TTX neutralized in vitro, and antibody administration 4 days prior to 1.3 x LD TTX challenge in vivo neutralization. The protective efficiency was antibody quality factor dependent and with the highest detoxifying immunological equivalent as high as 1 300 microg (TTX)/L(ascites) approximately, while the antibody apparent affinity being at the order of 10(-6) to 10(-7)M. These results suggested that chemical vaccine for haptenic TTX could successfully raise high humoral immune response and the antibodies could neutralize TTX effectively both in vitro and in vivo, antibody therapy would be the hopeful means for detoxification of TTX.     

Tetrodotoxin levels of the rough-skin newt, Taricha granulosa, increase in long-term captivity.

We investigated the persistence of the neurotoxin tetrodotoxin (TTX) in individual captive newts (Taricha granulosa) from the Willamette Valley of Oregon using a non-lethal sampling technique. We found that the TTX levels of newts held in the laboratory for 1 yr increased. TTX stereoisomer-analog profiles were not affected by captive husbandry. Levels of TTX were high in newts from our study population and we observed substantial within population variation in quantitative levels of TTX. Females possessed more TTX than males, but the response of TTX levels to captivity did not differ between females and males. The stability of TTX toxicity in newts is consistent with other amphibian species where TTX is present and may indicate that exogenous factors play a less important role in TTX toxicity of newts than previously thought.     

Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients.

Immunoaffinity chromatography using the monoclonal antibody (Tl-1) specific for tetrodotoxin (TTX) has been developed for isolating TTX from urine samples. By combining immunoaffinity chromatography with fluorometric high performance liquid chromatography (HPLC), it has become possible to detect a small amount of TTX in urine samples. The detection limit of TTX in urine was 2 ng/ml. By this combined method, TTX was detected in all the urine samples that were collected from poisoned patients during the week following TTX ingestion. The combination of immunoaffinity chromatography with HPLC was very useful in detecting TTX from the urine samples of poisoned patients for diagnosis of TTX-food poisoning.     

Low concentrations of tetrodotoxin interact with tetrodotoxin-resistant voltage-gated sodium channels

BACKGROUND AND PURPOSE: Tetrodotoxin (TTX) is used to distinguish between two classes of voltage-gated sodium channel (VGSC)--TTX sensitive (TTXS) and TTX resistant (TTXR). The resistance of TTXR VGSCs is thought to result from a low binding affinity of TTX, although at high TTX concentrations channel block does occur. Here, we show that, at concentrations below those which produce block, TTX can bind to TTXR VGSCs. EXPERIMENTAL APPROACH: Whole-cell voltage clamp recordings were made from dissociated rat dorsal root ganglion neurones that expressed both TTXS and TTXR sodium currents. Voltage-gated calcium currents were blocked by 10 microM extracellular lanthanum chloride. TTXS, but not TTXR, current was suppressed by using a holding potential of -50 mV, and the effect of TTX on the isolated TTXR current was explored. KEY RESULTS: Extracellular application of 0.5 microM TTX produced a 40% increase in TTXR current amplitude, a negative shift in the voltage-dependence of current activation (approximately -8 mV) and inactivation (approximately -10 mV) and increased rates of current activation and inactivation. The effect of TTX on current amplitude was dose-dependent (EC50 = 364 nM). Removal of lanthanum prevented the effect of TTX on TTXR current amplitude, whereas reducing extracellular calcium did not. CONCLUSIONS AND IMPLICATIONS: The findings are consistent with an interpretation that TTX relieves a tonic block of the TTXR VGSC by lanthanum. We conclude that TTX binds to the TTXR VGSC at low concentrations, without blocking it. This appears to be the first demonstration of a clear distinction between binding affinity and blocking potency of a channel-blocking agent.     

Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus).

The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6–4.0 MU of TTX. Higher neutralizing activity (7.2–12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish posions was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.