高效液相色谱法同时测定动物血浆中萘哌地尔及其主要代谢产物浓度

为研究大鼠口服萘哌地尔的药代动力学规律,建立了同时测定萘哌地尔及其去甲基代谢产物浓度的高效液相色谱法。以甲醇、乙腈以及0.02mol·L^-1磷酸二氢钾溶液为流动相,RP-C₁₈为固定相,普萘洛尔作内标,紫外定量波长240nm。此条件下内标物、去甲基物和原药的保留时间分别为6.2,7.6和10.3min;绝对回收率分别为97.4%,86.6%和86.1%。去甲基物和原药在10～800ng·ml^-1血浆浓度范围内线性关系良好,γ=0.9998,最低检测浓度分别为8ng·ml^-1和5ng·ml^-1;日内和日间的相对标准偏差(RSD)均分别小于6.3%和11.6%(n=5)。并用此法研究了大鼠单次口服萘哌地尔后两者的药时规律。     

液相色谱-电喷雾串联质谱法测定人血浆中班布特罗:在药代动力学研究中的应用

目的　建立液相色谱 串联质谱法测定人血浆中班布特罗浓度,研究中国受试者口服该药的动力学特点。方法　血浆样品经液 液萃取后,采用液相色谱电喷雾串联质谱法以选择离子反应监测(SRM)方式进行检测。结果班布特罗的线性范围为0.05 - 4.0ng·mL^-1 ,最低定量浓度为0.05ng·mL^-1 ,该法的日内及日间精密度(RSD)小于8% ,准确度(RE)在±9%范围内。18名中国健康受试者单剂量口服班布特罗10 mg后,主要药动学参数T_max,C_max, T_1/2和AUC_0-t分别为(2.3±1.3)h ,(3.95±2.20 )ng·mL^-1 ,(11.4±6.1)h和(26.85±11.77)ng·h·mL^-1 。结论　该法灵敏度高,操作简便、快速,适用于临床药代动力学研究     

高效液相色谱法测定大鼠血浆中伊立替康及其活性代谢产物SN-38的含量

目的:建立同时测定大鼠血浆中伊立替康(CPT-11)及其活性代谢产物7-乙基-10-羟基喜树碱(SN-38)浓度的高效液相色谱法。方法:以10-羟基喜树碱作为内标,先用7%高氯酸酸化血浆,再用7%高氯酸-乙腈(50∶50)沉淀蛋白。采用Hypersil C18色谱柱(4.6 mm×250 mm,5μm)进行分离;以0.05 mol·L-1的磷酸氢二钠-甲醇-三乙胺(50∶50∶0.025,磷酸调pH 3.0)为流动相;荧光检测波长:激发波长380 nm,发射波长550 nm。结果:大鼠血浆中CPT-11和SN-38线性范围分别是20～5000 ng·mL-1(r=0.9997)和2～500 ng·mL-1(r=0.9999)。两组分最低检出限分别为15 ng·mL-1和1.7 ng·mL-1。2组分平均相对回收率分别是98.7%和99.9%;平均绝对回收率分别87.2%和94.7%。2组分日内、日间精密度均小于12%。结论:本方法快速、简便、准确,灵敏度高,可用于CPT-11及其活性代谢产物SN-38药代动力学的研究。     

手性和非手性联用色谱法研究尼卡地平对映异构体兔体内过程的差异性

目的研究尼卡地平(NCD)对映体在家兔体内药代动力学和组织分布的差异性。方法生物样品在碱性条件下,正己烷-醋酸乙酯(1:1)提取,用手性和非手性联用色谱法进行分离分析。结果尼卡地平及其对映体分别在反相色谱系统及手性色谱系统中分离良好,浓度为55-550ng·mL^-1线性关系良好。对映体的平均日内、日间RSD分别为5.25%和8.97%,回收率分别为99.99%和97.10%;对映体间主要动力学参数T_max,C_max和AUC,S-NCD为(2.49±0.03)h,(134±2)ng·mL^-1和(1082±32)ng·mL^-1·h,R-NCD为(1.24±0.05)h,(109±2)ng·mL^-1和(778±22)ng·mL^-1·h;在主要脏器和细胞中S-NCD的浓度明显高于R-NCD。药代动力学和靶组织分布均有一定差异性。结论尼卡地平对映体兔体内过程包括代谢动力学和靶细胞浓度分布存在着立体差异性。     

高效液相色谱法测定人血清和尿中盐酸青藤碱浓度及药代动力学研究

用RP-HPLC法,以三唑仑为内标,反相C₁₈为分析柱,乙腈—0.01mol·L^-1磷酸二氢钠—四甲基乙二胺(46∶54∶0.22v/v)为流动相,磷酸调至pH6.9,检测波长263nm,测定血清和尿中盐酸青藤碱浓度,线性范围分别为6～480ng·mL^-1和0.06～3μg·mL^-1,平均回收率75.88%和91.35%,日内日间误差小于5%,最低检测浓度血清4ng·mL^-1,尿40ng·mL^-1。8名健康男性志愿者单次口服盐酸青藤碱片80mg,测定血清及尿浓度,该药符合二室开放模型,体内消除符合一级动力学消除过程,主要药代动力学参数:T_1/2α0.791±0.491h,T_1/2β9.397±2.425h,T_{max 1.040±0.274h,Cmax246.604±71.165ng·mL^-1,AUC 2651.158±1039.050ng·h·mL^-1,CL 0.033±0.01ng·mL^-1。}     

UPLC-MS/MS测定血浆中伊伐布雷定及其活性代谢产物的含量

目的 建立超高效液相色谱-串联质谱法测定人血浆中伊伐布雷定及其活性代谢产物(N-去甲伊伐布雷定)的含量。方法 选用Waters Acquity BEH C₁₈(50 mm×2.1 mm,1.7 μm)色谱柱,流动相为0.1%甲酸水溶液(A)-乙腈(B),梯度洗脱;流速为0.4 mL·min^-1;电喷雾离子源,多反应监测。伊伐布雷定：[M+H]⁺,m/z 469.3→177.2,N-去甲伊伐布雷定：[M+H]⁺,m/z 455.2→262.2,卡马西平：[M+H]⁺,m/z 237.1→194.2。结果 伊伐布雷定线性范围为0.2~100 ng·mL^-1(r=0.998 1),N-去甲伊伐布雷定线性范围为0.05~25 ng·mL^-1(r=0.993 1);两者日间、日内精密度均<15%,方法回收率>90%,稳定性较好。结论 该方法快速、灵敏、重复性好,适用于血浆中伊伐布雷定及其代谢产物含量测定。     

非那雄胺片的健康人体药动学研究

目的建立测定人体血浆中非那雄胺的HPLC-MS方法,并应用于健康志愿者体内药物动力学研究。方法18名健康志愿者单剂量口服非那雄胺片5mg,不同时间抽取静脉血,以建立的方法测定血药浓度。血样经乙腈沉淀蛋白后取上清液进样,乙腈-10mmol/L醋酸铵(含0.08%甲酸)(65∶35)为流动相,Nuc leodur C₁₈柱分离后进入质谱,采用电喷雾电离源正离子模式(ESI⁺)选择性离子监测(SIR)准分子离子峰,并用3P87软件估算药物动力学参数。结果非那雄胺浓度在1.53～306.70ng·mL^-1范围线性良好(r=0.999 8),日内日间精密度均小于5%,回收率大于95%。主要药物动力学参数t_max、C_max、AUC_0～24分别为(1.59±0.47)h,(47.16±14.78)ng·mL^-1,(319.29±122.52)ng·h·mL^-1。结论非那雄胺的体内过程符合二室开放模型,药物代谢个体差异较大。     

UPLC-MS/MS测定大鼠血浆中8-O-乙酰山栀苷甲酯浓度

目的 建立超高效液相串联质谱法快速测定大鼠体内8-O-乙酰山栀苷甲酯的浓度。方法 用乙腈沉淀蛋白方法处理血浆,色谱柱为ACQUITY UPLC BEH C₁₈柱(50 mm×2.1 mm,1.7 μm);流动相为乙腈-0.1%甲酸水,梯度洗脱,流速为0.4 mL·min^-1;用正离子多离子反应监测(MRM)扫描,内标为槲皮素。结果 血浆中8-O-乙酰山栀苷甲酯的线性范围为2.5~500 ng·mL^-1(r=0.997 9),最低定量限为0.5 ng·mL^-1。低、中、高3个浓度(3, 45和450 ng·mL^-1)的质控样品的日内、日间精密度RSD均<10%,3个浓度的相对回收率分别为(103.59±4.75)%, (98.68±4.62)%和(97.06±5.64)%。结论 该方法操作简便、快捷,灵敏度高,适于大鼠血浆中8-O-乙酰山栀苷甲酯浓度的测定及其药代动力学研究。     

高灵敏度HPLC测定大鼠血浆中益母草碱浓度及其药动学研究

目的 建立一种高灵敏度HPLC测定大鼠血浆中益母草碱浓度,并研究益母草碱在大鼠体内的药动学特征。方法 大鼠口服益母草碱混悬溶液（50 mg·kg^-1）后,不同时间点尾静脉采血,以苯甲酰精氨酸乙酯为内标,血浆样品经酸化后乙酸乙酯萃取,采用HPLC进行测定。色谱条件：采用Diamonsil C₁₈（250 mm×4.6 mm,5 μm）为色谱柱,以乙腈-0.02 mol·L^-1磷酸二氢钾缓冲溶液（pH 3.0）（22：78）为流动相,流速1.0 mL·min^-1,柱温35℃,检测波长277 nm。并利用PKS 1.0软件计算药动学参数。结果 益母草碱血浆浓度在0.05~1.5 μg·mL^-1内线性关系良好（r=0.999 1）。方法的定量下限（LLOQ）为0.05 μg·mL^-1（RSD=12.8%,n=5）;提取回收率为76.5%~82.5%;批内、批间准确度为96.9%~104.9%;日内、日间精密度均<10%;质控样品经反复冻融3次及-20℃放置1个月后均较稳定。大鼠口服益母草碱后,药-时曲线符合二室开放模型,主要药动学参数为t_max=0.95 h,C_max=0.51 μg·mL^-1,t_1/2=3.64 h,AUC_0-t=1.56 μg·mL^-1·h^-1,AUC_0-∞=1.78 μg·mL^-1·h^-1。结论 该方法准确度、灵敏度高,重复性好,可用于生物样品中益母草碱浓度的测定。     

小鼠血浆中马钱素的高效液相色谱测定法及药代动力学

目的建立测定小鼠血浆中马钱素浓度的高效液相色谱法,并研究马钱素在小鼠体内的药代动力学。方法色谱柱为C18柱,流动相甲醇-水(30∶70),流速为0.8 mL·min^-1,检测波长240 nm;血浆样品用固相萃取法预处理。结果线性范围0.01～5.00 μg·mL^-1。日内RSD<10%,日间RSD<15%,回收率86.0%～91.5%,最低定量浓度为10 ng·mL^-1。ig给药0.5 h后血浆药物浓度达峰值,C_max为6.8 μg·mL^-1,_t1/2α为26.1 min,_t1/2β为29.01 min。结论 该方法灵敏度高,操作方便,适用于马钱素的药代动力学研究;本品口服吸收快消除也快。     

沙利度胺对伊立替康及其代谢物SN-38在大鼠体内药动学的影响

目的:研究伊立替康合用沙利度胺后伊立替康及其代谢物SN-38在大鼠体内的药动学情况。方法:以健康♂SD大鼠为受试对象,随机分成伊立替康注射液10、20mg·kg-1单用组(对照组)及与沙利度胺(20mg·kg-1)合用组(实验组),给药后0.083、0.5、1.0、2.0、4.0、6.0、8.0、10、12h采集血样并测定伊立替康、SN-38血药浓度,采用DASver2.0软件拟合二者的药动学参数。结果:与对照组比较,伊立替康10mg·kg-1实验组伊立替康AUC0～t、Cmax显著增加(P<0.05),SN-38的AUC0～t降低(P<0.05);20mg·kg-1实验组伊立替康Cmax显著增加(P<0.05),SN-38的Cmax、AUC0～t显著降低(P<0.05)。结论:联用沙利度胺可以增加伊立替康AUC0～t同时减少SN-38的AUC0～t及Cmax。     

甲氧基聚乙二醇-聚己内酯共聚物载药7-乙基-10-羟基喜树碱纳米粒的制备及药动学参数的测定

目的：制备甲氧基聚乙二醇-聚己内酯（MePEG-PCL）共聚物载药7-乙基-10-羟基喜树碱（SN-38）纳米粒,并测定其在大鼠体内的药动学参数。方法：采用薄膜分散法制备MePEG-PCL共聚物载药SN-38纳米粒。将SD大鼠随机分为两组,分别静脉注射SN．38纳米粒及SN-38溶液制剂（作为对照）,采用HPLC法测定两种制剂给药后0．17、0．5、1、2、3、5、7、9、12小时的血药浓度,并用3p97药动学软件处理数据,计算药动学参数。结果：制得的MePEG-PCL共聚物载药SN-38纳米粒粒径为115nm,包封率大于90％,纳米粒分散液中SN-38质量浓度约为0．2g·L^-1。SN．38纳米粒和SN．38溶液制剂的代谢半衰期（t1/2β）分别为（8．8±1．4）和（1．4±0．3）h;AUC分别为（3．1±0．8）和（1．9±0．5）g·h·L^-1。结论：MePEG-PCL嵌段共聚物能够有效包载SN-38。与SN-38溶液制剂相比,MePEG-PCL共聚物载药SN．38纳米粒代谢半衰期更长,AUC更大,表明其具有长循环作用。     

Investigation into the Efficacy of Val-SN-38, a Valine-Ester Prodrug of the Anti-Cancer Agent SN-38

We recently reported that Val-SN-38, a novel valine ester prodrug of SN-38, had greatly improved the intracellular accumulation of SN-38 in MCF-7 cell line, probably through enhanced uptake via amino acid transporters. In the present study, the efficacy of Val-SN-38 was further investigated both in vitro and in vivo. It was found that the in vitro cytotoxic effect of Val-SN-38 was similar to that of SN-38. Moreover, Val-SN-38 exhibited an equal potency to that of SN-38 in survival experiments in vivo. Because these results seemed to be contrary to the previous finding, further investigation was performed to find out the underlying cause of the contradiction. As only the lactone form is known to have cytotoxic activity, the proportion of lactone in Val-SN-38 and SN-38 was determined, but no differences were found. However, it turned out that Val-SN-38 had poor stability compared with SN-38, which resulted in a decrease in beneficial efficacy for Val-SN-38. Overall, the present study showed that a valine-added prodrug approach could be advantageous provided that the stability of the compound can be ensured. We believe this is a noteworthy study that unravels the discrepancy between intracellular accumulation and efficacy of valine-added prodrug.     

Development and characterization of a novel liposome-based formulation of SN-38

SN-38, 7-ethyl-10-hydroxycamptothecin, is the active metabolite of Irinotecan (CPT-11), a topoisomerase I inhibitor commercially available as Camptosar^®. SN-38 is approximately 200–2000-fold more cytotoxic than CPT-11. Despite its promising anticancer potential, SN-38 thus far has not been used as an anticancer drug due to its poor solubility in any pharmaceutically acceptable solvents. In addition, SN-38 has low affinity to lipid membranes; it tends to precipitate in aqueous phase resulting in a very low drug-to-liposome entrapment. SN-38 also reversibly converts to an inactive open lactone ring structure at physiological pH. We have developed a novel, liposome-based SN-38 formulation (LE-SN-38). The formulation contains liposomes of uniform size distribution (<200 nm), and it is easy-to-use. Drug entrapment efficiency of the formulation is>95%. Long-term stability studies indicate that the lyophilized LE-SN-38 is physically and chemically stable for at least 6 months at 2–8 °C. In preclinical studies, LE-SN38 has shown promising results in terms of increased cytotoxicity against various tumor cell lines and better therapeutic efficacy towards xenograft mouse models compared to CPT-11.     

Convection-enhanced delivery of SN-38-loaded polymeric micelles (NK012) enables consistent distribution of SN-38 and is effective against rodent intracranial brain tumor models

Convection-enhanced delivery (CED) of therapeutic agents is a promising local delivery technique that has been extensively studied as a treatment for CNS diseases over the last two decades. One continuing challenge of CED is accurate and consistent delivery of the agents to the target. The present study focused on a new type of therapeutic agent, NK012, a novel SN-38-loaded polymeric micelle. Local delivery profiles of NK012 and SN-38 were studied using rodent brain and intracranial rodent brain tumor models. First, the cytotoxicity of NK012 against glioma cell lines was determined in vitro. Proliferations of glioma cells were significantly reduced after exposure to NK012. Then, the distribution and local toxicity after CED delivery of NK012 and SN-38 were evaluated in vivo. Volume of distribution of NK012 after CED was much larger than that of SN-38. Histological examination revealed minimum brain tissue damage in rat brains after delivery of 40?µg NK012 but severe damage with SN-38 at the same dose. Subsequently, the efficacy of NK012 delivered via CED was tested in 9L and U87MG rodent orthotopic brain tumor models. CED of NK012 displayed excellent efficacy in the 9L and U87MG orthotopic brain tumor models. Furthermore, NK012 and gadolinium diamide were co-delivered via CED to monitor the NK012 distribution using MRI. Volume of NK012 distribution evaluated by histology and MRI showed excellent agreement. CED of NK012 represents an effective treatment option for malignant gliomas. MRI-guided CED of NK012 has potential for clinical application.     

人血浆中伊立替康及其活性代谢物7-乙基-10羟基喜树碱浓度高效液相色谱-荧光测定方法的建立

目的:建立人血浆中伊立替康(CPT-11)及其代谢物7-乙基-10羟基喜树碱(SN-38)的浓度测定方法并进行方法学考证。方法:用Luna 5u CN100A(4.6 mm×150 mm,5μm)色谱柱,乙腈与醋酸铵缓冲溶液(50 mmol.L-1,pH4)为流动相梯度洗脱,CPT-11的检测波长为Ex/Em=368 nm/432 nm,SN-38的检测波长为Ex/Em=368 nm/535 nm。结果:CPT-11保留时间为(9.3±0.3)min,SN-38保留时间为(4.8±0.3)min。空白样品在CPT-11、SN-38及内标喜树碱出峰位置均无干扰。CPT-11在46.9～6 000.0nmol.L-1的范围内线性良好,SN-38在2.0～250.0nmol.L-1的范围内线性良好,r值均为0.998。低浓度点RSD均在20%内,其余浓度点的RSD均在15%内,准确度均在85%～115%之间。血浆样品长期冻存稳定性良好,反复冻融3次及提取后室温放置24 h条件下,样品浓度均无显著变化。结论:使用高效液相色谱-荧光检测方法简便,准确,灵敏,适用于伊立替康及其活性代谢物SN-38的血药浓度检测。     

Effect of erythropoietin, 5-fluorouracil and SN-38 on the growth of DLD-1 cells

Supplementation of recombinant human erythropoietin (rHuEpo) is one of the methods for the treatment of anemia. The influence of rHuEpo on proliferation or clonogenic growth of cancer cells is not clear and some of the published results are conflicting. The aim of this work was to study the effect of rHuEpo on colon cancer cells when given alone or in combination with cytostatics. Human colon adenocarcinoma cells (DLD-1) were cultured in medium with rHuEpo, 5-fluorouracil (5-FU) and an active metabolite of irinotecan (SN-38). Cell viability was determined using a hematocytometer and 0.4% (w/v) trypan blue dye. Cell proliferation was measured by the MTT assay. Expression of EpoR, Bax, Bcl-2 and Akt1 protein was assessed by Western blot. The results of this study indicate a dose-dependent inhibitory effect of rHuEpo on DLD-1 cell growth and proliferation. Moreover, the combined treatment of rHuEpo and cytotoxic agents such as 5-FU and SN-38 increases the antitumor action, which is indicated by decreases in proliferation in the MTT test, cell numbers and DNA synthesis. We found a significant increase in EpoR, Bcl-2 and Akt1 protein expression in all cells grown in medium containing 3 IU of rHuEpo. We observed that EpoR is constitutively expressed in DLD-1 cells. Our results indicate that rHuEpo acts via EpoR to directly inhibit DLD-1 cell growth and indirectly modulate the cytostatics effects of 5-FU and SN-38.     

SN-38 active loading in poly(lactic-co-glycolic acid) nanoparticles and assessment of their anticancer properties on COLO-205 human colon adenocarcinoma cells

Abstract

SN-38 is a highly effective drug against many cancers. The development of an optimal delivery system for SN-38 is extremely challenging due to its low solubility and labile lactone ring. Herein, SN-38 encapsulated in poly(d,l-lactide-co-glycolide) nanoparticles (NPs) is introduced to enhance its solubility, stability and cellular uptake. SN-38-loaded NPs prepared by spontaneous emulsification solvent diffusion (SESD) method had an average diameter of 310?nm, a zeta potential of ?9.69?mV and a loading efficiency of 71%. They were able to protect the active lactone ring of SN-38 against inactivation under physiological condition. A colorectal adenocarcinoma cell line (COLO-205) was used to assess the NPs effects on cytotoxicity and cellular uptake. Result showed a significant decreased cell proliferation and cell apoptosis. These results suggest that these SN-38-loaded NPs can be an effective delivery system for the treatment of colon cancer and potentially for other types of cancers.     

Potential Oral Delivery of 7-Ethyl-10-Hydroxy-Camptothecin (SN-38) using Poly(amidoamine) Dendrimers

Purpose  To investigate potential application of poly(amidoamine) (PAMAM) dendrimers for improving the delivery of SN-38. Methods  Complexes of SN-38 with generation 4 amine terminated PAMAM dendrimers were synthesized with varying amounts of drug. Stability of the complexes as well as influence of complexation on permeability across and cellular uptake by Caco-2 cells was evaluated. Results  The complexes were stable at pH 7.4 and drug was released at pH 5. A tenfold increase in permeability and more than hundredfold increase in cellular uptake of the complexes with respect to free SN-38 was observed. Conclusions  Studies suggest that complexation with PAMAM dendrimers has the potential to improve the oral bioavailability of SN-38.