不同内酯型比例9-硝基喜树碱对胆汁排泄的影响

目的考察9-硝基喜树碱(9-nitrocamptothecin,9-NC)不同形式(内酯型或羧酸盐型)给药对胆汁排泄的影响。方法建立HPLC测定胆汁中9-NC浓度的方法,比较不同内酯型比例的9-NC溶液以4 mg·kg?1剂量静脉注射后大鼠胆汁中原形药物的排泄量。结果静脉注射100%,75%,50%,25%,0%内酯型比例的9-NC在8 h内的胆汁累积排泄百分数分别为(7.03±2.23)%,(13.36±0.83)%,(22.68±4.83)%,(28.01±6.71)%,(32.65±2.82)%。结论 9-NC羧酸盐型更易经胆汁途径排泄。     

9-硝基喜树碱内酯型与羧酸盐型在大鼠体内的药动学与肾排泄研究

目的:比较静脉注射9-硝基喜树碱内酯型与羧酸盐型溶液后大鼠体内药动学和肾排泄情况。方法:采用HPLC法同时测定大鼠血浆中9-硝基喜树碱内酯型浓度与总(内酯型+羧酸盐型)浓度以及尿液中9-硝基喜树碱的总浓度。按4mg.kg-1剂量给大鼠静脉注射9-硝基喜树碱内酯型与羧酸盐型溶液,绘制药-时曲线,并采用DAS 2.0软件拟合药动学参数。按同剂量给大鼠静脉注射9-硝基喜树碱内酯型与羧酸盐型溶液,并在各时间段收集尿液,测定尿液中9-硝基喜树碱原形药物累积排泄量。结果:根据AUC计算,9-硝基喜树碱内酯型与羧酸盐型溶液给药以后内酯型的比例分别为(46.7±8.0)%和(8.8±2.5)%,两者的MRT分别为(21.6±2.1)min与(12.7±5.1)min,Vz分别为(0.91±0.16)L.kg-1与(0.56±0.13)L.kg-1,t1/2分别为(17.2±2.4)min与(13.3±3.9)min,差异均有显著性,但总量的AUC并无明显差别。9-硝基喜树碱羧酸盐型及内酯型给药后累积尿液排泄百分率为(30.3±6.4)%和(8.9±0.8)%。结论:9-硝基喜树碱内酯型与羧酸盐型的体内药动学过程存在显著差异,羧酸盐型给药后的肾排泄量远高于内酯型。     

共聚物胶束对硝基喜树碱内酯环稳定性的影响

目的 评价共聚物胶束对9-硝基喜树碱(9-NC)内酯环稳定性的影响.方法 采用HPLC法测定,以甲醇-PBS(70:30,pH6.5)为流动相,检测波长370 nm,流速1ml ·min-1.结果 内酯和羧酸盐形式的9-NC能完全分离;在PBS(pH7.4)中培养160 min后,载药胶束中具有抗肿瘤活性的9-NC内酯比例占80%,而无胶束包覆的原料药中内酯比例仅15%.结论 共聚物胶束能有效提高9-NC内酯环结构的稳定性.     

NGR多肽修饰的9-硝基喜树碱隐形脂质体药剂学性质与抗肿瘤作用研究

目的 考察NGR修饰前后9-硝基喜树碱(9-nitrocamptothecin,9-NC)隐形脂质体的药剂学性质与抗肿瘤作用。方法 采用薄膜-超声化法制备NGR修饰9-NC隐形脂质体,以未经NGR修饰的隐形脂质体作对照,考察包封率、体外释放度、内酯稳定性等药剂学性质;以HT1080为细胞模型,MTT法考察NGR修饰与未修饰9-NC隐形脂质体对细胞生长的影响,流式细胞实验比较NGR修饰与未修饰组的入胞效率。结果 NGR修饰对9-NC隐形脂质体的药剂学性质没有显著影响。修饰后的9-NC隐形脂质体对HT1080细胞的抑制率明显高于未修饰组(P<0.05),流式细胞实验显示修饰后的脂质体入胞效率显著提高。结论 NGR修饰能够显著提高9-NC隐形脂质体的抗肿瘤效果和入胞效率。     

9-硝基喜树碱在大鼠组织中的分布及内酯稳定性

考察9-硝基喜树碱(9-NC)静脉注射后在人鼠组织中的分布及内酯稳定性.建立了HPLC法间时测定组织和血浆中9-NC内酯浓度和总浓度.大鼠静脉注射9-NC溶液后测定各时间点组织中内酯浓度、总浓度和内酯比例.大多数组织中的9-NC内酯比例明显高于血浆;肝中的内酯比例最低,甚至低于血浆;血浆、肾和小肠中的内酯比例随时问延长而下降.9-NC在肝以外的组织中内酯稳定性显著优于血浆.     

9-硝基喜树碱内酯型在大鼠体内外的稳定性

目的考察9-硝基喜树碱内酯型在大鼠体内和离体大鼠血浆中的稳定性.方法建立利用HPLC法测定大鼠血浆中9-硝基喜树碱内酯型浓度和总浓度的方法;利用此法测定9-硝基喜树碱在离体大鼠血浆、全血及体内血浆中的内酯型比例变化以及大鼠尾静脉注射后不同时间点的内酯浓度和总浓度;并对体内外实验结果进行比较以确定影响血浆中内酯型稳定性的主要因素.结果9-硝基喜树碱内酯型在大鼠体内的稳定性显著优于体外,在体外全血中的稳定性显著优于血浆.结论血细胞具有稳定9-硝基喜树碱内酯型的作用;药物从血浆中的清除是影响体内大鼠血浆中9-硝基喜树碱内酯型比例的主要因素;9-硝基喜树碱内酯型浓度和总浓度在大鼠体内的药代动力学过程符合二室模型,而羧酸盐型浓度符合一室模型.     

9-硝基喜树碱内酯型在大鼠体内外的稳定性9-硝基喜树碱内酯型在大鼠体内外的稳定性

目的考察9-硝基喜树碱内酯型在大鼠体内和离体大鼠血浆中的稳定性。方法建立利用HPLC法测定大鼠血浆中9-硝基喜树碱内酯型浓度和总浓度的方法；利用此法测定9-硝基喜树碱在离体大鼠血浆、全血及体内血浆中的内酯型比例变化以及大鼠尾静脉注射后不同时间点的内酯浓度和总浓度；并对体内外实验结果进行比较以确定影响血浆中内酯型稳定性的主要因素。结果9-硝基喜树碱内酯型在大鼠体内的稳定性显著优于体外，在体外全血中的稳定性显著优于血浆。结论血细胞具有稳定9-硝基喜树碱内酯型的作用；药物从血浆中的清除是影响体内大鼠血浆中9-硝基喜树碱内酯型比例的主要因素；9-硝基喜树碱内酯型浓度和总浓度在大鼠体内的药代动力学过程符合二室模型，而羧酸盐型浓度符合一室模型。     

静脉注射9-硝基喜树碱脂质体后原形药物经大鼠胆汁的排泄

目的:考察静脉注射9-硝基喜树碱脂质体后原形药物经大鼠胆汁的排泄。方法:建立了利用HPLC法测定大鼠胆汁中9-硝基喜树碱浓度的方法;测定了9-硝基喜树碱在大鼠空白胆汁中的内酯型浓度、总浓度和内酯型比例的变化;测定了静脉注射3mg.kg-19-硝基喜树碱溶液和脂质体后大鼠胆汁中原形药物的排泄情况。结果:9-硝基喜树碱内酯型结构在胆汁中不稳定,内酯型浓度和比例迅速下降但总浓度保持恒定。静脉注射9-硝基喜树碱溶液和脂质体后12h胆汁中原形药物的累积排泄量分别为给药剂量的7.9%和8.1%。结论:脂质体包封对于9-硝基喜树碱静脉注射后的胆汁排泄没有显著影响     

静脉注射不同比例内酯型9-硝基喜树碱的荷瘤小鼠药动学和组织分布

采用摇瓶法测定内酯型和羧酸盐型9-硝基喜树碱(1)的油水分配系数,结果为145.5±2.96和0.034±0.26.建立了HPLC法同时测定小鼠组织和血浆中1的内酯型浓度和总浓度.静脉注射给予S180荷瘤小鼠不同比例的内酯型1溶液,结果显示给药时内酯型比例对其在小鼠体内的组织分布有明显影响.在血浆中,测得的内酯型浓度随给药时内酯型比例的变化而变化,而总浓度却与给药时内酯型比例关系不大.在肿瘤组织中,给药的内酯型1比例为100％和75％时,测得的内酯型和总量的AUC结果相近;其他各组测得的内酯型和总量的浓度均随给药时内酯型1比例(50％、25％和0)降低而降低.肝、肾组织中测得的内酯型比例与给予比例没有明显相关性(如肾)或呈现出负相关(如肝).这可能与肝、肾是羧酸盐型1的主要排泄途径有关.     

喜树碱类抗肿瘤药内酯型/羧酸盐型平衡研究进展

喜树碱类药物是抗肿瘤药物领域的研究热点之一。喜树碱类药物以内酯型和羧酸盐型2种形式存在,其中内酯型为公认的主要有效形式。文中分别阐述了pH、血液成分、药动学、制剂等多因素对喜树碱类药物内酯型/羧酸盐型的平衡影响,通过制剂手段可以提高具有生物活性的喜树碱类药物内酯型在体内的稳定性,延长药物在血浆中的滞留时间,提高抗癌活性,提示若能提高这类药物内酯型在体内靶部位浓度比,则能增强这类药物抗肿瘤的疗效。     

9-nitrocamptothecin polymeric nanoparticles: cytotoxicity and pharmacokinetic studies of lactone and total forms of drug in rats

The objective of this study was to evaluate the cytotoxicity and pharmacokinetics of total and lactone forms of 9-nitrocamptothecin (9-NC), an effective antineoplastic drug, after intravenous injection of drug incorporated into poly (DL-lactic-glycolic acid) nanoparticles (NPs). Drug-loaded NPs (9-NC.NP) were prepared by the nanoprecipitation method and examined for particle characteristics and in-vitro release in phosphate buffered saline. The best formulation showed a narrow size with an average diameter of 207+/-26 nm and a drug loading of more than 33.5%. The drug release profile showed a sustained 9-NC release up to 160 h. For a pharmacokinetic study, the concentration of 9-NC as the lactone form (9-NC.lac) and as the total of the lactone and carboxylate forms (9-NC.tot) in plasma was determined by using reverse-phase high performance liquid chromatography after intravenous administration of 9-NC.NP and a control solution to cannulated Wistar rats. In-vitro cytotoxic activity of 9-NC.NP and control solution was evaluated on the human ovarian cancer cell line (A2780sn) by MTT cell cytotoxicity assay. Results of in-vivo studies showed that NP encapsulation markedly increased the plasma concentration of both lactone and total forms of 9-NC compared with free drug. In comparison with free drug, NPs resulted in 3.63-fold and 5.40-fold increases in area under the plasma concentration-versus-time curve (AUC(0-infinity)) for lactone and total forms of 9-NC, respectively. The values of mean residence time and elimination half-life (T(1/2)) were also significantly higher for NPs than for free drug. The in-vitro cytotoxicity study revealed that the IC50 value of NPs decreased 10-fold compared with the drug solution. Prepared NPs described here were considered potentially useful in both stabilizing and delivering 9-NC and enhancing the efficacy of this drug for cancer treatment for which high drug retention in the body, protection from the drug-active lactone form, and gradual drug release appeared to be related.     

The Acidic Microclimate in Poly(lactide-co-glycolide) Microspheres Stabilizes Camptothecins

Purpose. The camptothecin (CPT) analogue, 10-hydroxycamptothecin (10-HCPT) has been shown previously to remain in its acid-stable (and active) lactone form when encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres (1). The purpose of this study was to determine the principal mechanism(s) of 10-HCPT stabilization. Methods. CPTs were encapsulated in PLGA 50:50 microspheres by standard solvent evaporation techniques. Microspheres were eroded in pH 7.4 buffer at 37°C. The ratio of encapsulated lactone to carboxylate was determined by HPLC as a function of time, initial form of drug encapsulated, fraction of co-encapsulated Mg(OH)₂, CPT lipophilicity, and drug loading. Two techniques were developed to assess the microclimate pH, including: i) measurement of H⁺ content of the dissolved microspheres in an 80:20 acetonitrile/H₂O mixture and ii) confocal microscopy of an encapsulated pH-sensitive dye, fluorescein. Results. The encapsulated carboxylate converted rapidly to the lactone after exposure to the release media, indicating the lactone is favored at equilibrium in the microspheres. Upon co-encapsulation of Mg(OH)₂, the trend was reversed, i.e., the lactone rapidly converted to the carboxylate form. Measurement of -log(hydronium ion activity) (pa*_H) of dissolved microspheres with pH-electrode and pH mapping with fluorescein revealed the presence of an acidic microclimate. From the measurements of H+ and water contents of particles hydrated for 3 days, a microclimate pH was estimated to be in the neighborhood of 1.8. The co-encapsulation of Mg(OH)₂ could both increase the pa*_H reading and neutralize pH in various regions of the microsphere interior. Varying the drug lipophilicity and loading revealed that the precipitation of the lactone could also stabilize CPT. Conclusions. PLGA microspheres prepared by the standard solvent evaporation techniques develop an acidic microclimate that stabilizes the lactone form of CPTs. This microclimate may be neutralized by co-encapsulating a base such as Mg(OH)₂, as suggested by previous work with poly(ortho esters) (2).     

Pharmacokinetics of lactone, carboxylate and total 9-nitrocamptothecin with different doses and administration routes in rats

9-Nitrocamptothecin (9-NC) is a newly developed poorly soluble derivative of camptothecin and has a wide spectrum of anticancer activity in preclinical evaluation. The effects of the dose and administration route on pharmacokinetics and lactone/carboxylate equilibrium of 9-NC were studied in rats. A single intravenous dose of 1.5, 3 or 6 mg/kg of 9-NC solution was given to male rats (n = 6 per dose level). In another study, a single dose of 6 mg/kg 9-NC solution was given orally to rats (n = 6). Plasma samples were drawn at predetermined intervals and the concentrations of lactone, carboxylate and total 9-NC were determined by a validated HPLC method. Pharmacokinetic analysis was performed using non-compartmental analysis. Analysis of variance showed that the pharmacokinetic characteristics of lactone, carboxylate and total 9-NC were all independent of dose (p > 0.05). Based on the AUC measurements, the lactone 9-NC constituted 52% +/- 4%, 49% +/- 6% and 55% +/- 6% of the circulating total 9-NC in rats after intravenous administration of 1.5, 3, 6 mg/kg 9-NC solution, respectively. After oral administration of 6 mg/kg, the pharmacokinetics parameters were significantly different from those of intravenous administration at the same dose (p < 0.05). The lactone ratio was 60% +/- 14%. The absolute bioavailability of lactone and total 9-NC were calculated to be 23.4% and 22.7%, respectively. In conclusion, the pharmacokinetics of lactone, carboxylate and total 9-NC are not dose-dependent. Lactone, carboxylate and total 9-NC are poorly absorbed following oral administration. Both the dose and the route of administration have little effect on the lactone/carboxylate equilibrium of 9-NC in rats in vivo. But the route of administration plays an important part on the pharmacokinetics of 9-NC.     

Effect of injection routes on pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin in rats

Pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin (9-NC) were compared after intravenous (i.v.) and intramuscular (i.m.) injection at a dose of 1.5 mg/kg 9-NC solution. The concentrations of three different forms of 9-NC, namely lactone, carboxylate and total 9-NC, were measured by HPLC analysis. Injection routes were demonstrated to have significant effect on pharmacokinetics of 9-NC. Compared with i.v. injection route, mean residence time (MRT) of 9-NC three forms was significantly prolonged following i.m. route (p < 0.05). The AUC_0–∞ ratios of i.m. to i.v. route were calculated to be 102 ± 43%, 273 ± 221% and 150 ± 62% for lactone, carboxylate and total 9-NC, respectively. Compared with i.v. injection route, although AUC_0–∞ was barely changed, MRT of lactone 9-NC was dramatically prolonged 4.5-fold after i.m. injection, which may account for the reported improved antitumor efficacy. However, the results of the present study also demonstrated that i.m. injection route increased both AUC_0–∞ and MRT of carboxylate 9-NC more significantly. Since the carboxylate form of CPT analogs including 9-NC is associated with their unwanted toxicity, i.m. injection route might lead to severe toxicity compared with i.v. route. Lactone/carboxylate equilibrium was also significantly influenced by injection routes. Based on the AUC_0–∞ measurements, the lactone 9-NC constituted 50 ± 8% and 32 ± 7% of circulating total 9-NC after i.v. or i.m. administration, respectively (p < 0.01).     

Transition Metal-Mediated Liposomal Encapsulation of Irinotecan (CPT-11) Stabilizes the Drug in the Therapeutically Active Lactone Conformation

Purpose To determine whether entrapped transition metals could mediate the active encapsulation of the anticancer drug irinotecan into preformed liposomes. Further, to establish that metal complexation could stabilize liposomal irinotecan in the therapeutically active lactone conformation. Materials and Methods Irinotecan was added to preformed 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/chol) liposomes prepared in CuSO₄, ZnSO₄, MnSO₄, or CoSO₄ solutions, and drug encapsulation was determined over time. The roles of the transmembrane pH gradient and internal pH were evaluated. TLC and HPLC were used to monitor drug stability and liposome morphology was assessed by cryo-TEM. Results Irinotecan was rapidly and efficiently loaded into preformed liposomes prepared in unbuffered (∼pH 3.5) 300 mM CuSO₄ or ZnSO₄. For Cu-containing liposomes, results suggested that irinotecan loading occurred when the interior pH and the exterior pH were matched; however, addition of nigericin to collapse any residual transmembrane pH gradient inhibited irinotecan loading. Greater than 90% of the encapsulated drug was in its active lactone form and cryo-TEM analysis indicated dark intravesicular electron-dense spots. Conclusion Irinotecan is stably entrapped in the active lactone conformation within preformed copper-containing liposomes as a result of metal–drug complexation.     

Preparation and characterization of novel polymeric micelles for 9-nitro-20(S)-camptothecin delivery.

9-Nitro-20(S)-camptothecin (9-NC) has achieved remarkable curative effect in anticancer research. However, the clinical application of 9-NC is largely hampered by its poor solubility and stability. In this paper, novel amphiphilic block copolymers derived from d,l-lactide, trimethylene carbonate, and methylated poly(ethylene glycol) (mPEG) (PECA) with different molecular weight were synthesized and characterized. Self-assembly PECA micelles loaded with 9-NC were prepared. The micelles were regular spheres with a diameter ranged from 20 to 120 nm. The critical micelle concentration (CMC) decreased with the increase of the hydrophobic components. The solubility of 9-NC was improved obviously with micelle encapsulation. The stability experiments proved that over 90% of 9-NC could keep its lactone form in micelle solution after incubating in phosphate-buffered saline for 100 min, while the corresponding proportion for free drug solution was 25%. The release of 9-NC was nearly zero-order after the burst release, and the long hydrophobic chain length led to slower release rate. The novel PECA copolymer micelles could be effective carriers to improve the solubility, stability, and release performance of 9-NC.     

HPLC法测定人血浆中9-硝基喜树碱内酯型浓度和总浓度

目的:建立以高效液相色谱法测定人血浆中9-硝基喜树碱内酯型浓度和总浓度的方法。方法:血浆样品采用—20℃甲醇快速沉淀蛋白后进行测定,其中色谱柱为shim-packCLS-ODS,流动相为甲醇∶1%三乙胺(冰醋酸调pH6.5)=55∶45,流速为1.0mL·min-1,检测波长370nm,进样量为50μL,柱温为40℃。测定总浓度的样品采用冰醋酸酸化上清液。并以该法测定了不同时间点离体人血浆中9-硝基喜树碱内酯型、羧酸盐型浓度和总浓度的变化。结果:9-硝基喜树碱血药浓度在0.10～10.0μg·mL-1范围内线性关系良好(r=0.9991),定量下限为0.10μg·mL-1,平均方法回收率、酸化回收率各为101.02%、101.46%。结论:本法操作简便、快速,适用于9-硝基喜树碱内酯结构稳定性的研究。