Inhibition of Autophagy Does Not Re-Sensitize Acute Myeloid Leukemia Cells Resistant to Cytarabine

Elevated activation of the autophagy pathway is currently thought to be one of the survival mechanisms allowing therapy-resistant cancer cells to escape elimination, including for cytarabine (AraC)-resistant acute myeloid leukemia (AML) patients. Consequently, the use of autophagy inhibitors such as chloroquine (CQ) is being explored for the re-sensitization of AraC-resistant cells. In our study, no difference in the activity of the autophagy pathway was detected when comparing AraC-Res AML cell lines to parental AraC-sensitive AML cell lines. Furthermore, treatment with autophagy inhibitors CQ, 3-Methyladenine (3-MA), and bafilomycin A1 (BafA1) did not re-sensitize AraC-Res AML cell lines to AraC treatment. However, in parental AraC-sensitive AML cells, treatment with AraC did activate autophagy and, correspondingly, combination of AraC with autophagy inhibitors strongly reduced cell viability. Notably, the combination of these drugs also yielded the highest level of cell death in a panel of patient-derived AML samples even though not being additive. Furthermore, there was no difference in the cytotoxic effect of autophagy inhibition during AraC treatment in matched de novo and relapse samples with differential sensitivity to AraC. Thus, inhibition of autophagy may improve AraC efficacy in AML patients, but does not seem warranted for the treatment of AML patients that have relapsed with AraC-resistant disease.     

Synthesis and transdermal permeation of novel N4-methoxypoly(ethylene glycol) carbamates of cytarabine

Background: Cytarabine is a deoxycytidine analogue commonly used in the treatment of hematological malignant diseases. Its clinical utility, however, is severely limited by its short plasma half-life because of the catabolic action of nucleoside deaminases. Method: In this study, N⁴-carbamate derivatives of cytarabine (1) were synthesized and evaluated for transdermal penetration because this mode of administration may circumvent its limitations. The synthesis of these compounds was achieved in a two-step process. First, the methoxypoly(ethylene glycol) was activated by p-nitrophenyl chloroformate. Second, the activated intermediates were reacted with cytarabine in the presence of N-hydroxysuccinamide to give the N⁴-methoxypoly(ethylene glycol) carbamate derivatives. The transdermal flux values of the N⁴-carbamates of cytarabine were determined in vitro by Franz diffusion cell methodology. Aqueous solubility and log D (pH 7.4) values were determined and assessed for correlation with transdermal flux values. Results: The synthesized carbamates, particularly, (9)–(13), showed increased solubility in both aqueous and lipid media. Log D values decreased as the oxyethylene chain lengthened. Conclusion: Although none of the derivatives showed significantly higher transdermal penetration than cytarabine (1), it should be mentioned that the mean for cytarabine N⁴-methoxyethyleneoxycarbamate (8) was 10 times higher and the median was 2 times higher.     

In vitro release dynamics of an anticancer drug from swellable gelatin nanoparticles

Gelatin (Type A) nanoparticles were prepared by a single W/O emulsion technique and characterized by infrared (IR) spectra, scanning electron microscopy (SEM), and particle size analysis. The IR spectra clearly confirmed the presence of gelatin and cytarabine in the loaded nanoparticles while the scanning electron micrograph (SEM) image depicts smooth surface, spherical shape and uneven size of nanoparticles (100–300 nm). The prepared nanoparticles were loaded with cytarabine, a well‐known anticancer drug, and the release dynamics of entrapped drug was investigated as a function of various experimental factors, such as percent loading of the drug, chemical architecture of the nanocarriers, and pH, temperature, ionic strength, and nature of the release medium. The nanoparticles were also studied for their water sorption capacity by optical microscopic method taking advantage of the aggregation of nanoparticles. The drug release process was analyzed kinetically using Ficks power law, and a correlation was established between the quantity of released drug and swelling of the nanoparticles. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 2320–2332, 2006     

Microencapsulation of cytarabine using poly(ethylene glycol)–poly(ε-caprolactone) diblock copolymers as surfactant agents

Background: The high water solubility and the low molecular weight of cytarabine (Ara-C) are major obstacles against its particulate formulation as a result of its low affinity to the commonly used hydrophobic polymers. Methods: Biodegradable cytarabine loaded-microparticles (Ara-C MPs) were elaborated using poly(?-caprolactone) (PCL) and monomethoxy polyethylene glycol (mPEG)–PCL diblock copolymer in order to increase the hydrophilicity of the polymeric matrix. For this purpose, a series of mPEG–PCL diblock copolymers with different PCL block lengths were synthesized. Compositions and molecular weights of obtained copolymers were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, size exclusion chromatography, and size exclusion chromatography–multi-angle laser light scattering. Ara-C MPs were prepared by double emulsion-solvent evaporation method. The effects of varying PCL block lengths on microparticle encapsulation efficiency, size, and zeta potential were evaluated. Results: Increasing the PCL block lengths of copolymers substantially increased the Ara-C encapsulation efficiency and the microparticle size but it decreased their zeta potential. Microparticles were spherical in shape, with a smooth surface and composed of homogenously distributed Ara-C-containing aqueous domains in the polymer matrix. The in vitro drug release kinetics of the optimized microparticles showed a hyperbolic profile with an initial burst release. Conclusion: These results showed the important role of the amphiphilic diblock copolymers as stabilizing agent in the encapsulation of Ara-C in PCL microparticles, suggesting their potential use for the microparticulate formulations of other small hydrophilic bioactive molecules.     

高压静电法制备载阿糖胞苷海藻酸钙-几丁聚糖微胶囊

采用海藻硅酸钠和几丁聚糖为原料制备海藻酸钙-几丁聚糖药物缓释微胶囊,初步考察了载阿糖胞苷微胶囊的制备以及不同分子量几丁聚糖、静/动态载药方式对阿糖胞苷释放性能的影响。     

阿糖胞苷衍生物的可控选择性酶促合成与表征

应用简单有效的酶促合成方法可控选择性地合成阿糖胞苷不同官能团的酰化衍生物。通过酶与溶剂之间的选择性调控实现了己二酸二乙烯酯对阿糖胞苷糖环上5′-OH和嘧啶环上4-N的可控选择性酰化,采用FTIR和NMR分析手段对所得到的衍生物的结构进行了确定,并考察了底物摩尔比,反应时间,反应温度对产率的影响。结果表明:脂肪酶Novozym435在无水丙酮中对C′-5位的伯羟基上显示较高的化学选择性,得到单一的5′-O-乙烯己二酰-阿糖胞苷衍生物,产率达到69%,而脂肪酶PS‘Amano’(PS)在无水吡啶中选择性地在N-4位的伯氨基上发生酰化,得到单一的4-N-乙烯己二酰-阿糖胞苷衍生物,产率达到58%。     

四苄基阿糖胞苷的合成

通过选用三苯甲基选择性保护阿拉伯糖5＇-位羟基、苄基保护4-位氨基和2’,3’-位羟基,于酸性条件下选择性脱除三苯甲基合成了制备阿糖胞苷5＇-位氨基酸酯类前药的关键中间体-四苄基阿糖胞苷,苄基保护基可经催化氢化顺利脱除。各步反应条件温和,收率高,操作简便。     

Scavenger receptor class BI (SR-BI) mediates uptake of CPX-351 into K562 leukemia cells

Purpose: CPX-351 is a liposomal formulation of cytarabine and daunorubicin encapsulated at a 5:1 molar ratio, for the treatment of acute myeloid leukemia. The Scavenger Receptor class B type I (SR-BI) plays an important role in mediating the uptake of high-density lipoproteins. The purpose of this study is to assess the role of the cell surface lipoprotein receptor SR-BI in the uptake of CPX-351 liposomes (Jazz Pharmaceuticals) into K562 leukemia cells.

Methods: K562 cells were pre-treated with 10?nM siRNA for 48?h and then treated with varying amount of CPX-351 for 24, 48 and 72?h. Cells were then collected and analyzed at 480/590?nm on a CytoFLEX Multicolour flow instrument to determine cellular uptake of daunorubicin. Experimental data were analyzed using two-way ANOVA with Bonferroni multiple comparisons. Significance was set at p?<?.05.

Results: K562 cells pre-treated with SR-BI siRNA for 48?h had a reduced SRB1 cell surface concentration (74–85%). Addition of CPX-351 at 10–50?nM followed by measurement of cellular daunorubicin at 48, 48 or 72?h showed a significantly lower percentage of daunorubicin positive population compared with control K562 cells (p?<?.05). There was significantly less daunorubicin taken up in the SR-BI knock-down cells across all drug concentrations and at all three time points, although there were no concentration-related trends.

Conclusions: These preliminary studies suggest that SR-BI may be one potential mechanism by which CPX-351 is taken up into K562 cells.     

联合应用化疗药物对三氧化二砷耐药白血病细胞的毒性实验研究

目的: 探讨化疗药物联合应用对三氧化二砷(As2O3)耐药白血病细胞(K562/AS2)的毒性作用。方法: 细胞毒实验采用MTT 法, 二药合用时细胞毒性作用采用Chou-Talalay 联合指数法分析, 细胞表面P-糖蛋白(P-gp)和细胞内柔红霉素(DNR)浓度测定采用流式细胞术测定。结果: K562/AS2 细胞对三氧化二砷、柔红霉素、鬼臼乙叉苷(VP16)、三尖杉酯碱(H)、米托蒽醌(NVT)和阿糖胞苷(Ara-C)的耐药倍数分别为7.4、2.9、3.8、3.6、2.8 和1.1。K562 细胞和K562/AS2 细胞的细胞表面P-gp 或细胞内任意荧光强度无显著的统计学意义(P >0.05)。As2O3 与DNR、VP16、H 或NVT 联合应用时, 对K562、K562/AS2 和P-gp 表达的白血病细胞(K562/A02)细胞的联合指数均大于1。异搏定与DNR 联合应用时, 对K562 和K562/AS2 细胞的联合指数均大于1, 但是对K562/A02 细胞的联合指数均小于1。结论: K562/AS2 细胞对As2O3、DNR、VP16 和NVT 耐药, 其机制与P-gp 表达无关。异搏定联合应用DNR 可以逆转K562/A02 对DNR 的耐药性, 不能逆转DNR 对As2O3耐药细胞的耐药性。As2O3 与DN、VP16、H 和NVT联合应用时, 对K562、K562/AS2 和K562/A02 细胞的毒性均为拮抗作用。     

Cytarabine trapping in poly(2-hydroxyethyl methacrylate-co-acrylamide) hydrogels: drug delivery studies

Copolymeric hydrogels of poly(2-hydroxyethyl methacrylate-co-acrylamide) [p(HEMA-co-A)] crosslinked with ethylene glycol dimethacrylate, with a high equilibrium degree of swelling (37–65 wt%) in saline solution (NaCl 0.9 wt%) were synthesized as devices for controlled release of cytarabine (ara-C). Two compositions of the copolymer, each with a different degree of crosslinking have been studied, HEMA80/A20 and HEMA60/A40. The antineoplasic drug was included in the feed mixture of polymerization, and discs 3.7 ± 0.4 mm thick and 11.8 ± 0.2 mm in diameter with 5–40 mg (1.0–8.3 wt%) of ara-C were obtained. The diffusion studies followed Fick's second law. The diffusion coefficients for swelling of the gels were between 3.60 × 10⁻¹¹ and 15.8 × 10⁻¹¹ m² s⁻¹; those for release of ara-C were between 0.31 × 10⁻¹¹ and 7.18 × 10⁻¹¹ m² s⁻¹. The activation energies for swelling were in the range 23.4–31.9 kJ mol⁻¹ and those for ara-C release were 42.2–61.6 kJ mol⁻¹; their values indicate that the drug release process depends on drug–matrix and drug–water interactions that are influenced by the aqueous solution content and the network size of the gels. Total release of the drug takes place between 17 h from H60/A40/E2 at 310 K and 6 days from H80/A20/E10 at 288 K. Ara-C degradation was not observed either during loading of the gels or during drug release. © 1999 Society of Chemical Industry