斑蝥酸钠逆转K562/AO2细胞多药耐药的机制初探

目的 观察斑蝥酸钠(SCA)对多药耐药的白血病细胞(K562/AO2)细胞是否有逆转作用,并初步探讨其逆转机制.方法 应用 MTT测定多柔比星(阿霉素,ADM)对K562/AO2细胞的半数抑制浓度(IC50);应用逆转录聚合酶链式反应(RT-PCR)检测K562、K562/AO2细胞表达mdr1基因水平;用流式细胞术(FCM)检测其P-gp蛋白表达的水平.结果 MTT结果显示,无毒剂量的SCA与ADM联合应用比单纯加ADM(同等剂量)对K562/AO2细胞的IC50降低了1.51倍;耐药细胞(K562/AO2)经SCA处理后mdr1基因与P-gp蛋白表达均下降(P＜0.05).结论 SCA对K562/AO2有一定的逆转作用,其机制可能与下调mdr1基因和P-gp蛋白表达有关.     

培美曲塞二钠的合成路线

培美曲塞二钠是唯一治疗恶性胸膜间皮瘤(MPM)的药物。本文根据不同的原料和中间体,通过图表形式归纳了培美曲塞二钠的合成路线。     

原卟啉二钠的抗鸭乙型肝炎病毒作用

目的:研究原卟啉二钠(PPN)抗鸭乙型肝炎病毒(DHBV)的作用。方法:采用先天感染DHBV的龙岩麻鸭为动物模型,分设模型对照组、拉米呋啶组和PPN组,各组按20 mg.kg-1.d-1灌胃给药10 d,斑点杂交法观察用药前、用药5 d、10 d及停药后3 d血清中DHBV DNA表达,HE染色观察肝脏组织病理学变化。结果:PPN组于用药后5 d1、0 d,血清DHBV DNA水平分别为1.36±0.261、.05±0.28,与模型对照组2.04±0.05、1.90±0.31比较明显降低(T5P<0.05、T10P<0.01),病理检查发现PPN组肝细胞点状坏死明显减轻,与模型对照组比较,差异有显著性(P<0.05)。结论:PPN有抑制DHBV DNA的作用,并具有明显的抗炎、保肝作用。     

原卟啉二钠体外对丙型肝炎病毒复制子的抑制作用

目的:探讨原卟啉二钠(PPN)体外对丙型肝炎病毒(HCV)的抑制作用。方法:用不同浓度PPN和利巴韦林处理HCV复制子细胞模型,MTT法检测药物的细胞毒性作用;荧光定量PCR检测HCV RNA。结果:PPN和利巴韦林浓度在1 mg·mL-1以下时,MTT法比色A值比较,实验组与对照组间差异均无显著性,无明显细胞毒性;PPN在1 mg·mL-1时对HCV RNA的抑制率为73.44%,差异有显著性(P<0.05),利巴韦林在1 mg·mL-1时对HCV RNA的抑制率为40.78%,差异无显著性,二者抑制HCV RNA的EC50分别为0.23 mg·mL-1和1.19 mg·mL-1。结论:PPN和利巴韦林浓度在1 mg·mL-1以下时对HCV Replicon细胞生长无毒性作用;PPN具有明显的体外抑制在HCV复制的作用,抑制作用强于利巴韦林。     

Kinetic validation of the use of carboxydichlorofluorescein as a drug surrogate for MRP5-mediated transport

Multidrug resistance protein-5 (MRP5, ABCC5) is a member of the ATP-binding cassette transporter superfamily that effluxes a broad range of natural and xenobiotic compounds such as cyclic GMP, antiviral compounds, and cancer chemotherapeutic agents including nucleoside-based drugs, antifolate agents and platinum compounds. In cellular assays, MRP5 transfectants are less fluorescent after incubation with 5-chloromethylfluorescein diacetate (CMFDA). The present study examines the uptake of a close fluorescent analog, carboxydichlorofluorescein (CDCF), and drug substrates into inside-out membrane vesicles prepared from MRP transfected cells. MRP5-mediated uptake of CDCF was ATP-dependent and GSH-independent and possessed a K_m of 12 μM and a V_max of 56 pmol/min/mg prot. Comparison of kinetic parameters with drug substrates such as methotrexate (MTX), pemetrexed (Alimta™), and the metabolite of 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (5-FdUMP) (K_m values of 0.3–1.3 mM) indicated that MRP5 has a 25–100-fold higher affinity for CDCF than for these drugs and that they share a common transport binding site. In addition, the potency of MRP5 inhibitors such as probenecid, MK571, and the phosphodiesterase 5 inhibitors correlated well between the uptake of CDCF and MTX. A survey of CDCF uptake by other MRPs revealed that MRP2 (ABCC2) also demonstrated ATP-dependent uptake with a K_m of 19 μM and V_max of 95.5 pmol/min/mg prot, while MRP1 (ABCC1) and MRP4 (ABCC4) had little to no uptake. Taken together, these data indicate that CDCF is a useful fluorescent drug surrogate with which to measure ATP-dependent MRP5-mediated transport.     

Attempted randomized controlled trial of pamidronate versus calcium and calcitriol supplements for management of steroid-induced osteoporosis in children and adolescents

OBJECTIVES: To describe an attempted interventional trial for glucocorticoid-induced osteoporosis in children and adolescents and to discuss the reasons for trial failure to inform future interventional studies in this important group of patients. METHODS: Prospective randomized controlled trial comparing the effect of bisphosphonate therapy with calcium and vitamin D supplementation on bone mineral accrual is described. For non-trial patients, retrospective analysis of the effect of calcium and vitamin D supplementation combined with bisphosphonate treatment on bone mineral accrual. RESULTS: Only 12 patients were enrolled in the trial over 4 years. Bisphosphonate recipients (n = 5) had a mean annual percentage increase in lumbar spine bone mineral density of 8.76 +/- 5.2% compared to 6.6 +/- 4.0% in the calcium/vitamin-treated group (difference not significant). Mean annual change in lumbar spine areal bone mineral density in non-trial patients (n = 11) was 3.72 +/- 2.5%. CONCLUSION: Conducting a randomized controlled trial in this group of corticosteroid users is difficult, given the unpredictable nature of the underlying disease and intermittent need for steroid treatment. The trial failed through inadequate recruitment combined with discontinued interventions.     

Disodium octaborate tetrahydrate (DOT) application and vacuum cleaning,a combined strategy to control house dust mites

BACKGROUND: The effectiveness of acaricides in homes is controversial. OBJECTIVE: To determine whether disodium octaborate tetrahydrate (DOT) combined with vacuuming lowers dust mite numbers and their allergens in carpets and sofas. METHODS: A 6-month study was carried out with 93 homes, which were randomized into three groups: (i). active, received DOT; (ii). placebo, received water; and (iii). control, received no application. Active and placebo homes were vacuumed weekly. Dust was collected from carpets and sofas at the start of the study and every 2 months thereafter and quantified for live, total mites, and mite allergen levels. RESULTS: At 2 months, live mite numbers in active carpets were 3 +/- 1, in placebo carpets 129 +/- 48, and in control carpets 177 +/- 39 mites/g. The corresponding numbers in sofas were 3 +/- 2, 81 +/- 31, and 134 +/- 45 mites/g, respectively (P < 0.001 active vs placebo and vs. control). Live mites in carpets and sofas remained lower in the active group at 6 months (P < 0.001). Total mites in active carpets decreased from 555 +/- 69 at baseline to 223 +/- 32 mites/g at 6 months (P < 0.001) and mite allergen levels from 1.36 +/- 0.13 to 0.85 +/- 0.16 microg/g (P < 0.001). Total mites in active sofas remained unchanged, but mite allergen levels decreased from 1.48 +/- 0.25 at baseline to 0.7 +/- 0.15 microg/g at month 6 (P < 0.05). CONCLUSION: DOT kills mites in carpets and sofas, and, combined with vacuuming, effectively reduces total mites in carpets and mite allergen levels in carpets and sofas.     

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.     

反相高效液相色谱-荧光检测法测定粉针剂中帕米膦酸二钠含量及有关物质

目的：建立帕米膦酸二钠粉针剂中含量及有关物质的检测方法。方法：采用反相高效液相色谱-荧光检测法，色谱柱为迪马Diamond C18（250mm×4．6mm，5μm），流动相为乙腈：0.4％EDTA的氢氧化钠溶液（21：79），检测波长为395nm（激发波长）和480nm（发射波长），流速为1mL／min。结果：帕米膦酸二钠浓度线性范围为0．6μg／mL--16．0μg/mL，日内及日间精密度均小于3．7％，检出灵敏度（S／N=3）小于3．0ng，平均回收率为101．0％，有关物质β-丙氨酸检出限量小于0．02％。结论：本法简单、灵敏、快速,可用于帕米膦酸二钠粉针剂中含量及有关物质的测定。