抗癌药米尔法兰的合成研究

米尔法兰为氨基酸氮芥类抗肿瘤药，临床上能治疗乳腺癌、卵巢癌和慢性淋巴细胞及粒细胞白血病等。本文通过对米尔法兰结构的分析，选择了以L-苯丙氨酸为原料的合成路线。实验表明，该路线具有操作简单、原料易得、反应条件温和等优点，反应总收率达32．4％。     

Hepatic artery infusion of high-dose melphalan at reduced flow during isolated hepatic perfusion for the treatment of colorectal metastases confined to the liver: A clinical and pharmacologic evaluation

Isolated hepatic perfusion (IHP) offers the advantage of high local drug exposure with limited systemic toxicity. To increase local drug exposure, we administered melphalan at a reduced flow in the hepatic artery during IHP (hepatic artery infusion, hepatic artery–portal vein perfusion, HI–HPP).     

人多发性骨髓瘤耐药细胞系MOL P22/ R 的 建立及其耐药机制

 目的　建立耐马法兰的人多发性骨髓瘤细胞系MOL P22/ R , 并对其生物学特性及耐药机制进 行初步探讨。方法　采用马法兰浓度梯度递增法,建立耐马法兰多发性骨髓瘤细胞系。检测两种细胞 的形态差异、生长曲线及倍增时间; 用药物敏感试验检测两种细胞对马法兰及多种化疗药物的半数抑 制浓度( IC50 ) 和耐药指数( R I) ;使用Western blot 比较两种细胞P2gp 、MRP 和FANCD2 的表达差异来 探讨可能的耐药机制。结果　成功建立了耐药指数为6. 03的MOL P22/ R 耐药细胞系,与MOL P22 细胞 相比,MOL P22/ R 耐药细胞异形明显; 耐药细胞倍增时间显著延长( P < 0. 05 ) ; MOL P22/ R 且对 ADM、CTX、DDP、VP216 有交叉耐药; MOL P22/ R 中FANCD2 的单泛素化表达较MOL P22 明显增加, 而P2gp 、MRP 在耐药细胞系中表达无升高。结论　MOL P22/ R 具有典型多药耐药特性,为进一步研究 耐药逆转途径提供了实验基础。FANCD2 蛋白单泛素化表达增强可能是MOL P22/ R 细胞产生获得性 耐药的主要机制之一。     

Interpreting cell cycle effects of drugs: the case of melphalan

Multiple effects usually occur in the cell cycle, during and after the exposure to a drug, while treated cells flowing through the cycle encounter G₁, S and G₂M checkpoints. We developed a simulation tool connecting the microscopic level of the cellular response in G₁, S and G₂M with the experimental data of growth inhibition and flow cytometry. We found that multiple—often not intuitive—combinations of cytostatic and cytotoxic effects can be in keeping with the observations. This multiplicity of interpretation can be strongly reduced by considering together data with different methods, ideally reaching a reconstruction of the underlying cell cycle perturbations. Here, we propose an experimental plan including a time course of DNA flow cytometry and absolute cell count measurements with several drug concentrations and a limited number of flow cytometric DNA-Bromodeoxyuridine and TUNEL analyses, coupled with computer simulation. We showed its use in the attempt to define the complete time course of the effects of melphalan on three cancer cell lines. After drug treatment, each subset of cells experienced blocks and lethality in all phases of the cell cycle, but the dynamics was different, the differences being strongly dose-dependent. Our approach allows a better appreciation of the complexity of the cell cycle phenomena associated with drug treatment. It is expected that such level of understanding of the time- and dose-dependence of the cytostatic and cytotoxic effects of a drug might support rational therapeutic design.     

Effects of intraocular irrigation with melphalan on rabbit retinas during vitrectomy

Purpose To investigate the toxic effects of perfusion of intravitreal melphalan during vitrectomy on the rabbit retina. Methods We performed electoretinography (ERG) in 18 eyes of 18 healthy albino rabbits before and after intraocular melphalan perfusion at concentrations of 5-, 10-, and 20-μg/ml during pars plana vitrectomy. Fellow eyes that underwent vitrectomy without melphalan served as controls. The histopathologic retinal changes were observed in both eyes of two rabbits from each group. Results In the 5-μg/ml perfusion group, the ERGs and histology showed no substantial changes compared with control fellow eyes during 28 days postoperatively. In the 10- and 20-μg/ml groups, the mean a-wave amplitude decreased to 52% and 31% respectively of the fellow eye; the mean b-wave amplitude decreased to 52% and 19% respectively. However, the peak implicit time of the a- and b-waves did not significantly differ in the 10- and 20-μg/ml groups during 28 days postoperatively. Histologic sections showed necrosis of the inner nuclear layer and thinning of the outer nuclear layer in the 10-μg/ml group. Loss of the outer nuclear layer and the photoreceptor layer and necrosis of the inner nuclear layer were observed in the 20-μg/ml group. Conclusion The intravitreal 5-μg/ml melphalan perfusion during vitrectomy appears to be nontoxic to the retina. This therapeutic modality might be a potential treatment for retinoblastoma with vitreous seeding. Grant Support: Supported in part by a grant from the Health and Welfare Ministry, Japan.     

The effect of timing on chemosensitization by clinical levels of SR-2508

The nitroimidazole SR-2508 is currently being tested clinically as a radiosensitizer. Its relatively low toxicity allows it to be used at higher doses than misonidazole so that its potential as a chemosensitizer is also of considerable interest. Multiple injections of SR-2508 were given to SCC VII/St tumor-bearing mice to achieve a clinically realistic plasma concentration of approximately 300 micrograms/ml over 8 hrs. Single doses of melphalan (L-PAM) or cyclophosphamide (CY) were given at different times after the first SR 2508 injection. With L-PAM, a delay of at least 2 hr was necessary before enhancement of L-PAM cytotoxicity was observed. A similar result was obtained when a simulation was carried out with SCC VII/St tumor cells in vitro. Results with CY were less clear, although the most consistent enhancement was observed when a 4 to 8 hr interval elapsed between the beginning of SR 2508 exposure and the CY injection. In general, although precise timing was not essential for enhancement, an interval of at least 4 hr is recommended between the administration of SR 2508 and either alkylating agent. This is particularly important for L-PAM where no enhancement would be expected if the drugs were given simultaneously.     

烷化剂马法兰耐药细胞系L1210／Mel的建立及其耐药机制的研究

应用逐步增加剂量的方法在体外建立两株耐不同剂量马法兰（Ｍｅｌ）的小鼠白血病细胞系，命名为Ｌ＿（１２１０）／Ｍｅｌ＿１及Ｌ＿（１２１０）／Ｍｅｌ＿２，它们分别对Ｍｅｌ耐药３．６倍及９．９倍，后者对氮芥及噻哌有明显交叉耐药，而对卡氮芥及阿霉素仍敏感。我们发现随着Ｍｅｌ耐药水平增加，其谷胱甘肽－Ｓ－转移酶（ＧＳＴ）总量及ＧＳＴα基因表达水平明显增加，而ＧＳＴπ轻度升高，多药耐药基因ＭＤＲ－１及ＧＳＴμ基因表达水平无明显升高，表明ＧＳＴα基因与马法兰耐药密切相关。此外，还检测了马法兰诱导的ＤＮＡ交联率，结果发现耐药株在４小时后ＤＮＡ交联宰低于敏感株，在２４小时ＤＮＡ交联得到完全恢复。这表明耐药株中ＤＮＡ损伤减少，而ＤＮＡ修复功能大大增强。     

Enhancement of melphalan activity by inhibition of DNA polymerase-α and DNA polymerase-β

Our previous studies exploring melphalan resistance in the human rhabdomyosarcoma xenograft TE-671 MR revealed elevation of DNA polymerase-α and DNA polymerase-β . The present study evaluated the alteration of melphalan activity in TE-671 (melphalan-sensitive) and TE-671 MR (melphalan-resistant) subcutaneous xenografts in nude mice after DNA polymerase-α was inhibited using aphidicolin glycinate (AG) and DNA polymerase-β was inhibited using dideoxycytidine (DDC). Administration of AG or DDC did not produce toxicity or demonstrate antineoplastic activity when given alone. AG (90 mg/m²) enhanced the activity of melphalan against TE-671, with growth delays increasing by 8.4, 15.8, and 21.2 days over the regimen with melphalan only. AG (180 mg/m²) only modestly increased melphalan activity against TE-671 MR, with the growth delays increasing from 9.6 and 12.1 days using melphalan alone to 12.1 and 14.5 days using melphalan plus AG. AG (180 mg/m²) plus melphalan (the dose lethal to 10% of animals) produced greater weight loss compared with melphalan alone, whereas DDC plus melphalan produced no additional toxicity. DDC modestly enhanced the activity of melphalan plus AG against TE-671 MR. AG plus O ⁶-benzylguanine did not increase the activity of 1,3-bis(2-chloroethyl)-1-nitrosourea against TE-671 or TE-671 MR. AG (90 mg/m² and 180 mg/m²) inhibited DNA polymerase-α to 80% and 72% of control in TE-671 and 64% and 37% in TE-671 MR, and DDC inhibited DNA polymerase-β to 59% in TE-671 and 48% in TE-671 MR. These results suggest a role for AG-mediated enhancement of melphalan activity, particularly in the treatment of newly diagnosed, melphalan-sensitive tumors. Received: 19 June 1995 / Accepted: 2 November 1995     

Anterograde versus retrograde isolated lung perfusion with melphalan in the WAG-Rij rat

Objective: Isolated lung perfusion (ILuP) is an experimental technique currently tested to increase the 5-year survival of 40% after surgical resection of pulmonary metastases from certain solid tumors. The standard technique of anterograde perfusion was compared with retrograde isolated lung perfusion in which the drug is introduced through the pulmonary veins while the effluent is collected from the pulmonary artery. Since the lung has a dual arterial circulation through the pulmonary artery and bronchial circulation, perfusion through the pulmonary veins can result in a more homogeneous distribution throughout the lung with subsequent higher melphalan concentration. Methods: We randomized 20 rats into two groups. Group one underwent anterograde isolated left lung perfusion while group two underwent retrograde isolated left lung perfusion. A dose of 2 mg/kg melphalan (MN) was administered to the lung at a flow of 0.5 mL/min during 30 min, followed by a 5-min washout with buffered hetastarch (BHE). The final melphalan lung concentration (FMLC) was determined in the hilum, at the apex, the mid-periphery and the base of the lung. Statistical analysis was done with an unpaired student's t-test. Results: Retrograde left ILuP resulted in a higher FMLC in the hilum (P<0.0001) and in the base of the lung (P=0.03), while anterograde ILuP induced a higher concentration at the apex of the lung (P=0.04). No difference was seen in the mid-peripheral area of the lung (P=0.92). Conclusions: In this experimental study, retrograde perfusion seems to increase final melphalan lung concentration in hilar and basal regions of the lung compared to anterograde perfusion.     

MMC方案预处理后异基因外周血造血干细胞移植治疗慢性粒细胞白血病

为进一步探讨慢性粒细胞白血病（ＣＭＬ）的治疗，对３例ＣＭＬ患者采用ＨＬＡ相合同胞兄妹异基因外周血干细胞移植治疗，预处理方案为马法兰（Ｍ）１７０ｍｇ／ｍ２×１，甲环亚硝脲（Ｍ）４００ｍｇ／ｍ２×１，环磷酰胺（Ｃ）６０ｍｇ／ｋｇ×２。供者造血干细胞动员用重组人粒细胞集落刺激因子３００μｇ／ｄ×６，血细胞分离机连续采集外周血单个核细胞，其中ＣＤ３４＋细胞为１２．８～２０．０×１０６／ｋｇ，ＣＦＵ－ＧＭ集落数为３．５～４．３×１０５／ｋｇ。结果显示：移植后患者中性粒细胞恢复达≥０．５×１０９／Ｌ，＋１４～＋２０天；血小板≥２．０×１０９／Ｌ在＋１６～＋３４天。移植后＋１００天骨髓染色体核型分析：２例完全嵌合，Ｐｈ１（－），１例部分嵌合（７３％为供者核型），外周血和骨髓检查正常。结果表明，ＨＬＡ相合同胞兄妹异基因外周血干细胞移植采集方便，重建造血功能快；ＭＭＣ预处理方案对白血病细胞的清除和保证移植物存活均有作用，且移植相关毒性低。