Comprehensive mutagenesis on yeast cytosine deaminase yields improvements in 5-fluorocytosine toxicity in HT1080 cells

Improved prodrug-activating enzymes have the potential to increase the therapeutic efficacy of gene-directed enzyme prodrug therapy (GDEPT). Yeast cytosine deaminase (yCD) is commonly used to convert the prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic 5-fluorouracil for GDEPT. Mutagenesis studies on yCD aimed at improving its application in GDEPT have been limited to subsets of residues or have sought to improve a single property of the enzyme. We performed comprehensive site-saturation mutagenesis (CSM) on yCD designed to create all 2,983 possible unique protein mutants with a single amino acid substitution. We identified active variants through Escherichia coli genetic complementation and screened these mutants, and combinations thereof, for increased ability to sensitize E. coli and HT1080 fibrosarcoma cells to 5-FC. Several mutants identified in this study showed increased sensitization ability for both E. coli and HT1080 cells indicating that CSM is an effective directed evolution tool for identifying unexpectedly beneficial mutations.     

Development of the First Oral Bioprecursors of Bis‐alkylguanidine Antimalarial Drugs

Plasmodium falciparum is responsible of the most severe form of malaria, and new targets and novel chemotherapeutic scaffolds are needed to fight emerging multidrug‐resistant strains of this parasite. Bis‐alkylguanidines have been designed to mimic choline, resulting in the inhibition of plasmodial de novo phosphatidylcholine biosynthesis. Despite potent in vitro antiplasmodial and in vivo antimalarial activities, a major drawback of these compounds for further clinical development is their low oral bioavailability. To solve this issue, various modulations were performed on bis‐alkylguanidines. The introduction of N‐disubstituents on the guanidino motif improved both in vitro and in vivo activities. On the other hand, in vivo pharmacological evaluation in a mouse model showed that the N‐hydroxylated derivatives constitute the first oral bioprecursors in bis‐alkylguanidine series. This study paves the way for bis‐alkylguanidine‐based oral antimalarial agents targeting plasmodial phospholipid metabolism.     

Synthesis and In Vitro Properties of Dexamethasone 21-Sulfate Sodium as a Colon-Specific Prodrug of Dexamethasone

ABSTRACT

We synthesized dexamethasone 21-sulfate sodium (DS) as a colon-specific prodrug of dexamethasone and investigated its properties. Introduction of a sulfate group to dexamethasone lowered the apparent partition coefficient from 52.5 to 0.27 in 1-octanol/pH 6.8 phosphate buffer at 37°C. DS was stable on incubation with buffer solutions of varied pH or with the upper intestinal contents of rats at 37°C for 24 h. On incubation with the cecal contents, DS was hydrolyzed by producing dexamethasone over 80% of the dose at 10 h. When DS was incubated with the cecal contents collected from trinitrobenzenesulfonic acid (TNBS)-induced colitic rats, the degree of prodrug hydrolysis and production of dexamethasone amounted to 70% of healthy rats. In comparison with prednisolone, hydrocortisone, and cortisone, dexamethasone was stable against bioinactivation by the cecal contents, a desirable property for the development of a colon-specific prodrug. These results demonstrated that DS might be delivered specifically to the colon as an intact form to produce dexamethasone in high yield, suggesting DS as a potential colon-specific prodrug of dexamethasone.     

姜黄素前药的研究进展

姜黄素前药的研究现状及进展,主要包括聚乙二醇(PEG)负载前药、胆固醇等细胞膜成分负载前药、表面活性剂样两亲分子负载前药、RNA片段负载前药、姜黄素-氨基酸生物结合物等.为姜黄素的进一步开发提供了依据.     

磷苯妥英的合成

以抗囊痫药物苯妥英为原料,通过合成3-羟甲基-苯妥英、3-氯甲基-苯妥英、3-羟甲基-苯妥英磷酸二苄酯、3-羟甲基-苯妥英磷酸酯等4种中间产物,最终得到目标产物磷苯妥英,总收率35.9%.     

Syntheses and biological activities of polymers containing methacryloyl‐2‐oxy‐1,2,3‐propanetricarboxylic acid or 5‐fluorouracil

A series of novel copolymers, poly(methacryloyl‐2‐oxy‐1,2,3‐propanetricarboxylic acid‐co‐exo‐3,6‐epoxy‐1,2,3,6‐tetrahydrophthalic acid) [poly(MTCA‐co‐ETAc)], poly(methacryloyl‐2‐oxy‐1,2,3‐propanetricarboxylic acid‐co‐hydrogenethyl‐exo‐3,6‐epoxy‐1,2,3,6‐tetrahydrophthalate) [poly(MTCA‐co‐HEET)], and poly(methacryloyl‐2‐oxy‐1,2,3‐propanetricarboxylic acid‐co‐α‐ethoxy‐exo‐3,6‐epoxy‐1,2,3,6‐tetrahydrophthaloyl‐5‐fluorouracil) [poly(MTCA‐co‐EETFU)], were prepared from corresponding monomers by photopolymerizations at 25°C for 48 h. The polymers were identified by FTIR, ¹H‐NMR, and ¹³C‐NMR spectroscopies. The number‐average molecular weights of the fractionated polymers determined by GPC were in the range from 9400 to 14,900 and polydispersity indices were 1.2–1.4. The in vitro IC₅₀ values of polymers against mouse mammary carcinoma (FM3A), mouse leukemia (P388), and human histiocytic lymphoma (U937) as cancer cell lines and mouse liver cells (AC2F) as a normal cell line were much higher compared to that of 5‐fluorouracil (5‐FU). The in vivo antitumor activities of monomers and polymers against mice bearing sarcoma 180 tumor cell line were better than those of 5‐FU. The inhibition of DNA replication and antiangiogenesis activities of MTCA and copolymers were better compared to those of 5‐FU. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 57–64, 2004     

替诺福韦前药设计合成及初步代谢

以替诺福韦（PMPA）为原料,经氨基保护,酰化,缩合,脱保护,成盐得到目标产物。共合成了八个化合物（Ⅲa～Ⅲh）,收率为16.1%～40.7%。探讨了制备化合物Ⅱ时,不同碱性试剂对化合物Ⅰ转化率的影响;考察了乙酸与前药母体成盐比例问题。得到了最优化的实验条件为：碱性试剂选用三乙胺;乙酸与前药母体成盐物质的量之比为0.74～0.93∶1。得到的化合物经ESI-MS、1HNMR对化合物进行了结构表征。通过初步活性研究筛选出一个潜在化合物Ⅲh（替诺福韦二羟丙酮乙二醇缩酮酯）,与富马酸替诺福韦二吡呋酯（TDF）相比,化合物Ⅲh体外大鼠和人血浆中稳定性分别提高了2倍和2.5倍。化合物Ⅲh在小鼠体内肝脏和肾脏中PMPA药时曲线下面积AUC(0-t)分别为(47314.75±10128.11)(μg•h)/L和(21670.64±8964.98) (μg•h)/L,而TDF在肝脏和肾脏中PMPA药时曲线下面积AUC(0-t)分别为(213269.79±10750.47) (μg•h)/L和(46379.24±3944.65)(μg•h)/L。该前药表现出一定的肝靶向性,并降低了肾毒性,具有一定的开发前景。