C16-Ceramide Nano-Liposomes Reverse Multi-Drug Resistance

 描述(由申请人提供)：由于脂类转运体P-糖蛋白(P-gp)异常表达而导致的多药耐药(MDR)细胞表现出广泛的生化变化，影响膜脂成分。尽管临床上做出了重大努力，但目前还没有有效的治疗方法来逆转P-gp介导的人类癌症的多药耐药。最近我们发现，由于丝氨酸棕榈酰基转移酶活性的增强，不同的MDR细胞表现出神经酰胺合成的从头合成途径的结构性激活，导致神经酰胺含量(1.7-1.9倍)和鞘磷脂水平(2-3倍)增加。对鞘磷脂紊乱的ADX细胞中柔红霉素耐药机制的研究表明，药物转运到柔红霉素敏感的亲本DC-3F细胞的细胞核，而在ADX MDR细胞中错误定位于大的细胞质空泡。此外，应用少量外源性酸性鞘磷脂酶生成内源性神经酰胺，或提供外源长链天然C16-神经酰胺，导致空泡柔红霉素快速移位到细胞核中(几分钟内)，并将细胞杀伤率提高25倍。我们推测，MDR耐药的部分原因是不能在囊泡运输系统中产生融合脂神经酰胺，该系统通常将柔红霉素运输到细胞核中，而外源性神经酰胺是可逆的。天然神经酰胺分为长链(C16：0-C20：0)和超长链(C22：0-C24：1)两类。我们最近发现不同种类的神经酰胺具有不同的生物学特性，其中长链C16：0神经酰胺具有促凋亡作用，而超长链C24：0、C24：1神经酰胺具有抗凋亡作用。我们现在发现，在纳米脂质体制剂中加入C16：0神经酰胺，但不包括其他天然神经酰胺种类，可以使柔红霉素从细胞质小泡快速转移到ADX MDR细胞的细胞核。基于这些数据，我们启动了与Chezy Barenholz的合作，他设计了Doxil，这是第一种成功的脂质体药物，将阿霉素系统地输送到肿瘤中。Doxil的专利保护最近已经到期。本申请的总体目的是通过三个特定的体外和体内目标为C16-神经酰胺多西环类脂质体的开发奠定基础，用于癌症治疗。这些研究的成功完成将导致药物开发的直接途径，因为Chezy Barenholz已经原则上同意与纪念斯隆-凯特琳癌症中心合作为临床生产C16-神经酰胺/柔红霉素脂质体。

 DESCRIPTION (provided by applicant): Cells with multidrug resistance (MDR) due to aberrant expression of the lipid transporter P-glycoprotein (P-gp) display a wide range of biochemical changes that affect membrane lipid composition. Despite significant clinical effort, no effective therapy currently exists to reverse P-gp mediated MDR in human cancers. Recently we discovered that distinct MDR cells exhibit constitutive activation of the de novo pathway of ceramide synthesis as a result of enhanced serine palmitoyltransferase activity, resulting in increased ceramide content (1.7-1.9 fold) and sphingomyelin levels (2-3 fold). Evaluation of the mechanism of daunorubicin resistance in sphingolipid-disordered ADX cells revealed that drug traffics to the nucleus of daunorubicin-sensitive parental DC-3F cells, whereas it mislocalizes into large cytoplasmic vacuoles in ADX MDR cells. Furthermore application of small amounts of exogenous acid sphingomyelinase to generate endogenous ceramide, or provision of exogenous long-chain natural C16-ceramide, result in rapid translocation of vacuolar daunorubicin into the nucleus (within minutes), and 25-fold enhanced cell kill. We posit that MDR chemoresistance results in part from failure to generate the fusogenic lipid ceramide in a vesicular trafficking system that normally transports daunorubicin into the nucleus, reversible by exogenous ceramide. Natural ceramides are categorized into long chain (C16:0-C20:0) and very long chain (C22:0-C24:1) species relative to the length of the N-acylated fatty acid at the second position of the sphingosine backbone. We recently showed that different ceramide species possess distinct biologic attributes with long chain C16:0 ceramide being pro-apoptotic, while very long chain C24:0, C24:1 ceramides are anti-apoptotic. We now show that inclusion of C16:0 ceramide, but no other natural ceramide species, in a nano-liposomal preparation permits rapid translocation of daunorubicin from cytoplasmic vesicles to the nucleus of ADX MDR cells. Based on these data we initiated a collaboration with Chezy Barenholz, who engineered Doxil, the first successful liposomal drug that delivers doxorubicin systemically to tumors. Patent protection for Doxil has recently expired. The overall purpose of this application is to set the groundwork for the development of C16-ceramide Doxil-like liposomes for cancer therapy through three specific in vitro and in vivo aims. Successful completion of these studies will resul in a direct path of drug development as Chezy Barenholz has agreed in principle to generate C16-ceramide/daunorubicin liposomes for the clinic in collaboration with Memorial Sloan-Kettering Cancer Center.