C16-Ceramide Nano-Liposomes Reverse Multi-Drug Resistance

 描述（由申请方提供）：由于脂质转运蛋白P-糖蛋白（P-gp）异常表达导致的多药耐药（MDR）细胞显示出影响膜脂质组成的广泛生化变化。尽管有显著的临床努力，但目前还没有有效的治疗方法来逆转人类癌症中P-gp介导的MDR。最近，我们发现，不同的MDR细胞表现出组成性激活的神经酰胺合成的从头途径的丝氨酸棕榈酰转移酶活性增强的结果，导致增加的神经酰胺含量（1.7-1.9倍）和鞘磷脂水平（2-3倍）。对鞘脂紊乱的ADX细胞中柔红霉素耐药机制的评估显示，药物转运至柔红霉素敏感的亲本DC-3F细胞的细胞核，而其在ADX MDR细胞中错误定位于大的细胞质空泡中。此外，应用少量的外源性酸性鞘磷脂酶来产生内源性神经酰胺，或提供外源性长链天然C16-神经酰胺，导致空泡柔红霉素快速易位到细胞核中（在几分钟内），和25倍增强的细胞杀伤。我们认为MDR耐药性部分是由于在囊泡运输系统中不能产生融合脂质神经酰胺所致，该系统通常将柔红霉素转运到细胞核中，可被外源性神经酰胺逆转。相对于鞘氨醇主链第二位的N-酰化脂肪酸的长度，天然神经酰胺分为长链（C16：0-C20：0）和极长链（C22：0-C24：1）种类。我们最近发现，不同的神经酰胺种类具有不同的生物属性，长链C16：0神经酰胺是促凋亡的，而非常长链C24：0，C24：1神经酰胺是抗凋亡的。我们现在表明，在纳米脂质体制剂中包含C16：0神经酰胺，但没有其他天然神经酰胺种类，允许柔红霉素从细胞质囊泡快速易位到ADX MDR细胞的细胞核。基于这些数据，我们开始与Chezy Barenholz合作，他设计了Doxil，这是第一种成功的脂质体药物，可将阿霉素全身性地递送到肿瘤中。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开发用于癌症治疗的C16-神经酰胺Doxil样脂质体奠定基础。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.