Leptomycin B Development for Cancer Therapy

描述(由申请人提供)： 长期目标：LMB提供了将聚酮类天然产物开发成具有新作用机制的抗癌药物的机会。这种微生物代谢产物通过与CRM1(syn.Exportin 1)，并抑制其在核膜上穿梭蛋白质复合体的能力。许多细胞信号传递过程可能会受到影响，包括对癌症发生和发展以及病毒复制的特定步骤至关重要的过程。LMB与用于治疗慢性粒细胞白血病(CML)的蛋白酪氨酸激酶抑制剂伊马替尼(Gleevec)具有协同作用和克服获得性耐药性的能力。这表明LMB可以与其他通过癌细胞特异性机制发挥作用的药物有效结合。 临床前研究一直受到LMB生产批次质量变化的阻碍，这可能是由于杂质或次要同系物造成的。这也可能影响了LMB在1994-95年度进行的第一阶段临床试验中的表现。为了克服这一局限，我们建议(I)开发一种可靠的发酵工艺来生产高纯度的LMB和(Ii)使用kosan的专有聚酮合成酶(PKS)基因操纵技术 在异源宿主中表达LMB生物合成基因或适当的工程形式，从而仅产生LMB。圣地亚哥大学和国家癌症研究所的合作者将在慢性粒细胞白血病的动物模型中评估格列卫与纯化的LMB联合给药的疗效，并在合适的动物系统中进行LMB的临床前药代动力学和毒理学研究。 一期研究的具体目标：(1)通过优化链霉菌ATCC 39366的发酵工艺，生产出化学纯度至少95%的LMB。(2)在天蓝色链霉菌中表达天然的LMB生物合成基因，或设计用于增强LMB选择性形成的工程形式，以专一性地生产LMB。(3)将纯化的LMB提供给UCSD和NCI合作者进行生物学评估。如果我们能够在本地生产商或天蓝色葡萄球菌中生产出质量一致的高纯度LMB，我们将继续进行LMB的临床前开发或在第二阶段研究中发现和开发LMB类似物。

DESCRIPTION (provided by applicant): Long-range goals: Leptomycin B (LMB) offers the opportunity to develop a polyketide natural product into a cancer drug with a novel mechanism of action. This microbial metabolite inhibits protein export from the cell nucleus by binding tightly to CRM1 (syn. exportin 1) and inhibiting its ability to shuttle protein complexes across the nuclear membrane. Numerous cell signalling processes can be affected, including ones essential to specific steps in the onset and development of cancer as well as viral replication. The ability of LMB to synergize with and overcome acquired resistance to imatinib (Gleevec), a protein tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML), has been demonstrated in vitro. This suggests that LMB could be combined effectively with other drugs that act by cancer cell specific mechanisms. Preclinical studies have been hampered by variability of the quality of LMB production lots, which were probably due to impurities or minor congeners. This may have also compromised the performance of LMB in a Phase I clinical trial carried out in 1994-95. To overcome this limitation, we propose (i) to develop a reliable fermentation process for producing highly pure LMB and (ii) to use Kosan's proprietary polyketide synthase (PKS) gene manipulation technology to express the LMB biosynthesis genes, or a suitably engineered form, in a heterologous host so as to produce only LMB. Collaborators at the University of San Diego and the National Cancer Institute will assess the efficacy of co-administration of Gleevec with purified LMB in an animal model of CML, and conduct preclinical pharmacokinetic and toxicology studies of LMB in a suitable animal system. Specific aims of Phase I research: (1) Produce LMB of at least 95% chemical purity by an optimized fermentation process with Streptomyces sp ATCC 39366. (2) Express the native LMB biosynthetic genes, or an engineered form designed to enhance the selective formation of LMB, in Streptomyces coelicolor so as to produce LMB exclusively. (3) Provide purified LMB to UCSD and NCI collaborators for biological evaluation. If we can produce highly pure LMB in consistent quality in the native producer or in S. coelicolor, we will proceed with preclinical development of LMB or the discovery and development of an LMB analog in Phase II research.