Synthesis and biological studies of multifunctional dendritic conjugates of synergistically acting therapeutic agents

许多有趣的候选药物由于其低水溶性、快速从体内消除或不能穿过细胞膜等特性而限制了其治疗应用。因此，研究如何提高这些潜在药物对细胞的靶向性是当前药物化学研究和开发的一个主要领域。在过去的几年里，一类具有多个侧分支的聚合物被称为树状大分子，作为一种有希望的增强和优化药物传递的手段而出现。使用树状大分子的主要优点是，许多药物分子可以连接到树状大分子外围的侧分支上，这样就可以向肿瘤细胞输送高剂量的药物。树状大分子的另一个优点是结构明确，大小和载药量都是已知的，这使得它们对制药工业很有吸引力。在这个项目中，我们建议合成含有天然化合物5-氨基乙酰丙酸（ALA）的树状大分子，该化合物用于癌症的光动力治疗（PDT）。这种治疗方法包括将光照射到肿瘤（如皮肤肿瘤）上，以激活肿瘤中的光敏药物，从而产生对癌细胞有毒的自由基。ALA被用于这种治疗，因为一旦进入细胞，它就会转化为一种被称为卟啉的光激活化合物。为了产生一个卟啉分子，八个ALA分子通过一系列细胞酶的反应结合在一起。然而，ALA不易被细胞吸收，这限制了它在肿瘤治疗中的治疗效果。对于治疗较厚的皮肤肿瘤，如结节性基底细胞癌，需要更好的穿透和更高的细胞卟啉水平贯穿整个肿瘤。在这个项目中，我们提出了一种提高细胞内ALA摄取和卟啉水平的新方法。新的ALA衍生物将被合成，其中ALA与铁结合化合物连接，这些铁结合化合物与ALA结合可以诱导比添加剂或协同作用更大的卟啉水平增强。这些铁结合或螯合化合物（HPOs）最初是在KCL开发的，用于治疗因代谢缺陷而导致体内铁积聚的患者。由于这些化合物能够降低细胞内的游离铁水平，它们也应该抑制光活性卟啉向光非活性形式（称为原血红素）的自然转化。因此，铁螯合可以导致细胞中光活性卟啉中间体的积聚，并可能改善治疗结果。由于卟啉是荧光的，我们能够使用荧光检测来证明卟啉水平的协同增强。我们最近对含有这两种药物的新化合物进行了原理验证研究，以证明这种方法的可行性。我们的目标是制备ALA和HPO分子的偶联物，从两种药物的小单偶联物开始增加到纳米颗粒大小的树状大分子。这些相对较小的分子通过酯键结合在树状大分子内，将使生物活性药物的高有效载荷能够共同递送到细胞中，并且避免了单独给药的需要，这将受到它们不同药理学性质的限制。较小的化合物将被测试表面或局部应用于组织，而较大的化合物将被设计用于口服和静脉注射。该项目将联合KCL、Essex和UCL的团队，在化学、生物化学和光生物学方面具有互补的技能和经验，并将利用MedPharm Ltd在药物配方和组织外植体研究方面的专业知识。虽然这项工作的重点是光动力治疗，但同样的原理可以作为多种药物治疗中其他药物的模板。

Many interesting drug candidates are limited in their therapeutic applications by properties such as low water solubility, rapid elimination from the body or inability to cross cell membranes. The investigation of ways to improve the targeting of these potential pharmaceuticals to a cell is therefore a major current area of medicinal chemistry research and development. Over the past few years a class polymers with multiple side-branches known as dendrimers have emerged as a promising means for enhancing and optimising drug delivery. The key advantage of using a dendrimer is that many drug molecules can be linked to the side-branches on the periphery of a dendrmer which can then deliver a high drug payload to a tumour cell for example. A further advantage is that the structure of a dendrimer is well-defined with a known size and drug loading, which makes them attractive to the pharmaceutical industry. In this project we propose to synthesise dendrimers containing a natural compound known as 5-aminolaevulinic acid (ALA), which is used for photodynamic therapy (PDT) of cancer. This treatment involves shining light onto a tumour (eg a skin tumour) to activate a photosensitising drug in the tumour resulting in the production of free radicals that are toxic to cancer cells. ALA is used for this therapy because once inside cells it is converted to a light-activated compound known as a porphyrin. To produce one porphyrin molecule, eight ALA molecules are combined through reactions involving a series of cellular enzymes. However ALA is not readily taken up by cells which limits its therapeutic efficacy in tumour treatment. For treating thicker skin tumours such as nodular basal cell carcinomas better penetration and higher cellular porphyrin levels throughout the tumour are required. In this project we propose a novel means of enhancing ALA uptake and porphyrin levels inside cells. New ALA derivatives would be synthesized in which ALA is linked with iron-binding compounds which, in combination with ALA, can induce a greater than additive or synergistic enhancement in porphyrin levels. These iron-binding or chelating compounds (HPOs) were developed at KCL originally to treat patients suffering from metabolic deficiencies which caused a build-up of iron in the body. Since these compounds are capable of reducing levels of free iron within cells, they should also inhibit the natural conversion of the photoactive porphyrin into a photoinactive form, called protohaem. Therefore iron chelation can result in a build-up of the photoactive porphyrin intermediate in the cells and potentially improve the therapeutic outcome. Since the porphyrin is fluorescent, we are able to use fluorescence detection to demonstrate the synergistic enhancement in porphyrin levels. We have recently carried out proof-of-principle studies on new compounds incorporating these two agents to demonstrate the feasibility of this approach. Our aim is to prepare conjugates of ALA and HPO molecules, starting from small single conjugates of both drugs increasing to nanoparticale size dendrimers. Incorporation of these relatively small molecules bound via ester linkages within dendrimers will enable a high payload of the bioactive agents to be codelivered to cells, and avoid the need to administer the drugs separately which would be limited by their differing pharmacological properties. The smaller compounds will be tested for surface or topical application on tissue, whereas the larger ones would be designed for oral and intravenous administration. The project would involve a concerted effort bringing together groups at KCL, Essex and UCL with complementary skills and experience in chemistry, biochemistry and photobiology, and would draw on expertise available at MedPharm Ltd in drug formulation and tissue explant studies. Although the work is focused on photodynamic therapy, the same principles may serve as a template for other agents in multiple drug therapy.