Synthesis of bone-selective osteogenic oxysterols

描述(申请人提供)：1000万美国人患有骨质疏松症，另有3400万人患有骨质疏松症并有发生骨质疏松症的风险。骨折是该病最重要的并发症，在老龄化人口中造成大量的发病率和死亡率，并造成巨大的社会经济损失。自20世纪60年代S以来，双膦酸类药物治疗产生了临床上的好处，如通过减缓破骨细胞性骨吸收来提高骨密度和降低骨折风险。现有的抗吸收药物存在副作用大、临床见效时间短等问题。需要新的和改进的骨质疏松症治疗干预战略，特别是在新的 安全地促进合成代谢骨生长的药物。目前，只有一种FDA批准的骨合成代谢药物Forteo(Teriparatide)在治疗骨质疏松症方面具有显著的临床益处，但出于安全考虑，其使用受到严格限制。多能间充质干细胞(MSCs)是多种细胞类型的前体，包括成骨细胞和脂肪细胞。新骨的形成是由MSCs的成骨分化驱动的，这一过程可以被年龄和其他有利于脂肪生成的因素破坏。Parhami等人。他们发现，当将特定的天然氧化甾醇应用于骨髓间充质干细胞时，可以诱导成骨，同时抑制其脂肪生成。最近，我们表征了一系列新的半合成的天然氧化甾醇类似物，这些化合物具有更好的性能。我们最先进的化合物OXY133在体外显示出更强的成骨分化能力，包括在小鼠和人类原代MSCs中，它还在大鼠脊柱融合模型中刺激体内强大的局部骨形成。在这里，我们建议开始在全身给药和骨靶向的背景下评估Oxy133作为骨合成代谢药的作用。我们的策略包括将OXY133与从四环素衍生的已知骨靶向制剂(BTA)偶联。我们预测，全身剂量的这种结合物将导致它们选择性地沉积在骨中，随后是酶连接物的水解和成骨剂OXY133以受控的速度释放到骨组织中。我们已经生产了这种结合物的第一个例子，OXY149，并确定它在C3H10T1/2细胞中保持着显著的成骨活性。在原理研究证明的基础上，我们建议通过在分子的不同位置上可调节的琥珀酸键和天冬氨酸酯键将BTA-接头连接到OXY133上，可能导致接头的不同程度的水解和骨亲和力，从而允许 用于优化这些特性。我们建议进行三个特定目标的研究：1)Oxy133-BTA共轭类似物的合成，2)Oxy133-BTA共轭类似物的羟基磷灰石结合能力的检测，以及3)Oxy133-BTA共轭类似物的成骨活性的检测。从这些研究中获得的信息将为未来研究Oxy133-BTA类似物在骨质疏松症动物模型中的治疗效果提供理论基础，最终可能导致适合于人类使用的新的骨合成代谢药物的开发。

DESCRIPTION (provided by applicant): Osteoporosis affects 10 million Americans and another 34 million are osteopenic and at risk for developing osteoporosis. Bone fractures, the most important complication of the disease, cause substantial morbidity and mortality in the aging population as well as significant socio-economic cost. Since the 1960's, bisphosphonate drug therapy has produced clinical benefits such as improved bone density and reduced fracture risk by slowing osteoclastic bone resorption. The existing anti-resorptive drugs are plagued with untoward side effects and limited duration of clinical benefits. New and improved strategies for therapeutic intervention in osteoporosis are needed, particularly in the area of new medicines that safely promote anabolic bone growth. Presently, there is only one FDA approved bone anabolic agent, Forteo (teriparatide) that confers significant clinical benefits in osteoporosis, but its use is severely restricted due to safety concerns. Multipotent mesenchymal stem cells (MSCs) are precursors of a variety of cell types, including osteoblasts and adipocytes. Formation of new bone is driven by osteoblastic differentiation of MSCs, a process that can be disrupted by age and other factors in favor of adipogenesis. Parhami et al. discovered that specific naturally- occurring oxysterols induce osteogenesis when applied to MSCs while inhibiting their adipogenesis. Recently, we have characterized a new series of semi-synthetic analogues of the natural oxysterols with improved properties. Our most advanced compound, OXY133, displays increased potency for osteogenic differentiation in vitro, including in mouse and human primary MSCs, and it stimulates robust localized bone formation in vivo in a rat spine fusion model. Here we propose to begin evaluating Oxy133 as a bone anabolic agent in the context of systemic administration and bone targeting. Our strategy involves conjugation of OXY133 to a known bone-targeting agent (BTA) derived from tetracycline. We predict that systemic dosing of such conjugates will result in their selective deposition in bone followed by enzymatic linker hydrolysis and release of the osteogenic agent, OXY133, at controlled rates into the bone tissue. We have produced the first example of such a conjugate, OXY149, and determined that it retains significant osteogenic activity in C3H10T1/2 cells. Expanding on this proof of principle study, we propose to conjugate the BTA-linker to OXY133 through tunable succinate and aspartate ester linkages in different positions of the molecule, likely to result in variable degrees of linker hydrolysis and bone affinity thus allowing for optimization of these properties. We propose to perform studies as part of three Specific Aims: 1) Synthesis of Oxy133-BTA conjugated analogues, 2) Examination of the hydroxyapatite binding capacity of Oxy133-BTA conjugated analogues, and 3) Examination of the osteogenic activity of Oxy133-BTA conjugated analogues. Information obtained from these studies will provide the rationale for future investigation of the therapeutic effects of Oxy133-BTA analogues in animal models of osteoporosis that may ultimately lead to the development of new bone anabolic agents fit for use in humans.