Preclinical development of a chemopreventive agent, 4-methylumbelliferone(4-MU) HHSN2512015000361 TORFP: 2018-E07 PP: 9/17/18 to 9/16/19

摘要： 4-甲基伞形酮(4-MU)(M.W.176)，又称紫草酮或7-羟基-4-甲基香豆素，是一种具有利胆和解痉剂作用的香豆素类化合物。在欧洲和亚洲，它是一种被批准用于治疗胆道运动障碍的药物，在那里，它也被用作改善肝脏健康的膳食补充剂。已知4-MU抑制透明质酸(HA)的生物合成，部分是通过与尿苷二磷酸(UDP)竞争，作为UDP-葡萄糖醛酸基转移酶(UGT)的底物。透明质酸的前体UDP-葡萄糖醛酸(UGA)和UDP-N-乙酰-氨基葡萄糖胺(UDP-N-乙酰氨基葡萄糖)在UGT的作用下分别转化为葡萄糖醛酸和N-乙酰氨基葡萄糖，经HA合成酶合成。通过抑制UGT，4-MU耗尽了UGA的胞液池，从而减少了HA的合成。4-MU也被证明可以降低HA合成酶的表达水平，尽管这些酶的下调对HA合成的影响尚不完全清楚。 在过去的十年中，4-MU被报道在体内外对多种癌症具有抗肿瘤和预防肿瘤的活性，包括前列腺癌、肾癌、胰腺癌、卵巢癌、结肠癌、肝癌和皮肤癌。这些抗肿瘤活性归因于4-MU抑制HA生物合成的能力，因为HA信号被认为在肿瘤的起始、生长、侵袭和新生血管中发挥关键作用。更高水平的HA和已被证明在更晚期和侵袭性的癌症中。 利用转基因前列腺癌(TRAMP)小鼠模型，Vinata Lokeshwar博士及其同事此前曾报道，每天口服4-MU(450 mg/kg/天)，从8周、12周或22周开始，一直持续到28周，以一种治疗时机依赖的方式显著降低前列腺癌的发生，而没有明显的毒性迹象。从8周或12周到28周，接受4-MU治疗的TRAMP小鼠前列腺组织学正常，有低度前列腺上皮内瘤变(PIN)，这被认为是8周龄TRAMP小鼠前列腺的特征，没有任何器官转移，而赋形剂对照组小鼠被发现患有前列腺癌，并有肾、肝和肺的转移性病变。即使在28周后停止4-MU的治疗，4-MU仍能在52周龄的裸鼠体内观察到肿瘤抑制作用。此外，在4-MU处理的小鼠中观察到的抗肿瘤效果与HA表达水平的降低有关，并伴随着前列腺组织中Ki67阳性细胞和微血管密度的减少以及E-钙粘附素表达水平的增加。这些发现支持4-MU作为前列腺癌和其他癌症的无毒口服化学预防药物的潜在用途。 4-MU的药代动力学和安全性之前已经在涉及健康志愿者和需要胆囊切除术的患者的临床试验中进行了研究。临床上已经证明，每天短期服用4MU 1200毫克至2400毫克具有良好的安全性。影响4-MU用途的不利因素是其较短的半衰期(~30分钟)和较低的口服生物利用度，据报道，在人体内的生物利用度不到3%。4-MU通过UGT或硫酸盐形式(4-MUS)被广泛代谢为葡萄糖醛酸(4-MUG)。葡萄糖醛酸化被认为占4-MU代谢的90%以上。4-MUG具有胆汁排泄作用，可能会从肠道重新吸收(肠肝循环)，最终被肾脏消除，尽管4-MUG的药代动力学(PK)尚未得到很好的研究。考虑到4-MU抗肿瘤作用模式的工作假说，需要非代谢形式的4-MU分子作为UGT的竞争底物，如果能够开发一种新的4-MU制剂来靶向淋巴系统，同时避免肝脏的首过代谢，其肿瘤预防活性可能会显著提高。这种淋巴靶向给药方法有望增加非肝脏器官中4-MU的浓度。 由于奥古斯塔大学佐治亚州医学院的Vinata Lokeshwar博士向NCI预防癌症临床前药物开发计划提交了申请，通过DCP资料库计划与预防协调开发了4-MU，4-MU肉豆蔻酸盐(MUM)的新配方。DCP资料库对MU进行的初步PK研究表明，在多次给药的小鼠研究中，4-MU及其代谢物MUG在膀胱中的积累量较高，其次是前列腺。这些数据表明，MUM可能在预防浸润性膀胱癌方面有用。目前的研究集中在新配方的4-MU，MUM的进一步开发上，包括利用膀胱肿瘤发生的临床前模型评价其PK和药效学(PD)谱以及肿瘤预防效果。

Abstract: 4-Methylumbelliferone (4-MU) (M.W. 176), also known as hymechromone or 7-hydroxy-4-methylcoumarin, is a coumarin derivative with choleretic and spasmolytic properties. It is an approved drug for the treatment of biliary dyskinesia in Europe and Asia, where it is also consumed as a dietary supplement to improve liver health. 4-MU is known to inhibit biosynthesis of hyaluronic acid (HA), in part by competing with uridine diphosphate (UDP) as a substrate for UDP-glucuronosyltransferase (UGT). HA is synthesized by HA synthases from its precursors, UDP-glucuronic acid (UGA) and UDP-N-acetyl-glucosamine, both of which are generated by the UGT-mediated transfer of UDP to glucuronic acid and N-acetyl-glucosamine, respectively. By inhibiting UGT, 4-MU depletes the cytosolic pool of UGA, thereby reducing HA synthesis. 4-MU has also been shown to reduce the expression levels of HA synthases, although the impact of downregulations of these enzymes on HA synthesis is not fully understood. For over the last decade, 4-MU has been reported to exert anti-tumor and tumor-preventive activities in vitro and in vivo against wide spectrum of cancers, including cancers of prostate, kidney, pancreas, ovary, colon, liver and skin. These antitumor activities have been attributed to the ability of 4-MU to inhibit HA biosynthesis, as HA signaling is known to play a critical role in tumor initiation, growth, invasion, and neovascularization. Higher levels of HA and HAS have been demonstrated in more advanced and aggressive cancers. Using the transgenic adenocarcinoma of the prostate (TRAMP) mouse model, Dr. Vinata Lokeshwar and colleagues have previously reported that daily oral administration of 4-MU (450 mg/kg/day by gavage), started at 8 weeks, 12 weeks, or 22 weeks and continued through 28 weeks of age, significantly reduced prostate tumorigenesis without signs of overt toxicity in a treatment-timing dependent manner. TRAMP mice treated with 4-MU from 8 weeks or 12 weeks through 28 weeks of age had histologically normal prostates with areas of low-grade prostatic intraepithelial neoplasia (PIN), which is considered characteristic of the 8-week old TRAMP mice prostate, without any organ metastasis, while the vehicle control mice were found to have invasive adenocarcinoma of the prostate with metastatic lesions in the kidney, liver, and lung. The tumor suppressive effect of 4-MU continued to be observed in the TRAMP mice through 52 weeks of age even though the 4-MU treatment was stopped after 28 weeks. Moreover, the antitumor efficacy observed in the 4-MU treated mice was associated with the decreased levels of HA expression, accompanied by reduced Ki67-positive cells and microvessel density counts with the increased level of E-cadherin expression in the prostate tissue. These findings supported the potential utility of 4-MU as a nontoxic oral chemopreventive agent for prostate and other cancers. Pharmacokinetics and safety of 4-MU have previously been studied in clinical trials involving healthy volunteers and patients requiring cholecystectomy. Excellent safety profiles have been clinically demonstrated for short-term daily administrations of 4MU at 1200 mg to 2400 mg per day. Factors negatively affecting the utility of 4-MU are its short half-life (~30 min) and low oral bioavailability, which has been reported to be less than 3% in humans. 4-MU is extensively metabolized to a glucuronic acid (4-MUG) via UGT or a sulfate form (4-MUS). Glucuronidation is believed to account for more than 90% of 4-MU metabolism. 4-MUG is biliary eliminated and likely undergoes reabsorption from the intestine (enterohepatic recycling) for eventual elimination by the kidney, although pharmacokinetics (PK) of 4-MUG has not been well studied. Considering the working hypothesis for the mode of antitumor action of 4-MU, which requires a non-metabolized form of 4-MU molecule to serve as a competitive substrate for UGT, its tumor preventive activity may be significantly improved if a new formulation of 4-MU can be developed to target the lymphatic system while avoiding the first pass metabolism in the liver. Such a lymphatic targeting drug delivery approach is expected to increase 4-MU concentrations in non-hepatic organs. As a result of an application to the NCI PREVENT Cancer Preclinical Drug Development Program submitted by Dr. Vinata Lokeshwar of the Medical College of Georgia, Augusta University, a new formulation of 4-MU, 4-MU myristate (MUM), has been developed through the DCP Repository Program in coordination with PREVENT. The preliminary PK study of MUM conducted by the DCP Repository demonstrated that higher accumulations of 4-MU and its metabolite MUG were demonstrated in the bladder followed by the prostate in a multi-dosing mouse study. The data suggested that MUM may be useful for the prevention of invasive bladder cancer. The current study is focused on the further development of the newly formulated 4-MU, MUM, including the evaluation of its PK and pharmacodynamics (PD) profiles and tumor preventive efficacy using a preclinical model of bladder tumorigenesis.