開發Piperazinedione系列化合物做為癌症治療新藥

Development of Piperazinedione as Novel Anticancer Drugs

王群力(Chun-li Wang)

指導教授 ： 鄭幼文

臺北醫學大學/藥學院/藥學系(碩博士班)/博士(2014年)

摘要

背景與研究動機: Azatyrosine在先前研究中被證實具有選擇性抑制癌細胞生長能力，本實驗室將azatyrosine進行結構優化得到雙胜肽，進一步結構修飾，得到piperazinedione系列化合物；本論文之目的在得到之piperazinedione進一步結構優化與臨床前開發。
材料與方法: 化學合成得到piperzainedione系列化合物藉由體外癌細胞生長抑制活性挑選最佳化合物後，送至美國癌症研究中心 (National Cancer Institute, NCI) 進行癌細胞生長抑制活性試驗 (NCI60 screening)。確認活性最佳之化合物進行臨床前開發，包含活體動物模式確認療效、藥物動力學試驗、早期配方設計及毒性/安全性試驗。
結果: 本論文主要成果為 (1) Piperazinedione類化合物結構最優化及活性篩選。此系列環狀azatyrosine衍生物具有強效抑制癌細胞生長活性，其中體外活性最佳之PE092002、化合物B4、B5抑制癌細胞生長活性 (GI50) 落在10-80 nM。(2) 活性最優之PE092002進行臨床前研究之專案管理。最優化合物PE092002被選為先導化合物進行後續藥理試驗及藥動學試驗；由美國癌症研究中心NCI60平台篩選中證實PE092002之強效及廣效癌細胞生長抑制活性 (45/60個細胞株GI50 < 10 nM)，同時透過COMPARE演算法推測其藥理作用機轉為抑制微管蛋白聚合反應造成mitotic arrest；此一結果在OncoPanel60 (Eurofin Scientific) 結果中獲得驗證，在52個細胞株都顯示具有導致mitotic arrest的效果；同時於體外細胞微管聚合反應試驗中證明。PE092002之癌細胞生長抑制活性在裸鼠腫瘤動物模式中獲得證實，在肺癌A549、血癌HL-60、肝癌Hep3B、Huh-7等細胞株異體移植試驗 (xenograft)中都證實其活體療效，並在Huh-7中求得ED50為8.47 mg/kg/PO/qd。後續PE092002臨床前開發包含 (1) 製程優化，將總產率由3%提升至50%；(2) 溶解度優化，將水溶液態溶解度自54 ng/mL提升二千-三千倍至110 - 203 μg/mL；(3) 單劑量藥動學試驗，PE092002之T1/2為PO 5.7 h，i.v. 6.2 h，生體可用率為9.3%，較TW01提高許多 (T1/2 PO 2.7 h，i.v. 2.34 h，生體可用率1.14%)；(4) 口服急毒性試驗結果大鼠 (公/母) >112 / 68.2 mg/kg，小鼠 100 / 181 mg/kg；(5) 體外受體結合抑制試驗，在10 μM下74個受體中有69個抑制比例低於50%。

結論: PE092002後續開發將按照IND規範，包含FDA guideline M3(R2)、ICH Guideline Topic S 9、FDA Guideline for Industry優先進行化合物品質管制及強化毒理試驗證據，以期早日邁向臨床試驗。本論文產出之PE092002曾獲選台灣生技整合育成中心 (Si2C) 評選跨部會新藥選題計畫獲核准，為25件申請案中獲選之三件之一，且為唯一學術界獲選案，另外正與中國化學製藥公司進行技術移轉授權當中。

關鍵字

癌症 ； 微管蛋白 ； 臨床前 ； 藥物開發 ； 哌嗪二酮

並列摘要

Background and purpose: Azatyrosine was shown to have selective cytotoxicity against cancer cells. We designed and synthesized cyclized dipeptide mimetics and tried to optimize the activity of dipeptide mimetic azatyrosinamides.
Material and methods: We designed and synthesized piperazinedione compounds and screening for in vitro tumor cell growth inhibition. The compounds were submitted to National Cancer Institute (NCI) for NCI60 panel screening. The optimal compound was selected for preclinical development, includes in vivo proof-of-concept, pharmacokinetic study, early formulation development and safety/toxicity study.
Result: (1) Structure optimization of piperazinedione series compounds and primary screening. The piperazinedione compounds showed profound anticancer activities. Among them, PE092002, compounds B4 and B5 exhibited in vitro GI50 at 12 to 80 nM with broad spectrum activities. PE092002 was selected as the lead for further pharmacological and pharmacokinetic studies. (2) The project management of pre-clinical development of most potent compound PE092002. PE092002 showed significant cancer cell line growth inhibition with GI50 12 nM. The result was confirmed in NCI60 platform which showed GI50 < 10 nM in 45 out of total 60 cancer cell lines. Through COMPARE algorithm, we predicted that the mechanism of action of PE092002 is tubulin polymerization inhibitor and will cause mitotic arrest in cellular study. This result was confirmed in OncoPanel60 study (by Eurofin Scientific) in which 52 out of 60 cell lines showed mitotic arrest. The in vitro potency was well transferred to in vivo animal model. PE092002 showed significant potency in xenograft model of lung cancer A549, leukemia HL-60 and hepatoma Hep3B and Huh-7 cell lines. ED50 of Huh-7 xenograft was obtained as 8.47 mg/kg/PO/qd.

The follow-up drug development includes (1) Process optimization: we improved overall yield from 15% to 50%; (2) Solubility optimization, which enhance water solution solubility by 2-3,000 times, from 54 ng/mL to 110 μg/mL (solid formulation)-203 μg/mL (liquid formulation); (3) Single-dose pharmacokinetic study. PE092002 showed improved pharmacokinetic properties with increased T1/2 (PO 5.7 h, i.v. 6.2 h) and bioavailability (9.3%) compared to compound 7 (T1/2 PO 2.7, i.v. 2.34 h and bioavailability 1.14%). (4) Oral acute toxicity study. PE092002 exhibit LD50 >112 / 68.2 mg/kg in male/female rats and 100 / 181 mg/kg in male/female mice. (5) In vitro receptor binding study. With 10 μM added, PE092002 showed only 5 out of 74 inhibition percentage > 50%, which means it is safe for the other 69 receptor.

Conclusion: The next step will be IND-orientated drug development. Based on FDA Guideline M3(R2), ICH Guideline Topic S 9, and FDA Guideline for Industry, future work should focuse on material quality and toxicity issue, which will be our main focus in follow-up development of PE092002. We had submitted this project to Si2C (Supra Integration and Incubation Center, Taiwan) and was approved by expert committee. Currently, we are processing the technical transfer of this project to China Chemical & Pharmaceutical Co., Ltd. for future development.

並列關鍵字

Cancer ； tubulin ； preclinical ； drug development ； piperazinedione

參考文獻

1. 台灣癌症登記中心資料，網址: http://tcr.cph.ntu.edu.tw/main.php?Page=A6.

2. Inouye. S., Shomura, T., Tsuruoka, T., Ogawa, Y., Watanabe, H., Yoshida, J., Niida, T. Chem. Pharm. Bull. 1975, 23:2669-2677. L-beta-(5-Hydroxy-2-pyridyl)-alanine and L-beta-(3-hydroxyureido)-alanine from Streptomyces.

3. Shindo-Okada, N., Makabe, O., Nagahara, H., Nishimura, S. Mol. Carcinog. 1989, 2:159-167. Permanent conversion of mouse and human cells transformed by activated ras or raf genes to apparently normal cells by treatment with antibiotic azatyrosine.

4. Monden, Y., Hamano-Takaku, F., Shindo-Okada, N., Nishimura, S. Ann. N. Y. Acad. Sci.1999, 886:109-121. Azatyrosine-Mechanism of action for conversion of transformed phenotype to normal.

5. Burke, T. R., Yao, Z. –J., Ye, B., Miyoshi, K., Otaka, A., Wu, Li., Zhang, Z. –Y. Bioorg. Med. Chem. Lett. 2001, 11:1265-1268. Phospho-azatyrosine, a less effective protine-tyrosine phosphatase substrate than phosphotyrosine.