發展低氧與組織蛋白酶B依序活化之雙重保護前驅藥物

Development of Double-capped Prodrugs Sequentially Activated by Hypoxia and Cathepsin B

林欣漢(Xin-Han Lin)

指導教授 ： 王宗興

國立臺灣大學/理學院/化學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202100699

摘要

大部分化療藥物同時會干擾一般細胞及癌細胞的功能，造成不良反應使得療效因此受限。伏立諾他(SAHA)及阿黴素(doxorubicin)為FDA核准的癌症治療藥物，但他們同時皆被指出具有包含心毒性的嚴重副作用。在我們的研究當中，我們使用雙重保護的策略以增進前驅藥物的選擇性。首先以Lys-Lys雙肽及對胺基苄醇(p-aminobenzyl alcohol)遮蔽伏立諾他的羥胺酸(hydroxamic acid)，並將雙肽進一步衍生為對硝基苄胺甲酸酯(p-nitrobenzyl carbamate (PNB))。相似的策略也應用於阿黴素胺醣3號碳上的胺基。雙重保護前驅藥物上的遮蔽基團可被還原酶及組織蛋白酶B依序移除，這些還原酶將會表達於低氧之腫瘤，而組織蛋白酶B亦為癌細胞診斷及治療具前景之標的，無論是腫瘤低氧抑或組織蛋白酶B皆與腫瘤進程(tumor progression)及癌症轉移(cancer metastasis)有關。
我們以酵素評估了五種受到不同遮蔽組合之胜肽對於組織蛋白酶B的反應性並以高效能液相層析法(HPLC)監測，相較於原先的Lys-Lys雙肽以及在P2位置衍生PNB基團，在Lys-Lys雙肽的P1、P2位置同時衍生PNB或t-BOC基團，或者只在P1位置上衍生PNB基團皆可減緩組織蛋白酶B對於雙肽醯胺鍵的水解速率。我們亦藉由大腸桿菌之硝基還原酶評估了由伏立諾他及阿黴素所衍生的單重及雙重保護前驅藥物在低氧條件下的反應性，並進一步在海拉細胞(Hela)及人源肝癌細胞(HepG2)兩種癌細胞株當中評估了單重保護前驅藥物在常氧及低氧環境下細胞毒性的差異。

關鍵字

雙重保護前驅藥物 ； 伏利諾他 ； 阿黴素 ； 低氧 ； 組織蛋白酶B

並列摘要

Most chemotherapeutic drugs interfere functions common to normal and tumorous cells, resulting in adverse side effects and thereby limited therapeutic efficacy. SAHA and doxorubicin are FDA-approved drugs for cancer treatment, but they are both pointed out serious side effect including cardiotoxicity. In our work, the “double-capping” strategy was utilized to increase the selectivity of the prodrugs. We firstly masked hydroxamic acid group of SAHA by a lysine-lysine dipeptide with a self-immolative PABA linker, and further derived the dipeptide with p-nitrobenzyl carbamate groups. A similar strategy was also applied to modification of doxorubicin, and the amino group on C3’ of amino sugar moiety was masked by the double-capping group. The masking groups of double-capped prodrugs could be sequentially cleaved by reductases expressed in tumor hypoxia and cathepsin b (CTSB), a promising target in cancer diagnosis and therapy. Both hypoxia and CTSB are associated to tumor progression and cancer metastasis.
We evaluated reactivity of 5 peptide models with different capping combination by in vitro enzymatic test monitored by high performance liquid chromatography (HPLC). We confirmed that derivation of ε-amine to p-nitrobenzyl (PNB) or t-BOC carbamate on both P1 and P2 site or PNB on P1 site can slow down the hydrolytic rate of scissile amide bond by CTSB, compared with original Lys-Lys dipeptide and Lys-Lys with PNB modification on P2. We also assessed the reactivity of the single- and double-capped prodrugs derived from SAHA and doxorubicin in hypoxia by means of E.coli nitroreductase (NTR). Furthermore, we compared the cytotoxicity of single-capped prodrugs in normoxic and hypoxic environment in Hela and HepG2 cancer cell line.

並列關鍵字

double-capped prodrugs ； SAHA ； doxorubicin ； hypoxia ； cathepsin b

參考文獻

1. Zhao, L.; Zhang, B., Doxorubicin induces cardiotoxicity through upregulation of death receptors mediated apoptosis in cardiomyocytes. Sci Rep 2017, 7, 44735.

2. Pugazhendhi, A.; Edison, T.; Velmurugan, B. K.; Jacob, J. A.; Karuppusamy, I., Toxicity of Doxorubicin (Dox) to different experimental organ systems. Life Sci 2018, 200, 26-30.

3. Duvic, M.; Talpur, R.; Ni, X.; Zhang, C.; Hazarika, P.; Kelly, C.; Chiao, J. H.; Reilly, J. F.; Ricker, J. L.; Richon, V. M.; Frankel, S. R., Phase 2 trial of oral vorinostat (suberoylanilide hydroxamic acid, SAHA) for refractory cutaneous T-cell lymphoma (CTCL). Blood 2007, 109 (1), 31-9.

4. Gryder, B. E.; Sodji, Q. H.; Oyelere, A. K., Targeted cancer therapy: giving histone deacetylase inhibitors all they need to succeed. Future Med Chem 2012, 4 (4), 505-24.

5. Thorn, C. F.; Oshiro, C.; Marsh, S.; Hernandez-Boussard, T.; McLeod, H.; Klein, T. E.; Altman, R. B., Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet Genomics 2011, 21 (7), 440-6.