Chlorotoxin 是從以色列金蠍所萃取出來的毒素，他是屬於一種神經毒素。他是由36個胺基酸所組成的胜肽，分子量大小是3.9kDa。Chlorotoxin對於運用在癌症方面的治療是非常有幫助的，因為他可以專一性的結合到癌細胞的表面，所以目前他已經被當作是一種顯影劑，用來幫助手術時辨認腫瘤位置。有很多文獻也顯示，chlorotoxin是可以穿越血腦障壁的，這就代表他是可以攜帶藥物到腦部的。也有很多CTX系列的藥物已經通過人體臨床試驗。Glucagon-like peptide-1 (GLP-1)是一種腸促胰素激素，會去促進胰島素的合成。在最近的研究當中，GLP-1在動物實驗上對於阿茲海默症是具有保護的作用的。RR是屬於disintegrin的一個種類，他可以抑制血小板的凝集並且不會造成出血性的副作用。所以我們認為將他攜帶到腦部之後，可能會對治療腦部中風具有很大的幫助。所以在我的實驗當中，我將CTX做為一個攜帶藥物的載體，攜帶我們所要治療的藥物，通過血腦障壁去治療腦部的疾病。我目前已經成功的做出以及表現出CTX的融合蛋白，包括了CTX-GLP-1-HIS、2GLP-1-HSA-CTX、GLP-1-HSA-CTX、CTX-RR、CTX-ARLDDL還有CTX-KG。為了確認CTX融合蛋白是否有表現，使用了西方點墨法來做確認。另外我們也會用Alexa 488，將他標記在我們所要測試的蛋白質上面，借由計算這些標定上瑩光的蛋白穿過人類微血管內皮細胞的量，來了解這些CTX 融合蛋白是否可以穿過血腦障壁。2GLP-1-HSA-CTX、GLP-1-HSA-CTX以及CTX-GLP-1可以用來治療神經退化性疾病，CTX-RR可以被用來治療治療中風。CTX-ARLDDL及CTX-KG可以被用來對抗神精母膠質細胞瘤。以上這個實驗所得到的結果，可以被當作一個篩選藥物是否可以通過血腦障壁的藥物的一個平台。

Chlorotoxin (CTX) is a 36-amino-acid with four disulfide-bond peptide from the venom of the Israeli scorpion Leiurus quinquestriatus, which has potential therapeutic applications in the treatment of cancer. Its ability to preferentially bind to tumor cells has been harnessed to develop an imaging agent to help visualize tumors during surgical resection. Many reports also showed that CTX is able to cross the blood brain and tissue barriers, suggesting that it can deliver drugs to the brain. Indeed, several analogs of CTX have reached clinical trials. Glucagon-like peptide-1 (GLP-1) is an incretin hormone, which was originally found to increase insulin synthesis and secretion. Recently it was reported that GLP-1 also showed good protective effects in animal models of Alzheimer's disease. RR is a disintegrin mutant with potent activity in inhibiting platelet aggregation and with high safety index. We also found that RR is a potential therapeutic agent for ischemic stroke. In this study we proposed to use CTX as a carrier to deliver protein therapeutics to the brain and will fuse it with them. To date, I have successfully expressed six CTX-fusion proteins, including CTX-GLP-1, 2GLP-1-HSA-CTX, GLP-1-HSA-CTX, CTX-RR, CTX-ARLDDL, and CTX-KG. The expression of CTX proteins was confirmed with western blot. The internalization of CTX fusion proteins will be examined by quantifying cellular uptake of fluorophore-labeled peptides with Alexa Fluor or Cy5.5. CTX-GLP-1, 2GLP-1-HSA-CTX, and GLP-1-HSA-CTX will be the potential agents for the treatment of neurodegenerative diseases. CTX-RR protein will be potentially used for the treatment of ischemic stroke. CTX-ARLDDL and CTX-KG will be the potential anti-glioblastoma agents. The results of this study will serve as the basis for the design of protein therapeutics, which can cross the blood-brain-barrier to treat brain diseases.

Ali, S., Alam, M., Abbasi, A., Undheim, E., Fry, B., Kalbacher, H., and Voelter, W. (2016). Structure-Activity Relationship of Chlorotoxin-Like Peptides. Toxins 8, 36.
Allam, A.R., Sridhar, G.R., Thota, H., Babu, C.S., Prasad, A.S., and Divakar, C. (2006). Alzheimer’s disease and Type 2 diabetes mellitus: the cholinesterase connection? Lipids in Health and Disease 5.
Baggio, L.L., Huang, Q., Brown, T.J., and Drucker, D.J. (2004). A Recombinant Human Glucagon-Like Peptide (GLP)-1-Albumin Protein (Albugon) Mimics Peptidergic Activation of GLP-1 Receptor-Dependent Pathways Coupled With Satiety, Gastrointestinal Motility, and Glucose Homeostasis. Diabetes 53, 2492–2500.
CHEN, J., BAI, G., CAO, Y., GAO, Z., ZHANG, Q., ZHU, Y., and YANG, W. (2014a). One-Step Purification of a Fusion Protein of Glucagon-Like Peptide-1 and Human Serum Albumin Expressed in pichia pastoris by an Immunomagnetic Separation Technique. Bioscience, Biotechnology, and Biochemistry 71, 2655–2662.
Dardevet, L., Rani, D., Aziz, T., Bazin, I., Sabatier, J.-M., Fadl, M., Brambilla, E., and De Waard, M. (2015). Chlorotoxin: A Helpful Natural Scorpion Peptide to Diagnose Glioma and Fight Tumor Invasion. Toxins 7, 1079–1101.
Deshane, J., Garner, C.C., and Sontheimer, H. (2003). Chlorotoxin Inhibits Glioma Cell Invasion via Matrix Metalloproteinase-2. Journal of Biological Chemistry 278, 4135–4144.
Femminella, G.D., and Edison, P. (2014). Evaluation of neuroprotective effect of glucagon-like peptide 1 analogs using neuroimaging. Alzheimer’s & Dementia 10, S55–S61.
Femminella, G.D., Bencivenga, L., Petraglia, L., Visaggi, L., Gioia, L., Grieco, F.V., de Lucia, C., Komici, K., Corbi, G., Edison, P., et al. (2017). Antidiabetic Drugs in Alzheimer’s Disease: Mechanisms of Action and Future Perspectives. Journal of Diabetes Research 2017, 1–7.
GAO, Z., BAI, G., CHEN, J., ZHANG, Q., PAN, P., BAI, F., and GENG, P. (2014). Development, Characterization, and Evaluation of a Fusion Protein of a Novel Glucagon-Like Peptide-1 (GLP-1) Analog and Human Serum Albumin in. Bioscience, Biotechnology, and Biochemistry 73, 688–694.
Gejl, M., Gjedde, A., Egefjord, L., Møller, A., Hansen, S.B., Vang, K., Rodell, A., Brændgaard, H., Gottrup, H., Schacht, A., et al. (2016). In Alzheimer’s Disease, 6-Month Treatment with GLP-1 Analog Prevents Decline of Brain Glucose Metabolism: Randomized, Placebo-Controlled, Double-Blind Clinical Trial. Frontiers in Aging Neuroscience 8.
Gejl, M., Brock, B., Egefjord, L., Vang, K., Rungby, J., and Gjedde, A. (2017a). Blood-Brain Glucose Transfer in Alzheimer’s disease: Effect of GLP-1 Analog Treatment. Scientific Reports 7.
Gejl, M., Brock, B., Egefjord, L., Vang, K., Rungby, J., and Gjedde, A. (2017b). Blood-Brain Glucose Transfer in Alzheimer’s disease: Effect of GLP-1 Analog Treatment. Scientific Reports 7.
Hansen, H.H., Fabricius, K., Barkholt, P., Kongsbak-Wismann, P., Schlumberger, C., Jelsing, J., Terwel, D., Termont, A., Pyke, C., Knudsen, L.B., et al. (2016). Long-Term Treatment with Liraglutide, a Glucagon-Like Peptide-1 (GLP-1) Receptor Agonist, Has No Effect on β-Amyloid Plaque Load in Two Transgenic APP/PS1 Mouse Models of Alzheimer’s Disease. PLOS ONE 11, e0158205.
Holscher, C. (2013). Central effects of GLP-1: new opportunities for treatments of neurodegenerative diseases. Journal of Endocrinology 221, T31–T41.
JACOBY, D.B., DYSKIN, E., YALCIN, M., KESAVAN, K., DAHLBERG, W., RATLIFF, J., JOHNSON, E.W., and MOUSA, S.A. (2010). Potent Pleiotropic Anti-angiogenic Effects of TM601, a Synthetic Chlorotoxin Peptide. ANTICANCER RESEARCH 8.
Jalewa, J., Sharma, M.K., Gengler, S., and Hölscher, C. (2017). A novel GLP-1/GIP dual receptor agonist protects from 6-OHDA lesion in a rat model of Parkinson’s disease. Neuropharmacology 117, 238–248.
Janson, J., Laedtke, T., Parisi, J.E., O’Brien, P., Petersen, R.C., and Butler, P.C. (2004). Increased Risk of Type 2 Diabetes in Alzheimer Disease. Diabetes 53, 474–481.
Jayaraman, A., and Pike, C.J. (2014). Alzheimer’s Disease and Type 2 Diabetes: Multiple Mechanisms Contribute to Interactions. Current Diabetes Reports 14.
Kandimalla, R., Thirumala, V., and Reddy, P.H. (2017). Is Alzheimer’s disease a Type 3 Diabetes? A critical appraisal. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1863, 1078–1089.
Kasai, T., Nakamura, K., Vaidyanath, A., Chen, L., Sekhar, S., El-Ghlban, S., Okada, M., Mizutani, A., Kudoh, T., Murakami, H., et al. (2012). Chlorotoxin Fused to IgG-Fc Inhibits Glioblastoma Cell Motility via Receptor-Mediated Endocytosis. Journal of Drug Delivery 2012, 1–10.
Li, C., Wen, A., Shen, B., Lu, J., Huang, Y., and Chang, Y. (2011). FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method. BMC Biotechnology 11, 92.
McCarty, D.J. (2017). Albiglutide for the treatment of type 2 diabetes mellitus: The Nurse Practitioner 42, 10–11.
Nazareth, A.M. de (2017). Type 2 diabetes mellitus in the pathophysiology of Alzheimer’s disease. Dementia & Neuropsychologia 11, 105–113.
Ojeda, P.G., Henriques, S.T., Pan, Y., Nicolazzo, J.A., Craik, D.J., and Wang, C.K. (2017). Lysine to arginine mutagenesis of chlorotoxin enhances its cellular uptake. Biopolymers 108, e23025.
Pennington, M.W., Czerwinski, A., and Norton, R.S. (2018). Peptide therapeutics from venom: Current status and potential. Bioorganic & Medicinal Chemistry 26, 2738–2758.
Sridhar, G.R. (2015). Emerging links between type 2 diabetes and Alzheimer’s disease. World Journal of Diabetes 6, 744.
Tai, J., Liu, W., Li, Y., Li, L., and Hölscher, C. (2018). Neuroprotective effects of a triple GLP-1/GIP/glucagon receptor agonist in the APP/PS1 transgenic mouse model of Alzheimer’s disease. Brain Research 1678, 64–74.
Utkin, Y.N. (2015). Animal venom studies: Current benefits and future developments. World Journal of Biological Chemistry 6, 28.
Vahl, T.P., Paty, B.W., Fuller, B.D., Prigeon, R.L., and D’Alessio, D.A. (2003). Effects of GLP-1-(7–36)NH 2 , GLP-1-(7–37), and GLP-1- (9–36)NH 2 on Intravenous Glucose Tolerance and Glucose-Induced Insulin Secretion in Healthy Humans. The Journal of Clinical Endocrinology & Metabolism 88, 1772–1779.
Xu, W., Yang, Y., Yuan, G., Zhu, W., Ma, D., and Hu, S. (2015). Exendin-4, a Glucagon-Like Peptide-1 Receptor Agonist, Reduces Alzheimer Disease-Associated Tau Hyperphosphorylation in the Hippocampus of Rats With Type 2 Diabetes. Journal of Investigative Medicine 63, 267–272.
Yarchoan, M., and Arnold, S.E. (2014). Repurposing Diabetes Drugs for Brain Insulin Resistance in Alzheimer Disease. Diabetes 63, 2253–2261.
Yu, H., Wang, Q., Sun, Y., Shen, M., Li, H., and Duan, Y. (2015). A New PAMPA Model Proposed on the Basis of a Synthetic Phospholipid Membrane. PLOS ONE 10, e0116502.
Zhang, Y., Sun, B., Feng, D., Hu, H., Chu, M., Qu, Q., Tarrasch, J.T., Li, S., Sun Kobilka, T., Kobilka, B.K., et al. (2017). Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein. Nature 546, 248–253.
洪至頡 (2013). 探討Chlorotoxin之特定胺基酸對抑制細胞電容之影響 = Identification of the Residues of Chlorotoxin Involved in Inhibition of Cell Capacitance
盧璟毅 (2014). Chlorotoxin之第二迴圈、N端及C端於抑制膠質瘤細胞遷移中的角色 = The roles of the Loop-2, N- and C -termini of chlorotoxin in the inhibition of glioma cell migration.