臺灣博碩士論文加值系統

目前臨床上對於蛋白質和多肽類藥物之給藥方式仍以注射為主，在長期治療下，往往會對病患的身心和經濟上造成重大痛苦和負擔。因此，本研究目的為建立一有效口服傳遞系統以提高蛋白質藥物的吸收。利用乳化液 (emulsion) 的形式做為藥物傳遞系統，是目前廣為研究的方式之一。其中，油包水-水包油型 (water-in-oil-in-water, W/O/W) 的複合乳化液已被證實可將蛋白質藥物包裏於內部的水相，可避免被腸胃道的酵素所降解，並且可增進親水性藥物在體內的吸收性。因此本論文欲探討蛋白質藥物顆粒球細胞生長刺激素 (Granulocyte colony-stimulating factor, G-CSF) 經W/O/W 複合乳化法包裏後，對小鼠口服吸收性的影響。
試驗結果顯示，使用兩階段式的乳化方式所建構出的 W/O/W 乳化液，不論是在顆粒大小 (直徑 2 μm)、包覆率 (40±5%)、穩定性 (耐酸性 pH 1.2，2hr 及耐高溫 60°C，5天)、穿透率 (30分鐘內滲透入 Caco-2 cell monolayer) 及體外毒性試驗上，皆顯示此乳化液的配方有傾向去幫助小鼠體內吸收的效果。進一步藉由大腸桿菌表現系統 (E. coli expression system) 產製具有活性之重組人類 G-CSF (rhG-CSF) 蛋白，並包裏入上述乳化液中，測試其對小鼠的口服生物利用率。經實驗結果顯示，連續七天餵予不同劑量的乳化液或水溶液劑型之 rhG-CSF，在第七天的六小時後觀察，相較於餵食空白乳化液的對照組，高劑量 rhG-CSF (2,500 μg/kg/day) 包埋處理組的白血球數有顯著的增加 (p < 0.01)；但乳化劑包埋與不包埋的兩組相互比較，兩者則無差異性。口服十二個小時後，所有餵食 rhG-CSF 的小鼠，其白血球數皆有顯著的提升 (p < 0.01)。此外，對餵食乳化劑的小鼠進行腸道組織切片的染色觀察與穿透腸黏膜細胞 Caco-2 cell 的測試，W/O/W 乳化劑對小鼠腸道組織並不會造成損害且可促進其穿透率。由本次試驗證實，W/O/W 乳化液可促進 rhG-CSF 於腸道黏膜細胞的穿透率、耐酸性、耐高溫及包覆率的效果，並且對小鼠的腸道組織不會造成損害。因此，在未來或許可運用此劑型做為蛋白質的口服傳遞系統。

Since the injection administration as the peptides and proteins delivery system, it imposes discomfort and inconvenience on patients, particularly for a long-term treatment. Therefore, the goal of this thesis is to find out an oral delivery system for improving the absorption of therapeutic protein drugs.
Emulsion is one of strategies known delivery vehicles for those oral medicines. Water-in-oil -in-water (W/O/W) multiple emulsion has been known to be possible to protect protein drugs from enzyme degradation and enhance the absorption of hydrophilic drugs to systemic circulation. Therefore, we tried to use granulocyte colony-stimulating factor (G-CSF) as the indicator to test the emulsion formulation for the oral delivery system in the study.
In this two-step emulsification procedure, the data of the droplet size (2 μm of diameter), encapsulation efficiency (40±5%), stability (at pH 1.2 for 2 hrs and 60°C for 5 days), permeability (into Caco-2 cell monolayer with 30 min) and in vitro cytotoxicity studies all showed that the emulsion formulation was indicated to promote the absorption of proteins drugs by oral administration. Thus, we used the E. coli expression system to express and purify the recombinant human G-CSF (rhG-CSF). And then, rhG-CSF was incorporated into the inner aqueous phase of the emulsion for the test of oral bioavailability. After 6 hrs of oral administration of various dosages of rhG-CSF in solution or in emulsion for 7 days, the proliferations of leukocytes in the two treatments with the highest dose of rhG-CSF (2,500 μg/kg/day) were significantly higher than the blank emulsion (p < 0.01). However, two of them, rhG-CSF in emulsion and not in emulsion, were not different. After 12 hrs of oral administration, mice, administrated by 50, 500, and 2500 μg/kg/day rhG-CSF emulsion, all presented a significant elevation in leukocyte numbers when compared to the blank emulsion (p < 0.01). Finally, it was indicated about the advantages of the W/O/W multiple emulsions with encapsulation efficiency, penetrating ability, and stability to resist acid pH (1.2) and high temperature (60°C). In the future, it could be potential for protein drugs delivery by oral administration.

誌謝 1
中文摘要 5
Abstract 7
Introduction 9
Chapter 1 Literature review 12
1.1 The development and delivery systems of protein drugs 12
1.1.1 The development of protein drugs 12
1.1.2 The approaches for non-invasive delivery of protein drugs 13
1.1.3 The obstacles of developing oral formulations for protein drugs 15
1.2 Strategies of the oral delivery for protein drugs 17
1.2.1 Chemical modifications 17
1.2.2 Protease inhibitors 18
1.2.3 Absorption enhancers 19
1.2.4 Colloidal carrier systems 20
1.3 Water-in-oil-in- water (W/O/W) multiple emulsions 30
1.3.1 The property of W/O/W multiple emulsions 30
1.3.2 The emulsifiers 30
1.4 Experimental framework 36
Chapter 2 Materials and methods 37
2.1 Objective 37
2.2 Preparation of the water-in-oil-in-water (W/O/W) multiple emulsion 38
2.2.1 Morphology observation 40
2.2.2 Droplet size analysis 40
2.3 Stability evaluation of the W/O/W emulsion 41
2.3.1 The pH-dependent stability of emulsions in simulated fluids 41
2.3.2 Accelerated stability studies 41
2.4 Penetrating pathway of EGFP into the Caco-2 cell monolayer 42
2.5 Gene construction of the recombinant human granulocyte colony stimulating factor (rhG-CSF) 43
2.5.1 Cell lines and isolation of RNA 43
2.5.2 Complimentary DNA (cDNA) cloning 43
2.5.3 Construction of the rhG-CSF expression vector 43
2.6 Expression and purification of rhG-CSF 45
2.6.1 Expression of rhG-CSF in E. coli 45
2.6.2 Isolation of inclusion bodies 46
2.6.3 Purification and refolding of rhG-CSF 47
2.6.4 Analysis of the expressed rhG-CSF 47
2.7 Measurement of rhG-CSF bioactivity 48
2.7.1 In vitro assay of cell proliferation 48
2.7.2 In vivo assay of leukocyte proliferation 49
2.8 Protein encapsulating efficiency 50
2.9 In vitro study to evaluate the cytotoxicity of emulsion 51
2.10 In vivo study to evaluate the bioactivity 52
2.11 Histological evaluation of intestinal tissue 52
2.12 Statistical analysis 53
Chapter 3 Result 54
3.1 Morphology, droplet size and encapsulating efficiency of the W/O/W multiple emulsions 54
3.2 The pH-dependent and accelerated stability of the W/O/W emulsion 54
3.3 Penetrated pathway of EGFP into the Caco-2 cell monolayer 55
3.4 Construction of expression vector of rhG-CSF 56
3.5 Expression and purification of rhG-CSF in E. coli 56
3.6 Bioactivity of rhG-CSF 57
3.7 In vitro study to evaluate the cytotoxicity of emulsion 58
3.8 In vivo study to evaluate the bioactivity 58
3.9 Histological observation of intestinal tissue 59
Chapter 4 Discussion 77
Chapter 5 Conclusion 82
Reference 83

Aboofazeli, R., Patel, N., Thomas, M., and Lawrence, M.J. (1995). Investigations into the formation and characterization of phospholipid microemulsions. IV. Pseudo-ternary phase diagrams of systems containing water-lecithin-alcohol and oil; the influence of oil. International journal of pharmaceutics 125, 107-116.
Aliabadi, H.M., and Lavasanifar, A. (2006). Polymeric micelles for drug delivery. Expert Opin Drug Deliv 3, 139-162.
Arrieta-Molero, J.F., Aleck, K., Sinha, M.K., Brownscheidle, C.M., Shapiro, L.J., and Sperling, M.A. (1982). Orally Administered Liposome-Entrapped Insulin in Diabetic Animals A Critical Assessment. Horm Res 16, 249-256.
Artursson, P., Palm, K., and Luthman, K. (1996). Caco-2 monolayers in experimental and theoretical predictions of drug transport. Advanced drug delivery reviews 22, 67-84.
Aungst, B. (1994). Permeability and metabolism as barriers to transmucosal delivery of peptides and proteins. Drugs and the pharmaceutical sciences 62, 323-343.
Baby, A.R., Santoro, D.M., Velasco, M.V.R., and dos Reis Serra, C.H. (2008). Emulsified systems based on glyceryl monostearate and potassium cetyl phosphate: Scale-up and characterization of physical properties. International journal of pharmaceutics 361, 99-103.
Bai, Y., Ann, D.K., and Shen, W.C. (2005). Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent. Proceedings of the National Academy of Sciences 102, 7292-7296.
Banerjee, D., Flanagan, P., Cluett, J., and Valberg, L. (1986). Transferrin receptors in the human gastrointestinal tract. Relationship to body iron stores. Gastroenterology 91, 861.
Barichello, J.M., Morishita, M., Takayama, K., and Nagai, T. (1999). Encapsulation of hydrophilic and lipophilic drugs in PLGA nanoparticles by the nanoprecipitation method. Drug development and industrial pharmacy 25, 471-476.
Batrakova, E.V., Li, S., Li, Y., Alakhov, V.Y., Elmquist, W.F., and Kabanov, A.V. (2004). Distribution kinetics of a micelle-forming block copolymer Pluronic P85. Journal of Controlled Release 100, 389-397.
Bernkop-Schnurch, A. (2000). Chitosan and its derivatives: potential excipients for peroral peptide delivery systems. International journal of pharmaceutics 194, 1-13.
Boyd, J., Parkinson, C., and Sherman, P. (1972). Factors affecting emulsion stability, and the HLB concept. J Colloid Interface Sci 41, 359–370.
Bundgaard, H., and Moss, J. (1995). Prodrug derivatives of thyrotropin-releasing hormone (TRH) (Google Patents).
Cantrell, M., Anderson, D., Cerretti, D., Price, V., McKereghan, K., Tushinski, R., Mochizuki, D., Larsen, A., Grabstein, K., and Gillis, S. (1985). Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor. Proceedings of the National Academy of Sciences 82, 6250-6254.
Carino, G., Jacob, J., and Mathiowitz, E. (2000). Nanosphere based oral insulin delivery. Journal of Controlled Release 65, 261-269.
Cho, Y., Kim, S., Bae, E., Mok, C., and Park, J. (2008). Formulation of a Cosurfactant-Free O/W Microemulsion Using Nonionic Surfactant Mixtures. Journal of food science 73, 115-121.
Creighton, T. (1993). Proteins, structures and molecular properties.
Cunnah, D., Ross, G., and Besser, G.M. (1986). Management of cranial diabetes insipidus with oral desmopressin (DDAVP). Clinical Endocrinology 24, 253-257.
Demetri, G.D., and Griffin, J.D. (1991). Granulocyte colony-stimulating factor and its receptor. Blood 78, 2791-2808.
DiBiase, M.D., and Morrel, E.M. (1997). Oral delivery of microencapsulated proteins. Protein Delivery: Physical Systems, 255.
Dogru, S.T., Calis, S., and Oner, F. (2000). Oral multiple w/o/w emulsion formulation of a peptide salmon calcitonin: in vitro-in vivo evaluation. Journal of Clinical Pharmacy & Therapeutics 25, 435-443.
Engel, R.H., Riggi, S.J., and Fahrenbach, M.J. (1968). Insulin: intestinal absorption as water-in-oil-in-water emulsions. Nature 219, 856.
Engels, T., Forster, T., and Von Rybinski, W. (1995). The influence of coemulsifier type on the stability of oil-in-water emulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects 99, 141-149.
Flanagan, J., and Singh, H. (2006). Microemulsions: a potential delivery system for bioactives in food. Critical reviews in food science and nutrition 46, 221-237.
Florence, A.T., and Whitehill, D. (1983). Stability and stabilization of water-in-oil-in-water multiple emulsions (American Chemical Society).
Francis, M.F., Cristea, M., and Winnik, F.M. (2004). Polymeric micelles for oral drug delivery: Why and how. Pure and applied chemistry 76, 1321-1336.
Friberg, S., Larsson, K., and Sjoblom, J. (2004). Food emulsions (Marcel Dekker Inc).
Fujii, S., Yokoyama, T., Ikegaya, K., Sato, F., and Yokoo, N. (1985). Promoting effect of the new chymotrypsin inhibitor FK-448 on the intestinal absorption of insulin in rats and dogs. The Journal of pharmacy and pharmacology 37, 545.
Fukunaga, M., Miller, M.M., and Deftos, L.J. (1991). Liposome-entrapped calcitonin and parathyroid hormone are orally effective in rats. Hormone and metabolic research Hormon-und Stoffwechselforschung Hormones et metabolisme 23, 166.
Gallarate, M., Trotta, M., Battaglia, L., and Chirio, D. (2008). Preparation of solid lipid nanoparticles from W/O/W emulsions: Preliminary studies on insulin encapsulation. Journal of Microencapsulation 99999, 1-9.
Garcia-Garcia, E., Andrieux, K., Gil, S., and Couvreur, P. (2005). Colloidal carriers and blood–brain barrier (BBB) translocation: A way to deliver drugs to the brain? International journal of pharmaceutics 298, 274-292.
Garcia-Ochoa, F., Santos, V.E., Casas, J.A., and Gomez, E. (2000). Xanthan gum: production, recovery, and properties. Biotechnology advances 18, 549-579.
Garti, N. (1999). What can nature offer from an emulsifier point of view: trends and progress? Colloids and Surfaces A: Physicochemical and Engineering Aspects 152, 125-146.
Goldberg, M., and Gomez-Orellana, I. (2003). Challenges for the oral delivery of macromolecules. Nature reviews Drug discovery 2, 289.
Guo, J., Ping, Q., and Chen, Y. (2001). Pharmacokinetic behavior of cyclosporin A in rabbits by oral administration of lecithin vesicle and sandimmun neoral. International journal of pharmaceutics 216, 17-21.
Hartmann, L., Tschetter, L., Habermann, T., Ebbert, L., Johnson, P., Mailliard, J., Levitt, R., Suman, V., Witzig, T., and Wieand, H. (1997). Granulocyte colony-stimulating factor in severe chemotherapy-induced afebrile neutropenia, pp. 1776-1780.
Hoffman, A. (2002). Hydrogels for biomedical applications. Advanced drug delivery reviews 54, 3-12.
Iwanaga, K., Ono, S., Narioka, K., Kakemi, M., Morimoto, K., Yamashita, S., Namba, Y., and Oku, N. (1999). Application of surface-coated liposomes for oral delivery of peptide: effects of coating the liposome's surface on the GI transit of insulin. Journal of pharmaceutical sciences 88.
Jiao, J., and Burgess, D. (2003). Rheology and stability of water-in-oil-in-water multiple emulsions containing Span 83 and Tween 80. The AAPS Journal 5, 62-73.
Jung, T., Kamm, W., Breitenbach, A., Kaiserling, E., Xiao, J.X., and Kissel, T. (2000). Biodegradable nanoparticles for oral delivery of peptides: is there a role for polymers to affect mucosal uptake? European Journal of Pharmaceutics and Biopharmaceutics 50, 147-160.
Kaparissides, C., Alexandridou, S., Kotti, K., and Chaitidou, S. (2006). Recent advances in novel drug delivery systems. Journal of Nanotechnology, Online 2, 1-11.
Kataoka, K., Harada, A., and Nagasaki, Y. (2001). Block copolymer micelles for drug delivery: design, characterization and biological significance. Advanced drug delivery reviews 47, 113-131.
Kipnes, M., Dandona, P., Tripathy, D., Still, J.G., and Kosutic, G. (2003). Control of postprandial plasma glucose by an oral insulin product (HIM2) in patients with type 2 diabetes. Diabetes Care 26, 421.
Kompella, U.B., and Lee, V.H.L. (2001). Delivery systems for penetration enhancement of peptide and protein drugs: design considerations. Advanced drug delivery reviews 46, 211-245.
Leone-Bay, A., Ho, K.K., Agarwal, R., Baughman, R.A., Chaudhary, K., DeMorin, F., Genoble, L., McInnes, C., Lercara, C., and Milstein, S. (1996). 4-[4-[(2-Hydroxybenzoyl) amino] phenyl] butyric acid as a novel oral delivery agent for recombinant human growth hormone. J Med Chem 39, 2571-2578.
Leone-Bay, A., Paton, D.R., and Weidner, J.J. (2000). The development of delivery agents that facilitate the oral absorption of macromolecular drugs. Medicinal Research Reviews 20.
Li, A.P. (2001). Screening for human ADME/Tox drug properties in drug discovery. Drug discovery today 6, 357-366.
Li, H., An, J.H., Park, J.S., and Han, K. (2005). Multivesicular liposomes for oral delivery of recombinant human epidermal growth factor. Archives of pharmacal research 28, 988-994.
Li, H., Song, J.H., Park, J.S., and Han, K. (2003). Polyethylene glycol-coated liposomes for oral delivery of recombinant human epidermal growth factor. International journal of pharmaceutics 258, 11-19.
Lieschke, G., Grail, D., Hodgson, G., Metcalf, D., Stanley, E., Cheers, C., Fowler, K., Basu, S., Zhan, Y., and Dunn, A. (1994). Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 84, 1737.
Lin, C., and Wang, K. (2003). The fuel properties of three-phase emulsions as an alternative fuel for diesel engines. Fuel 82, 1367-1375.
Lowman, A.M., Morishita, M., Kajita, M., Nagai, T., and Peppas, N.A. (1999). Oral delivery of insulin using pH-responsive complexation gels. Journal of pharmaceutical sciences 88, 933-937.
Lv, F.F., Li, N., Zheng, L.Q., and Tung, C.H. (2006). Studies on the stability of the chloramphenicol in the microemulsion free of alcohols. European Journal of Pharmaceutics and Biopharmaceutics 62, 288-294.
Lyons, K.C., Charman, W.N., Miller, R., and Porter, C.J.H. (2000). Factors limiting the oral bioavailability of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion. International journal of pharmaceutics 199, 17-28.
Mackay, M. (1990). Delivery of recombinant peptide and protein drugs. Biotechnology & genetic engineering reviews 8, 251.
Mahato, R.I., Narang, A.S., Thoma, L., and Miller, D.D. (2003). Emerging trends in oral delivery of peptide and protein drugs. Critical reviews in therapeutic drug carrier systems 20, 153-214.
Mataumoto, S., and Kang, W. (1989). Formation and applications of multiple emulsions. Journal of Dispersion Science and Technology 10, 455-482.
Mathiowitz, E., Jacob, J.S., Jong, Y.S., Carino, G.P., Chickering, D.E., Chaturvedi, P., Santos, C.A., Vijayaraghavan, K., Montgomery, S., and Bassett, M. (1997). Biologically erodable microspheres as potential oral drug delivery systems.
Matsumoto, S. (1986). W/O/W-type multiple emulsions with a view to possible food application 1. Journal of Texture Studies 17, 141-159.
Matsumoto, S., Kita, Y., and Yonezawa, D. (1976). An attempt at preparing water-in-oil-in-water multiple phase emulsions. Journal of colloid and interface science 57, 353-361.
Matsuzaki, G., Li, X., Ohyama, Y., and Nomoto, K. (1996). Kinetics of serum granulocyte-colony stimulating factor (G-CSF) concentration and G-CSF receptor expression during G-CSF treatment of cyclophosphamide-treated mice. International journal of immunopharmacology 18, 363-369.
Matsuzawa, A., Morishita, M., Takayama, K., and Nagai, T. (1995). Absorption of insulin water-in-oil-in-water emulsion from an enteral loop in rats. Biological & pharmaceutical bulletin 18, 1718-1723.
Modi, N.B. (1994). Pharmacokinetics and pharmacodynamics of recombinant proteins and peptides. Journal of controlled release 29, 269-281.
Mokrushin, S.G. (1962). Thomas Graham and the Definition of Colloids. Nature 195, 861.
Morishita, M., Goto, T., Nakamura, K., Lowman, A.M., Takayama, K., and Peppas, N.A. (2006). Novel oral insulin delivery systems based on complexation polymer hydrogels: Single and multiple administration studies in type 1 and 2 diabetic rats. Journal of Controlled Release 110, 587-594.
Morishita, M., Matsuzawa, A., Takayama, K., Isowa, K., and Nagai, T. (1998). Improving insulin enteral absorption using water-in-oil-in-water emulsion. International Journal of Pharmaceutics 172, 189-198.
Muschiolik, G. (2007). Multiple emulsions for food use. Current Opinion in Colloid & Interface Science 12, 213-220.
O'Hagan, D. (1994). Novel delivery systems for oral vaccines (CRC).
Oba, N., Sugimura, H., Umehara, Y., Yoshida, M., Kimura, T., and Yamaguchi, T. (1992). Evaluation of an oleic acid water-in-oil-in-water-type multiple emulsion as potential drug carriervia the enteral route. Lipids 27, 701-705.
Ohvo-Rekila, H., Ramstedt, B., Leppimaki, P., and Peter Slotte, J. (2002). Cholesterol interactions with phospholipids in membranes. Progress in lipid research 41, 66-97.
Okochi, H., and Nakano, M. (2000). Preparation and evaluation of w/o/w type emulsions containing vancomycin. Advanced drug delivery reviews 45, 5-26.
Ongpipattanakul, B., Burnette, R., Potts, R., and Francoeur, M. (1991). Evidence that oleic acid exists in a separate phase within stratum corneum lipids. Pharmaceutical research 8, 350-354.
Onuki, Y., Morishita, M., and Takayama, K. (2004). Formulation optimization of water-in-oil-water multiple emulsion for intestinal insulin delivery. Journal of Controlled Release 97, 91-99.
Pan, Y., Li, Y., Zhao, H., Zheng, J., Xu, H., Wei, G., Hao, J., and Cui, F. (2002). Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo. International journal of pharmaceutics 249, 139-147.
Park, K., Shalaby, W., and Park, H. (1993). Biodegradable hydrogels for drug delivery (CRC Press).
Patton, J.S., and Bossard, M.J. (2004). Drug delivery strategies for proteins and peptides from discovery and development to life cycle management. Drug Deliv Technol 4, 73–77.
Peracchia, M.T., Fattal, E., Desmaele, D., Besnard, M., Noel, J.P., Gomis, J.M., Appel, M., d’Angelo, J., and Couvreur, P. (1999). StealthR PEGylated polycyanoacrylate nanoparticles for intravenous administration and splenic targeting. Journal of Controlled Release 60, 121-128.
Pisani, J., and Bonduelle, Y. (2006). Opportunities and barriers in the biosimilar market: evolution or revolution for generics companies (BioPharm Knowledge Publishing, London).
Qiu, Y., and Park, K. (2001). Environment-sensitive hydrogels for drug delivery. Advanced drug delivery reviews 53, 321-339.
Quintanar-Guerrero, D., Allemann, E., Fessi, H., and Doelker, E. (1998). Preparation techniques and mechanisms of formation of biodegradable nanoparticles from preformed polymers. Drug development and industrial pharmacy 24, 1113.
Ritschel, W.A. (1991). Microemulsions for improved peptide absorption from the gastrointestinal tract. Methods and findings in experimental and clinical pharmacology 13, 205-220.
Russell-Jones, G.J. (2004). Use of targeting agents to increase uptake and localization of drugs to the intestinal epithelium. Journal of drug targeting 12, 113.
Schmalfuss, U., Neubert, R., and Wohlrab, W. (1997). Modification of drug penetration into human skin using microemulsions. Journal of Controlled Release 46, 279-285.
Sehgal, A. (2004). Peptides: markets and trends. In: Peptide Thrapeutics – Applications in the Treatment of Human Disease.
Sethia, S., and Squillante, E. (2004). In vitro-in vivo evaluation of supercritical processed solid dispersions: Permeability and viability assessment in Caco-2 cells. Journal of pharmaceutical sciences 93.
Shichiri, M., Shimizu, Y., Yoshida, Y., Kawamori, R., Fukuchi, M., Shigeta, Y., and Abe, H. (1974). Enteral absorption of water-in-oil-in-water insulin emulsions in rabbits. Diabetologia 10, 317-321.
Shinoda, K., Araki, M., Sadaghiani, A., Khan, A., and Lindman, B. (1991). Lecithin-based microemulsions: phase behavior and microstructure. The Journal of Physical Chemistry 95, 989-993.
Shinoda, K., Yoneyama, T., and Tsutsumi, H. (1980). Evaluation of emulsifier blending. Journal of Dispersion Science and Technology 1, 1-12.
Shively, M.L. (1997). Multiple emulsions for the delivery of proteins. Pharm Biotechnol 10, 199-211.
Silva-Cunha, A., Cheron, M., Grossiord, J.L., Puisieux, F., and Seiller, M. (1998). W/O/W multiple emulsions of insulin containing a protease inhibitor and an absorption enhancer: biological activity after oral administration to normal and diabetic rats. International Journal of Pharmaceutics 169, 33-44.
Silva-Cunha, A., Grossiord, J.L., Puisieux, F., and Seiller, M. (1997). W/O/W multiple emulsions of insulin containing a protease inhibitor and an absorption enhancer: preparation, characterization and determination of stability towards proteases in vitro. International Journal of Pharmaceutics 158, 79-89.
Singh, R., Singh, S., and Lillard, J. (2008). Past, present, and future technologies for oral delivery of therapeutic proteins. Journal of pharmaceutical sciences 97.
Sinha, V.R., and Trehan, A. (2003). Biodegradable microspheres for protein delivery. Journal of controlled release 90, 261-280.
Sood, A., and Panchagnula, R. (2001). Peroral route: an opportunity for protein and peptide drug delivery. Chem Rev 101, 3275-3303.
Stoll, B.R., Leipold, H.R., Milstein, S., and Edwards, D.A. (2000). A mechanistic analysis of carrier-mediated oral delivery of protein therapeutics. Journal of Controlled Release 64, 217-228.
Takahashi, Y., Yoshida, T., and Takahashi, T. (1990). Process for producing a W/O/W type multiple emulsion for medicines, cosmetics, etc (Google Patents).
Takeuchi, H., Matsui, Y., Yamamoto, H., and Kawashima, Y. (2003). Mucoadhesive properties of carbopol or chitosan-coated liposomes and their effectiveness in the oral administration of calcitonin to rats. Journal of Controlled Release 86, 235-242.
Tang, F., and Borchardt, R.T. (2002). Characterization of the efflux transporter (s) responsible for restricting intestinal mucosa permeation of an acyloxyalkoxy-based cyclic prodrug of the opioid peptide DADLE. Pharmaceutical research 19, 780-786.
Tenjarla, S. (1999). Microemulsions: an overview and pharmaceutical applications. Critical reviews in therapeutic drug carrier systems 16, 461.
Thomas, J., Liu, F., and Link, D.C. (2002). Mechanisms of mobilization of hematopoietic progenitors with granulocyte colony-stimulating factor. Curr Opin Hematol 9, 183-189.
Vanz, A., Renard, G., Palma, M., Chies, J., Dalmora, S., Basso, L., and Santos, D. (2008). Human granulocyte colony stimulating factor(hG-CSF): cloning, overexpression, purification and characterization. Microbial Cell Factories 7, 13.
Vaziri, A., and Warburton, B. (1994). Some preparative variables influencing the properties of W/O/W multiple emulsions. Journal of microencapsulation 11, 649-656.
Veronese, F.M., and Pasut, G. (2005). PEGylation, successful approach to drug delivery. Drug discovery today 10, 1451-1458.
Vilhardt, H., and Lundin, S. (1986). In vitro intestinal transport of vasopressin and its analogues. Acta Physiologica Scandinavica 126, 601-607.
Wang, L., Ma, J., Pan, W., Toledo-Velasquez, D., Malanga, C., and Rojanasakul, Y. (1994). Alveolar permeability enhancement by oleic acid and related fatty acids: evidence for a calcium-dependent mechanism. Pharmaceutical research 11, 513-517.
Widera, A., Kim, K., Crandall, E., and Shen, W. (2003). Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers. Pharmaceutical research 20, 1231-1238.
Yamada, K., Murakami, M., Yamamoto, A., Takada, K., and Muranishi, S. (1992). Improvement of intestinal absorption of thyrotropin-releasing hormone by chemical modification with lauric acid. J Pharm Pharmacol 44, 717-721.
Yamaguchi, T., Kogi, M., Yamamoto, Y., and Hayakawa, T. (1997). Bioassay of human granulocyte colony-stimulating factor using human promyelocytic HL-60 cells. Biological & pharmaceutical bulletin 20, 943-947.
Yamamoto, A., Iwata, A., Tuchiya, K., Katsumata, A., Oishi, K., Saito, T., Tsujimoto, H., Hasegawa, A., and Ueda, S. (2001). Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor. Gene 274, 263-269.
Yamamoto, A., Taniguchi, T., Rikyuu, K., Tsuji, T., Fujita, T., Murakami, M., and Muranishi, S. (1994). Effects of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. Pharmaceutical research 11, 1496-1500.
Yuan, X., Harada, A., Yamasaki, Y., and Kataoka, K. (2005). Stabilization of Lysozyme-Incorporated Polyion Complex Micelles by the [omega]-End Derivatization of Poly (ethylene glycol)? Poly ([alpha],[beta]-aspartic acid) Block Copolymers with Hydrophobic Groups. Langmuir 21, 2668-2674.
Zhang, Y., Bryant, J., Charles, A., Boado, R.J., and Pardridge, W.M. (2004). Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer (AACR), pp. 3667-3677.
Zhou, X. (1994). Overcoming enzymatic and absorption barriers to non-parenterally administered protein and peptide drugs. Journal of controlled release 29, 239-252.