Rabbit as an Animal Model for Pharmacokinetics Studies of Enteric Capsule Contains Recombinant Human Keratinocyte Growth Factor Loaded Chitosan Nanoparticles

Author(s): Palanirajan V. Kumar*, Marwan A. Abdelkarim Maki, Yeong S. Wei, Lee M. Tatt, Manogaran Elumalai, Shiau-Chuen Cheah, Bharathy Raghavan, Abu Bakar Bin A. Majeed

Journal Name: Current Clinical Pharmacology
Continued as Current Reviews in Clinical and Experimental Pharmacology

Volume 14 , Issue 2 , 2019

Graphical Abstract:


Background: Recombinant human keratinocyte growth factor (rHuKGF) has gained considerable attention by researchers as epithelial cells proliferating agent. Moreover, intravenous truncated rHuKGF (palifermin) has been approved by Food and Drug Administration (FDA) to treat and prevent chemotherapy-induced oral mucositis and small intestine ulceration. The labile structure and short circulation time of rHuKGF in-vivo are the main obstacles that reduce the oral bioactivity and dosage of such proteins at the target site.

Objective: Formulation of methacrylic acid-methyl methacrylate copolymer-coated capsules filled with chitosan nanoparticles loaded with rHuKGF for oral delivery.

Methods: We report on chitosan nanoparticles (CNPs) with diameter < 200 nm, prepared by ionic gelation, loaded with rHuKGF and filled in methacrylic acid-methyl methacrylate copolymercoated capsules for oral delivery. The pharmacokinetic parameters were determined based on the serum levels of rHuKGF, following a single intravenous (IV) or oral dosages using a rabbit model. Furthermore, fluorescent microscope imaging was conducted to investigate the cellular uptake of the rhodamine-labelled rHuKGF-loaded nanoparticles. The proliferation effect of the formulation on FHs 74 Int cells was studied as well by MTT assay.

Results: The mucoadhesive and absorption enhancement properties of chitosan and the protective effect of methacrylic acid-methyl methacrylate copolymer against rHuKGF release at the stomach, low pH, were combined to promote and ensure rHuKGF intestinal delivery and increase serum levels of rHuKGF. In addition, in-vitro studies revealed the protein bioactivity since rHuKGFloaded CNPs significantly increased the proliferation of FHs 74 Int cells.

Conclusion: The study revealed that oral administration of rHuKGF–loaded CNPs in methacrylic acid-methyl methacrylate copolymer-coated capsules is practically alternative to the IV administration since the absolute bioavailability of the orally administered rHuKGF–loaded CNPs, using the rabbit as animal model, was 69%. Fluorescent microscope imaging revealed that rhodaminelabelled rHuKGF-loaded CNPs were taken up by FHs 74 Int cells, after 6 hours’ incubation time, followed by increase in the proliferation rate.

Keywords: Recombinant human keratinocyte growth factor, chitosan nanoparticles, proliferation, fluorescence imaging, protein delivery, pharmacokinetics, bioavailability.

Hattori Y, Yamasaki M, Konishi M, Itoh N. Spatially restricted expression of fibroblast growth factor-10 mRNA in the rat brain. Brain Res Mol Brain Res 1997; 47(1-2): 139-46.
[http://dx.doi.org/10.1016/S0169-328X(97)00044-2] [PMID: 9221911]
Ohuchi H, Nakagawa T, Yamamoto A, et al. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development 1997; 124(11): 2235-44.
[PMID: 9187149]
Guo L, Degenstein L, Fuchs E. Keratinocyte growth factor is required for hair development but not for wound healing. Genes Dev 1996; 10(2): 165-75.
[http://dx.doi.org/10.1101/gad.10.2.165] [PMID: 8566750]
Park JW, Hwang SR, Yoon IS. Advanced Growth Factor De-livery Systems in Wound Management and Skin Regeneration. Molecules 2017; 22: 1259.
Kitsberg DI, Leder P. Keratinocyte growth factor induces mammary and prostatic hyperplasia and mammary adenocarcinoma in transgenic mice. Oncogene 1996; 13(12): 2507-15.
[PMID: 9000125]
Finch PW, Mark Cross LJ, McAuley DF, Farrell CL. Palifermin for the protection and regeneration of epithelial tissues following injury: new findings in basic research and pre-clinical models. J Cell Mol Med 2013; 17(9): 1065-87.
[http://dx.doi.org/10.1111/jcmm.12091] [PMID: 24151975]
Ishikawa A, Kudo M, Nakazawa N, et al. Expression of keratinocyte growth factor and its receptor in human endometrial cancer in cooperation with steroid hormones. Int J Oncol 2008; 32(3): 565-74.
[http://dx.doi.org/10.3892/ijo.32.3.565] [PMID: 18292933]
Beaven AW, Shea TC. Recombinant human keratinocyte growth factor palifermin reduces oral mucositis and improves patient outcomes after stem cell transplant. Drugs Today (Barc) 2007; 43(7): 461-73.
[http://dx.doi.org/10.1358/dot.2007.43.7.1119723] [PMID: 17728847]
Blijlevens N, Sonis S. Palifermin (recombinant keratinocyte growth factor-1): a pleiotropic growth factor with multiple biological activities in preventing chemotherapy- and radiotherapy-induced mucositis. Ann Oncol 2007; 18(5): 817-26.
[http://dx.doi.org/10.1093/annonc/mdl332] [PMID: 17030544]
Morris J, Rudebeck M, Neudorf S, et al. Safety, pharmacoki-netics, and efficacy of palifermin in children and adolescents with acute leukemias undergoing myeloablative therapy and allogeneic hematopoietic stem cell transplantation: a pediatric blood and marrow transplant consortium trial. Biol Blood Marrow Transplant 2016; 22(7): 1247-56.
[http://dx.doi.org/10.1016/j.bbmt.2016.02.016] [PMID: 26968792]
Pinakini K, Bairy K. Palifermin: A keratinocyte growth factor for oral mucositis. Indian J Pharmacol 2005; 37(5): 338.
Gullotti E, Yeo Y. Extracellularly activated nanocarriers: a new paradigm of tumor targeted drug delivery. Mol Pharm 2009; 6(4): 1041-51.
[http://dx.doi.org/10.1021/mp900090z] [PMID: 19366234]
Hobbs SK, Monsky WL, Yuan F, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci USA 1998; 95(8): 4607-12.
[http://dx.doi.org/10.1073/pnas.95.8.4607] [PMID: 9539785]
Sun M, Sun B, Liu Y, Shen QD, Jiang S. Dual-color fluores-cence imaging of magnetic nanoparticles in live cancer cells using conjugated polymer probes. Sci Rep 2016; 6: 22368.
[http://dx.doi.org/10.1038/srep22368] [PMID: 26931282]
Nishimori H, Kondoh M, Isoda K, Tsunoda S, Tsutsumi Y, Yagi K. Silica nanoparticles as hepatotoxicants. Eur J Pharm Biopharm 2009; 72(3): 496-501.
[http://dx.doi.org/10.1016/j.ejpb.2009.02.005] [PMID: 19232391]
Moser F, Hildenbrand G, Müller P, et al. Cellular uptake of gold nanoparticles and their behavior as labels for localization microscopy. Biophys J 2016; 110(4): 947-53.
[http://dx.doi.org/10.1016/j.bpj.2016.01.004] [PMID: 26910431]
Shang L, Nienhaus K, Jiang X, et al. Nanoparticle interactions with live cells: Quantitative fluorescence microscopy of nanoparticle size effects. Beilstein J Nanotechnol 2014; 5: 2388-97.
[http://dx.doi.org/10.3762/bjnano.5.248] [PMID: 25551067]
Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul 2001; 41(1): 189-207.
[http://dx.doi.org/10.1016/S0065-2571(00)00013-3] [PMID: 11384745]
Chang SJ, Niu GC, Kuo SM, Chen SF. Preparation and preliminary characterization of concentric multi-walled chitosan microspheres. J Biomed Mater Res A 2007; 81(3): 554-66.
[http://dx.doi.org/10.1002/jbm.a.31084] [PMID: 17133452]
Wang JJ, Zeng ZW, Xiao RZ, et al. Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomedicine 2011; 6: 765-74.
[PMID: 21589644]
Cao JN, Zhou J. Progress in antitumor studies of chitosan. Chin J Biochem Pharm 2005; 26(2): 127-37.
Xia G, Liu Y, Tian M, et al. Nanoparticles/thermosensitive hydrogel reinforced with chitin whiskers as a wound dressing for treating chronic wounds. J Mater Chem 2017; 5(17): 3172-85.
Hans ML, Lowman AM. Biodegradable nanoparticles for drug delivery and targeting. Curr Opin Solid State Mater Sci 2002; 6(4): 319-27.
Pan Y, Li YJ, Zhao HY, et al. Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo. Int J Pharm 2002; 249(1-2): 139-47.
[http://dx.doi.org/10.1016/S0378-5173(02)00486-6] [PMID: 12433442]
Calvo P, Remuñan-López C, Vila-Jato JL, Alonso MJ. Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines. Pharm Res 1997; 14(10): 1431-6.
[http://dx.doi.org/10.1023/A:1012128907225] [PMID: 9358557]
Fan W, Yan W, Xu Z, Ni H. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf B Biointerfaces 2012; 90: 21-7.
[http://dx.doi.org/10.1016/j.colsurfb.2011.09.042] [PMID: 22014934]
Lee MK, Kim MY, Kim S, Lee J. Cryoprotectants for freeze drying of drug nano-suspensions: effect of freezing rate. J Pharm Sci 2009; 98(12): 4808-17.
[http://dx.doi.org/10.1002/jps.21786] [PMID: 19475555]
Varshosaz J, Eskandari S, Tabbakhian M. Freeze-drying of nanostructure lipid carriers by different carbohydrate poly-mers used as cryoprotectants. Carbohydr Polym 2012; 88(4): 1157-63.
Zhang HL, Wu SH, Tao Y, Zang LQ, Su ZQ. Preparation and characterization of water-soluble chitosan nanoparticles as protein delivery system. J Nanomater 2010; 1.
KGF (FGF-7) Human SimpleStep ELISA® Kit. In: abcam, editor. 2016.
Lachman L, Lieberman HA, Kanig JL. The theory and prac-tice of industrial pharmacy. Philadelphia: Lea & Febiger 1976.
Cole ET, Scott RA, Connor AL, et al. Enteric coated HPMC capsules designed to achieve intestinal targeting. Int J Pharm 2002; 231(1): 83-95.
[http://dx.doi.org/10.1016/S0378-5173(01)00871-7] [PMID: 11719017]
Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.
[http://dx.doi.org/10.1016/0022-1759(83)90303-4] [PMID: 6606682]
van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM. A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 1994; 174(1-2): 311-20.
[http://dx.doi.org/10.1016/0022-1759(94)90034-5] [PMID: 8083535]
Ferrari M, Fornasiero MC, Isetta AM. MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 1990; 131(2): 165-72.
[http://dx.doi.org/10.1016/0022-1759(90) 90187-Z] [PMID: 2391427]
Gerlier D, Thomasset N. Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 1986; 94(1-2): 57-63.
[http://dx.doi.org/10.1016/0022-1759(86)90215-2] [PMID: 3782817]
Scudiero DA, Shoemaker RH, Paull KD, et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988; 48(17): 4827-33.
[PMID: 3409223]
Chitra K, Annadurai G. Synthesis and characterization of dye coated fluorescent chitosan nanoparticles. J Acad Ind Res 2012; 15: 199.
[http://dx.doi.org/10.3390/ijms160920943] [PMID: 26340627]
Palanirajan VK, Marwan AM, Mhd LT, Yeong SW, Lee MT, Abu Bakar BM. Detection of Formation of Recombinant Hu-man Keratinocyte Growth Factor Loaded Chitosan Nanoparti-cles Based on its Optical Properties. Curr Nanosci 2018; 14(2): 127-35.
Palanirajan VK, Marwan AM, Lee MT, Yeong SW, Abu Bakar BM, Eds. UV-spectroscopy Method for Detecting the Chi-tosan Nanoparticles Formation. Proceedings of the Interna-tional Conference of Applied Nanotechnology and Nanoscience. 2017 Oct 18-20; Rome, Italy. 293.
Konturek SJ, Brzozowski T, Konturek JW, Slomiany BL. Growth factors in gastric mucosal integrity, protection and healing of acute and chronic ulcerations. In The stomach 1993; pp. 159-76.
Qian Z, Dougherty PG, Pei D. Targeting intracellular protein-protein interactions with cell-permeable cyclic peptides. Curr Opin Chem Biol 2017; 38: 80-6.
[http://dx.doi.org/10.1016/j.cbpa.2017.03.011] [PMID: 28388463]
Wang T, Wang L, Li X, et al. Size-dependent regulation of intracellular trafficking of polystyrene nanoparticle-based drug delivery carriers. ACS Appl Mater Interfaces 2017; 9(22): 18619-25.
[http://dx.doi.org/10.1021/acsami.7b05383] [PMID: 28497682]
MacParland SA, Tsoi KM, Ouyang B, et al. Phenotype deter-mines nanoparticle uptake by human macrophages from liver and blood. ACS Nano 2017; 11(3): 2428-43.
[http://dx.doi.org/10.1021/acsnano.6b06245] [PMID: 28040885]
Liang J, Yan H, Puligundla P, Gao X, Zhou Y, Wan X. Appli-cations of chitosan nanoparticles to enhance absorption and bioavailability of tea polyphenols: A review. Food Hydrocoll 2017; 69: 286-92.
Patra CN, Priya R, Swain S, Jena GK, Panigrahi KC, Ghose D. Pharmaceutical significance of Eudragit: A review. Future J Pharm Sci 2017; 3(1): 33-45.
Dai J, Nagai T, Wang X, Zhang T, Meng M, Zhang Q. pH-sensitive nanoparticles for improving the oral bioavailability of cyclosporine A. Int J Pharm 2004; 280(1-2): 229-40.
[http://dx.doi.org/10.1016/j.ijpharm.2004.05.006] [PMID: 15265562]
Kumar J, Newton AMJ. Rifaximin - Chitosan Nanoparticles for Inflammatory Bowel Disease (IBD). Recent Pat Inflamm Allergy Drug Discov 2017; 11(1): 41-52.
[http://dx.doi.org/10.2174/1872213X10666161230111226] [PMID: 28034350]
Li L, Jiang G, Yu W, et al. Preparation of chitosan-based multifunctional nanocarriers overcoming multiple barriers for oral delivery of insulin. Mater Sci Eng C 2017; 70(Pt 1): 278-86.
[http://dx.doi.org/10.1016/j.msec.2016.08.083] [PMID: 27770892]
Banerjee A, Lee J, Mitragotri S. Intestinal mucoadhesive devices for oral delivery of insulin. Bioeng Transl Med 2016; 1(3): 338-46.
[http://dx.doi.org/10.1002/btm2.10015] [PMID: 29313019]
Yin L, Wang Y, Wang C, Feng M. Nano-reservoir Bioadhesive Tablets Enhance Protein Drug Permeability Across the Small Intestine. AAPS PharmSciTech 2017; 18(6): 2329-35.
[http://dx.doi.org/10.1208/s12249-016-0709-6] [PMID: 28116599]
Zhang J, Zhu X, Jin Y, Shan W, Huang Y. Mechanism study of cellular uptake and tight junction opening mediated by goblet cell-specific trimethyl chitosan nanoparticles. Mol Pharm 2014; 11(5): 1520-32.
[http://dx.doi.org/10.1021/mp400685v] [PMID: 24673570]

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Article Details

Year: 2019
Published on: 19 November, 2018
Page: [132 - 140]
Pages: 9
DOI: 10.2174/1574884714666181120103907

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