INVESTIGATIONS INTO THE TRANSPORT PROPERTIES OF ANGIOTENSIN PEPTIDES CHUA HUI LEE NATIONAL UNIVERSITY OF SINGAPORE 2004 INVESTIGATIONS INTO THE TRANSPORT PROPERTIES OF ANGIOTENSIN PEPTIDES CHUA HUI LEE (B Sc (Pharm.) (Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE (PHARMACY) DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2004 To my family ACKNOWLEDGEMENTS My sincere appreciation goes to my supervisor, Associate Professor Go Mei Lin, for her guidance and support during the course of this research I am grateful to my co-supervisor, Associate Professor Sim Meng Kwoon in the Department of Pharmacology, whose advice has been invaluable Special thanks are extended to Assistant Professor Seetharama D.S Jois in the Department of Pharmacy, for his assistance with the interpretation of CD and NMR spectra, as well as with the operation of Insight II I am also thankful to the National University of Singapore for awarding me the Research Scholarship, and all the lecturers, technical staff, fellow students and friends in the Department of Pharmacy for their help and friendship, in particular, Mr Bong Yong Koi, Miss Liu Jining, and Mr Wu Xiang I would also like to show my appreciation to those technical staff and friends in the Department of Biological Science and the Department of Pharmacology, who have helped me along Last but not least, I would like to express my heart-felt gratitude to my family members, especially my parents, and Mr Kang Tse Siang, for their patience and understanding I am deeply grateful to them for sharing my laughter, joys and frustration, and I would like to share this triumph moment with them i TABLE OF CONTENTS TITLE ACKNOWLEDGEMENT i TABLE OF CONTENTS ii-v SUMMARY vi-vii CONFERENCE PRESENTATIONS viii TABLE OF CONTENTS SECTION ONE: INTRODUCTION 1-15 1.1 Transport of Oligopeptides Across the Intestinal Epithelium 1.1.1 Paracellular transport of oligopeptides 1.1.2 Transcellular transport of oligopeptides 1.1.3 Active transport of oligopeptides 10 1.1.4 Transcytosis of oligopeptides 10 1.1.5 Membrane translocation of oligopeptides 12 1.2 Angiotensin Peptides SECTION TWO: AIM OF THESIS 13 16-17 SECTION THREE: TRANSPORT OF ANGIOTENSIN PEPTIDES ACROSS THE Caco-2 CELL MONOLAYERS 18-53 3.1 Introduction 18 ii 3.2 Experimental 19 3.2.1 Materials 19 3.2.2 Purification of crude des-Asp angiotensin I 20 3.2.3 Caco-2 cell culture 21 3.2.3.1 Culture of Caco-2 cells 21 3.2.3.2 Cryopreservation of Caco-2 cells 22 3.2.3.3 Revival of Caco-2 cells 23 Monitoring integrity of Caco-2 cell monolayers 23 3.2.4.1 Transepithelial electrical resistance (TEER) 23 3.2.4.2 Transport of lucifer yellow 24 3.2.4 3.2.5 3.2.6 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess viability of Caco-2 cells 25 Peptide stability 26 3.2.6.1 Stability of the peptides in HBSS- HEPES buffer (pH 7.4) 26 3.2.7 3.2.8 3.3 3.2.6.2 Metabolic stability of the peptides 26 Transport of peptides across Caco-2 cell monolayers 27 3.2.7.1 Transport of non- radiolabelled peptides 27 3.2.7.2 Transport of radiolabelled peptides 28 3.2.7.3 Transport of peptides in the presence of inhibitors 29 Statistical analysis 30 Results and Discussion 30 3.3.1 Monitoring integrity of Caco-2 cell monolayers 30 3.3.2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to 3.3.3 assess viability of Caco-2 cells 34 Peptide stability 37 iii 3.3.4 Transport of peptides across Caco-2 cells 39 3.3.4.1 Transport of DAA-I across Caco-2 cell monolayers 40 3.3.4.2 Transport of Ang III across the Caco-2 cell monolayers 45 3.3.4.3 Transport of Ang IV across Caco-2 cell monolayers 3.4 Conclusion 48 51 SECTION FOUR: EFFECT OF PEPTIDE STRUCTURE ON TRANSPORT PROPERTIES OF ANGIOTENSIN PEPTIDES 54-97 4.1 Introduction 54 4.2 Experimental 55 4.2.1 Materials 55 4.2.2 Circular dichroism (CD) spectroscopy 55 4.2.3 Nuclear magnetic resonance (NMR) spectroscopy 56 4.2.4 Molecular modelling 56 4.2.5 Determination of lipophilicity by reversed phase HPLC 58 4.2.6 Calculation of hydrogen bonding potential 59 4.3 Results and Discussion 59 4.3.1 59 Determination of solution conformation 4.3.1.1 Determination of solution conformation by circular dichroism (CD) spectroscopy 60 4.3.1.2 Determination of solution conformation by nuclear magnetic resonance (NMR) spectroscopy 4.3.1.3 Molecular modelling of the angiotensin peptides 4.3.2 69 79 Determination of physicochemical and size parameters of the angiotensin peptides 88 iv 4.3.3 Structure- transport correlations of angiotensin peptides 91 4.3.4 Conclusion 95 SECTION FIVE: CONCLUSION 98-99 APPENDICES AI-AIX BIBLIOGRAPHY I-X v SUMMARY The therapeutic potential of peptides is limited by poor oral bioavailability due to their susceptibility to enzymatic degradation in the gastrointestinal tract and unfavourable physicochemical properties Des- Asp angiotensin I (DAA-I) is an example of a physiological peptide which can be potentially used for the treatment of cardiovascular diseases (namely, the attenuation of post- infarction injuries and neointima growth in catheter- injured arteries) Although preliminary studies suggest that DAA-I is able to transverse the intestinal epithelium, the extent of transport and its transport route have not been investigated in detail By systematically examining the transport properties of DAA-I across Caco-2 monolayers, this project serves to address these issues in greater detail DAA-I is structurally related to angiotensin III (Ang III) and angiotensin IV (Ang IV) In this investigation, we are interested in comparing the transport properties of these structurally related peptides and examining how their transport would be affected by physicochemical properties (lipophilicity, hydrogen- bonding potential, charge) and structure (area, volume, conformation) The metabolic stability and the transport properties of these peptides were examined using the Caco-2 cell monolayers as a surrogate model for the intestinal epithelium Lipophilicity was evaluated experimentally by reverse phase chromatography The secondary structure of the peptides were investigated using circular dichroism and 1H-NMR and the data subsequently used together with a molecular modelling software to construct probable low energy conformation(s) of the peptide Physicochemical properties like volume, polar surface area, hydrogen bonding potential, ClogP were obtained from these minimised conformations vi The angiotensin peptides exhibited limited permeability (P app ≈ 10 -8 to 10 -9 cm/sec) across the Caco-2 cell monolayers They were largely stable to enzymatic degradation in the presence of the Caco-2 cells Different routes of transport were observed for the peptides The transport of DAA-I shows many characteristics of transcellular diffusion, in contrast to Ang IV which is transported by an energy-requiring pathway More than one route is implicated for Ang III, depending on concentration of the peptide Spectroscopic data and molecular modelling suggest the presence of secondary structures in Ang III and Ang IV, but not in DAA-I Ang III appears to have more conformational options than Ang IV As to whether the transport properties of the peptides are related to their physicochemical and structural characteristics, there is support from the present findings that transport is influenced by the conformational characteristics of the peptides vii Transport of Ang IV (1 nM) 444 ± 14 369 ± 57 (+ 1.25 mM EDTA) AVI Figure Separation and analysis of angiotensin peptides Figure 4A Elution of N-Benzoyl-Gly-His-Leu (internal standard), Ang III, Ang IV and DAA-I 1.9 Ang III Absorbance 1.4 Internal standard 0.9 Ang IV DAA-I 0.4 -0.1 10 15 20 Time/ minutes Figure 4B HPLC separation of the peptide solution DAA-I after metabolism study in the presence of cells 0.9 0.7 Absorbance DAA-I 0.5 Ang IV 0.3 0.1 -0.1 10 15 20 Time/ minutes AVII Figure 4C Identification of metabolite peak by ESI-MS Figure 4D HPLC separation of the peptide solution Ang III after metabolism study in the presence of cells 2.5 2.0 Absorbance Ang III 1.5 1.0 0.5 -0.1 10 12 Time/ minutes 14 16 18 20 AVIII Figure 4E HPLC separation of the peptide solution Ang IV after metabolism study in the presence of cells 2.5 2.0 Absorbance Ang IV 1.5 1.0 0.5 -0.1 10 12 14 16 18 20 Time/ minutes The amount of non- radiolabelled peptides transported across the Caco-2 cell monolayers was quantified by HPLC in the presence of an internal standard (N-Benzoyl-Gly-His-Leu) (Sigma Aldrich Corporation) (St Louis, MO) AIX BIBLIOGRAPHY AX BIBLIOGRAPHY Lee V.H.L., Yamamoto A Penetration and enzymatic barriers to peptides and protein absorption Adv Drug Del Rev., 4: 171-207 (1990) Bladon C.M Pharmaceutical Chemistry: Therapeutic aspects of biomacromolecules (ed Bladon C.M.), John Wiley & Sons Ltd, England, Chapter 1: 1-7 (2002) Parker K.L., Schimmer B.P Pituitary Hormones and Their Hypothalamic Releasing Factors Goodman and Gilman’s the pharmacological basis of therapeutics 10th edition (ed Hardman JG, Limbird LE, Gilman A.G.) Chapter 56: 1555 (2002) a Gao J., Winslow S.L., Velde D.V., Aubé J., Borchardt R.T Transport characteristics of peptides and peptidomimetics: II Hydroxyethylamine bioisostere-containing peptidomimetics as substrates for the oligopeptide transporter and P-glycoprotein in the intestinal mucosa J Peptide Res., 57: 361373 (2001) b Pauletti G.M., Gangwar S., Siahaan T.J., Aubé J., R.T Borchardt Improvement of oral peptide bioavailability: peptidomimetics and prodrug strategies Adv Drug Del Rev., 27: 235-256 (1997) c Rich D.H Prasad J.V., Sun C.Q., Green J., Mueller R., Houseman K., MacKenzie D., Malkovsky M New hydroxyethylamine HIV protease inhibitors that suppress viral replication J Med Chem., 35: 3803-3812 (1992) d Roberts N.A., Martin J.A., Kinchington D., Broadhurst A.V., Craig J.C., Duncan I.B., Galpin S.A., Handa B.K., Kay J., Krohn A., Lambert R.W., Merrett J.H., Mills J.S., Parkes K.E.B., Redshaw S., Ritchie A.J., Taylor D.L., Thomas G.J., Machin P.J Rational design of peptide-based HIV proteinase inhibitors Science, 248: 358-361 (1990) I a Shimizu M., Tsunogai M., Arai S Transepithelial transport of oligopeptides in the human intestinal cell, Caco-2 Peptides, 18(5): 681-687 (1997) b Loukas S., Varoucha D., Zioudrou C., Streaty R.A., Klee W.A Opioid activities and structure of α-casein-derived exorphins Biochemistry, 22: 4567-4573 (1983) c Ariyoshi Y Angiotensin-converting enzyme inhibitors derived from food proteins Trends Food Sci Technol., 4: 139-144 (1993) d Migliore-Samour D., Jolles P Casein, a prohormone with an immunomodulating role for the newborn? Experientia, 44: 188-193 (1988) e Fiat A.M., Levy-Toledano S., Caen J.P., Jolles P Biologically active peptides of casein and lactotransferrin implicated in platelet function J Dairy Res., 56: 351355 (1989) Yamamoto N., Akino A., Takano T Antihypertensive effect of peptides derived from casein by an extracellular proteinase from lactobacillus helveeticus CP700 J Dairy Sci., 77: 917-922 (1994) Schusdziarra V., Schick R., Fuente A., Holland A., Brantl V., Pfeiffer E.F Effect of βcasomorphins and analogs on insulin release in dogs Endocrinology, 112: 1948-1951 (1983) Schusdziarra V., Schick R., Fuente A., Specht J., Klier M., Brantl V., Pfeiffer E.F Effect of β-casomorphins on somatostatin release in dogs Endocrinology, 112: 885-889 (1983) a Reuss L., Wills N.K., Lewis S.A Epithelial transport proteins Epithelial transport: a guide to methods and experimental analysis (ed Wills N.K.), Chapman and Hall London., Chapter 2: 21-49 (1996) b Tukker J.J Characterization of transport over epithelia barriers Cell culture models of biological barriers (ed Lehr C.M.) Chapter 4: 52-61 (2002) II 10 Yee S., Day W.W Applications of Caco-2 monolayers in drug discovery and development Handbook of drug metabolism (ed Woolf T.F.), Marcel Dekker, Inc., 507522 (1999) 11 Pauletti G.M., Okumu F.W., Borchardt R.T Effect of size and charge on the passive diffusion of peptides across Caco-2 cell monolayers via the paracellular pathway Pharm Res., 14(2):164-168 (1997) 12 Werner U., Kissel T., Stüber W Effects of peptide structure on transport properties of seven thyrotropin releasing hormone (TRH) analogues in a human intestinal cell line (Caco-2) Pharm Res., 14(2): 246-250 (1997) 13 Lang V.B., Langguth P., Ottiger C., Wunderli-Allenspach H., Rognan D., RothenRutishauser B., Perriar J.C., Lang S., Biber J., Merkle H.P Structure-permeation relations of met-enkephalin peptide analogues on absorption and secretion mechanisms in Caco-2 monolayers J Pharm Sci., 86(7): 846-853 (1997) 14 Satake M., Enjoh M., Nakamura Y., Takano T, Kawamura Y., Arai S., Shimizu M Transepithelial transport of the bioactive tripeptide, Val-Pro-Pro, in human intestinal Caco-2 cell monolayers Biosci Biotechnol Biochem, 66: 378-384 (2002) 15 Artursson P Cell cultures as models for drug absorption across the intestinal mucosa Crit Rev Ther Drug Carrier Syst., 8(4): 305-330 (1991) 16 Burton P.S., Conradi R.A., Hilgers A.R., Ho N.F.H., Maggiora L.L The relationship between peptide structure and transport across epithelial cell monolayers J Controlled Release, 19: 87-98 (1992) 17 Goodwin J.T., Conradi R.A., Ho N.F.H., Burton P.S Physicochemical determinants of passive membrane permeability: role of solute hydrogen-bonding potential and volume J Med Chem., 44(22): 3721 (2001) III 18 Goodwin J.T., Conradi R.A., Ho N.F.H., Burton P.S Physicochemical determinants of passive membrane permeability: role of solute hydrogen-bonding potential and volume J Med Chem., 45(10): 2122 (2002) 19 Paterson A.D., Conradi R.A., Hilgers A.R., V.T.J., B.P.S A non-aqueous partitioning system for predicting the oral absorption of peptides Quant Struct.- Act Relat., 13: 4-10 (1994) 20 Kim D.C., Burton P.S., Borcharct R.T A correlation between the permeability characteristics of a series of peptides using an in vitro cell culture model (Caco-2) and those using an in situ perfused rat ileum model of the intestinal mucosa Pharm Res., 10: 1710-1714 (1993) 21 Stenberg P., Luthman K., Artursson P Prediction of membrane permeability to peptides from calculated dynamic molecular surface properties Pharm Res., 16(2): 205-212 22 Artursson P., Palm K., Luthman K Caco-2 monolayers in experimental and theoretical predictions of drug transport Adv Drug Del Rev., 22: 67-84 (1996) 23 Kramer W Identification of identical binding polypeptides for cephalosporins and dipeptides in intestinal brush border membrane vesicles by photoaffinity labelling Biochem Biophys Acta, 905: 65-74 (1987) 24 Herrera-Ruiz D., Knipp G.T Current perspectives on established and putative mammalian oligopeptide transporters J Pharm Sci., 92(4): 691-714 (2003) 25 Gardner M.L Gastrointestinal absorption of intact proteins Ann Rev Nutr., 8: 329-350 (1988) 26 Shen W.C., Wan J., Ekrami H Enhancement of polypeptide and protein absorption by macromolecular carriers via endocytosis and transcytosis Adv Drug Del Rev., 8: 93-119 (1992) IV 27 Sai Y., Kajita M., Tamai I., Wakama J., Wakamiya T., Tsuji A Adsorptive-mediated endocytosis of a basic peptide in enterocyte-like Caco-2 cells Am J Physiol., 275:G514520 (1998) 28 Tamai I., Sai Y., Kobayashi H., Kamata M., Wakamiya T., Tsuji A Structureinternalization relationship for adsorptive-mediated endocytosis of basic peptides at the blood-brain barrier J Pharmacol Exp Ther., 280: 410- 415 (1997) 29 Persiani S., Shen W.C., Increase of poly (L-Lysine) uptake but not fluid phase endocytosis in neuraminidase pretreated Madin-Darby canine kidney (MDCK) Life Sci., 45: 26052610 (1989) 30 Green M., Loewenstein P.M Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein Cell, 55(6): 1179-1188 (1998) 31 Joliot A., Pemelle C., Deagostini-Bazin H., Prochiantz A Antennapedia homeobox peptide regulates neural morphogenesis Proc Natl Acad Sci USA, 88(5): 1864-1868 (1991) 32 Derossi D., Joliot A.H., Chassaing G., Prochiantz A The third helix of the Antennapedia homeodomain translocates through biological membranes J Biol Chem., 269(14): 10444-10450 (1994) 33 Lundberg P., Langel U A brief introduction to cell penetrating peptides J Mol Recognit., 16(5): 227-233 (2003) 34 Oehlke J., Beyermann M., Wiesner B., Melzig M., Berger H., Krause E., Bienert M Evidence for extensive and non-specific translocation of oligopeptides across plasma membranes of mammalian cells Biochim Biophys Acta, 1330: 50-60 (1997) 35 Langel U Cell-penetrating peptides, processes and applications (ed Langel U.) CRC Press: Boca Raton, FL (2002) 36 Futaki S., Goto S., Sugiura Y Membrane permeability commonly shared among argininerich peptides J Mol Recognit., 16(5): 260-264 (2003) V 37 Thomas W.G., Mendelsohn F.A Angiotensin receptors: form and function and distribution Int J Biochem Cell Biol., 35(6): 774-779 (2003) 38 Moeller I., Allen A.M., Chai S.Y., Zhuo J., Mendelsohn F.A Bioactive angiotensin peptides J Hum Hypertens., 12(5): 289-93 (1998) 39 Albiston A.L., McDowall S.G., Matsacos D., Sim P., Clune E.R., Mustafa T., Lee J., Mendelsohn F.A.O., Simpson R.J., Connolly L.M., and Chai S.Y Evidence that the angiotensin IV (AT4) receptor is the enzyme insulin-regulated aminopeptidase J Biol Chem., 276: 48623-48626 (2001) 40 a Braszko J.J., Kupryszewski G., Witczuk B., Wisniewski K Angiotensin II-(3-8)hexapeptide affects motor activity, performance of passive avoidance and a conditioned avoidance response in rats Neurosci., 27: 777-783 (1988) b Wright J.W et al Angiotensin II(3-8) (Ang IV) hippocampal binding: potential role in the facilitation of memory Brain Res Bull 32: 497-502 (1993) 41 Chen W.S., Sim M.K., Go M.L Structure-activity and structure-binding studies of desAsp1-angiotensin I analogues on the rabbit pulmonary artery Regul Pept., 106: 39-46 (2002) 42 Albiston A.L., McDowall S.G., Matsacos D., Sim P., Clune E., Mustafa T., Lee J., Mendelsohn F.A., Simpson R.J., Connolly L.M., Chai S.Y Evidence that the angiotensin IV (AT(4)) receptor is the enzyme insulin-regulated aminopeptidase, J Biol Chem., 276: 48623-48626 (2001) 43 Palczéwski K., Kumasaka T., Hori T, Behnke C.A., Motoshima H., Fox B.A., Le Trong I., Teller D.C., Okada T., Stenkamp R.E., Yamamoto M., Miyano M Crystal structure of rhodopsin: a G protein-coupled receptor, Sci., 289:739-745 (2000) 44 Curnow K.M., Pascoe L., Davies E., White P.C., Corvol P., Clauser E Alternatively spliced human type angiotensin II receptor mRNAs are translated at different efficiencies and encode two receptor isoforms Mol Endocrinol., 9: 1250-1262 (1995) VI 45 Sim M.K., Qui X.S Angiotensins in plasma of hypertensive rats and human Regul Pept 111: 179-182 (2003) 46 Local drug set to enter world market The Straits Times, 14th Nov 2003 47 Sim M.K., Min L Effects of des-Asp-angiotensin I on experimentally-induced cardiac hypertrophy in rats Int J Cardiol., 63(3): 223-227(1998) 48 Matsufuji H., Matsui T., Ohshige S., Kawasaki T., Osajima K., Osajima Y Antihypertensive effects of angiotensin fragments in SHR Biosci Biotechnol Biochem., 59(8): 1398-1401 (1995) 49 Xu C Effects of angiotensin IV on cardio-hypertrophy in rats and rats’ cardiomyocytes, (2002) 50 Chen W.Q., Tang F., Horie K., Borchardt R.T Caco-2 cell monolayers as a model for studies of drug transport across human intestinal epithelium Cell culture models of biological barriers, in- vitro test systems for drug absorption and delivery (ed Lehr C.M.), Chapter 10: 143-163 (2002) 51 Ingels F.M., Augustijns P.F Biological, pharmaceutical, and analytical considerations with respect to the transport media used in the absorption screening system, Caco-2 J Pharm Sci., 92(8): 1545-1558 (2003) 52 Artursson P., Palm K., Luthman K Caco-2 monolayers in experimental and theoretical predictions of drug transport Adv Drug Del Rev., 56: 27-43 (2001) 53 Bruan A., Hämmerle S., Suda K., Rothern-Rutishauser B., Günthert M., Krämer S.D., Wunderli-Allenspach H Cell cultures as tools in bio-pharmacy Eur J Pharm Sci., 11(S2): S51-S60 (2000) 54 Behrens S., Kissel T Do cell culture conditions influence the carrier-mediated transport of peptides in Caco-2 cell monolayers? Eur J Pharm Sci., 19: 433-442 (2003) VII 55 Artursson P., Johan K., Ocklind G., Schipper N Studying transport processes in absorptive epithelia Epithelial cell culture, a practical approach (ed Shaw A.J.), Chapter 6: 111-135 (1996) 56 Tamura K., Bhatnagar P.K., Takata J.S., Lee C.P., Smith P.L., Borchardt R.T Metabolism, uptake, and transepithelial transport of the diastereomers of Val-Val in the human intestinal cell line, Caco-2 Pharm Res., 13(8): 1213-1218 (1996) 57 Tamura K., Lee C.P., Smith P.L., Borchardt R.T Metabolism, uptake and transepithelial transport of the stereoisomers of Val-Val-Val in the human intestinal cell line, Caco-2 Pharm Res., 13(11): 1663-1667 (1996) 58 Yee S In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man- fact or myth Pharm Res.,14(6): 763-766 (1997) 59 Tim M Rapid Colorimetric Assay for Cellular Growth and Survivial: Application to Proliferation and Cytotoxicity Assays J Immunological Methods, 65: 55-63 (1983) 60 Howell S., Kenny A.J., Turner A.J A survey of membrane peptidases in two human colonic cell lines, Caco-2 and HT-29 Biochem J., 284: 595-601 (1992) 61 Gan L.S., Hsyu P.H., Pritchard J.F., Thakker D Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers Pharm Res., 10(12), 1722- 1725 (1993) 62 Min L., Sim M.K., Xu X.G., Effects of des-aspartate-angiotensin I on angiotensin IIinduced incorporation of phenylalanine and thymidine in cultured rat cardiomyocytes and aortic smooth muscle cells Regul Pept., 95: 93-97 (2003) 63 Gan L.S., Niederer T., Eads C., Thakker D Evidence for predominantly paracellular transport of thyrotropin- releasing hormone across Caco-2 cell monolayers Biochem Biophys Res Commun., 197(2): 771- 777 (1993) VIII 64 Walter E and Kissel T Transepithelial transport and metabolism of thyrotropin- releasing hormone (TRH) in monolayers of a human intestinal cell line (Caco-2): evidence for an active transport component? Pharm Res., 11(11): 1575- 1580 (1994) 65 Artursson P Epithelial transport of drugs in cell culture I: a model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells J Pharm Sci., 79(6): 476-482 (1990) 66 Burton P.S., Conradi R.A., Hilgers A.R., Ho N.F.H Evidence for a polarized efflux system for peptides in thi apical membrane of Caco-2 cells Biochem Biophys Res Commun., 190(3): 760-766 (1993) 67 Knipp G.T., Vander Velde D.G., Siahaan T.J., Borchardt R.T The effect of β- turn structure on the passive diffusion of peptides across Caco-2 cell monolayers Pharm Res., 14(10): 1332-1340 (1997) 68 Toppmeyer D.L., Slapak C.A., Croop J., Kufe D.W Role of P-glycoprotein in dolastatin 10 resistance Biochem Pharmacol., 48(3): 609-612 (1994) 69 Comer J.A.E High-throughput measurements of log D and pKa Drug Bioavailability, Estimation of Solubility, Permeability, Absorption and Bioavailability (ed Van de Waterbeemd J., Lennernäs H., Artursson P), Chapter 2: 21-46 (2003) 70 Stein W.D The Molecular Basis of Diffusion Across Cell Membranes The movement of molecules across cell membranes, Academic Press NY, Chapter 3: 65-125 (1967) 71 Zhou H., Moore G.J., Vogel H.J Proton NMR studies of angiotensin II and its analogs in aqueous solution J Protein Chem., 10: 333-343 (1991) 72 Lintner K., Fermandjian S., Fromageot P pH titration effects on the CD spectra of angiotensin II, truncated peptides and other analogues: aromatic region FEBS letters, 56(2): 366-369 (1975) IX 73 Lintner K., Fermandjian S., Fromageot P., Khosla M.C., Smeby R.R., Bumpus F.M Circular dichroism studies of angiotensin II and analogues: effects of primary sequence, solvent, and pH on the side-chain conformation Biochem., 16(4) 806-812 (1977) 74 Macquaire F., Baleux F., Giaccobi E., Huynh-Dinh T., Neumann J.M., Sanson A Peptide secondary structure induced by a micellar phospholipidic interface: proton NMR conformational study of a lipopeptide Biochem., 31(9): 2576-2582 (1992) 75 Milon A., Miyazawa T., Higashijima T Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogs by proton nuclear magnetic resonance: correlation between activities and membrane-bound conformations Biochem., 29(1): 65-75 (1990) 76 Sharan R., Rubas W., Kolling W.M., Ghandehari H Molecular modelling of arginineglycine-aspartic acid (RGD) analogs: relevance to transepithelial transport J Pharm Sci., 4(1): 32-41 (2001) 77 Palm K., Luthman K., Ungel A.L., Strandlund G., Artursson P Correlation of drug absorption with molecular surface properties J Pharm Sci., 85(1): 32-39 (1996) 78 Pauletti G.M., Gangwar S., Wang B., Borchardt R.T Esterase- sensitive cyclic prodrugs of peptides: evaluation of a phenylpropionic acid promoiety in a model hexapeptide Pharm Res., 14(1): 11-17 (1997) 79 Pauletti G.M., Gangwar S., Okumu F.W., Siahaan T.J., Stella V.J., Borchardt R.T Esterase-sensitive cyclic prodrugs of peptides: evaluation of an acyloxyalkoxy promoiety in a model hexapeptide Pharm Res., 13: 1615-1623 (1996) 80 Gangwar S., Jois S.D.S., Siahaan T.J., Vander Velde D.G., Stella V.J., Borchardt R.T The effect of conformation on membrane permeability of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide Pharm Res., 13: 1657-1662 (1996) 81 Stenberg P., Luthman K., Ellens H., Lee C.P., Smith P.L., Lago A., Elliot J.D., Artursson P Prediction of the intestinal absorption of the endothelin receptor antagonists using three theoretical methods of increasing complexity Pharm Res., 16(10): 1520-1526 X [...]... from their breakdown, have been found to possess ACE inhibitory activities 48 15 SECTION TWO AIM OF THESIS i 2 AIM OF THESIS The aim of this thesis is to obtain a better understanding of the transport properties of the angiotensin peptide DAA-I across the intestinal epithelium DAA-I is a physiological peptide involved in the pathophysiology of the cardiovascular system The therapeutic potential of DAA-I... route h Transcellular route The various routes and mechanisms of transfer across the intestinal epithelium are well described 9 Table 1 summarises some of the salient features of the mechanisms underlying the transport routes across the epithelium The transport of a solute across the intestinal epithelium is determined by the physicochemical characteristics of both the solute and the cellular barrier In... PRESENTATION 1 Annual meeting of American Association of Pharmaceutical Scientists (AAPS), 2002 The Transport of Angiotensin III, IV and Des- Asp Angiotensin I through Polarised Monolayers of Caco-2 Cells, Abstract Number: AM02-02131 2 Asian Association of Schools of Pharmacy (AASP), 2004 Transport Properties of Angiotensin Peptides 3 Peptides Transport of angiotensin peptides across the Caco-2 monolayer (Accepted... represents the transport of the peptide into the polar head group region (interface) of the lipid bilayer, a process described earlier by log PC octanol The authors found good correlation between PSA d and the permeability of 9 19 oligopeptides belonging to three homologous series On the other hand, other reports indicate that the usefulness of PSA d is restricted to homologous series of peptides and... and Ang IV) on the Caco-2 cell monolayers and to characterise their transport properties under different conditions of temperature, concentration and in the presence of selected inhibitors Prior to these investigations, it is necessary to establish methods to validate the integrity of the Caco-2 cell monolayers, quantify the amount of peptide transported, evaluate the stability of the peptides to enzymatic... phase from the apolar membrane interior The partitioning of the peptide into this interface is driven by the hydrophobic effect (about 70 % of the hydrophobic force associated with the peptide is expended as it moves from the aqueous phase into this region) as well as the extent of hydrogen bonding between the peptide and the polar head groups in the interface The hydrogen bonding potential of a peptide... conclusions being drawn on the transport kinetics of small peptides and peptidomimetics 24 This is because there are several other putative peptide transporters (for example, the peptide histidine transporters) expressed in the intestinal epithelium and they are likely to contribute to the transport of oligopeptide as well 1.1.4 Transcytosis of oligopeptides In transcytosis, the solute is first enclosed... by a vesicle formed by the invagination of the apical membrane The vesicle crosses the cytosol and fuses with the basolateral membrane, depositing the solute on the other side of the epithelial barrier 25, 26 If the solute is released in the cytosol, the process is described as endocytosis 10 The solute may be internalised by binding specifically to receptor- like molecules on the membrane surface Various... arising from the use of different media in transport experiments 51 Cell culture conditions like time in culture, membrane support and seeding density have been noted to influence the morphology, formation of tight junctions, expression of peptide transporters and the efflux pump protein of the Caco-2 cells 54 The purpose of this section is to evaluate the permeability of the angiotensin peptides (DAA-I,... The significance of these peptides lies in their potential as delivery vehicles for bioactive molecules, namely, the ability to deliver their “cargo” without disturbing the stability of the cell membrane, demonstrable effective delivery in vivo in a variety of cell types and the absence of antigenic and immunogenic side effects 33 The mechanism of the translocation process is still widely debated The ... SECTION TWO AIM OF THESIS i AIM OF THESIS The aim of this thesis is to obtain a better understanding of the transport properties of the angiotensin peptide DAA-I across the intestinal epithelium DAA-I... modelling of the angiotensin peptides 4.3.2 69 79 Determination of physicochemical and size parameters of the angiotensin peptides 88 iv 4.3.3 Structure- transport correlations of angiotensin peptides. .. Number: AM02-02131 Asian Association of Schools of Pharmacy (AASP), 2004 Transport Properties of Angiotensin Peptides Peptides Transport of angiotensin peptides across the Caco-2 monolayer (Accepted