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Effect of lipidization on transport and uptake of peptides across rat alveolar epithelial cell monolayers

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Effect of lipidization on transport and uptake of peptides across rat alveolar epithelial cell monolayers

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Content EFFECT OF LIPIDIZATION ON TRANSPORT AND UPTKAKE OF PEPTIDES ACROSS RAT ALVEOLAR EPITHELIAL CELL MONOLAYERS by Rana Bahhady A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (PHARMACEUTICAL SCIENCES) May 2003 Copyright 2003 Rana Bahhady Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 1416533 UMI UMI Microform 1416533 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY O F S O U T H E R N CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 9 0 0 0 7 This thesis, written by i?&m6......S.M M AH}L.................... under the direction of H&C....T hesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of The Graduate School, in partial fulfillment of the requirements for the degree of Dtan Date Augtf.st. 12j . . 2003. THESIS COMMITTEE 'hairi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS My thanks and gratitude is to my parents who provided me with the opportunity to experience what I have had these two years in the M asters Program. My appreciation extends to my advisor, Dr. Shen, who provided guidance and advice throughout my project and to Daisy, who provided technical support. My thanks is to Dr. Okamoto and Dr. Kim for being my committee members and providing me with positive feedback. And thanks finally, to the wonderful friends I have made in the lab and the “graduate common room” for their ideas and care. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS A C K N O W L E D G E M E N T S..........................................................................ii LIST OF T A B L E S ...........................................................................................iv LIST OF F IG U R E S..........................................................................................v A B S T R A C T .......................................................................................................vii I. B A C K G R O U N D ........................................................................................ 1 1.1 Alveolar epithelial cell monolayer model...................................1 1.2 Peptide and protein transport across whole lung and cultured lung epithelial cell monolayer models..................5 1.3 Surfactant proteins............................................................................ 8 1.4 Lipidization........................................................................................ 10 II. M A TE R IA L S & M E T H O D S.............................................................. 15 2.1 Peptide lipidization and iodination................................................. 15 2.2 Primary culture of rat alveolar epithelial cell monolayers (AEC)...................................................................... 17 2.3 Transport and uptake studies across A E C ............................... 18 2.3.1 In serum-free media........................................................ 18 2.3.2 In media containing serum............................................19 2.4 Analysis of transported peptide......................................................20 III. R E SU L T S & D IS C U S S IO N ................................................................21 3.1 Transport and uptake in serum-free media....................................21 3.2 Transport and uptake in various media conditions.......................33 IV. C O N C L U S IO N S..................................................................................... 41 R E FE R E N C E S..................................................................................................43 B IB L IO G R A P H Y ............................................................................................48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1: Permeability o f various peptides and their lipidized analogues....................4 Table 2: Trichloroacetic acid precipitation for calcitonin (CT) and lipidized calcitonin (LCT) in various media conditions................................................. 34 Table 3: Trichloroacetic acid precipitation for calcitonin (CT) and lipidized calcitonin (LCT) transported across AECI or AECII in various media conditions.................................................................................................. 37 Table 4: Trichloroacetic acid precipitation for transported calcitonin (CT) dosed in CMII, CMII plus 20mM NH4C1, and CMII plus 50mM NH4C1......................................................................................................... 40 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 1: Structure o f surfactant protein A (SP-A).......................................................9 Figure 2: Lipidization o f cysteine containing protein using a water-soluble derivative o f palmitic acid (Pal-CPD)...........................................................12 Figure 3: Structures o f desompressin (DP) and palmitoyl-cysteine desmopressin (DPP)............................................................................................ 12 Figure 4: Calcitonin (CT) and lipidized calcitonin (LCT).......................................... 13 Figure 5: Transport A) and uptake B) as a function o f apical concentration of desmopressin (DP) and lipidized desmopressin (DPP) across type I alveolar epithelial cell m onolayer..................................................................22 Figure 6: Transport A) and uptake B) as a function o f apical concentration of desmopressin (DP) and lipidized desmopressin (DPP) across type II alveolar epithelial cell m onolayer...................................................................23 Figure 7: Time courses o f transport o f 2 pg/ml A) desompressin (DP) and B) lipidized desompressin (DPP) in presence and lOmM glutathione placed in basolateral compartment o f A ECI...........................25 Figure 8: Time courses o f transport o f 2 pg/ml A) desompressin (DP) and B) lipidized desompressin (DPP) in presence and lOmM glutathione placed in basolateral compartment o f A EC II......................... 26 Figure 9: Size exclusion chromatography using G-10 in DMF to analyze DP transport across AECII in A) absence and B) presence o f lOmM glutathione...............................................................................................27 Figure 10: Size exclusion chromatography using G-10 in DMF to analyze DPP transport across AECII in A) absence and B) presence o f lOmM glutathione.........................................................................................28 Figure 11: Size exclusion chromatography using G-10 in DMF to analyze DP transport across AECII in A) presence o f protease inhibitors and B) presence o f protease inhibitors and lOmM glutathione...........................................................................................................29 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 12: Size exclusion chromatography using G-10 in DMF to analyze DP transport across AECII in A) presence o f protease inhibitors and B) presence o f protease inhibitors and lOmM glutathione...........................................................................................................30 Figure 13: Transport A) and uptake B) as a function o f apical concentration o f enkephalin (ENK) and lipidized enkephalin (051) across type I alveolar epithelial cell m onolayer.................................................................. 31 Figure 14: Time courses o f transport A) and uptake B) o f 2.5 pg/ml enkephalin (ENK) and lipidized enkephalin (051) across type I alveolar epithelial cell monolayer...............................................................32 Figure 15: Transport o f 2 pg/ml calcitonin (CT) and lipidized calcitonin (LCT), across alveolar epithelial cells type II (AECII) when dosed in SFM and C M ................................................................................................. 34 Figure 16: Transport o f 2 pg/ml calcitonin (CT) and lipidized calcitonin (LCT), across alveolar epithelial cells type II (AECII) when dosed in SFM, SM, C M II...........................................................................................35 Figure 17: A) Transport and B) uptake o f calcitonin across alveolar epithelial cell monolayers type I (AECI) and type II (AECII) in SFM, NBS, CMI, CM II.......................................................................................36 Figure 18: Transport o f 3 pg/mj calcitonin across AECII in 3x, lx, O.lx CMII, in 3x, lx CMI, and in SFM .............................................................................. 38 Figure 19: Transport A)and uptake B) o f calcitonin at apical concentration o f 3 pg/ml dosed in CMII, CMII plus 20mM NH4CI, CMII plus 50mM NH4CI in A ECII.................................................................................... 40 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Reversible aqueous lipidization (REAL) is a process whereby a fatty acid is linked to a peptide via either a disulfide or amide bond. Reversible lipidization has shown to enhance absorption and bioavailability o f various peptides when administered subcutaneously (1, 2). In this study, alveolar epithelial cells (AEC) were used as an in vitro model for type I (AECI) or type II (AECII) since they express various transporters, caveoli and other biological characteristics, making them suitable to study uptake and transport o f peptides. The aim o f the study was to demonstrate whether lipidization affects the uptake and transport o f desmopressin, enkephalin and calcitonin across AECI or AECII. It also provided some insights into the importance o f using conditioned media from cultured type II-like AEC on the transport o f calcitonin across the alveolar epithelial cell monolayer. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I. BACKGROUND 1.1 Alveolar epithelial cell monolayer model The lung is divided into two parts: the conducting airway region and the gas exchange region. The gas exchange region is composed o f alveolar sacs which consist o f a monolayer o f epithelial cells and these sacs are located in proximity to the pulmonary capillary endothelial cells. Thus the human lung provides an expansive absorptive surface area o f about 143 m (3) which is highly permeable and enriched by an adjacent bloodstream. Morphometric studies o f mammalian lung indicate that the alveolar epithelium is composed o f large, squamous type I cells which cover greater than 90% o f the area o f this tissue boundary but comprise only 9% o f total lung cells, and smaller, cuboidal, surfactant-producing type II pneumocytes seen in the alveolar comers and cover up about 3-5% o f the alveolar surface, but accounts for 15% o f total lung cells (4, 5). Renewal o f the squamous alveolar epithelium usually follows injury o f the lung and occurs by the division of the type II cells with subsequent transformation to type I epithelial cells(6). Therefore, it is the type II cells that are important for integrity and normal distribution o f cuboidal and squamous cells in the lung. Similarly, in vitro primary cultures o f rat alveolar epithelial type II cells on specific filters such as tissue culture-treated Nuclepore filters (4), result in transdifferentiation o f type II cells into type I cells. Differentiation in vitro follows a course similar to that o f in vivo after lung injury. Type II cells form confluent cell monolayers starting from around day three o f culture (7) and henceforth begin exhibiting traits that resemble type I cells. 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M orphologically, the cells acquire thin cytoplasmic extensions, protuberant nuclei and lamellar bodies (4, 8). Biochemically, the cells progressively lose their ability to produce surfactants but gain the expression o f markers such as aquaporin-5 and caveolin-1 (8,9). Some studies, however, required the maintenance of type II alveolar epithelium rather than terminal differentiation into type I. In this study, to grow the type II cells, keratinocyte growth factor (KGF) was added in the culture media (8,10). KGF is an epithelial mitogen and one o f its roles in vivo includes the repair o f damaged tissues such as the alveolar epithelium (11). W hen added to rat alveolar type II epithelial cell culture in vitro, KGF was also able to promote retention o f the type II phenotype and partially to reverse transdifferentiation from type II cell to type I cell phenotype in primary culture (8). For transport study o f peptides across an alveolar type II cell monolayer model, it was necessary to add KGF no later than culture day three. Because human alveolar epithelial type I cell monolayers show a resemblance to rat alveolar cells regarding differentiation, resistance, and transport o f hydrophilic molecules ( 12), the primary cultured rat alveolar epithelial cell monolayers are well regarded as a good substitute for human pneumocytes. In fact, the culturing o f rat alveolar cells on appropriate substratum has proven to be a successful model for studying drug transport across the lung epithelium, mainly due to the ability o f the monolayer to retain its tight junctions and biological characteristics. Therefore, rat alveolar epithelial cell monolayers are suitable for mechanistic studies of biochemical and cell biological functions as well as drug transport and uptake in the 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pulmonary delivery route (13). The transepithelial electrical resistance (TEER) is a pertinent measurement o f the tight junctions as well as the transcellular membrane resistances. The tight junctions are the main barrier for the paracellular pathway, where a decreased resistance means leaky barrier, which allows the escape o f greater number o f molecules via the loosened tight junctions. In general, TEER falling between 1500-2500 Qcm is an indication o f relatively tight barrier which allows reliable studying o f drug transport across the lung epithelium. Nevertheless, studies have shown that permeability o f peptides with differing molecular weights is inversely related to TEER and that a minimum cut o ff value for TEER (14) should be set for the alveolar epithelium below which the epithelium should not be used for transport studies. Dodoo et al showed that, for rat alveolar epithelial cell monolayers, there is a continual decline in permeability with increasing TEER up to a value o f 2000 Qcm , and that solute or peptide permeabilities remain more or less constant when measured using monolayer o f TEER > 2000 £2cm2 (15). Besides cell integrity, there are other factors that contribute to the transport of peptides across cultured alveolar epithelial cell monolayers. The size o f peptides, for example, has been found to be a principal determinant for how rapid their fluxes occur a peptide’s flux across the alveolar epithelial barrier (16), where an inverse relation was shown to exist between the size o f peptides and their rates o f transport. Hydrophilicity, on the other hand, as shown by Dodoo et al, does not have a pronounced effect as molecular weight does and therefore affects permeability to a lesser degree. Permeability is dependent on the likelihood o f a molecule to permeate 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. through the pores o f the monolayer, where Dodoo et al showed that two populations o f pores exist, namely cylindrical pores o f diameter 1.5nm made by the tricellular junctions and larger pores o f diameter 20 nm present as a consequence o f defects in the monolayers (15). Our study showed an inverse relation between permeability and size (Table 1): with enkephalin being the highest and calcitonin the lowest. The corresponding lipidized peptide showed a slight decrease in permeability than parent peptide. Table 1. Permeability o f various peptides and their lipidized analogues Peptide Permeability (cm. sec'1 ) Enkephalin 2 .8* 10'6 Lipidized Enkephalin 2.06*1 O '6 Desmopressin 1.5*10'7 Lipidized Desmopressin 0.62* 10'8 Calcitonin 2.08*10'8 Lipidized Calcitonin 1.8* 10'8 It is still controversial as to what cut off would be for peptide molecular weight and whether transport is transcellular or paracellular. Some studies predict that peptides with molecular weight less than 40 kDa probably traverse the alveolar epithelium mainly via paracellular pathway (17, 18). One must not ignore the fact that though the major route o f transport o f small peptides is paracellular, transcellular transport may also have its contribution. Peptide charge also has a significant effect on 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. permeability where the alveolar cell monolayers appear to be cation selective, so the higher the charge number the greater the permeability that may result (15). The study by Dodoo et al confirmed that the in vitro simulation o f the lung provides a suitable prediction for in vivo transport o f peptides via the lung and that either the transcellular or paracellular routes may be utilized depending on the peptide size, hydrophilicity, charge specific transcellular routes. 1.2 Peptide and protein transport across whole lung and cultured lung epithelial cell monolayers The lung provides a large surface area for drug absorption and transport. However, similar to the intestine, the cells lining lung airspaces also contain receptors, transporters, enzymes and other features that increase the complexity of drug transport studies. Utilizing the whole lung approach, Folkesson et al showed that aerosolized analogue o f vasopressin (DP, M W t 1067) when delivered via the respiratory tract, resulted in a measurable DP concentration greater than the case o f oral delivery (19). Insulin delivery via the pulmonary route has been studied since 1971 where experiments have been carried out on animals such as rabbit, pig and rat using aerosolized or intratracheal administration with various formulations. Okumura et al demonstrated that aerosolized insulin via rat lung had a relative bioavailability similar to that o f subcutaneous administration (20). Calcitonin delivery via the lung has been studied to a lesser extent; but a study by Patton et al reported an absolute bioavailability o f about 17% when calcitonin solution was 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. intratracheally instilled into rats (21). Kobayashi et al, also showed that when salmon calcitonin was instilled intratracheally to rats, a remarkable enhancement in absorption resulted when particular protease inhibitors such as: chymostatin, bacitracin, phosphoramidon and absorption enhancers such as: oleic acid and polyomyethylene oleyl ether dissolved or suspended with the peptide formulation (22). However due to complexity o f the whole lungs and experimental limitations, this whole lung approach provided various unknowns which made data interpretation difficult. More recently, the development o f an in vitro protocol to isolate and culture alveolar epithelial cell monolayers has made a pivotal change in the investigation o f peptide and protein uptake and transport across the lung epithelium. The alveolar epithelial cell culture was utilized to study the transport o f various peptides. Morimoto et al demonstrated that Gly-D-Phe and Gly-L-Phe crossed the monolayer via passive diffusion since their permeabilities were close to that of mannitol ( 1.8*10'7cm/sec) and their transport lacked concentration or direction dependence (23). Gly-D-Phe was able to traverse the monolayer intact, whereas Gly-L-Phe was susceptible to cellular metabolism by aminopeptidase activity; and when 3pM actinonin, an aminopeptidase inhibitor, was added to the apical donor media, the concentration o f L-Phe (metabolite) was reduced by 50% (23). Interestingly, this decrease was observed only when the inhibitor was placed in the apical donor media rather than the basolateral fluid indicating the presence o f higher aminopeptidase activity at the mucosal surface o f the alveolar epithelium. In another 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. study, W ang et al used met-enkephalin and [D-Ala2]met-enkephalinamide to test peptide transport across AECI. They demonstrated that when incubated with AECI, met-enkephalin showed a lower hydrolysis rate than other tissues, however, [D- Ala2]met-enkephalinamide was resistant to aminopeptidase and showed absence o f metabolites in the receiver compartment as well as increased rate o f delivery over met-enkephalin (24). In a similar study, Yamahara et al showed that vasopressin (AVP), when translocated from apical to basolateral sides, was degraded to a significantly greater extent than that for basolateral to apical transport (25). Furthermore, the apical presence o f camostat mesylate, an aminopeptidase inhibitor, increased the fraction o f intact AVP appearing in the basolateral receiver fluid. Use o f alveolar epithelial cell cultures has allowed a more thorough follow up o f transport pathways for various proteins. Studies have shown that albumin (-67 kDa) has about two to three orders o f magnitude greater flux than 70 kDa dextrans (17). The albumin absorption is saturable with a higher Papp in the apical to basolateral direction than the opposite direction. A recent study showed that a possible explanation is due to the presence o f gp60 on the alveolar epithelium which mediates the absorption o f albumin in a manner that is neither explained by passive diffusion nor nonspecific adsorptive endocytosis (26). M oreover, the addition of filipin, a caveolar disrupting agent, or the stimulation of the alveolar epithelial cells by cross-linking gp60 with anti-gp60, both abolished the uptake or internalization of albumin. Since caveolae have long been recognized to exist in alveolar epithelial type I cells (27, 26), it is possible that after binding to gp60, albumin was driven into 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the cell by these invaginations. Immunoglobulin G (IgG) is transported across several epithelial barriers including the lung alveolar epithelium. A recent study (28) showed unidirectional fluxes of IgG that were slowed down after lowering the temperature or adding excess unlabeled rat Fc. The study, therefore, indicated that IgG traversed the alveolar epithelial cell monolayer via FcRn-mediated trancytosis. It is therefore apparent that the lung epithelium is a dynamic surface that provides transport facilitation as well as transport obstacles. 1.3 Surfactant proteins M any components are secreted into the lung airspaces by various pulmonary cells, however, our study specifically uses alveolar epithelial cells o f either type I or type II phenotypes. From the previous section, it is obvious that a number of proteases exist in the lung epithelium such as aminopeptidase, carboxypeptidase and endopeptidase. In addition, protein and phospholipid surfactants are also produced, specifically by type II AECs (29). Surfactants, in fact, are one o f the markers used to identify AEC II (7). They are responsible for decreasing the surface tension, which is changed during breathing cycles and is highest at the end o f expiration. The mixture o f lipid and protein surfactants found at the interface o f the alveolar aqueous film and air, are spread across the lung surface leading to decreased alveolar surface tension. The absence o f surfactants in babies leads to an increased surface tension along the alveolar epithelium resulting in a subsequent collapse and death o f the epithelial cells, a disease known as respiratory distress syndrome (RDS). There are 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. four types o f protein surfactants: SP-A, SP-B, SP-C and SP-D. SP-A, a heterogeneous protein with a molecular weight between 34000-36000 (30), is of most interest since it is the major component o f surfactant proteins estimated to be between 0.1-1% (31). The structure o f SP-A is similar to that o f mannose binding lectin (Fig. 1) and may therefore play a role in the clearance o f bacteria (32). Since SP-A also associates with lipids, there is a possibility o f the existence o f a lipid- binding domain (29). SP-A 2 0 n m Figure 1. Structure o f surfactant protein A (SP-A). SP-A synthesis occurs in the endoplasmic reticulum where it is assembled into a collagen-like triple helix by the action o f protein disulphide isomerase (33) and hydroxylated by prolylhydroxylase, two necessary steps for ensuring the formation of a stable SP-A. SP-A is then transported via Golgi apparatus towards the lamellar bodies where it is stored. The fate o f SP-A follows two different pathways. In the first case, SP-A is internalized by alveolar macrophages via a mannose-dependent mechanism and degraded at the lysosomes (34). In the second route, SP-A is internalized by AEC II via a receptor-mediated process where binding was shown to be a saturable process and was inhibited by excess unlabelled SP-A (35). 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Internalization, in the latter case, did not show a significant degradation suggesting that SPA may be recycled (35). SP-A has also been found in coated pits, endosomes and multi vesicular bodies at a proximity to the lamellar bodies (36). Due to its unique spreading properties in the lung environment, it has been proposed to use surfactant as a carrier for several therapeutic agents (37). One study showed that surfactant-tobramycin mixture administered intratracheally to mice suffering respiratory infection from Klebsiella pneumonia proved more effective in protecting mice from death due to respiratory infection than either agents administered alone because surfactant increased the spatial lung distribution o f tobramycin and lead to higher lung clearance o f the drug (38). Surfactant improved lobular distribution o f adenovirus delivered beta-galactosidase expression, where the activity o f the gene expression was significantly greater when surfactant was used instead o f saline (39). Therefore, surfactant as a drug carrier may be an agent of choice for improving drug delivery and transport enhancement via lung. 1.4 Lipidization Numerous strategies have been used to protect peptides against degradation by proteases which are either present in the cytosol or are membrane-bound to the epithelial cells o f various organs including the intestine and lungs. Protease inhibitors are one way o f tackling the problem o f peptide and protein digestion, however, including these agents into peptide formulations, may cause side effects upon long usage. Alternatively, the formation o f peptide analogues may provide a 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. useful strategy to circumvent the enzymatic barrier associated with the delivery of peptides across various mucosal epithelium. A study by Kahns et al, showed that increasing the lipophilicity of desmopressin by esterification o f its tyrosine phenolic group protected it from degradation by particular proteolytic enzymes when added to rabbit liver homogenate (40). The study also showed that the esterification leads to an increased transport o f the desmopressin carboxylic esters, in particular the pivalate ester, across Caco-2 cells when compared to desmopressin. Another peptide o f interest is enkephalin, where a study by Wang et al, showed that when transported across AECI, M et-enkephalin was completely degraded in contrast to its analogue (D -A la2) M et-enkephalinamide which was found to be resistant to the degradative enzymes in pneumocytes (24). Reversible aqueous lipidization (REAL) is a novel approach, which has been reported to increase uptake (41), decrease proteolytic degradation and prolong plasma half-life o f particular peptide drugs (2). The process, shown in Fig 2, involves conjugation o f a water soluble, thiol-reactive agent, A-palmitoyl cysteinyl 2-pyridyl disulfide (Pal-CPD) to a sulfur containing (cysteine) peptide such as calcitonin or desmopressin via a reversible disulfide linkage (42). The end result is a soluble peptide-fatty acid conjugate, in this case palmitic acid, which can be released under reductive environment to the original bioactive parent peptide. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. <CVs. \ = N S - + (^proteirf^)— g(-| -----^ (^p ro teirT ^- s s . HOOC ^ ----- (CH2)1 4 CH3 ^>— N 0 HOOC ^ ----- (CH2)1 4 CH3 0 Figure 2. Lipidization o f cysteine containing protein using a water-soluble derivative o f palmitic acid (Pal-CPD). The first lipidized peptide, Bowman-Birk protease inhibitor (BBI), an 8 kDa polypeptide with anti-carcinogenic activity, demonstrated 140-fold higher uptake in Caco-2 cells and retained biological activity (2). In a more recent study, desmopressin, a nanopeptide with one cysteine and positive net charge, was conjugated to palmitic acid using the same approach described above to form lipidized dipalmitoyl desmopressin (DPP) (Fig.3) (1). SCH2CH2CO-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2 DP Pal Pal Cys Cys SCH2CH2CO-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH2 DPP Figure 3. Structures o f desompressin (DP) and palmitoyl-cysteine desmopressin (DPP) 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Desompressin (DP) is an analogue o f vasopressin which is an anti-diuretic hormone released by the posterior lobe o f the pituitary gland. As a therapeutic agent, desmopressin is used in treating diabetes insipidus, primary nocturnal enuresis, hemophilia and Type I Von W illebrand’s disease (43). W hen DP and DPP were injected subcutaneously in Brattleboro rats, which are rats that exhibit diabetes insipidus, DPP showed a 250-fold enhancement o f anti-diuretic potency compared to DP (1). An intravenous injection showed that in the first 20 minutes, DP showed a rapid decrease in plasma concentration, whereas DPP concentration increased steadily, followed by a decrease similar to that o f DP. Since the disulfide ring structure in DP is essential for biological activity, then the original peptide must have been regenerated from DPP by reduction (in the liver). Furthermore, cell culture studies using Caco-2 cells, showed that the cellular uptake, which includes intracellular and membrane-bound, was 33-fold higher for DPP when compared to DP (41). Thus, the process of lipidization was able to provide a higher bioavailability in case o f subcutaneous administration and a higher uptake in case of Caco-2 cell cultures. Similarly, calcitonin, a hormone consisting o f 32 amino acids and having a positive net charge, was also lipidized using the same approach as DP to form lipidized calcitonin (LCT). 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys- Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys- Leu-Gln- Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly- S er-Gly-Thr-Pro-NH2 Figure 4. Calcitonin (CT) and lipidized calcitonin (LCT) Calcitonin suppresses bone resorption by inhibiting osteoclast activity which cause bone digestion and calcium release into blood. Calcitonin is therefore used to treat Paget’s disease and postmenopausal osteoporosis (44). Currently calcitonin is used as either subcutaneous injection or nasal spray. Subcutaneous injections are undesirable, since calcitonin treatment requires long-term therapy, and nasal sprays upon extended use cause nasal irritation. Therefore, an oral formulation o f calcitonin is an appropriate and desirable substitute. In a recent study, W ang et al showed that lipidized calcitonin administered orally to ovariectomized rats, resulted in a significant decrease o f deoxypyridinoline, a biomarker o f bone resorption, whereas unmodified calcitonin showed no such effect (45). The lipidized peptide was therefore able to show an enhancement o f oral calcitonin bioavailability. Leucine enkephalin (Mwt 642), a pentapeptide with a neutral charge, was also lipidized. However due to the absence o f a sulfur containing amino acid, the CT Pal Pal Cys LCT 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lipidization process conjugated the amino terminal o f enkephalin to one long fatty acid chain via maleic anhydride and cysteine. Contrary to enkephalin, lipidized enkephalin, when administered orally, demonstrated an increased stability against enzymatic hydrolysis and an enhanced GI absorption and analgesic activity in formalin-injected mice (46). 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II. MATERIALS AND METHODS 2.1 Peptide lipidization and iodination The peptides used in this study included: desmopressin (Bachem, CA), enkephalin (Sigma, CA) and calcitonin (Penta Biotech, CA). Desmopressin (DP) and calcitonin (CT) were both lipidized with palmitic acid via a disulphide bond using a cysteine linker to form palmitoyl desmopressin (DPP) and palmitoyl calcitonin (LCT), respectively. The lipidization in both instances was carried out as described by Ekrami et al (42). Enkephalin (ENK), on the other hand, was lipidized with palmitic acid via an amide bond to the a-am ino group o f the peptide using maleic anhydride and cysteine as linkers (46). The palmitic acid-lipidized enkephalin (051), was a gift from Dr. Jeff Wang. Each peptide and its derivative were then radiolabeled with 1 2 5 I (ICN, Irvine, CA) via the standard chloramine-T method o f iodination (47). Briefly, appropriate columns were packed before a day. Sephadex G-10 (Amersham Biosciences, NJ) soaked in phosphate buffer saline (PBS) was used for DP, G-15 (Sigma, CA) in dimethylformamide (DMF) for DPP, G-25 in PBS for CT, LH20 in DMF for LCT and acetonitrile for ENK and 051. Chloramine-T (8mg/0.5mL) (), an oxidizing agent responsible for iodide substitution on the meta position o f the tyrosine ring, was first added for ten minutes followed by sodium metabisulfite (Sigma, CA) (4.8mg/0.5mL), a reducing agent responsible for terminating the reaction. After ten minutes potassium iodide (Sigma, CA) (lOmg/mL) was added to complex any excess radiolabeled iodide in the reaction 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mixture. The peptide was run through the appropriate column and 1 mL was pre fraction was collected for thirteen fractions. To determine the fraction containing the peptide, 0.5 ul from each test-tube was assayed for radioactivity using Packard Gamma Counter (Packard Instruments, Meriden, CT). 2.2 Primary culture of rat alveolar epithelial cell monolayers Primary rat alveolar epithelial cells were obtained from the Will Rogers Institute Pulmonary Research Center at USC. Purification o f AEC followed the procedure described by Borok et al. Briefly, specific pathogen-free male Sprague- Dawley rat lungs were perfused via the pulmonary artery and lavaged with Ca / 9 + Mg -free Ringer’s solution. The lungs were then instilled with porcine pancreatic elastase (2.5 U/mL, W orthington Biochemical, Lakewood NJ) for 20 minutes at 37°C. The lung tissue blocks were then minced and sequentially filtered through 100, 40 and 10 pm Nitex membranes. Alveolar macrophages were removed from the crude cell mixture by IgG panning (48). These partially purified alveolar type II cells (pneumocytes) were then seeded onto tissue culture-treated 12 mm polycarbonate filter (Transwell 3401, 0.4 pm pore size, Costar Coming, Cambridge, MA) at a density o f 1.5 million cells/cm in culture medium composed o f serum-free defined media (SFM) supplemented with 10% newborn bovine serum. The cells were fed with serum enriched media (SM) on days three and five and the transport experiment was done on day six or seven. Alveolar epithelial cells are primarily isolated as those with type II cell morphology, but they differentiate into type I cell- 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. like phenotype starting from day three. To prevent transdifferentiation, human keratinocyte growth factor (hKGF, Calbiochem, San Diego, CA) was used for the purpose o f testing transport across type II-like cells. A final concentration of lOng/ml o f KGF was added on day three to the media bathing both apical and basolateral compartments. To ensure cell viability, transepithelial resistance (TEER) measurements were taken on days three and five using an epithelial voltage- ohm meter (EVOM, W orld Precision Instruments, Sarasota, FL). Resistance of about 1500 Gem2 or greater was an indication o f integrity for both the tight junctions and cells. 2.3 Transport and uptake studies across rat alveolar epithelial cell monolayers 2.3.1 In serum-free media (SFM) On day six, cells were first washed with SFM and incubated for ten minutes at 37 °C to remove any proteins that might interfere with the experiment. Cells were dosed with the respective iodinated compound or its lipidized derivative. Concentration and/or time-dependant experiments were carried out for 1 2 5 I- desmopressin, 1 2 5 I-enkephalin and 1 2 5 I-calcitonin along with their lipidized analogue to assess type o f transport and suitable concentration. Prior to sample collection, resistance o f the cells was measured to ensure cell viability. Apical to basolateral transport was assessed by collecting all the basolateral media (1.5ml) and using the Packard Gamma Counter for radiation count. For uptake studies, the 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Transwell filter where the cell monolayers are adhered, was washed three times with 4 °C PBS (pH 7.4). The filter was excised and radioactivity associated with the adhering cell monolayer was measured as an indication o f uptake. In some cases, transport studies used a final concentration o f 3mM glutathione (Sigma, CA) which was added at the start o f experiment incubation to the basolateral media as a reducing agent that could possibly help separate the peptide from the fatty acid. Glutathione did not significantly alter the cell TEER. Some transport studies also used protease inhibitors to assess whether degradation o f the peptides was due to endopeptidase or exopeptidases associated with the alveolar cell membrane or present within the cells. Protease inhibitors added at the beginning o f experiment incubation to the apical side o f the Transwell were, 0.0145mM pepstatin (Sigma, CA) and 0.028mM N-p- Tosyl-L-phenylalanine chloromethylketone -TPCK- (Sigma, CA) for surface peptidase and in a separate experiment, 0.5mM Leupeptin (Sigma, CA) for lysosomal peptidase. 2.3.2 In media containing serum This section investigated the effect o f using media other than SFM on the transport o f calcitonin and lipidized calcitonin. Alveolar epithelial cells were fed on days three and five and the experiment was carried out on day seven. The apical media referred to as conditioned media from AEC I (CMI) or conditioned media from AECII (CMII) was collected and centrifuged using J-6B centrifuge (Beckman, CA) at 2000 rpm, JS-4.2, 4°C for 20 minutes and in other cases it was centrifuged in 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. centricon YM-50 (Millipore Corporation, Bedford, MA) using RC5 centrifuge (Sorvall Instruments, CA) at 5000 rpm, SS-34, 4°C for 30 minutes. After dosing the media with radiolabeled peptide, the cells were incubated for three hours and the procedure o f measuring transport and uptake o f the peptide followed that described in section 2.3.1. 2.4 Analysis of transported peptide To determine whether the transported peptide present in the basolateral side was degraded or intact, an appropriate analysis method was carried out. Desmopressin (Mwt 1067) and its lipidized analogue were analyzed using G-10 in DMF size exclusion column chromatography and collected using M icro fractionator FC-80K (Gilson Medical Electronics, Middleton, WI). Fractions o f 1 ml were collected and measured for radioactivity using Gamma Counter. Leu-enkephalin (Mwt 642) and its lipidized analogue, however, were unsuitable for size exclusion chromatography analysis and an alternative method, such as HPLC, was not provided by our lab. Calcitonin (Mwt 3432) was analyzed using 15% trichloroacetic (TCA) precipitation. TCA was added to the samples which were kept on ice for 30 minutes and then centrifuged at 2235 x g for 20 minutes at 4°C. The supernatant layer was aspirated leaving the precipitate, which indicated amount o f intact peptide, whose radioactivity was measured using the gamma counter. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. III. RESULTS AND DISCUSSION 3.1 Transport and uptake in serum free media Uptake and transport studies were carried out for desmopressin and enkephalin dosed in serum free media. Fig. 5 showed that in AECI, DP had a slightly higher transport than DPP; but on the other hand DPP showed a significantly greater uptake. In AECII, DP and DPP showed similar patterns to AECI transport and uptake (Fig.6). However the uptake o f DPP was higher in type I cells due to the greater surface area provided by the thin monolayer. The transport rate o f DP in AECI and AECII w asl.39 and 0.61 respectively whereas DPP showed no significant difference in rate o f transport (Fig.7 and Fig.8). Previous studies have shown that DP is transported mainly paracellularly (25) and that could account for the difference in rate o f transport since it would be easier for DP to traverse the tight junctions of the flat AECI rather than the columnar AECII. In addition, AECII exhibited 1.5-fold higher TEER values than AECI throughout experiment periods. The transport of DPP, a lipophilic molecule, was detained probably due to entrapment in the cell membrane via the two lipid chains o f palmitic acid conjugated to DP. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 35 - = 5 30 - I 25 - c t 20 o a _ in lo « 10 10 15 25 0 5 20 cone (|jg/ml) —♦— DP - ■ - D P P B) 35 n a o > c 15 - 10 - (0 a 3 0 10 5 15 20 25 cone (pg/ml) Figure 5. Transport A) and uptake B) as a function o f apical concentration of desmopressin (DP) and lipidized desmopressin (DPP) added to type I alveolar epithelial cell monolayer. The cells were incubated at 37°C with 0.2, 0.6, 2, 6 and 20 pg/ml DP and DPP for three hours. 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 18 1 =• 16 ' ■s 14 - I 12~ c m - r o Q. (A c 2 10 15 0 5 cone (jjg/ml) B) 9 8 = 7 0 1 6 i>5 ■ J T 4 « 3 2 1 0 a 3 0 5 10 15 cone (pg/ml) Figure 6. Transport A) and uptake B) as a function o f apical concentration of desmopressin (DP) and lipidized desmopressin (DPP) added to type II alveolar epithelial cell monolayer. The cells were incubated at 37°C with 0.3, 3, and 10 pg/ml DP and DPP for five hours. The adsorption o f DPP onto the cell membrane was probably due to the lack o f an extracellular reductive environment, although previous groups have reported the expression o f some cell surface-associated redox enzymes such as protein disulfide isomerase (49). Therefore, the possibility o f DPP being adsorbed on the 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. membrane was tested by the addition o f glutathione to the basolateral media. Glutathione (GSH) is a reducing agent present intracellularly at a concentration o f 1- 10 mM (50). There are several transporters responsible for transporting GSH which are present either apically or basolaterally. Adding GSH to the basolateral compartment may allow it to desorb S-S bonded substances at the membranes and liberate membrane-adsorbed DPP via reduction o f the disulfide bond. Four hour incubation, showed no increase in DP transport and only a slight increase in DPP transport for both AECI and AECII (Fig.7, Fig.8) in presence o f glutathione. This maybe due to a GSH transporter present in the basolateral side o f the alveolar epithelial cell, however, no studies have addressed the presence o f GSH transporter in AEC and so the explanation remains questionable. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 10 8 6 2 0 0 2 4 6 time(hr) •DP -DP+glut B) 3.00 „ 2.50 " a > 2.00 O ) c t 1.50 o § ■ 1.00 r e ~ 0.50 0.00 0 1 2 3 4 5 time (hr) Figure 7. Time courses o f transport o f 2 pg/ml A) desompressin (DP)and B) lipidized desompressin (DPP), in the presence o f lOmM glutathione placed in basolateral compartment o f AECI. -♦— DPP DPP+glut 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) O O ) c tT o a 0 ) B) o > t o a (A C 3 2.5 2 1 0.5 0 2 4 6 0 time (hr) -DP -DP+Glu 3 2.5 2 1.5 1 0.5 0 2 4 6 0 •DPP ■ DPP+Glu time (hr) Figure 8. Time courses o f transport of 2 pg/ml A) desompressin (DP) and B) lipidized desompressin (DPP), in the presence o f lOmM glutathione in basolateral compartment o f AECII. Analysis o f the basolateral compartment showed that most o f the transported DP and DPP were degraded products. In AECII, the percentage o f intact DP showed no difference in absence or presence o f glutathione (Fig.9); whereas the total intact amount o f DPP increased slightly and doubled from 0.18 to 0.36 in the latter case (Fig. 10). 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 600 38% 0.88ng 500 400 s 300 < - > 200 100 -100 sample no B) 37% 0.73ng 400 300 s 200 Q. ° 100 -100 sample no Figure 9. Size exclusion chromatography using G-10 in DMF to analyze DP transport across AECII in A) absence and B) presence o f lOmM glutathione. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) s a o 600 500 400 300 200 100 0 -100 11.64% 0.18ng A 0 1 0 O C M sample no 50 B) 1200 - | 1000 16.3% 0.36ng 800 S 600 - O 400 200 - -200 sample no Figure 10. Size exclusion chromatography using G-10 in DMF to analyze DPP transport across AECII in A) absence and B) presence o f lOmM glutathione. In a separate experiment, pepstatin, a non-selective aspartic protease inhibitor cleaving Tyr-Phe containing peptides and TPCK, a serine protease inhibitor, when either o f the two was added to apical compartment, an increase o f intact DP in basolateral media (1.55ng) was observed and upon addition o f glutathione no enhancement was shown (0.8lng) (Fig. 11). On the hand, the amount o f intact DPP increased in presence o f protease inhibitors and glutathione, rather than when the protease inhibitors were present alone (Fig. 12). A possible explanation is that DPP 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bound to the membrane was exposed to membrane proteases longer than DP which was able to escape paracellularly and adding the protease inhibitors only enhanced the escape o f intact DP. However, the effect o f protease inhibitors on DPP was seen only when GSH was added which probably decreased the time o f exposure to proteases due to liberating the entrapped DPP from the cellular membranes. A) 2000 1500 12.8% 1.55ng C L 1000 500 0 0 10 20 30 40 sample no B) 2000 1500 12.2% 0.81ng 1000 s C L o 500 -500 sample no Figure 11. Size exclusion chromatography using G-10 in DMF to analyze DP transport across AECII in A) presence o f protease inhibitors and B) presence o f both protease inhibitors (TPCK and pepstatin) and lOmM glutathione. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) 900 800 700 600 2 500 O 400 300 200 100 10.67% 0.24ng 40 50 10 20 30 0 sample no. B) 2000 22.2% 0.86ng 1500 I 1000 o 500 30 40 0 10 20 sample no. Figure 12. Size exclusion chromatography using G-10 in DMF to analyze DPP transport across AECII in A) presence o f protease inhibitors and B) presence of protease inhibitors and lOmM glutathione. Leupeptin, a lysosomal peptidase inhibitor, had no effect on transport or changing the amount o f intact peptide in the basolateral compartment, indicating that if DPP was partially transported by transcytosis, it was not due to degradation at lysosomes. This conforms to the results by Yamahara et al who showed that leupeptin had no effect in protecting vassopressin from degradation (25). 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Uptake and transport studies for enkephalin (ENK) and lipidized enkephalin (051) were carried out. The studies showed that lipidized ENK, unlike DPP, had both a higher uptake and transport than ENK when tested at different concentrations over one hour period (Fig. 13). A) 250 = 200 S 150 r g. 100 (A c 10 15 5 0 B) v S ( 0 -Enk -051 conc(pg/ml) 25 20 15 10 5 0 10 15 5 0 -Enk -051 cone (|jg/ml) Figure 13. Transport A) and uptake B) as a function o f apical concentration of enkephalin (ENK) and lipidized enkephalin (051) added to type I alveolar epithelial cell monolayer. The cells were incubated at 37°C with 0.5, 2.5, and 10 pg/ml ENK and 051 for one hour. Furthermore, when incubated over a period o f five hours, the uptake o f ENK was low and constant. By contrast, uptake o f 051 was linearly increased for the first 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. three hours followed by a plateau in the next two hours (Fig. 14). The lipidized ENK showed 7 times higher uptake than ENK, but the rate o f transport was comparable at 20.94 ng/hr and 28.66 ng/hr, respectively. Normally, as observed with desmopressin and calcitonin, peptides have higher transport than uptake whereas their lipidized analogues show higher uptake than transport. However, because 051 demonstrated higher transport than ENK and higher transport value compared to its uptake, this suggests that a transport system other than paracellular diffusion may be involved. A) J r o o. (A c r c 150 100 - 50 - ~ r 3 1 2 4 5 -Enk -051 time (hr) B) o 10 8 o > 6 0 ) 4 rc a 2 3 0 -Enk -051 0 1 2 3 4 5 time (hr) Figure 14. Time courses of transport A) and uptake B) of 2.5 pg/ml enkephalin (ENK) and lipidized enkephalin (051) added to apical fluid o f type I alveolar epithelial cell monolayer. The cells were incubated at 37°C for five hours. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Studies have shown the presence o f caveolin-coated pits in alveolar epithelial type I cells which might be involved in the transcytosis o f some molecules (8, 27). Due to its partial lipophilicity, 051 had a higher chance to bind to the membrane than ENK which probably traversed the epithelium paracellularly. This, however, does not suggest that transport is restricted to transcellular pathway since at a particular concentration o f 051, the total amount o f peptide transported was 10 times greater than that taken up. 3.2 Transport and uptake of calcitonin in various media conditions All the previous experiments were carried out in serum free media (SFM) to eliminate the interference of serum components; however, in part II o f the study we wanted to investigate whether using conditioned media which was collected from apical compartment o f day 5 to day 7 cultured alveolar cells, would have an effect on transport. Due to cell activity, various substances are released into the apical or basolateral media. In fact, when calcitonin and lipidized calcitonin were dosed in either serum free or conditioned media obtained from AECII, the total radiolabled flux o f both peptides was increased significantly in the latter case. The conditioned media was centrifuged at 2000rpm, JS-4.2 rotar for 20 minutes at 4 °C (cent-1) or at 5000 rpm, SS-34 rotar for 30 minutes at 4 °C in Centricon-50 (cent-2). Fig. 15 shows a significantly increased transport in cent-1 but no such effect in cent-2. Therefore, the conditioned media, but not the serum free media or the media containing 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. substances less than 30kDa, was able to provide a condition for increasing the transport o f calcitonin and its lipidized analogue. 5 i — 4 -I SFM cent-1 cent-2 Figure 15. Transport o f calcitonin (CT) and lipidized calcitonin (LCT) studied at apical donor concentration o f 2 pg/ml across AECII, when dosed in fresh serum free media (SFM), type II apical conditioned media (cent-1) centrifuged at 2000 rpm, JS- 4.2 at 4°C for 20 minutes, type II apical conditioned media (cent-2) centrifuged with Centricon-50 at 5000 rpm, SS-34, at 4°C for 30 minutes. Judging from the TCA precipitation studies, the percentage o f intact peptide was higher in the SFM rather than the cent-1 and cent-2. However, the total intact proteins in the receiver compartment was higher for cent-1 transported peptide (Table 2). Table 2. Trichloroacetic acid precipitation for calcitonin (CT) and lipidized calcitonin (LCT) in various media conditions. Values for percentage o f peptide precipitated (%ppt) due to TCA, total amount o f transported radiolabeled peptide (total) and total amount o f intact radiolabeled peptide (intact) are shown. %ppt total (ng) intact (ng) CT SFM 33.08 0.59 0.20 cent-1 11.66 3.46 0.40 cent-2 16.91 0.84 0.14 LCT SFM 19.92 0.47 0.09 cent-1 14.14 2.01 0.28 cent-2 16.34 0.92 0.15 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Next, we studied the possible effect of both newborn bovine serum (SM) and KGF, calcitonin and lipidized calcitonin were dosed in various media (Fig. 16). It is quite evident that there was higher rates o f transport o f CT than LCT when dosed in the type II cell conditioned media (CMII), although the increase in both cases was due neither to SM nor KGF, suggesting that certain components present in CMII were responsible for the significant increase in the peptide transport. T □ CT |1 L C T SM SM +K G F SFM CMII Figure 16. Transport o f calcitonin (CT) and lipidized calcitonin (LCT), measured at apical donor concentration o f 2ug/ml across AECII. CT and LCT were dosed in newborn bovine serum (SM), SM with KFG, serum free media (SFM), and centrifuged conditioned media from type II cells (CMII). The use o f various media did not significantly alter the uptake o f calcitonin, except that it was highest in SFM, perhaps due to the absence o f interfering serum components. To see whether the increase in transport o f calcitonin was different in AECI and AECII, an experiment was carried out where both cell types were cultured and the apical media was used as dosing solutions. In Fig. 17, conditioned media from AECI (CMI) showed no difference in calcitonin transport between type I 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and type II cell monolayers. However, when CMII was used as apical fluid o f type I and type II cell monolayers, an increased transport o f calcitonin was seen only for the latter cell monolayers. This is a preliminary proof that might indicate the presence o f a secreted substance from type II cells which has an effect on calcitonin transport in AECII. The TCA results (Table 3) in case o f AECI showed that, though the percentage o f precipitate was lower for CMII (28.59%) than CMI (40.25%), the amount o f total peptide transported across AECI did not increase in case o f CMII, 2.08ng and 2.36ng, respectively. However, this increase was seen when CMII was used as an apical fluid for AECII cell monolayers, where a similar percent of precipitate was obtained with CMII (14.15%) and CMI (13.87%), yet a greater amount o f total peptide was transported in the former condition (3.10ng) compared to the latter (2.01ng). This suggests that increase o f transport was specific to CMII used on AECII. The uptake was highest in SFM when compared to the serum containing media. A) □ AECI ■ AECII SFM NBS CMI CMII 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B) 2.0 n 0.8 □ AECI ■ AECII SFM NBS CMI CMII Figure 17. Transport A) and uptake B) o f calcitonin measured at apical donor concentration o f 2ug/ml across alveolar epithelial cell monolayers exhibiting type I (AECI) and type II (AECII) phenotypes in serum free media (SFM), newborn bovine serum (NBS), conditioned apical media from AECI (CMI) and conditioned apical media from AECII (CMII). Table 3.Trichloroacetic acid precipitation for calcitonin (CT) and lipidized calcitonin (LCT) transported across AECI or AECII in various media conditions. %ppt total (ng) intact (ng) CT(AECI) SFM 37.05 2.53 0.95 NBS 42.02 2.56 1.09 CMI 40.25 2.36 0.99 CMII 28.59 2.08 0.60 CT(AECII) SFM 15.32 1.59 0.25 NBS 22.14 1.67 0.35 CMI 13.87 2.01 0.32 CMII 14.15 3.10 0.40 Furthermore, aliquots from CMII were taken and diluted with SFM to form three different concentrations o f CMII apical fluids: 3x, lx and O.lx (A,B and C, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. respectively). Similarly, CMI was diluted once with SFM obtaining apical fluids of two concentrations: 3x and lx (E and F, respectively). _ 18 = 16 o £ 14 1 > 12 - ~ 1 0 J o 8 a _ < /> 6 ro 4 ~ 2 0 A B C D E F treatment Figure 18. Transport o f calcitonin at apical donor concentration o f 3pg/ml dosed in AECII in: A) 3x CMII, B) lx CMII, C) 0.3x CMII, D) serum free medium, E) 3x CMI, F) lx CMI. Fig. 18 shows that as CMII became more dilute (A, B and C), a gradual decrease in calcitonin transport was noted. However, as CMI became more dilute (E and F), no significant decrease was observed. This might be explained as follows: when the serum concentration was decreased, the secreted substance (which acts to increase the transport o f the peptide), was also diluted, leading to a significant drop in calcitonin in the receiver compartment. On the other hand, a decrease o f a lesser extent was observed when calcitonin was dosed in decreasing concentrations o f CMI as shown in E and F (Fig. 18). There are two explanations for the increased transport o f calcitonin across AECII when using CMII. One possible explanation is that due to the secretion o f a larger amount o f proteases in type II cells than type I cells, calcitonin is subjected to greater degradation in CMII and hence higher transport. TCA precipitates in Table 3 show a higher percentage o f transported intact peptide in SFM and CMI rather than CMII, however analyzing dosed CMI and CMII 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. indicated that degradation o f calcitonin over five hours in either media was equal. Therefore, degradation o f calcitonin in CM was occurring due to factors in AECII rather than proteases in CMII. The other possible explanation resides in the fact that AECII are known to secrete various surfactants, which may augment the transport of calcitonin. As CMII was diluted, so was the amount o f surfactants present leading to a decrease o f calcitonin transport. Mason et al have shown that medium collected for 48hr from culture days 5 to 7 contained about 1250 ng/ml SP-A when AEC were incubated with KGF (51). To determine where calcitonin degradation was taking place, ammonium chloride was used as a pH-changing agent since it is able to diffuse into the endosome and neutralize the acidic environment, which is necessary for lysosomal hydrolase activity. Ammonium chloride had no effect on changing TEER values. The addition o f 20mM or 50mM ammonium chloride had no effect on total amount o f transported calcitonin, but uptake showed a slight increase (Fig. 19). TCA assay showed that ammonium chloride decreased both the percentage of precipitated calcitonin as well as the amount o f intact peptide. This maybe due to the high ammonium chloride concentration that may have caused an alteration of vesicular trafficking leading to an overall decrease o f peptide exocytosis and higher cellular uptake. 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A) T T : .. ' * > ’ ■ CMII CMII+20NH4CI CMII+50NH4CI B) S 0.6 CMII CMII+20NH4CI CMII+50NH4CI Figure 19. Transport A) and uptake B) o f calcitonin at apical concentration o f 3ug/ml dosed in CMII, CMII plus 20mM NH 4 CI, CMII plus 50mM NH 4 CI in AECII. Table 4. Trichloroacetic acid precipitation for transported calcitonin (CT) dosed in CMII, CMII plus 20mM NH4C1, and CMII plus 50mM NH4C1. %ppt total (ng) in ta ct(n g ) CT CM 10.59 11.40 1.22 CM+ 20mM NH4CI 8.11 11.08 0.90 CM+ 50mM NH4CI 6.15 11.05 0.80 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IV. Conclusions The lipophilic nature o f the plasma membrane poses an obstacle for the transcellular transport o f peptides and proteins that are hydrophilic in nature and which can pass mainly via the passive diffusional routes provided by the tight junctional regions. Since this paracellular pathway provides only a small fraction of the total epithelial surface area, the former transport mechanism, i.e. transcellular, remains more desirable. The presence o f both membrane bound and intracellular peptidases is yet another barrier for peptide transport which leads to increased peptide degradation. Lipidization o f peptides was therefore intended to increase lipophilicity and size, leading to an enhancement o f transcellular transport, as well as to protect from degradative enzymes allowing higher peptide bioavailability. The current study showed that lipidized peptides had significantly higher uptake than parent peptide when tested in alveolar epithelial cell monolayer culture. However, it is hard to make a general conclusion since the nature of transport and uptake was different for peptides o f differing number o f amino acids and differing net charges. It is worthwhile, though, to dwell on the possibility of utilizing such behaviour of increased uptake to treat surface related lung diseases or moreover to target particular surface proteins or invaginations that would help internalize the peptide and move it across the lung epithelial barrier. The study also demonstrated that dosing a particular peptide, in this case calcitonin, in conditioned media, i.e. media collected from cultured AECs rather than fresh serum free or serum containing media, resulted in a significantly higher 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transport o f peptide. This has not been demonstrated before, but if one disregards the possibility o f degradation by membrane-associated peptidases, the reason may be related to the presence o f a particular protein produced by the cultured AECII which helps transport the peptide via a transcellular pathway, leading to a higher transport rate. If in-depth research does in fact show the presence o f factor(s) that help transport protein or peptides, studies utilizing the factor(s) as a drug delivery agent for peptide transport could prove worthwhile. 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. References 1. W ang J, Shen D, Shen W. Preparation, purification, and characterization o f a reversibly lipidized desmopressin with potentiated anti-diuretic activity. Pharm Res. 16: 1674-1679 (1999). 2. Honeycutt L, Wang J, Ekarami H, Shen WC. Comparison o f pharmacokinetic parameters o f a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate. Pharm Res. 13:1373-1377 (1996). 3. M atsukawa Y, Yamahara H, Yamashita F, Lee VHL, Crandall ED, Kim JK. Rates o f protein transport across rat alveolar epithelial cell monolayers. J Drug Targeting. 7: 335-342 (2000). 4. Cheek JM, Evans MJ, Crandall ED. Type I cell-like morphology in tight alveolar epithelial monolayers. Exp Cell Res. 184: 375-387(1989). 5. Crapo JD, Barry BE, Gehr P, Bachofen M, W eibel ER. Cell number and cell characteristics o f the normal human lung. Am Rev Respir Dis. 125: 332- (1982). 6. Adamson IR, Bowden DH. Derivation o f type 1 epithelium from type 2 cells in the developing rat lung. 32: 736-745 (1975). 7. Kim KJ, M alik AB. Protein transport across the lung epithelial barrier. Am J Physiol Lung Cell Mol Physiol. 284: 247-259 (2003). 8. Borok Z, Lubman RL, Danto SI, Ahang XL, Zabski SM, Kin LS, Lee DM, Agre P, and Crandall ED. Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression o f aquaporin 5. Am J Respir Cell Mol Biol. 18: 554- 561 (1998). 9. Campbell L, Hollins AJ, Al-Eid A, Newman GR, von Ruhland C, Gumbleton M. Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures. Biochem Biophys Res Commun. 262: 744-751 (1999). 10. Isakson BE, Lubman RL, Seedorf GJ, Boitano S. M odulation o f pulmonary alveolar type II cell phenotype and communicatin by extracellular matrix and KFG. Am J Physiol Cell Physiol. 281: C1291-C1299 (2001). 11. Urlich TR, Yi ES, Longmuir S, Yin S, Blitz R, Morris CF, Housley RM, Pierce GF. Keratinocyte growth factor is a growth factof for type II pneumocytes in vivo. J Clin Invest. 93: 1298-1306 (1994). 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12. Elbert KJ, Schafer UF, Shaafers HJ, Kim KJ, Lee VH, Lehr CM. M onolayers of human alveolar epithelial cells in primary culture for pulm onary absorption and transport studies. Pharm Res. 16:601-608 (1999). 13. Kwang-Jin K, Borok Z, Crandall ED. A useful in vitro model for transport studies o f alveolar epithelial barrier. Pharm Res. 18: 253-255 (2001). 14. Adson A, Raub TJ, Burton PS, Barsuhn CL, Hilgers AR, Audus KL, Ho FH. Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J Pharm Sci. 83: 1529- 1536 (1994). 15. Dodoo AN, Bansal SS, Barlow DJ, Bennet FC, H ider RC, Lansley AB, Lawrence MJ, M arriott C. Use o f alveolar cell monolayers o f varying electrical resistance to measure pulmonary peptide transport. J Pharm Sci. 89: 223-231 (2000a). 16. Dodoo AN, Bansal S, Barlow DJ, Bennet FC, Hider RC, Lansley AB, Lawrence MJ and M ariott C. Systematic investigations o f the influence o f molecular structure on the transport o f peptides across cultured alveolar cell monolayers. Pharm Res. 17: 7-14 (2000b). 17. M atsukawa Y, Yamahara H, Lee VH, Crandall ED, Kim KJ. Horseradish peroxidase transport across rat alveolar epithelial cell monolayers. Pharm Res. 13: 1331-1335 (1996) 18. M atsukawa Y, Lee VH, Crandall ED, Km KJ. Size-dependent dextran transport across rat alveolar epithelial cell monolayers. J Pharm Sci. 86: 305-309 (1997). 19. Folkesson HG, W estrom BR, Dahlback M, Lundin S, Karlson BW. Passage of aerosolized BSA and the nonapeptide dDAVP via the respiratory tract in young and adult rats. Exp Lung Res. 18:595-614 (1992). 20. Okumura K, Iwaka S, Tsuguchika Y, Komada F. Intratracheal delivery of insulin absorption from solution and aerosol by rat lung. Int J Pharm. 88: 63-73 (1992). 21. Patton JS, Trinchero P, Platz RM. Bioavailability o f pulmonary delivered peptides and proteins: alpha a-interferon, calcitonin and parathyroid hormone. J Contr Rel. 28:79-85 (1994). 22. Kobayashi S, Kondo S, Juni K. Study on pulmonary delivery o f salmon calcitonin in rats: effects o f protease inhibitors and absorption enhancers. Pharm Res. 11:1239-43 (1994). 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23. M orimoto K, Yamahara H, Lee VHL, Kim KJ. Dipeptide transport across rat alveolar epithelial cell monolayers. Pharm Res. 10: 1668-1674(1993). 24. W ang L, Toledo-Velasquez D, Schwegler-Berry D, Ma JK, Rojanasakul Y. Transport and hydrolysis o f enkephalins in cultured alveolar epithelial monolayers. Pharm Res. 10: 1662-1667(1993). 25. Yamahara H, Morimoto K, Lee VHL, Kim KJ. Effects o f protease inhibitors on vasopressin transport across rat alveolar epithelial cell monolayers. Pharm Res. 11: 1617-1622(1994). 26. John TA, Vogel SM, Minshall RD, Ridge K, Tiruppathi C, M alik AB. Evidence for the role o f alveolar epithelial gp60 in active transalveolar albumin transport in the rat lung. J Physiol 533: 547-559 (2001). 27. Gumbleton M. Caveolae as potential macromolecule trafficking compartments within alveolar epithelium. Adv Drug Del Rev. 49: 281-300 (2001). 28. Fandy TE, Lee VHL, Crandall ED, Kim KJ. Transport characteristics of immunoglobulin G (IgG) across rat alveolar epithelial cell monolayers (abstract). Am J Respir Crit Care Med. 163: A572 (2001). 29. W eaver ET, W hitsett JA. Function and regulation o f expression o f pulmonary surfactant-associated proteins. Biochem J. 273: 249-264(1991). 30. W hitsett JA, Hull W, Ross G, W eaver T. Characteristics o f human surfactant- associated glycoproteins A. PediatrR es. 19:501-508 (1985). 31. Benson B, Hawgood S, Schilling J, Clements J, Damm D, Cordell B, White RT. Nucleotide and amino acid sequences o f pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption. Proc. Natl. Acad. Sci. 84: 66-70 (1987). 32. Sidobre S, Puzo G, Rivieare M. Lipid-restricted recognition o f mycobacterial lipoglycans by human pulmonary surfactant protein A: a surface-plasmon-resonance study. Biochem. J. 365: 89-97 (2002). 33. Freedman RB. Protein disulfide isomerase: multiple roles in the modification o f nascent secretory proteins. Cell. 57: 1069-1072 (1989). 34. W intergerst E, Manz-Keinke H, Plattner H, Schlepper-Schafer J. The interaction o f a lung surfactant protein (SP-A) with macrophages is mannose dependent. Eur J Cell Biol. 50:291-298 (1989). 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35. Kuroki Y, M ason RJ, Voelker DR. Alveolar type II cells express a high-affmity receptor for pulmonary surfactant protein A. Proc Natl Acad Sci. 85: 5566-5570 (1988). 36. Kalin M, Socher R. Internalization o f pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells. J Histochem Cytochem. 38: 483-92 (1990). 37. Haitsma JJ, Lachmann U, Lachmann B. Exogenous surfactant as a drug delivery agent. Adv Drug Del Rev. 47: 197-207 (2001). 38. A. van’t Veen, Gommers D, Verbrugge SJ, W ollmer P, M outon JW, Kooij PP, Lachmann B. Lung clearance o f intratracheally instilled 99mTc-tobramycin using pulmonary surfactant as vehicle. Br. J. Pharmacol. 126: 1091-1096 (1999). 39. Katkin JP, Husser RC, Langston C, W elty SE. Exogenous surfactant enhances the delivery o f recombinant adenoviral vectors to the lung. Hum. Gen Ther. 8: 171- 176 (1997). 40. Kahns AH, Buur A, Bundgaard H. Prodrugs o f peptides: synthesis and evaluation o f various esters o f desmopressin (dDAVP). Pharm Res. 10: 68-74 (1993). 41. W ang J, W u D, Shen W-C. Structure-activity relationship o f reversibly lipidized peptides: studies o f fatty acid-desmopressin conjugates. Pharm Res. 19: 609-614 (2002). 42. Ekrami MH, Kennedy RA, Shen WC. W ater-soluble fatty acid derivatives as acylating agents for reversible lipidization o f polypeptides. FEBS Letters. 371: 283- 286 (1995). 43. Physicians’ Desk Reference. 51st edition. 778-779 (1997). 44. Physicians’ Desk Reference. 51st edition. 403-406 (1997). 45. W ang J, Chow D, Heiati H, Shen WC. Reversible lipidization for the oral delivery o f salmon calcitonin. J Cont Release, (in press) 46. W ang J, Hogenkamp D, Tran M, Li WY, Yoshimura R, Hohnstone T, Gee KW, Shen WC. Reversible lipidization for the oral delivery o f leu-enkephalin. Abstract for 30th Annual Meeting o f CRS (2003). 47. McConahey, PC and Dixon, FJ. Radioiodination of proteins by the use o f the chloramine-T method. M ethods Enzymol. 70: 221-247 (1980). 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48. Borok Z, Danto SI, Zabski SM, Crandall ED. Defined medium for primary culture de novo o f adult rat alveolar epithelial cells. In Vitro Cell Dev Biol Anim. 30A: 99-104 (1994). 49. Donoghue N, Yam PT, Jiang XM, Hogg PJ. Presence o f closely spaced protein thiols on the surface o f mammalian cells. Protien Sci. 9: 2436-2445 (2000) 50. Hammon CL, Lee TK, Ballatori N. Novel roles for glutathione in gene expression, cell death and membrane transport o f organic solutes. J Hepatology. 34: 946-954 (2001). 51. M ason RJ, Lewis MC, Edeen KE, Mccormick-Shannon K, Neilsen LD, Shannon JM. M aintenance o f surfactant protein A and D secretion by rat alveolar type II cells in vitro. Am J Physiol Lung Cell Mol Physiol. 282: L249-L258 (2002). 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bibliography A. van’t Veen, Gommers D, Verbrugge SJ, W ollmer P, M outon JW, Kooij PP, Lachmann B. Lung clearance o f intratracheally instilled 99mTc-tobramycin using pulmonary surfactant as vehicle. Br. J. Pharmacol. 126: 1091-1096(1999). Adamson IR, Bowden DH. Derivation o f type 1 epithelium from type 2 cells in the developing rat lung. 32: 736-745 (1975). Adson A, Raub TJ, Burton PS, Barsuhn CL, Hilgers AR, Audus KL, Ho FH. Quantitative approaches to delineate paracellular diffusion in cultured epithelial cell monolayers. J Pharm Sci. 83: 1529- 1536 (1994). Benson B, Hawgood S, Schilling J, Clements J, Damm D, Cordell B, White RT. Nucleotide and amino acid sequences o f pulmonary surfactant protein SP 18 and evidence for cooperation between SP 18 and SP 28-36 in surfactant lipid adsorption. Proc. Natl. Acad. Sci. 84: 66-70 (1987). Borok Z, Danto SI, Zabski SM, Crandall ED. Defined medium for primary culture de novo o f adult rat alveolar epithelial cells. In Vitro Cell Dev Biol Anim. 30A: 99- 104 (1994). Borok Z, Lubman RL, Danto SI, Ahang XL, Zabski SM, Kin LS, Lee DM, Agre P, and Crandall ED. Keratinocyte growth factor modulates alveolar epithelial cell phenotype in vitro: expression o f aquaporin 5. Am J Respir Cell Mol Biol. 18: 554- 561 (1998). Campbell L, Hollins AJ, Al-Eid A, Newman GR, von Ruhland C, Gumbleton M. Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures. Biochem Biophys Res Commun. 262: 744-751 (1999). Cheek JM, Evans MJ, Crandall ED. Type I cell-like morphology in tight alveolar epithelial monolayers. Exp Cell Res. 184: 375-387(1989). Crapo JD, Barry BE, Gehr P, Bachofen M, Weibel ER. Cell number and cell characteristics o f the normal human lung. Am Rev Respir Dis. 125: 332- (1982). Dodoo AN, Bansal SS, Barlow DJ, Bennet FC, Hider RC, Lansley AB, Lawrence MJ, M arriott C. Use o f alveolar cell monolayers o f varying electrical resistance to measure pulmonary peptide transport. J Pharm Sci. 89: 223-231 (2000a). 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Dodoo AN, Bansal S, Barlow DJ, Bennet FC, Hider RC, Lansley AB, Lawrence MJ and M ariott C. Systematic investigations o f the influence o f m olecular structure on the transport o f peptides across cultured alveolar cell monolayers. Pharm Res. 17: 7-14 (2000b). Donoghue N, Yam PT, Jiang XM, Hogg PJ. Presence o f closely spaced protein thiols on the surface o f mammalian cells. Protien Sci. 9: 2436-2445 (2000) Ekrami MH, Kennedy RA, Shen WC. Water-soluble fatty acid derivatives as acylating agents for reversible lipidization o f polypeptides. FEBS Letters. 371: 283- 286 (1995). Elbert KJ, Schafer UF, Shaafers HJ, Kim KJ, Lee VH, Lehr CM. Monolayers o f human alveolar epithelial cells in primary culture for pulm onary absorption and transport studies. Pharm Res. 16:601-608 (1999). Fandy TE, Lee VHL, Crandall ED, Kim KJ. Transport characteristics of immunoglobulin G (IgG) across rat alveolar epithelial cell monolayers (abstract). Am J Respir Crit Care Med. 163: A572 (2001). Folkesson HG, W estrom BR, Dahlback M, Lundin S, Karlson BW. Passage of aerosolized BSA and the nonapeptide dDAVP via the respiratory tract in young and adult rats. Exp Lung Res. 18:595-614(1992). Freedman RB. Protein disulfide isomerase: multiple roles in the modification of nascent secretory proteins. Cell. 57: 1069-1072 (1989). Gumbleton M. Caveolae as potential macromolecule trafficking compartments within alveolar epithelium. Adv Drug Del Rev. 49: 281-300 (2001). Haitsma JJ, Lachmann U, Lachmann B. Exogenous surfactant as a drug delivery agent. Adv Drug Del Rev. 47: 197-207 (2001). Hammon CL, Lee TK, Ballatori N. Novel roles for glutathione in gene expression, cell death and membrane transport o f organic solutes. J Hepatology. 34: 946-954 (2001). Honeycutt L, W ang J, Ekarami H, Shen WC. Comparison o f pharmacokinetic parameters o f a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate. Pharm Res. 13:1373-1377 (1996). Isakson BE, Lubman RL, Seedorf GJ, Boitano S. M odulation o f pulm onary alveolar type II cell phenotype and communicatin by extracellular matrix and KFG. Am J Physiol Cell Physiol. 281: C1291-C1299 (2001). 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. John TA, Vogel SM, Minshall RD, Ridge K, Tiruppathi C, M alik AB. Evidence for the role o f alveolar epithelial gp60 in active transalveolar albumin transport in the rat lung. J Physiol 533: 547-559 (2001). 35. Kuroki Y, M ason RJ, Voelker DR. Alveolar type II cells express a high-affmity receptor for pulmonary surfactant protein A. Proc Natl Acad Sci. 85: 5566-5570 (1988). Kahns AH, Buur A, Bundgaard H. Prodrugs o f peptides: synthesis and evaluation of various esters o f desmopressin (dDAVP). Pharm Res. 10: 68-74 (1993). Kalin M, Socher R. Internalization o f pulmonary surfactant into lamellar bodies of cultured rat pulmonary type II cells. J Histochem Cytochem. 38: 483-92 (1990). Katkin JP, Husser RC, Langston C, Welty SE. Exogenous surfactant enhances the delivery o f recombinant adenoviral vectors to the lung. Hum. Gen Ther. 8: 171-176 (1997). Kim KJ, Borok Z, Crandall ED. A useful in vitro model for transport studies of alveolar epithelial barrier. Pharm Res. 18: 253-255 (2001). Kim KJ, M alik AB. Protein transport across the lung epithelial barrier. Am J Physiol Lung Cell Mol Physiol. 284: 247-259 (2003). Kobayashi S, Kondo S, Juni K. Study on pulmonary delivery o f salmon calcitonin in rats: effects o f protease inhibitors and absorption enhancers. Pharm Res. 11: 1239- 43 (1994). Mason RJ, Lewis MC, Edeen KE, M ccormick-Shannon K, Neilsen LD, Shannon JM. Maintenance o f surfactant protein A and D secretion by rat alveolar type II cells in vitro. Am J Physiol Lung Cell Mol Physiol. 282: L249-L258 (2002). M atsukawa Y, Lee VH, Crandall ED, Km KJ. Size-dependent dextran transport across rat alveolar epithelial cell monolayers. J Pharm Sci. 86: 305-309 (1997). M atsukawa Y, Yamahara H, Lee VH, Crandall ED, Kim KJ. Horseradish peroxidase transport across rat alveolar epithelial cell monolayers. Pharm Res. 13: 1331-1335 (1996) Matsukawa Y, Yamahara H, Yamashita F, Lee VHL, Crandall ED, Kim KJ. Rates o f protein transport across rat alveolar epithelial cell monolayers. J Drug Targeting. 7: 335-342 (2000). McConahey, PC and Dixon, FJ. Radioiodination o f proteins by the use o f the chloramine-T method. Methods Enzymol. 70: 221-247 (1980). 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Morimoto K, Yamahara H, Lee YHL, Kim KJ. Dipeptide transport across rat alveolar epithelial cell monolayers. Pharm Res. 10:1668-1674(1993). Okumura K, Iwaka S, Tsuguchika Y, Komada F. Intratracheal delivery o f insulin absorption from solution and aerosol by rat lung. Int J Pharm. 88: 63-73 (1992). Patton JS, Trinchero P, Platz RM. Bioavailability o f pulmonary delivered peptides and proteins: alpha a-interferon, calcitonin and parathyroid hormone. J Contr Rel. 28: 79-85 (1994). Physicians’ Desk Reference. 51st edition. 778-779 (1997). Physicians’ Desk Reference. 51st edition. 403-406 (1997). Sidobre S, Puzo G, Rivieare M. Lipid-restricted recognition o f mycobacterial lipoglycans by human pulmonary surfactant protein A: a surface-plasmon-resonance study. Biochem. J. 365: 89-97 (2002). Urlich TR, Yi ES, Longmuir S, Yin S, Blitz R, Morris CF, Housley RM, Pierce GF. Keratinocyte growth factor is a growth factof for type II pneumocytes in vivo. J Clin Invest. 93: 1298-1306 (1994). Wang J, Chow D, Heiati H, Shen WC. Reversible lipidization for the oral delivery o f salmon calcitonin. J Cont Release, (in press) Wang J, Hogenkamp D, Tran M, Li WY, Yoshimura R, Hohnstone T, Gee KW, Shen WC. Reversible lipidization for the oral delivery of leu-enkephalin. Abstract for 30th Annual Meeting o f CRS (2003). Wang J, Shen D, Shen W-C. Preparation, purification, and characterization o f a reversibly lipidized desmopressin with potentiated anti-diuretic activity. Pharm Res. 16: 1674-1679(1999). Wang J, W u D, Shen W-C. Structure-activity relationship o f reversibly lipidized peptides: studies of fatty acid-desmopressin conjugates. Pharm Res. 19: 609-614 (2002). Wang L, Toledo-Velasquez D, Schwegler-Berry D, M a JK, Rojanasakul Y. Transport and hydrolysis o f enkephalins in cultured alveolar epithelial monolayers. Pharm Res. 10: 1662-1667(1993). W eaver ET, W hitsett JA. Function and regulation of expression o f pulmonary surfactant-associated proteins. Biochem J. 273: 249-264(1991). 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. W hitsett JA, Hull W, Ross G, W eaver T. Characteristics o f human surfactant- associated glycoproteins A. PediatrRes. 19:501-508 (1985). W intergerst E, M anz-Keinke H, Plattner H, Schlepper-Schafer J. The interaction o f a lung surfactant protein (SP-A) with macrophages is mannose dependent. Eur J Cell Biol. 50: 291-298 (1989). Yamahara H, Morimoto K, Lee VHL, Kim KJ. Effects o f protease inhibitors on vasopressin transport across rat alveolar epithelial cell monolayers. Pharm Res. 11: 1617-1622(1994). 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Bahhady, Rana (author)

Core Title Effect of lipidization on transport and uptake of peptides across rat alveolar epithelial cell monolayers

School Graduate School

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Health Sciences, Pharmacology,OAI-PMH Harvest

Language English

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Advisor Shen, Wei-Chiang (committee chair), Kim, (Dr.) (committee member), Okamoto, Curtis T. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-303319

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