$6 Abstracts

The human intestinal ceil Caco-2 was used as an in vitro model for gene transfer to the gagmimeetinal tract. Caco-2 monolayers develop tight jrmctiom and form a significant diffusion barrier [2]. They are moq~holngically polar and develop brush border membranes at their apical surface. By infecting at various times after seeding, we found that Ad.RSVnt/acZ could indeed infect Caco-2 tormelayers with a marked preference for none~nfluent and undiffetetuiatnd cellS. Infection of Caco-2 ~ grown on permeable supports with the adenoviral vector containing the IL-Ira gene (Ad.RSVll.-Ira) resultnd in clearly delectable amounts of IL-Ira protein socreJed into the apical as well as basolateral comparmtetu. Secretkm of IL-lra ptotein has been maintained for at least 16 days after infection. Average secretion to the basolateral compartment, which would be the target side for IBD, was calculated to be about 10 rig/day. Additionally, the cell viability and integrity of the monulayers were not affected by infection with adanoviral vectors. In summary, we showed that adenoviral vectors were staHe to a simulated luminal envirotLmunt and were able to infect and express a transduced gene over the lifetime of the model Caco-2 m~-~ulayer. Furthermore. the R,-Ira protein was exprlmsed at therapeutic levels, Thus, we feel that the Caco-2 cell line serves as an effective /n vitro model system for developing adenoviral therapy in the treatment of IBD.

R e f e r ee : L F. Comin¢lli, C.C. Nast, A. Duchini, M. Lee. Gg~'eeateaology 1992, 103:65-71 2. I~ Artursson. Crit. Rcv, The. Drag Calri~ Sy'M. 1991, 8:305-330

P 2

CHARACTERIZATION OF 0PARACELLULAR-)TRANSPORT PROPERTIES OF I E c - I g AND CACO-2

E. Duizero W.H. Sterthuls, AH.Pennings, p.J.van Bladeren, and I.P. Groten TNO Nutrition and Food Research Institute, Division of Toxicology, P.O.Box 360, 3700 A J, Zeiat, The Nethoflands.

Enzymatic degradation is one of major ban-iers to the nasal absorption of pepfides. It was previously shown that coadministratlrm with proteolytic enzyme inhibitors such as aptulinin enhanced the abanqgion I. However, these broad spectrum Inhihitors may act on enzymes that am not involved in the degradation, and cause unfavecable side effects, Tberefom, inhibiturs that are more specific to peptide degradations are preferable.

Specificities in peptide degradation were studied using homogeantes of human nasal mneosa. An analysis of the degradation products revealed that many peptides such as calcitonth, somatos~alln and PTH were peeferentially cleaved at the C-lermina~ of lysines. This result suggested that protection against this cleavage may specifically inhibit such degradations. The lysth¢ side chain analogues were then considered as candidates for the inltibitors and their inhibitory effects on peptide degradations in human nasal mucosa homog~ate were studied. Among the compounds examined, tranexamic acid or p-aminomethyl benzoic acid, which has an amino-alkyl ring smtotore (NH2-(CH~.-CtHs-R , NH2-(CHz).-C6HIx-R , n=l-3), gave the highest inhibitions in the homoganate studies. AS tranexamie acid or p-aminomcthyl benzoic acid failed to inhibit the cieavages at sites other than the C-terminal of the lysines. their action seemed to be specific.

Because enzyme inhibitors that where selected from the nasal mucosa homngcoates studies gave enhanced nasal peptide absorption in rabbits (2), these compounds are expected to work in in vivo .studies with humans. Furthermore, they am already in clinical use as antifibdnoly- tic agents. Therefore they may be promising absovptinn enhancers for humans in view of their efficacy and safety.

References: I. K. Morimoto, et al. (1995), latJ.pharm. 113:1-8 2. M. Dohl. y. Nishibe, et al. (1993), ProcJnt.Symp. Delivert of Protein Drugs, 97-100

To date, several human colonic carcinoma cell lines 0¢I'-29, T84, Caco-2) are used to study the intestinal transport of compounds in vitro, However, severs] hydrophllic compounds, of which intestinal uptake in vivo was found, am not traraported across these colonic ceils. This migha 13e caused by exclusion from the paraculhthtr pathway through greater tightness of the tight junctions in colonic cells compared to that of small intestinal ceils. The aim of this study was to compare transport propeaies of the rat small intestinal cell line IEC-18 to those of Caco-2.

Caco-2 and IEC-Ig were cultured on 12 or 24.5 mm Transwell polycarbunate fdters (0.4 pin pore size) under standard conditions. Both cell lines formed a confluent monolayer as confirmed by electrical resistance measurements CI'ERc~2= 800 Ohm/era 2, TERt~: i,= 80 Ohnffeen ~) and fluorescence microscopy. Transport studies were performed in DMEM with 1% BSA instead of FCS in apical to basolateral direction. Radioactively labelled tracers were added at start concenU'ations of 10 pM, except Cd (1 gM), and sm'nples were taken basolate- raly between 10 rain and 3 hours.

The apparent permeability coefficients (P,,~ were determined according to the following equation: P.~= PIA*C o (cm/s), P = permeability rate (tool/s), Co= Initial apical concentration of test substance (mol/ml), A = area of filter with monolayer(cma).

Papp (x 10 ~ urals) compound I EC- 18 Caco-2 PEG-4GO0 0. 35 0. I 1 Marmltol 3,49 0.14 Cadmium 8.19 0.96 Acetyl salycilic acid i7. | 3.64 Propranohil 36.6 30.6

The compounds are ordered according to expected increasing importance of the transceilular transport pathway. P~pranolol has a comparable high p ~ for both cells, indicating mainly transcellalar transport, while PEG-4000 and marmhol are mainly transported paracellulady.

The higher Papp values of IEC-18 than of Caco-2 for PEG~IOC0 and Mannitol corresponds with the TER values of both cell lines. This might indicate that the fight junctional complex of the rat ileal crypt cell IEC-18 is looser than that of the colonic Caco-2 cells. To look at this more closely we immunolabelled the tight junctional protein ZO-I and F-actin in both cell lines. Images of the fluorescence labelled cells revealed a clear actin belt in the differentiated Caco-2 cells, whereas this actin belt was barely visible in the undifferentiated IEC-lg cells. However, the cellular disttihtuion of ZO-1 was similar for both ceils. Molpbu- meUic analysis of this ZO-1 label did not reveal any difference in intensity of the fluorescen- ce signal.

Exposure of monolayers of IEC-18 and Caco-2 to 0.4 mM palmitoylcamithine (PCC) resulted in a reduced TER and increased P.~ for Manmtol and PEG-4000 for both cell lines. However, the intensity and eeUalar distribution of ZO-I and the distribution of F-actin were dramatically changed in [EC-18 but not in Caco-2. This might indicate that the role of ZO-I and F-actin in the regulation of the permeability of the paracellalar pathway differs in IEC-18 and Caco-2 cells.

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SPECIFIC INHIBITORS FOR THE ENZYMATIC DEGRADATION OF PEPTIDES IN H U M A N NASAL MUCOSA

Y. Nishibu, M. Dohi, y . Makino, Y. Suzuki and T. Fujii DDS Researeh Laboratory, Teijin Limited. 4-3-2, Asabigako. Hinoshi. Tokyo 191, Japan

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T H E XENOPUS LUNG AS A M O D E L FOR ALVEOLAR DRUG TRANSPORT: ASSESSMENT OF B I O E L E C T R I C A L P A R A M E T E R S

M. Bartels and C.M. Lehr Department of Biopharmaceulics and Pharmaceutical Technology, University of the Saarland, Saarbrllckco, Germany

Introduction The fast progress in gane engineering techniques pt~lucea a large amount of new peptide ~ 1 protein drags which require novel, preferably non invasive delivery mutes. The lung is interesting for this purpose due to of its large surface, lack of degrading enzymes an a very thin epithelium in the alveolar region. The amphibian lung may serve as an interesting model of the mammalian lung, because of its simple sac-like molphology but its similar histology and physiology.

Material and Methods Excised lungs from Xenous laevis were mounted in a Grass-type Ussing chamber system, Both sides of the chamber were filled with an amphibian ringer solution and stirred by a gas lift system with carhogun (O~/CO z, 95:5) One pair of electrodes serves to measure the generated potential of the lung tissue (PD), another pair to measure the short circuit cunens (1SC), The electrical data acquisition system, consisting of a voltage clamp unit (VCC MC6, Physiological lnctmments, San Diego, USA) and a Macintosh-beased computer system, allows to simultaneously control up to six tissues. The voltage clamp can also be program- med to give regular pulses to the tissue in order to calculate the transcpithelial electrical resistance (TEER).

Results The potential difference (PD): The active transport of ions through the tissue generates a potential difference between the two sides of the lung tissue. This voltage was measured in the open mode of the voltage clamp. Afler mounting the tissue, the value increased during the first half hour to 6 to 12 mV (mean= 9.6. S.D.= 3.7, n= 14). This voltage did not decrease for more than 10% within the following 3 J, hours. The shun-circuit current (ISC): The ISC of the tissue is equivalent Io the net charge of the actively transported ions. It was measured in the voltage-clamp mode. In this mode the voltage of the tissue is clamped to zero by delivering a counter-current through the electro- des. This current exactly corresponds to the value of the current actively Wamponed by the tissue. The clamping to zero hence prevents a passive lr~aspon of solutes along electrical gradients. For the Xenopus lung an ISC of 7.5-18 pA/crn 2 (11.81 + 2.15, n=18) was found. The tissue resistance ('VEER): The tissue resistance of the lung tissue was calculated by the use of the pulsing feature of the voltage.ciamp. The fixed voltage of the pulse was divided by the resulting ISC-peak in the cltanp-tonde. TEER was found in a range between 400-1000 Ohoffcra 2 (552 + 89, n=lS). Transport of Na-Fhioresoein and correlation to TEER: Na-Fluorescein was used as marker for paracelialar transport . The transport rate in both directions was determined with a Miliipore Cytofluor II Fluorescence Reader. As expected, the apparent permeability of the trattsport marker was inversely correlated to the "VEER. The fife span of the excised lung tissue in Us.sing chamber:. There are several methods to check the viability of the mounted tissue, The decrease of the PD or ISC is indicative for a ceasing of active ion transport by the epithelium. So these values give a direct measure of tissue viability. As a control, addition of Triton X-100, immediately reduced ISC to zero. The appearance of the enzyme lactate dehydtogenzse in the medium is another equivalent to the size of cell damage. The maximal relative damage after 3 h was only 10 % greater than at the beginning of the experiment.