390

in diffusion chambers together with a low-molecular marker, 5’Cr-EDTA, and a protein, ovalbumin.

Materials and Methods: The colitis was induced via a rec- tal injection of 12 mg oxazolone dissolved in 250 ,uI 50% ethanol [ 11. Forty-eight h after the induction, the rats were anaesthetized and the severity of the colitis was assessed. All the animals for further studies had ulcerations, edema and infiltration of inflammatory cells in the distal colon. For permeability studies, pieces were taken from the proximal and distal colon, and the distal ileum. The intestinal segments mounted in diffusion chambers [ 21 had an exposed area of 1.78 cm* and were bathed on both sides with a modified Krebs buffer solution that was continuously oxygenated and circu- lated. The marker molecules ovalbumin and “Cr-EDTA were introduced on the mucosa-side and during 120 min, samples were taken every 20 min from the serosa-side to measure the marker appearance.

Results: No permeability differences were found between colitic and control rats for the two molecules in the distal co- lon (the inflamed segment) or the proximal colon. By con- trast, an increased permeability was found in the distal ileum in colitic rats.

Conclusions: The results indicate that experimentally in- duced colon-inflammation may affect the small-intestinal tissue in sites with no signs of inflammation, by an increased permeability to both macromolecules (ovalbumin) and smaller molecules (‘ICr-EDTA).

References

1 G.P. Morris, P.L. Beck, MS. Herridge, W.T. Depew, M.R. Szewczuk, J.L. Wallace, Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroen- terology, 96 (1989) 795-803.

2 G.M. Grass, S.A. Sweetana, In vitro measurements of gastrointestinal tissue permeability using a new diffusion cell. Pharm. Res. 5 (1988) 45-52.

Pll Effects of ovalbumin on the [“C]mannitol passage through Caco-2 monolayers. L. Lindberg, N. Pantzar and B.R. Wes- triim, Lund University, Department of Animal Physiology, Helgonaviigen 3B, S-223 62 Lund, Sweden

Background: The permeation of the low molecular weight marker [ i4C] mannitol and the macromolecular marker ovalbumin was studied through CaCo-2 cell monolayers cul- tured on Transwell filters.

Methods: The CaCo-2 cells were cultured on Transwell til- ters during up to 30 days [ 11. The marker molecules [ “C]mannitol (5 pi/ml, specific activity 49.3 mCi/mmol) and ovalbumin (25 mg/ml) were added together or alone either to the apical or to the basolateral chamber and the permeability was determined by measuring the marker ap- pearance in the receiver chamber during 4 h.

Results: The passage of [ ?Z]mannitol (without any oval- bumin in the marker solution) was five-times higher from the apical to the basolateral side than from the basolateral to the apical side. The apical to basolateral passage of ovalbu-

min was too low to be detected by electroimmunoassay (low- est detection limit 0.1 pg/ml x h-’ ), while the basolateral to apical passage was considerably higher and showed a time- dependent increase. Ovalbumin increased the apical to ba- solateral passage of [ 14C] mannitol through monolayers cul- tured less than 21 days, while no such effect could be seen after 25 days. Moreover, the basolateral to apical passage of [ “‘Clmannitol was increased by the presence of ovalbumin irrespective of the culture time. The passage increased ap- proximately 60-times through monolayers cultured for 25 days.

Conclusions: The greater [ “‘C]mannitol passage in the ap- ical to basolateral direction may be a result of the surface en- largement by the microvilli on the apical side and the water- flux. The CaCo-2 monolayer appears to be sensitive to ovalbumin when it is cultured less than 21 days and when ovalbumin is applied on the basolateral side. This sensitivity of the basolateral side can be due to the morphology, the high protein concentration or the ovalbumin molecule it self. This has to be evaluated before further permeability studies of proteins are performed.

References

1 P. Artursson, Epithelial transport of drugs in cell culture. 1. A model for studying the passive diffusion of drugs over intestinal absorptive (CaCo-2) cells. J. Pharm. Sci., 79 (1990) 476-482.

P12 Absorption enhancement of hydrophilic compounds by verapamil in Caco-2 cell monolayers. M. Sakai, A.B.J. Noach, M.C.M. Blom-Roosemalen, A.G. de Boer and D.D. Breimer, Leiden/Amsterdam Center for Drug Research, Division of Pharmacology, Center for Bio-Pharmaceutical, Leiden Vni- versity, P.O. Box 9503, 2300 RA Leiden, The Netherlands

The transport of hydrophilic compounds, like peptides, over epithelial cell layers is restricted to the paracellular transport route. This route is impeded by the tight junctions on the ap- ical side of the cells. It is known that extra- as well as intra- cellular calcium ions play a role in the barrier function of the tight junctions. However, it is not known if lowering intra- cellular calcium could influence tight junctional permeabil- ity. Verapamil is a well-known drug which blocks calcium en- try into the cell through calcium channels in cardiac and muscle cells. Also stimulation of water, sodium and chloride absorption into epitbelial cells can be induced by verapamil treatment which might induce ‘solvent drag absorption’ of hydrophilic drugs.

In the present investigations we used filter-grown mono- layers of the human intestinal epithelial cell line Caco-2 as a model for intestinal epithelium. As hydrophilic model com- pounds Fluorescein-Na (M.,, 376) (Flu) and FITC-labelled dextran (M,,, 4000) (FD-4) were used. Transepithelial elec- trical resistance (TEER) and transmonolayer permeability were used as measures for the enhancing effects, while trypan blue exclusion was used as a measure for the damaging effect of racemic verapamil.

391

Up to 300 PM verapamil caused an increase in TEER, whereas from concentrations of 700 PM onwards an abrupt dose-dependent drop in TEER was observed which was fully reversible within 2 h after removal of the compound. A sec- ond treatment of verapamil gave a much larger effect on TEER than the first treatment. At these concentrations no cell death could be observed. At concentrations of 1 mM ver- apamil caused severe damage to the cell layers.

At a concentration of 700 .uM, the transport of Flu was en- hanced 13- and 26-fold after the first and the second verapa- mil treatment, while the transport of FD-4 was increased 4- and 6-fold, respectively.

TEER experiments with the R- and S-verapamil enantiom- ers point in the direction that no enantioselective mechanism is involved.

P13 Absorption enhancement of hydrophiiic compounds by ra- cemic verapamil after rectal adminis~tjon in rats. A.B.J. Noach, M.C.M. Blom-Roosemalen, A.G. de Boer and D.D. Breimer, Leiden/Amsterdam Center for Drug Research, Divi- sion of Pharmacology, Center for Bio-Pharmaceutical Sci- ences, Leiden University, P. 0. Box 9503, 2300 RA Leiden, The Nether~an~

Absorption of hydrophilic compounds, like peptides, from the intestinal lumen into the blood is restricted to the para- cellular transport route. This route is impeded by the tight junctions on the apical side of the cells. Among the factors which are involved in maintaining this barrier is calcium. In an earlier study we investigated the effect of the calcium channel blocking agent verapamil on the paracellular perme- ability of hydrophilic model compounds through epithelial Caco-2 cell monolayers.

In the present study we verified in vivo the promising ef- fect of verapamii, which was found in the vitro study.

As a hydrophilic model compound FITC-labelled dextran (M, 4000) (FD-4) was used. Male Wistar rats ( 180-200 g) were cannulated in a carotid artery for blood sampling and a device was introduced in the rectum for the administration of FD-4 solution with and without verapamil. In order to study the kinetic profile of FD-4 a carotid artery (blood sam- pling) and a jugular vein canula (administration) were implanted.

The kinetics of FD-4 after i.v. administration of 10 or 1 mg FD-4 could be described by a two-compartment model with a terminal half-life of approx. 36 min.

Rectal administration was performed as a bolus dose (24 s) or as a rectal infusion (32 min) of 200 ~1 solution contain- ing 10 mg FD-4. A concentration of 700 PM verapamil, as was used in the in vitro experiments, did not show any effect. At a concentration of 7 mM verapamil, a very slight enhance- ment of the FD-4 uptake could be seen after bolus adminis- tration. After rectal infusion the effect was larger although still small. A major problem with these experiments is that the variation in effect between animals is quite large.

The data indicate that verapamil is in vivo not a very effec- tive absorption enhancer.

P14 Gastrointestinal absorption and plasma clearance of va- eopressin anatogues in the rat. S. Lundin” and H.G. Folkes- sonb, Departments of “Clinical Pharmacolo~ and bAnimat Physiology, Lund University, Lund, Sweden

The gastrointestinal absorption of a series of vasopressin anafogues with enhanced enzymatic stability was determined in chronically catheterized, conscious rats. The following peptides were used: [Mpa’, D-A$]-vasopressin (dDAVP), [Mpa’, Asn4, D-Arg8]-vasopressin (4-Asn-dDAVP), [Mpa’, Val“, D-Arg’]-vasopressin (4-Val-dDAVP), [ Mpa’, (CH,), Ala4, o-Arg*]-vasopressin (4-(CH-,), Ala-dDAVP), [ Mpa’, Tyr*(Et), D-Arg’j-vasopressin (2-Tyr(Et)-dDAVP). The peptides were administers by gavage and blood samples were taken for 3 h. In another series of experiments plasma clear- ance rates (Cl,) were determined using the constant infusion method. Plasma concentrations were measured by use of a cross-reacting dDAVP antiserum.

The bioavailability of all peptides was below 0.1 I, ranging from 0.0 13 to 0.046%. The Cl, values were between 4.3 (2- Tyr(Et)-dDAVP) and 14.7 ml kg-’ min-’ (CAsn-dDAVP). There was no apparent relationship between peptide hydro- philicity and either Cl, or bioavailability.

It can be concluded that, in the rat, the bioavailability of dDAVP is lower than in other species. This could be attrib- uted to a higher Cl, rather than a lower intestinal permeabil- ity. Therefore, the use of rat intestinal segments may prove to be a good model with which to predict intestinal permea- bility in man. Furthermore, it is obvious that small molecular changes, i.e., single amino acid substitutions or derivatiza- tions, have profound effects on Cl, and consequently on pep- tide bioavailability.

P15 The adjuvant effect of polyacryl starch microparticles with conjugated human serum albumin. L. Deglinga.’ and P. Stj~~kvistb.‘, aDepartment of Pharma~~tics, University of Uppsala, Box 580, S-751 23 Uppsala, Sweden: bDepartment of Pharmaceutical Biochemistry, University of Uppsala, Box 5 78, S- 75125 Uppsala, Sweden: ‘Division ofpharmacy, Med- ical Products Agency, Box 26, S-751 03 Uppsala, Sweden

Background: New safer vaccines which consist of purified proteins and peptides are often weakly immunogenic. To be effective, these new vaccines require adjuvants, that act non- specifically to increase the immune response to the specific antigen. Thus, there is a need for development of safe and effective adjuvants for human use that elicit both a humoral and cellular immune response.

Adjuvans appear to function with different mechanisms. One mechanism is the creation of a depot at the site of injec- tion which prolongs the release of antigen, e.g., mineral oil (Freund’s incomplete adjuvants, FIA). Another mechanism is targeting of antigen to cells of the immune system, i.e., macrophages, e.g., microparticular carrier systems f 1 ] _

Polyacryl starch microparticles is a drug carrier with a typ- ical distribution to macrophages in the reticuloendothelial system (RES). Starch microparticles are not immunogenic themseives, and do not induce an immune response against entrapped homologous proteins [ 21. The microparticles can