Compositions and methods for enhanced transepithelial and transendothelial transport or active agents
m comprising: (1) a carrier molecule selected from the group consisting of transferrin receptor ligands to which an active agent of interest may be conjugated; and (2) a transport enhancing agent (e.g., monensin or BFA) which enhances transendothelial or transepithelial transport of the transferrin receptor ligand/active agent conjugate via the TfR&#39;s. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The invention may be better understood with reference to the accompanying drawings, in which: 
     FIGS. 1A and 1B illustrate the levels of transcytosis of  125  I-Tf and HRP achieved in filter-grown MDCK cells, respectively; 
     FIGS. 2A and 2B illustrate the results of pulse-chase experiments monitoring processing of cell-associated  125  I-Tf with and without addition of bufeldin A (BFA), respectively; 
     FIG. 3 illustrates changes in the specific binding of  125  I-Tf to the basal and apical membranes upon treatment with BFA; 
     FIGS. 4A-4C illustrate the results of experiments to determine the effects of BFA on TfR-mediated uptake of  59  Fe, FIG. 4A showing uptake from the apical membrane, 4B uptake from the basolateral membrane and 4C transcytosis in the basal-to-apical direction; 
     FIGS. 5A and 5B illustrate the results of experiments to determine the effects of monensin and NH 4  Cl on the transcytosis of HRP-SS-Tf (FIG. 5A) and HRP (FIG. 5B); 
     FIGS. 6A and 6B illustrate the results of experiments to determine the effects of BFA on the transcytosis of HRP-SS-Tf and HRP, respectively; and 
     FIG. 7 illustrates the elution pattern of transcytosed TF-SS-HRP and a control standard in Sephacryl S-200 gel chromatography. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention involves the use of non-specific and specific-membrane binding ligands as well as the use of agents which can perturb intracellular protein sorting processes to significantly increase the transport of a macromolecular protein across the cells via endocytotic and transcytotic mechanisms. 
     During the last decade, most of the studies on drug transepithelial transport have been carried out at tissue and organ levels. For example, various types of intestinal samples, such as everted intestine preparations, intestinal ring slices and mucosal sheets have been used. Because of such factors as cellular heterogeneity, complexity in geometric structure, inaccessibility of the serosal side of the epithelium, and limited viability of epithelial cells in the isolated tissues, it is very difficult to use these methods for specific mechanistic studies on transepithelial transport of the drugs. 
     Purified brush border membrane vesicles, basolateral membrane vesicles and isolated intestinal epithelial cells have also been used to study drug transport. Although membrane vesicles offer an excellent model to study transport properties of solutes without the interference of cellular metabolism, they may not be suitable for studies on the endocytosis and transcytosis of macromolecules because of lack of membrane internalization. In addition, results from these membrane vesicles may not correlate well with those from viable cells. Isolated intestinal epithelial cell is a very good model to study the cellular binding, uptake and transport of drugs, but it suffers from short viability; therefore, the practical use of this type of cell is limited. 
     Cultured filter-grown epithelial cells, on the other hand, show many advantages as a model system. For example, the viability of the cell can be maintained for almost as long as it is needed, and studies on the interaction between peptides/proteins and cells can be carried out readily without the potential interference from other mucosal structures which are normally found in tissue samples. In addition, filter-grown epithelial cells allow easy access to both apical and basal side of the cell. The experimental data obtained is generally more reliable and easy to interpret, because of the relative homogeneity of the cell population. Other desirable properties, such as high sensitivity, reproducibility, and small sample requirements, are also intrinsic to cultured epithelial cell models. 
     Several epithelial cell lines, such as dog kidney epithelial cells and human colon epithelial cells, have been developed in culture [see, e.g., Chantret, I., Barbat, A., Dussaulx, E., Bratain, M. G., and Zweibaum, A., (1988) Cancer Research 48, 1936-1942; Rodriguez-Boulan, E. and Nelson, W. J. (1989) Science 245, 718-725]. For model purposes, the MDCK and Caco-2 cell lines have been recognized as particularly useful. 
     The MDCK cell line displays many features of in vivo epithelial cells when grown under cultured conditions. In addition, its morphogenesis and polarity have been extensively characterized in the literature [see, e.g., Gonzalez-Mariscal, L. (1985) J. Membr. Biol. 86, 113-125; Rodriguez-Boulan, supra]. This cell line was originally derived from dog kidney epithelial cells, and has been recently characterized as a model system for transepithelial drug transport studies [Cho, M. J., Thompson, D. P., Cramer, C. T. (1989) Pharm. research. 6, No. 1]. The MDCK cell line shows brushborder, lateral spaces, polarity of the cellular plasma membranes, and appropriate enzyme makers [Rodriquez-Boulan, supra]. This cell line also displays a good apical cell to cell tight junctions as indicated by very high transepithelial electrical resistance (TEER, about 3000 ohms cm 2 ) [Gonzalez-Mariscal, supra]. 
     The Caco-2 cell line is the only one among twenty human colon carcinoma epithelial cell lines tested that shows spontaneous enterocytic differentiation, as characterized by a polarization of the cell layer with the formation of domes and the presence of an apical brush border [Chantret, supra]. It has been shown that when grown-in filter membranes, Caco-2 cells also exhibit many intestinal epithelial characteristics, i.e., brush-border microvilli, brush border enzymes, and tight junctions [Pinto, M., Robine-Leon, S., Appay, M. D. et al. (1983) Biology of the Cell 47, 323-330; Grasset, E., Pinto, M., Dussaulx, E., Zweibaum, A. and Desjeux, J. F. (1984) Am. J. Physiol. 247 (Cell Physiol. 16), C260-C267; Hidalgo, I. J., Raub, T. J., and Borchardt R. T. (1989) Gastroenterology 96, 736-749]. The brush border of these cells has been shown to contain many hydrolases such as sucrase-isomaltase, lactase, alkaline phosphatase, aminopeptidase N, and dipeptidylpeptidase IV [Chantret, supra]. Brushborder enzymes indigenous to the adult human small intestine are increasingly produced during growth [Hidalgo, supra]. So far, this cell line has been used as a model for many studies relating to gastrointestinal drug absorptions [Hidalgo, supra; Artursson, P. (1990) J. Pharm. Sci. 79, 476-482; Heyman, M., Crain-Denoyelle, A. M., Nath, S. K., and Desjeux, J. F. (1990) J. Cell Physiol. 143, 391-395; Wilson, G., Hassan, I. F., Dix, C. J., Williamson, Shah, R., Mackay, M. (1990) J. Controlled Release 11, 25-40]. One drawback of this cell line is that it does not offer as high a TEER as the MDCK cell line does (its TEER is only about 500 ohms cm 2 ); nonetheless, it is a better physiological model for human intestinal epithelial cells. 
     Among several candidates, transferrin was selected as a model ligand for use in conjugation of active agents to be transported. Tf receptors exist in high amounts and in a highly polarized manner in epithelial cell models. Moreover, the recycling accuracy of Tf in MDCK cells is about 99% [Fuller, S. D. and Simmons, K. (1986) J. Cell Biol. 103, 1767-1779]; therefore, the chemical-induced Tf transcytosis as well as changes in receptor recycling and distribution can be easily and accurately detected. Furthermore, because Tf undergoes a rapid recycling with a half life of 3-15 min after internalization and its intracellular routing excludes a lysosomal compartment [Dautry-varsat, A., Ciechanover, A. and Lodish, H. F. (1983) Proc. Natl. Acad. Sci. USA 80, 2258-2262; Klausner, R. D., Ashwell, G., Van Renswoude, J., Harford, J. B. and Bridges, K. R. (1983) Proc. Natl. Acad. Sci. USA 80: 2263-2266], high efficiency of transport and limited intracellular degradation can be expected if its transcytosis is induced. In addition, monoclonal antibodies specific to the TfR are known and commercially available (for example, from Dako Corporation, Santa Barbara, Calif. and Boehringer Mannhein Biochemicals, Indianapolis, Ind.). Of course, it is equally possible to use conventional methods to generate polyclonal or monoclonal antibodies to the TfR or other receptors directly for use in formation of suitable conjugates. 
     For the purpose of studying macromolecular transport across epithelial cells, horseradish peroxidase (HRP) was selected as a probe protein. This protein does not naturally occur in mammalian cells, it has a fairly large molecular weight (MW=40,000) which is a good representative of potential protein drugs, and it is an enzyme whose enzymatic activity can be easily detected at a low concentrations (10 -9  g/ml) [Worthington Manual (1988) pp. 254-260, Worthington Biochemical Corporation, Freehold, N. J.]. HRP is thus a very useful marker for determining if the protein transported via the endocytotic and transcytotic pathway still maintains its biological activity. 
     The Tf transported via the enhanced transcytotic pathway showed a specific binding affinity for TfR on K562 cells, indicating that its biological activity was retained. This enhanced Tf transcytosis was not due to drug-induced leakage of or damage to the cell monolayers, because the same drug-treatment did not increase the transport of the fluid phase marker HRP (FIGS. 5B and 6B). The addition of unlabeled Tf and incubation at 25° C. strongly reduced the enhanced transcytosis, indicating that the transcytosis is a receptor-mediated active process. 
     The present invention demonstrates for the first time that the receptor-mediated Tf transcytosis can be enhanced to a high extent in epithelial cells. Furthermore, Tf was also shown to carry covalently-linked HRP across the cells via an enhanced transcytotic pathway and the increase of HRP transcytosis was about 10 to 25 fold, respectively (FIGS. 5A and 6A). These data demonstrate the feasibility of using this enhanced transcytotic pathway to deliver other macromolecules across epithelial cells. A salient feature of the present invention is the use of transport accelerators, such as monensin and BFA, to achieve acceptable levels of conjugate transport. 
     Monensin and NH 4  Cl are known for their effects on dissipating the pH gradients across acidic organelle membranes and alkalizing the intracellular acidic compartments, including endosomes, lysosomes, and the Golgi apparatus [Maxfield, F. R. (1982) J. Cell Biol. 95, 676-681; Mollenhauer, H. H., Morre, D. J. and Rowe, L. D. (1990) Biochimica et Biophysica Acta 1031, 225-246]. However, their mechanisms of action are quite different. Monensin, a hydrophobic ionophore, acts by partitioning into the lipid bilayer and exchanging H+ with cations, especially Na+ [Mollenhauer, supra] between the membranes. NH 4  Cl, on the other hand, acts by simple diffusion of NH 3  across the membrane and accumulation of NH 4  + in the acidic lumen [DeDuve, C. (1983) Eur. J. Biochem. 137, 391-397]. Since acidification in endosomes plays a crucial role in the dissociation of ferric ions from Tf as well as the sorting of TfR-Tf complexes to the recycling pathway [Dautry-Varsat, supra], both NH 4  Cl and monensin have been shown to prevent the release of Tf from the cells [Klausner, supra; Stein, B. S., Klaus, G. B., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772]. The increased Tf accumulation in MDCK and Caco-2 cells in presence of NH 4  Cl and monensin, therefore, can be explained by effects of the drugs on acidic organelles. 
     NH 4  Cl (5 mill) has been shown to elevate the intracellular pH in MDCK cells to a the same degree as monensin (6 μM) [Maxfield, supra]. In experiments, NH 4  Cl at concentrations as high as 10 and 20 mM induced an increase in Tf transcytosis which was less than 1/7 of that by monensin (6 μM). These results suggest that the enhanced Tf transcytosis by monensin is unlikely solely due to elevation of the intracellular pH. 
     The fact that the same increase of cellular accumulation of Tf by NH 4  Cl and monensin treatment does not give same increase in the transcytosis of Tf implies that monensin enhanced Tf transcytosis does not occur via passive diffusion. If the transcytosis is governed by a passive diffusion processes, the same amount of Tf accumulated in both monensin- and NH 4  Cl-treated cells would have resulted in the same extent of diffusion of Tf from an intracellular pool to the culture medium. 
     On the other hand, the slight increase in Tf transcytosis in NH 4  Cl-treated cells (Table 4) as compared to control cells may be explained by the elevated intracellular pool of Tf. It is possible that the highly accumulated Tf in NH 4  Cl treated cells can diffuse more readily across the cells as compared to that in control cells. 
     It is know that heavy membrane traffic from the cell surface to stacked Golgi cisternae and secretory granules or vacuoles in regulated and non-regulated secretory cells [Herzog, V. and Farquhar, M. G. (1977) Proc. Natl. Acad. Sci. USA 745, 5073-5077; Farquhar, M. G. (1978) J. Cell Biol. 77, R35-42] can occur. This plasmalemma to Golgi traffic is probably related to the recycling of the membrane which can be used as containers for the packaging of secretory products [Farquhar, M. G. (1981) Methods Cell Biol. 23, 399-427; Farquhar, M. G., in Membrane Recycling, Evered, D. and Collins, G. M. eds., Pitman Books Ltd., London 157-174 (1982)]. It has been shown that the recycling plasmalemmal TfR can visit most, if not all Golgi sub-compartments [Woods, J. W., Doriaux, M. and Farquhar, M. G. (1986) J. Cell Biol. 103, 277-286], and that Golgi-associated post-translational modification of TfR as well as Tf can occur [Snider, M. D. and Rogers, O. C. (1985) J. Cell. Biol. 100, 826-834]. Although the role of the Golgi-associated modification of TfR and Tf has not been elucidated, it is speculated that this modification may determine the subsequent intracellular routing of the TfR-Tf complexes. Such a Golgi-associated modification of proteins has been shown to be responsible for the targeting of lysosomal enzyme proteins to the lysosomal compartments [Pohlmann, R. S., Waheed, A., Hasilik, A. and van Figura, K. (1982) J. Biol. Chem. 257, 5323-5325; Goldberg D. E. and Kornfeld, S. (1983) J. Biol. Chem. 258, 3159-3165]. 
     In addition to the elevation of pH, monensin has many other effects [see Mollenhauer, supra], and its principal action site has been proven to be within the Golgi complex. It has been shown that monensin can specifically disassembles the trans-Golgi apparatus by its ionophore function [Stein, 1984, supra; Stein, B. S. and Sussman, H. H. (1986) J. Biol. Chem. 261, 10319-10331]. 
     Brefeldin A (BFA) is a fungal metabolite known to perturb secretory protein transport from the ER to the cell surface [Harri, E., Loeffler, W., Sing, H. P., Stahelin, H. and Tamm, C. (1963) Helv. Chim. Acta 46, 1235-1243; Takatsuki, A., and Tamura, G. (1985) Agric. Biol. Chem. 49, 889-902; Misumi, Y., Misumi, Y., Miki, K., Takatsuki, A., Tamura, G., and Ikehara, Y. (1986) J. Biol. Chem. 261, 11398-11403; Magner, J. A., and Papagianner, E. (1988) Endocrinology 122, 912-920; Fujiwara, T. Oda, K, Yokota, S., Takatsuki, A., and Ikehara, Y. (1988) J. Biol. Chem. 263, 18545-18552]. Pursuant to one preferred embodiment of the present invention, BFA is employed to markedly enhance receptor-mediated transcytosis of Tf (and any agent associated therewith) and cause transferrin receptor (TfR) redistribution in MDCK epithelial cells. 
     On the other hand, monensin has also been shown to radically slow the intracellular transport of newly synthesized secretory proteins and their residing membrane vesicles [Tartakoff, A. M. (1983) Cell 32, 1026-1028]. BFA also has been shown to curtail the vesicular transport of secretory proteins from the ER via cis- and trans-Golgi elements to the cell surface, possibly by its effects on blocking the association of B-COP, a non-clathoursin coating protein, to the Golgi membrane and preventing the formation of non-clathoursin coated vesicles budding from the cis-Golgi apparatus [Orci, L., Tagaya, M., Amherdt, M., Perrelet, A., Donaldson, J. G., Lippincott-Schwartz, J., Klausner, R. D. and Rothman, J. E. (1991) Cell 64, 1183-1195]. Therefore, as an alternative mechanism, the inhibition of outwardly directed vesicle transport by monensin and BFA may indirectly affect the traffic pattern of incoming TfR-Tf containing endocytotic vesicles, presumably by decreasing the interaction between the two types of vesicles. 
     The underlying mechanism for BFA-induced missorting and transcytosis of the TfR are not fully understood at the present time. BFA has been shown curtail secretory protein transport to the cell surface [Takatsuki et al., supra; Misumi et al., supra; Magner et al., supra; Fujiwara et al., supra], to interfere with protein traffic between the ER and the Golgi complexes [Misumi et al., supra; Oda, K., Hirose, S., Takami, N., Misummi, Y., Takatsuki, A., and Ikehara, Y. (1987) FEBS Lett. 214, 135-138; Perkel, V. S., Liu, A. Y., Miura, Y., and Magner, J. A. (1988) Endocrinology 123, 310-318; Kato, S., Ito, S., Noguchi, T., and Naito, H. (1989) Biochim. Biophys. Acta 991, 36-43] and to cause a rapid and reversible dysfunction of cis- and trans-Golgi complexes (Fujiwara et al., supra; Lippincott-Schwartz, J. Yuan, L. C., Bonifacino, J. S. and Klausner, R. D. (1989) Cell 56, 801-813; Ulmer, J. B., and Palade, G. E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6992-6996; Ulmer, J. B., and Palade, G. E. (1991) J. Biol. Chem. 266, 9173-9179; Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H., Yuan, L. C., and Klausner, R. D. (1990) Cell 60, 821-836]. BFA has also been shown to perturb the intracellular trafficking of endocytosed ricin and to inhibit ricin cytoxicity [Yoshida, T., Chen, C., Zhang, M., and Wu, H. C. (1991) Experimental Cell Research 192, 389-395). However, little is known about the effect of BFA on the transcellular trafficking of plasma membrane-associated proteins. 
     Pursuant to one preferred embodiment of the present invention, BFA is employed to markedly increase the receptor-mediated transcytosis of Tf and alter the intracellular trafficking of the TfR. Since the TfR exists in many types of epithelial and endothelial cells [Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C., and Mason, D. Y. (1984) Nature 312, 162-163.; Banerjee, D., Flanagan, P. R., Cluett, J., and Valberg, L. S. (1986) Gastroenterol. 91, 861-869], including the endothelium of brain capillaries [Fishman, J. B., Rubin, J. B., Handrahan, J. V., Connor, J. R., and Fine, R. E. (1987) J. Neurosci. Res. 18, 299-304.; Pardridge, W. M., Eisenberg, J., and Yang, J. (1987) Metabolism 36, 892-895], this embodiment of the inventive method permits an increase in the transepithelial or transendothelial movement of receptor bound ligands or ligand-drug conjugates. In this regard, it is of interest to note that TfR has been considered to be a potential carrier for drug delivery across the blood-brain barrier [Pardridge, W. M., Triguero, D., and Buciak, J. I. (1990) Endocrinol. 126, 977-984]. It is, however, only through the use of the inventive combination of a cell-surface receptor ligand/active agent conjugate with a transcytosis accelerator (e.g., a conjugate of the active agent with Tf or TfR antibodies in combination with BFA) that acceptable rates of transcytosis are achieved; in the absence of an agent which accelerates transcytosis, unacceptably low delivery rates are achieved. 
     Conventional enhancers used to increase the passive diffusion of drug molecules suffer from a drawback of being nonspecific, i.e., once the perturbation of cell membrane occurs and cellular tight junctions have been disrupted by the enhancers, the increased permeability of the cell monolayer will not only allow the intended peptide/protein drug, but also other undesired compounds to pass across the monolayer. The absorption of these undesired compounds may cause toxicity and other side effects. BFA and monensin, however, have been found only to increase the transcytosis of Tf, and not EGF, asialoglycoproteins and fluid phase marker HRP, indicating that these enhancers are receptor-specific and that non-relevant substances will not be able to pass across the cells via the enhanced transcytotic pathway. 
     Most non-specific absorption enhancers act at a relatively high concentration and in a dose-dependent manner [Muranishi, S. (1990) Therapeutic Drug Carrier Systems 7, 1-33]. It has been reported that in order to achieve a 3.5-fold increase in the oral absorption of insulin from rat, the amount of salicylate as an enhancer at the site of absorption has to be 6 mg/rat or higher (up to 15 mg/rat, about a 5 mg/ml concentration) [Nishihata, T., Rytting, J. H., Kamada, A. and Higuchi, T. (1981) Diabetes 30, 1065]. According to the present invention, however, it has been determined that a 25- to 50-fold increase in TfR-mediated Tf transepithelial transport could be achieved with concentration of monensin or BFA of 4 μg/ml and 1.6 μg/ml, respectively, which is about 1000 times less in scale than that of conventional enhancers. While the following discussion focuses on the combination of TfR-ligand conjugate/BFA, it should of course be understood that the present invention is by no means limited to this particular combination of cell-surface receptor ligand conjugate and accelerator. For example, as noted supra, other types of cell-surface receptors are well known to undergo transcytosis with their associated ligands. For example, it is contemplated according to the present invention that the following cell-surface receptors may also be exploited by introducing the active agent in the form of a conjugate with the corresponding cell-surface receptor ligand: asialoglycoprotein receptors, low-density lipoprotein (LDL&#39;s) receptors and major histocompatibility complex (MHC) epitope receptors. Similarly, it is known in the art that enhancement (i.e., acceleration) of transcytosis of receptor/ligand complexes may be achieved with a variety of different agents, depending of course upon the particular receptor/ligand complex under consideration. Thus, in principle the present invention may employ as an enhancer of transcytosis an agent that influences or interferes with intracellular protein trafficking (e.g., within the endoplasmic reticulum, endosomes and/or Golgi apparatus) in a manner such that transcytosis of the receptor/ligand complex is enhanced. Accordingly, while a preferred embodiment is exemplified herein, the present invention provides a powerful technique for transmembrane and transcellular molecular transport of active agents which is limited only by the ability to identify an appropriate transport agent/accelerator combination targeted to the cell(s) of interest. In view of the differential distribution of particular receptors, moreover, the present invention provides the ability to selectively target cells or organs with great precision. As a consequence, it must be understood that following the teachings herein, it is now possible to overcome one of the primary obstacles to delivery of active agents (and in particular, peptides and proteins) in an essentially routine manner. As would of course be immediately apparent to those of skill in the art, formation of the ligand/active agent complex may be effected by a wide range of different techniques available to those working in the field; these techniques include not only covalent conjugate formation (e.g., the formation of a disulfide bridge between the active agent and the cell-surface receptor ligand), but also ionic and non-covalent conjugate complex formation (e.g., as exemplified by biotin/avidin complex formation, etc.). The choice of suitable technique for ligand/active agent conjugate formation may readily be made in an empirical manner, and would depend upon such obvious factors as the chemical nature of the ligand and the active agent, and the physicochemical and pharmacological properties of the conjugate (e.g., whether the conjugate per se have therapeutic utility or the active agent must be separated from the ligand for optimum therapeutic efficacy). Techniques for forming conjugates between active agents and suitable carriers are well known in the art of drug transport, and thus any suitable technique for forming a covalent, ionic or non-covalent conjugate between an active agent and a cell-surface receptor ligand as transport agent would be contemplated as within the scope of the present invention. 
     The invention may be better understood by reference to the following examples which are intended for purposes of illustration only and are not to be construed as in any way limiting the scope of the present invention, which is defined in the claims appended hereto. 
     EXAMPLES 
     Abbreviations used throughout are as follows: BFA, brefeldin A; EGF, epidermal growth factor; ER, endoplasmic reticulum; HA-MEM, Eagle&#39;s minimum essential medium with 0.1% of human albumin; PBS, phosphate-buffered saline, pH 7.4; TEER, transepithelial electrical resistance; Tf, transferrin; TfR, transferrin receptors. 
     EXAMPLE 1 
     Transcytosis of  125  I-Tf and HRP in Filter-Grown MDCK Cells 
     Strain I MDCK cells were grown on 0.45-μm pore size polycarbonate filters in Transwells (Costar, Cambridge, Mass.) as has been described in the literature [Shen, W.-C., Wan, J., and Shen, D. (1990) Biochem. Biophys. Res. Commun. 166, 316-323]. Monolayers which displayed a transepithelial electrical resistance (TEER) of more than 2500 ohms cm 2  (as measured by an epithelial voltohmmeter, EVOM, World Precision Instruments, West Haven, Conn.) were used in experiments. 
     To remove endogenous Tf, MDCK cells used in all of the experiments were pre-treated as follows: confluent cell monolayers were rinsed 3 times with serum-free MEM, followed by incubation with serum-free MEM containing 1 mg/ml human albumin (fraction V, Sigma Chemical Co., St. Louis, Mo.) (HA-MEM) at 37° C. for 1 hr. After the incubation, the medium was replaced with fresh HA-MEM. The TEER of the cell monolayers was not changed by this pre-treatment. 
     Human apo-Tf (Sigma Chemical Co., St. Louis Mo.) was first loaded with iron [Larrick, J., and Cresswell, P. (1979) Biochim. Biophys. Acta 583, 483-490] and then labeled with  125  I using the Iodo-Beads (Pierce Chemical Co. Rockford, Ill.) method as suggested by the supplier. Briefly, 0.7 mCi of Na 125  I (ICN Radiochemicals, Irvine, Calif.) was incubated at 25° C. with 2 Iodo-beads for 5 in 0.22 ml of phosphate buffer (0.1M, pH 7.4), and then 20 μl of diferritransferrin (25 mg/ml) was added to the beads in the buffer solution. The solution was allowed to sit at 25° C. for 10 min and then any non-incorporated iodine was removed by filtration through a 20×0.5-cm Sephadex G-25 column (Pharmacia Fine Chemicals, Piscataway, N.J.). Apo-Tf was labeled with  59  Fe by a previously reported method [Timchak, L. M., Kruse, F., Marnell, M. H., and Draper, R. K. (1986) J. Biol. Chem. 261. 14154-14159] . Eluted  59  Fe-Tf and  125  I-Tf fractions showed specific radioactivity levels of 10 4  cpm/μg and 5×10 5  cpm/μg, respectively. 
       125  I-Tf (1.5 μg/ml),  59  Fe-Tf (6 μg/ml), or HRP (3 μg/ml) was added with or without unlabeled Tf at a 200- to 300-fold molar excess to the apical (1.5 ml) or the basal (2.5 ml) medium of filter-grown MDCK cells and BFA (1.6 μg/ml) was added to the basal medium as well. At various time intervals, the basal or apical media containing transcytosed  125  I-Tf or  59  Fe-Tf was collected and an equal volume of fresh HA-MEM was added as replenishment. The  125  I-Tf in the collected samples was subjected to 15% TCA precipitation and following centrifigation, the radioactivity in protein pellets was measured. For the media containing transported HRP, the HRP enzymatic activity was determined [Worthington Enzyme Mamual (1972) pp. 43-45, Worthington Biochem. Co., Freehold, N.J.]. After the last sample collection, cell monolayers were rinsed extensively with 4° C. serum-free MEM and then removed together with the filter membrane from the Transwell. The radioactivity level in the entire filter membrane containing  125  I-Tf or  59  Fe-Tf was counted in a Packard gamma counter. 
     Filter-grown MDCK cells were treated with BFA (1.6 μg/ml) in the basal medium at 37° C. for about 2 hours. After treatment, cells were rinsed extensively and the chambers were refilled with cold HA-MEM.  125  I-Tf (1.5 μg/ml) with or without unlabeled Tf (350 μg/ml) was then added to either the apical or basal medium. Binding of  125  I-Tf to the cells was measured at 4° C. for 3 hours. Following binding, the cells were rinsed extensively with cold HA-MEM and the cell-associated radioactivity was counted. The binding of  125  I-Tf to the membranes in the presence of unlabeled Tf was treated as the non-specific binding of  125  I-Tf to the membranes. The difference between the binding of  125  I-Tf and  125  I-Tf plus a large excess of unlabeled Tf to either the apical or the basal membrane of MDCK cells at 4° C. was considered to be the TfR-specific binding of  125  I-Tf. 
       125  I-Tf (1.5 μg/ml) was added either with or without BFA (1.6 μg/ml) to the basal medium of filter-grown MDCK cells. Cells were incubated with the medium at 37° C. for 2 hr, after which the media was removed. Cells were then rinsed extensively with 4° C. serum-free medium to remove any unbound  125  I-Tf. Cell-associated  125  I-Tf, which included internalized and membrane-bound  125  I-Tf, was chased at 37° C. in fresh HA-MEM medium or in the medium containing BFA (1.6 μg/ml). Both apical and basal medium were collected at various time intervals during the chase period, and the collected media was subjected to 15% TCA precipitation and the radioactivity in the TCA insoluble pellet was counted.  125  I-Tf recovered from the basal medium was taken as a measure as of Tf recycling, while radioactivity appearing in the apical medium was used as a measure of Tf transcytosis. 
     FIG. 1 illustrates transcytosis of  125  I-Tf and HRP in filter-grown MDCK cells.  125  I-Tf (1.5 μg/ml) either with or without unlabeled Tf (350 μg/ml) was added to the apical (AP) or basal (BL) medium of filter-grown MDCK cells. FIG. 1A shows the transcytosis of  125  I-Tf in control cells (◯  ) and in BFA-treated cells (BFA:1.6 μg/ml) (   ) in either the apical-to-basal (   ) or basal-to-apical (◯  ) direction. When BFA was present in the basal medium, the transcytosis of  125  I-labeled Tf ( 125  I-Tf) was markedly increased in both apical-to-basal and basal-to-apical directions (FIG. 1A), while the transcytosis of the fluid phase marker HRP remained unchanged (FIG. 1B). Briefly, cells were incubated with the medium at 37° C. for various time intervals and the BL or AP medium containing transcytosed  125  I-Tf was collected. A constant volume of fresh medium was added to the cells and collected samples were subjected to 15% TCA precipitation. The radioactivity in the protein pellets after centrifugation was counted in a gamma counter and converted to the amount of protein using the specific radioactivity of  125  I-Tf as 5×10 5  cpm/μg. Transcytosis of  125  I-Tf in the presence of unlabeled Tf (non-specific transport) was less than 5% of that in the absence of unlabeled Tf (total transport). The difference between total and the non-specific transport is considered to be TfR-specific transport, as presented in the graph. FIG. 1B shows the transcytosis of HRP in BFA-treated and control cells. HRP (3 μg/ml) was added to either the apical or basal medium, and the HRP activity that appeared in the opposite medium to administration was measured using a HRP enzymatic assay. Each point represents the mean of triplicate cell monolayers with a standard deviation which is either indicated as a bar or is smaller than the size of the symbol. Each cell monolayer on the filter consisted of approximately 3×10 6  cells. The enhanced transcytosis of  125  I-Tf was reduced by competition with excess unlabeled Tf. 
     As further demonstrated by pulse-chase experiments, the intracellular processing of cell-associated  125  I-Tf was profoundly altered by BFA.  125  I-Tf (1.5 μg/ml) either with (FIG. 2B) or without (FIG. 2A) BFA (1.6 μg/ml) was added to the basal medium of filter-grown MDCK cells. Cells were incubated with the medium at 37° C. for 2 hours and then both apical and basal media were removed. Cells were then extensively rinsed with cold serum-free medium to remove unbound  125  I-Tf. Cell-associated  125  I-Tf (control cells: 19.2±0.2 ng/filter; BFA-treated cells: 22.6±0.3) was chased at 37° C. either in fresh HA-MEM medium or in the medium containing BFA (1.6 μg/ml). At different time intervals after the chase, both apical and basal medium were collected and subjected to 15% TCA precipitation. The radioactivity in the TCA-insoluble protein pellet was counted.  125  I-Tf recovered from the basal medium was taken as recycled  125  I-Tf (◯), and  125  I-If that was recovered from apical medium was taken as amount as trancytosed (). Each point represents the mean of triplicate cell monolayers with a standard deviation which is either indicated as a bar or is smaller than the size of the symbol. Each cell monolayer on the filter consisted of approximately 3×10 6  cells. As is apparent from FIG. 2, 99% of internalized  125  I-Tf was recycled to the basal medium and only 1% was transcytosed to the apical medium in control cells (FIG. 2A), but the ratio shifted to 70% recycling and 30% transcytosis in BFA-treated cells (FIG. 2B). 
     Exposure to BFA also led to a redistribution of TfR&#39;s, as demonstrated by the changes in the specific binding of  125  I-Tf to the basal and apical membrane (FIG. 3). Filter-grown MDCK cells were treated (hatched column) or not treated (open column) with BFA (1.6 μg/ml) in the basal medium at 37° C. for 5 hr. After the treatment, both the apical and basal media were removed, and cells were rinsed extensively and then reincubated with HA-MEM at 4° C.  125  I-Tf (1.5 μg/ml) was added either with or without unlabeled Tf (350 μg/ml) to the apical or basal medium. Binding of  125  I-Tf to the cells was carried out at 4° C. for 3 hr. Following binding, both apical and basal medium were removed and cells were rinsed extensively with 4° C. HA-MEM to remove any unbound  125  I-Tf. The cell-associated radioactivity was counted, and the difference between the total and non-specific binding (in presence of the unlabeled Tf) of  125  I-Tf was taken as TfR-specific binding as presented in the graph. Each column represents the mean of triplicate cell monolayers with a standard deviation which is indicated as a bar or is smaller than the size of the symbol. Each cell monolayer on the filter consisted of approximately 3×10 6  cells. In control cells the amount of  125  I-Tf specifically bound to the basal and apical membranes was 5.8 ng and 0.5 ng/3×10 6  cells/filter, respectively. In BFA treated cells, these levels were shifted to 2.8 ng (basal) vs. 0.9 ng (apical) changing the ratio of basal to apical binding from 12:1 to 3:1. 
     The effects of BFA on TfR-mediated uptake of  59  Fe were also evaluated (FIG. 4).  59  Fe-Tf (6 μg/ml) was added either with or without unlabeled Tf (1 mg/ml) to either the apical or basal medium of MDCK cells. Cells were either treated () or not treated with BFA (1.6 μg/ml) (◯). After incubation at 37° C. for various time periods, the medium containing transcytosed Tf was collected and counted in a gamma counter. Cell monolayers were then rinsed extensively with cold medium to remove free  59  Fe-Tf and then the cell associated- 59  Fe was counted. Tf-mediated uptake of  59  Fe, i.e., the difference between the total uptake and the uptake in presence of large excess of unlabeled Tf is presented in the graph. FIG. 4A shows Tf-mediated uptake of  59  Fe from the apical membrane; FIG. 4B, safe uptake from the basolateral membrane; and FIG. 4C,  59  Fe transcytosis in the basal-to-apical direction in control and BFA-treated cells. Transcytoses in the apical-to-basal direction in both cells were very similar to that in the basal-to-apical direction and are not presented. Ferric ions associated with Tf are efficiently extracted inside endosomes and accumulate in the cytosol following endocytosis [Klausner, R. D., van Renswoude, J. Kempf, C., Rao, K., Bateman, J. L., and Robbins, A. R. (1984) J. Cell Biol. 98, 1098-1101]. This process was not altered by BFA treatment, as indicated by the fact that  59  Fe was not transcytosed in either control or BFA treated cells (FIG. 4C). Thus,  59  Fe uptake can be used as an index of total Tf endocytosis occurring either from the basolateral or apical membranes. As was expected, very little endocytosis occurred from the apical surface of control cells (FIG. 4A). Surprisingly, BFA dramatically increased apical endocytosis and reduced endocytosis from the basal surface (FIG. 4A and B). 
     The extent of transcytosis of Tf from the basolateral to the apical surface in MDCK monolayers is very small, and apical-to-basal transcytosis is even less measurable (FIG. 1). This difference, as has also been shown by others [Fuller, S. D., and Simons, K. (1986) J. Cell Biol. 103, 1767-1779.; Lippincott-Schwartz, J. Yuan, L. C., Bonifacino, J. S. and Klausner, R.d. (1989) Cell 56, 801-813], reflects the fact that Tf receptors are predominantly localized on the basolateral membrane. Such asymmetries in receptor distribution have been documented in several instances [Simons, K., and Fuller, S. D. (1985) Annu. Rev. Cell Biol. 1, 243-288; Rodriguez-Boulan, E., and Nelson, W. J. (1989) Science 245, 718-725]. BFA markedly enhances Tf transcytosis in both apical-to-basal and basal-to-apical directions. This enhancement was not accompanied by a concurrent increase in the transcytosis of HRP, a fluid-phase marker, indicating that it was not caused by BFA-induced leakage or damage to the monolayer. Instead, this enhancement is specific to Tf receptor-mediated transport, as indicated by the fact that the transcytosis of  125  I-Tf was decreased by the presence of excess unlabeled Tf. The transcytosis of ligands mediated by other receptors, such as EGF and asialoglycoprotein, was not affected by the same treatment. 
     The striking enhancement induced by BFA-treatment on Tf transcytosis must be due to a missorting of internalized Tf-TfR complexes, for several reasons: (1) the ratio of transcytosis vs. recycling of total cell-associated Tf was shifted from 1% vs. 99% in untreated cells to 30% vs. 70% in BFA-treated cells (FIG. 2A and 2B); (2) a two-fold increase in TfR levels was found in the apical membrane of BFA-treated cells (FIG. 3); and (3) a concomitant eight-fold increase in TfR-mediated  59  Fe uptake from the apical membrane was also observed (FIG. 4A). 
     The difference between the increase in TfR level and the increase in TfR-mediated uptake of  59  Fe from the apical membrane may imply that BFA only affects the intracellular routing of the Tf-TfR complex, but not the machinery which controls the polarity of cell membrane. It is likely that once the basolaterally internalized TfR is misdirected to the apical membrane it will be retrieved rapidly from the cell surface. Consequently, residence of the TfR on the apical membrane will be transient and the increase of TfR levels that can be detected at any given time point is significantly less than the increase of overall  59  Fe uptake from the apical membrane. In contrast, the decrease in  59  Fe uptake and TfR levels in the basal membrane in BFA-treated cells are 42% and 50%, respectively (FIG. 4B and FIG. 3). Thus, perhaps BFA only inhibits recycling but does not change the default location of the TfR on the basal membrane. The dynamic movement of the remaining TfR&#39;s on the basal membrane is not changed and therefore the level of  59  Fe uptake is proportionally related to the level of TfR is present on the membrane. 
     It is known that BFA has no effect on endocytosis, endosome acidification, lysosomal function, or the trans-Golgi network [Misumi et al., supra; Yoshida et al., supra]. This is consistent with the finding reported herein that the cellular extraction of  59  Fe from internalized  59  Fe-Tf, presumably inside acidic endosomes, still normally occurs in BFA-treated cells (FIG. 4). Therefore, it is likely that BFA exerts its effect not by interfering with internalization, but rather by disrupting intracellular processing of the Tf-TfR complex. It has been shown that after internalization, a fraction of Tf-TfR passes through most, if not all, Golgi subcompartments [Fishman, J. B., and Fine, R. E. (1987) Cell 48, 157-164; Stein, B. S., and Sussman, H. H. (1986) J. Biol. Chem. 261, 10319-10331.; Woods et al., supra] and that post-translational modification of the Tf-TfR complex occurs in the Golgi apparatus [Regoeczi, E., Chindemi, P. A. and Debanne, M. T. (1984) Can. J. Biochem. Cell Biol. 62, 852-858; Snider et al., supra]. Perturbation of the Golgi cisternae by BFA may disrupt the processing of Tf-TfR and result in a missorting of the complex into a transcytotic pathway. On the other hand, BFA has been shown to curtail the vesicular transport of secretory proteins from the ER via cis- and trans-Golgi elements to the cell surface. Possibly, BFA effects the association of β-COP, a non-clathrin coating protein, to the Golgi membrane thus preventing the formation of non-clathrin coated vesicles budding from the cis-Golgi apparatus [Orci et al., supra]. Therefore, as an alternative mechanism, this inhibition of outwardly directed vesicular transport by BFA may indirectly affect the intracellular trafficking of incoming TfR containing endocytotic vesicles, presumably by decreasing the interaction between these two types of vesicles. 
     EXAMPLE 2 
     Transcytosis of HRP-SS-Tf in Filter-Grown MDCK Cells 
     For preparation of HRP-SS-Tf, 1.6 μmol of SPDP in 50 μl of ethanol was added slowly to 0.4 μmol of diferritransferrin in 0.64 ml PBS. In a separate reaction, 3.2 μmol of SPDP in 100 μl of ethanol was added to 0.8 μmol of HRP in 1 ml PBS, pH 7.5. Both reaction mixtures were gently stirred at 25° C. for 1 hour and then dialyzed overnight in 1 L PBS at 4° C. SPDP-modified HRP was reduced by 100 μl of 1M dithiothreitol (DTT) at 25° C. and then purified by Sephadex G-50 gel filtration. The purified sulfhydryl-containing HRP was added to SPDP-modified Tf and the reaction mixture was gently stirred for 3 hr at 25° C. and then overnight at 4° C. The unconjugated HRP and Tf were removed by Sephadex G-100 gel filtration. Fractions containing TF-SS-HRP conjugate were identified by HRP activity measurement and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-Page) with a 7.5% gel. After staining with coomassie Blue, the conjugate showed as a major band in SDS-PAGE with an apparent molecular mass of 120 kDa. This major band disappeared when DTT was added to the conjugate before the electrophoresis, confirming that HRP and Tf were linked by a disulfide linkage. The molar ratio of HRP to Tf in the conjugate was 1.29:1 as estimated by the measurement of absorbance at 403 nm for HRP and 280 nm for the total protein. 
     Transport of the HRP covalently coupled to Tf via a disulfide linkage (HRP-SS-Tf) across epithelial cells in presence of monensin, NH 4  Cl and BFA was measured. Transcytosis of TF-SS-HRP was monitored by HRP enzymatic activity. HRP enzymatic activity was determined by the literature method [Worthington Enzyme Manual (1972), Worthington Biochem. Co., Freehold, N.J., pp. 43-45]. 
     FIG. 5 illustrates the effects of monensin and NH 4  Cl on the transcytosis of HRP-SS-Tf (FIG. 5A) and HRP (FIG. 5B) in filter-grown MDCK cells. HRP-SS-Tf (3 μg HRP/ml) and HRP (3 μg/ml), alone or together with either unlabeled Tf (1 mg/ml), monensin (6 μM) or NH 4  Cl (20 mM), was added to the basal medium. After incubation at 37° C. for various time periods, the HRP enzymatic activity appearing in the apical medium was measured. The data is presented as the mean ±S.D., n=3. FIG. 5A shows that the transcytosis of control Tf-SS-HRP in the basal-to-apical direction occurred at a very low rate of 0.05 ng/filter/hours, which is expected because it was shown that  125  I-Tf transcytosis was very low. However, the presence of monensin (6 μM) increased the transcytosis of TF-SS-HRP by about 9-fold. This enhanced transcytosis was TfR-mediated because the addition of a large excess of unconjugated Tf reduced the transcytosis by about 90%. The presence of NH 4  Cl (20 mM) increased the transcytosis of TF-SS-HRP by about 2-fold. FIG. 5B shows that the presence of monensin only increased the transcytosis of HRP-SS-Tf, but not that of free HRP, indicating that the enhanced transcytosis is not due to the drug-induced increase of cellular permeability. 
     FIG. 6 shows the effects of BFA on the transcytosis of HRP-SS-Tf (FIG. 6A) and HRP (FIG. 6B) in filter-grown MDCK cells. HRP-SS-Tf (3 μg HRP/ml) and HRP (3 μg/ml), alone or together with either unlabeled Tf (1 mg/ml) and BFA (1.6 μg/ml), was added to the apical medium. After incubation at 37° C. for various time periods, the HRP enzymatic activity in the basal medium was measured. Again, the data is presented as the mean ±S.D., n=3. FIG. 6A shows that presence of BFA increased the transcytosis of HRP-SS-TF in the apical-to-basal direction in MDCK cells by about 25-fold. This enhanced transcytosis was reduced by more than 90% upon addition of unconjugated free Tf, indicating that it was due to a receptor-mediated mechanism. FIG. 6B shows that the presence of BFA does not affect the transcytosis of free HRP. 
     The integrity of transcytosed TF-SS-HRP was demonstrated by retention of HRP enzymatic activity as indicated in FIGS. 5 and 6, and also by Sephacryl S-200 gel chromatography (FIG. 7). The medium containing transcytosed HRP-SS-Tf was concentrated using a Centricon-10 filter unit, and was then loaded onto the Sephacryl S-200 column (1.5×70 cm). Fractions containing HRP activity were determined. The resultant elution profile was compared with that of the standard Tf-SS-HRP and HRP from the same column. The transcytosed TF-SS-HRP was eluted off from the column at fractions which coincided with those of intact Tf-SS-HRP. 
     From the foregoing description, one skilled in the art can readily ascertain the essential characteristics of the invention and, without departing from the spirit and scope thereof, can adapt the invention to various usages and conditions. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient, and although specific terms have been employed herein, they are intended in a descriptive sense and not for purposes of limitation.