Compositions and Methods for Enhanced Transepithelial and Transendothelial Transport or Active Agents

Patent Citations (1)

Non-Patent Literature (105)

• Artursson, P., "Epithelial Transport of Drugs in Cell Culture", J. Pharm. Sci. 79:476-482 (1990).
• Balch, W. E., "Biochemistry of Interorganelle Transport", J. Biol. Chem. 264:16956-16968 (1989).
• Banerjee, D. Flanagan et al., "Transferrin Receptors in the Human Gastrointestinal Tract: Relationship to Body Iron Stores", Gastroenterology 91:861-869 (1986).
• Breitfeld, P. P. et al., "Sorting Signals, Current Opinion", Cell Biol. 1:617-623 (1989).
• Chantret, I. et al., "Epithelial Polarity, Villin Expression, and Enterocytic Differentiation of Cultured Human Colon Carcinoma Cells: A survey of Twenty Cell Lines", Cancer Research48:1936-1942 (1988).
• Cho, M. J. et al., "The Madin Carby Canine Kidney (MDCK) Epithelial Cell Monolayer as a Model Cellular Transport Barrier", Pharm. Research. 6, No. 1.
• Dautry-varsat, A. et al., "pH and the Recycling of Transferring During Receptor Mediated Endocytosis", Proc. Natl. Acad. Sci. USA 80:2258-2262 (1983).
• Davis, C. G. et al., "The Low Density Lipoprotein Receptor", J. Biol. Chem. 262:4075 (1987).
• DeDuve, C., "Lysosomes Revisited", Eur. J. Biochem., 137:391-397 (1983).
• Farquhar, M. G., "Recovery of Surface Membrane in Anterior Pituitary Cells. Variations in Traffic Detected with Anionic and Cationic Ferritin", J. Cell Biol. 77:/R35-42 (1978).
• Farquhar, M. G. "Membrane Recycling in Secretory Cells: Implications For Traffic of Products and Specialized Membranes Within the Golgi Complex", Cell. Biol. 23:399-427 (1981).
• Fishman, J. B. et al., "Receptor-Mediated Transcytosis of Transferring Across the Blood Brain Barrier", J. Neuroscience Res. 18:299-304 (1987).
• Fishman, J. B. and Fine, R. E., "A Trans Golgi-Derived Exocytic Coated Vesicle Can Contain Both Newly Synthesized Cholinesterase and Inernalized Transferrin", Cell 48:157-164 (1987).
• Friden, P. M. et al., "Anti-transferrin Receptor Antibody and Antibody-drug Conjugates Cross the Blood-brain Barrier", Proc. Natl. Acad. Sci USA 88:4771-4775 (1991).
• Fujiwara, T. et al., "Brefeldin A Causes Disassembly of the Golgi Complex and Accumulation of Secretory Protein in the Endoplasmic Reticulum", J. Biol. Chem. 263:18545-18552.
• Fuller, S. D., and Simons, K., "Transferrin Receptor Polarity and Recycling Accuracy in Tight and Leaky Strains of Madin-Darby Canine Kidney Cells", J. Cell. Biol. 103:1767-1779 (1986).
• Ghitescu, L. et al., "Specific Binding Sites for Albumin Restricted to Plasmalemmal Vesicles of Continuous Capillary Endothelium: Receptor-mediated Transcytosis", J. Cell. Biol. 102:1304-1311 (1986).
• Glenney, J. R. et al., Cell 52:675 (1988).
• Goldberg, D. E. and Kornfeld, S., "Evidence for Extensive Subcellular Organization of Asparagine-linked Oligasaccharide Processing and Lysosomal Enzyme Phosphorylation", J. Biol. Chem. 258:3159-3165 (1983).
• Gonzalez-Mariscal, L., "Tight Junction Formation in Cultured Epithelial Cells (MDCK)", J. Membr. Biol. 86:113-125 (1985).
• Grasset, E. et al., "Epithelial Properties of Human Colonic Carcinoma Cell Line Caco-2: Electrical Parameters", Am. J. Physiol. 247:C2560-C267 (1984).
• Herzog, V. and Farquhar, M. G., "Luminal Membrane Retrieved After Exocytosis Reaches Most Golgi Chisternea in Secretory Cells", Proc. Natl. Acad. Sci. USA 74:5073-5077 (1977).
• Heyman, M. et al., "Quantification of Protein Transcytosis in the Human Colon Carcinoma Cell Line CaCo-2", J. Cell Physiol. 143:391-395 (1990).
• Hidalgo, I. J. et al., "Characterization of the Human Colon Carcinoma Cell Line (Caco-2) As a Model System for Intestinal Epithelial Permeability", Gastroenterology 96:736-749 (1989).
• Jeffries, A. E. et al., "Transferrin Receptor of Endothelium of Brain Capillaries", Nature 312:162-163 (1984).
• Kato, S. et al., "Effects of Brefeldin A on the Synthesis and Secretion of Egg White Proteins in Primary Cultured Oviduct Cells of Laying Japanese Quail", Biochem. Biophy. Acta 991:36-43 (1989).
• King, G. L. and Johnson, S. M., "Receptor-Mediated Transport of Insulin Across Endothelial Cells", Science 227:1583-1586 (1985).
• Klausner, R. D. et al., "Binding of Apotransferrin to K562 Cells; Explanation of the Transferrin Cycle", Proc. Natl. Acad. Sci. USA 80:2263-2266 (1983).
• Klausner, R. D. et al., "Failure to Release Iron from Transferrin in a Chinese Hamster Ovary Cell Mutant Pleiotropically Defective in Endocytosis", J. Cell Biol. 98:1098-1101 (1984).
• Larrick, J. and Cresswell, P., "Transferrin Receptors on Human B and T Lymphoblastoid Cell Lines", Biochem. Biophys. Acta 583:483-490 (1979).
• Lippincott-Schwartz, J. et al., "Rapid Redistribution of Golgi Proteins into the ER in Cells Treated with Brefeldin A: Evidence for Membrane Cycling from Golgi to ER", Cell 56:801-813 (1989).
• Lippincott-Schwartz, J. et al., "Rapid Redistribution of Golgi Proteins into the ER in Cells Treated with Brefeldin A: Evidence for Membrane Cycling from Golgi to ER", Cell 60:821-836 (1990).
• Magner, J. A. and Papagianner, E., "Blockade by Brefeldin A of Intracellular Transport of Secretory Proteins in Mouse Pituitary Cells: Effects on the Biosynthesis of Thyrotropin and Free a-Subunits", Endocrinology 122:912-920 (1988).
• Maratos-Flier, E. et al., "Receptor-Mediated Vectorial Transcytosis of Epidermal Growth Factor by Madin-Darby Canine Kidney Cells", J. Cell Biol. 105:1595-1601 (1987).
• Maxfield, F. R., "Weak Bases and Ionophores Rapidly and Reversibly Raise the pH of Endocytic Vesicles in Cultured Mouse Fibroblasts", J. Cell Biol. 95:676-681 (1982).
• Misumi, Y. et al., "Novel Blockade by Brefeldin A of Intracellular Transport of Secretory Proteins in Cultured Rat Hepatocytes", J. Biol. Chem. 261:11398-11403 (1986).
• Mollenharer, H. H. et al., "Alteration of Intracellular Traffic by Monensin; Mechanism, Specificity and Relationship to Toxicity", Biochimica et Biphysica Acta 1031:225-246 (1990).
• Mostov, K. E. and Simister, N.E., "Transcytosis", Cell 43:389-390 (1985).
• Muranishi, S., "Absorption Enhancers", CRC Critical Review in Therapeutic Drug Carrier Systems 7:1-33 (1990).
• Neuwelt, E. A. et al., "Delivery of Melanoma-associated Immunoglobulin Monoclonal Antibody and Fab Fragments to Normal Brain Utilizing Osmotic Blood-Brain Barrier Disruption", Cancer Res. 48:4725-4729 (1988).
• Neuwelt, E. A. et al., "Developments in the Diagnosis and Treatment of Primary CNS Lymphoma", Cancer 58:1609-1620 (1986).
• Nishihata, T. et al., "Enhanced Intestinal Absorption of Insulin in Rats in Presence of Sodium 5-methoxy salicylate", Diabetes 30:1065 (1981).
• Oda, K. et al., "Brefeldin A Arrests the Intracellular Transport of a Precursor of Complement C3 Before Its Conversion Site in Rat Hepatocytes", FEBS Lett. 214:135-138 (1987).
• Orci, L. et al., "Brefeldin A, a Drug that Blocks Secretion, Prevents the Assembly of Non-clthrin-coated Buds on Golgi Cisternae" Cell 64:1183-1195 (1991).
• Pardridge, W. M. et al., "Human Blood Brain Barrier Transferrin Receptor", Metabolism 36:892-895 (1987).
• Pardridge, W. M. et al., ".beta.-Endorphin Chimeric Peptides: Transport Through the Blood-Brain Barrier In Vivo and Cleavage of Disulfide Linkage by Brain", Endocrinol. 126:977-984 (1990).
• Perkel, V. S. et al., "Brefeldin A Inhibits Oligosaccharide Processing of Glycoproteins in Mouse Hypothyroid Pituitary Tissue at Several Subcellular Sites (42682)", Endocrinology 123:310-318 (1988).
• Pinto, M. et al., "Enterocyte-like Differentiation and Polarization of the Human Colon Carcinoma Cell Line Caco-2 in Culture", Biol. of the Cell 47:323-330 (1983).
• Pohlmann, R. A. et al., "Synthesis of Phosphorylated Recognition Marker in Lysosomal Enzymes is Located in the cis Part of Golgi Apparatus", J. Biol. Chem. 257:5323-5325 (1982).
• Princiotto, J. V., "Difference Between the Two Ino-binding Sites of Transferrin", Nature 255:87-88 (1975).
• Regoeczi, E. et al., "Partial Resialylation of Human Asialotransferrin Types 1 and 2 in the Rat", Can. J. Biochem. Cell Biol. 62:852-858 (1984).
• Rodewald, R., "Distribution of Immunoglobulin G Receptors in the Small Intestine of the Young Rat", J. Cell Biol. 85:18-32 (1980).
• Rodman, J. S. et al., "Endocytosis and Transcytosis", Current Opinion in Cell Biology 2:644-672 (1989).
• Rodriquez-Boulan, E. and Nelson, W. J., "Morphogenesis of the Polarized Epithelial Cell Phenotype", Science 245:718-725 (1989).
• Rodriquez-Boulan, E. and Salas, P. J. I., "External and Internal Signals for Epithelial Cell Surface Polarization", Annu. Rev. Physiol. 51:741-754 (1989).
• Shen, W.-C. et al., "Proteolytic Processing in a Non-lysosomal Compartment is Required for Transcytosis of Protein-Polylysine Conjugates in Cultured Madin-Darby Canine Kidney Cells", Biochem. Biophy. Res. Comm. 166:316-323 (1990).
• Simons, K. and Fuller, S. D., "Cell Surface Polarity in Epithelia", Annul. Rev. Biol. 1:243-288 (1985).
• Snider, M. D. and Rogers, O. C., "Intracellular Movement of Cell Surface Receptors After Endocytosis: Resialylation of Asialo-Transferrin Receptor in Human Erythroleukemia Cells", J. Cell Biol. 100:826-834 (1985).
• Stein, B. S. et al., "Complete Inhibition of Transferrin Recycling by Monensin in K562 Cells", J. Biol. Chem. 259:14762-14772 (1984).
• Stein, B. S. and Sussman, H. H., "Demonstration of Two Distinct Transferrin Receptor Recycling Pathways and Transferrin-independent Receptor Internalization in K562 Cells", J. Biol. Chem. 261:10319-10331 (1986).
• Takatsuki, A. and Tamura, G., "Brefeldin A, A Specific Inhibitor of Intracellular Translocation of Vesicular Stomatitis Virus G Protein: Intracellular Accumulation of High Mannose Type G Protein and Inhibition of its Cell Surface Expression", Agric. Biol. Chem. 49:889-902 (1985).
• Tartakoff, A. M., "Perturbation of Vesicular Traffic With the Carboxylic Ionophore Monensin", Cell 32:1026-1028 (1983).
• Timchak, L. M. et al., "A Thermosensitive Lesion in a Chinese Hamster Cell Mutant Causing Differential Effects on the Acidification of Endosomes and Lysosomes",J. Biol. Chem. 261:14154-14159 (1986).
• Triguero, D. et al., "Capillary Depletion Method for Quantification of Blood-Brain Barrier Transport of Circulating Peptides and Plasma Proteins", J. Neurochem 54:1882-1888 (1990).
• Ulmer, J. B. and Palade, G. E., "Effects of Brefeldin A on the Processing of Viral Enveope Glycoproteins in Murine Erythroleukemia Cells", J. Biol. Chem. 266:9173-9179 (1991).
• Underdown, B. J., "Structural and Functional Aspects of IgA transcytosis", Immunological Investigations 18:287-297 (1989).
• Wilson, G. et al., "Transport and Permeability
• This invention was made with government support under 5 RO1 CA34798-08 awarded by the National Institute of Health. The government has certain rights in the invention.
• The present invention relates to methods and compositions for use in the delivery of active agents, such as proteins and peptides, across epithelial and endothelial cell walls.
• Many active agents, including most protein and peptide-based drugs, can only be administered via parenteral routes (e.g., intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intraarterial, and other routes of injection). Although these routes of drug delivery remain most effective, especially for patients who suffer from life-threatening diseases, they induce low compliance from patients. There are two primary reasons for this limited compliance. First, many drugs (and in particular, protein and peptide drugs) have very short half lives in vivo, and multiple injections per day are often necessary to maintain effective drug levels in the blood. Second, many patients (especially those who suffer from chronic diseases such as diabetes and other congenital or acquired metabolic disorders) need to take the medicine for their entire lives. It is difficult for these patients to accept a drug therapy regimen which requires daily multiple injections for an extended period of time.
• Alternative, non-invasive routes to deliver active agents (i.e., delivery of the drug via oral, nasal, transdermal and other non-parenteral routes) are receiving increasing attention. Non-invasive delivery provides convenience and selfcontrollability. Due to the specific physicochemical properties associated with many active agents (in particular, peptides and proteins), however, most of the active agents of current interest are poorly absorbed when given non-invasively; therefore, when administered in the heretofore-known manner, many of these agents are simply not effective. Although intense research has been carried out in this field in recent years, limited success has been achieved. It remains a great challenge to design and develop an effective and non-invasive delivery system for active agents of current interest, and in particular the peptides and proteins which have become available for potential therapeutic use with the advent of, e.g., recombinant DNA technology. It is envisioned that were an effective delivery system available, the clinical value of peptide- and protein-based pharmaceuticals would be dramatically increased and many patients would benefit accordingly.
• Non-invasive routes for drug delivery mainly include the oral, nasal, pulmonary, rectal, buccal, vaginal, transdermal and ocular routes. On the surface of entry to all these routes there exists a continuous mucous membrane of the epithelial cell layer. Before non-invasively delivered drugs can gain access to this epithelial cell monolayer and reach the target situs, there are several obstacles which must be overcome. The active agent must be dissolved and released from the dosage form. Moreover, the drugs must survive the harsh environment of the lumen (e.g., low pH and in the presence of proteolytic and hydrolytic enzymes), and then diffuse across the unstirred microclimate surrounding the epithelium. After reaching the surface of the cell, factors such as residence time at the surface, molecular size, membrane partition coefficient and other physicochemical properties of the drugs, as well as the structural morphology and biological function of the cells, also affect their penetration across the epithelium. Furthermore, during and after transcellular transport, the drugs still face possible enzymatic degradation and immunological and metabolic clearances, such as first-pass clearance in liver. All these factors can potentially block the drug from reaching its final target situs.
• Among all of these possible factors, enzymatic degradation and the poor penetration of the drug across epithelial and endothelial cells have been considered to be the major rate limiting steps. The enhancement of macromolecule penetration across epithelial cells is, in particular, a burdensome problem preventing the exploitation of otherwise promising active agents.
• The barrier properties of epithelial and endothelial cells exist primarily for two reasons. Adjacent cells are tightly connected by tight junctions which only allow very small water soluble molecules to pass through (paracellular transport). In addition, the membrane of the cells is composed of a hydrophobic lipid bilayer which is poorly permeable to hydrophilic or charged molecules. Indeed, most peptides and proteins are hydrophilic macromolecules, and therefore their diffusion across a tightly sealed hydrophobic cell monolayer is almost negligible. Indeed, epithelial and endothelial cells within mammalian tissues are generally regarded as protective barriers of the body against potentially harmful environmental conditions, and the formation of junctional complexes, especially a so-called tight junction, between epithelial or endothelial cells represents a major mechanism for the construction of these cellular barriers. Unfortunately, these cellular barriers also represent a formidable obstacle for the delivery of drugs to target tissues, especially drugs having large molecular weights such as peptides and proteins.
• When drug molecules gain access to the apical membrane of epithelial and endothelial cells, they may pass through the layer of cells by four possible mechanisms (FIG. 2); (a) partitioning into the lipid membrane and diffusing across the cell, a process driven by a chemical potential difference of the compound between two sides of the membrane; (b) passing through water-filled channels, most likely the paracellular junctions between adjacent cells; (c) combining with specific membrane-bound carriers, either energy-independent facilitated diffusion or energy-dependent carrier-mediated transport; and (d) specific or non-specific endocytotic and trascytotic membrane vesicles. Whereas compounds with a molecular weight of less than 1000 can in many instances be transported adequately by any of the above mechanisms, macromolecular drugs (e.g., peptides and proteins), due to their hydrophilic nature, can be transported only by paracellular or by endocytotic and transcytotic mechanisms.
• Under physiological conditions, peptides and proteins are normally digested or degraded into small fragments before they are absorbed. Certain macromolecules are known to cross epithelial or endothelial cell walls and gain access to the systemic circulation, albeit generally in very small amounts. The absorption of these molecules is usually either via the paracellular or transcellular pathway. In the paracellular pathway, which occurs only in leaky epithelial cells (e.g., the cells in infant GI-tract, cells in diseased states, or certain cells treated with penetration enhancers), macromolecules are able to pass through the leaky junctions between the cells. The practical use of this route is limited because it does not occur in normal adult tissues. In the transcellular pathway, macromolecules are internalized by cells via membrane-derived vesicles and subsequently transported to the other side of the cells. For purposes of providing a transport method of general applicability, attention has accordingly been focused on the enhancement of macromolecule transepithelial transport via these endocytotic and transcytotic mechanisms.
• Endocytosis is the most important process for the cellular uptake and transport of macromolecules in mammalian cells. For example, the transport of nutrients, the regulation of hormone actions, the control of metabolic reactions and processing of antigens all involve endocytosis. In addition, endocytosis is also involved in the transmembrane transport of many toxins and viruses. In endocytosis, exogenous macromolecules, either presented in the surrounding fluid phase or bound to the cell surface, are internalized by the cell via the formation of plasma membrane-derived vesicles. These vesicles with enclosed molecules can be processed through various intracellular organelles and then targeted to their final destinations.
• Less than 10% of total cell membrane proteins show specific affinities for certain molecules. These special membrane proteins are usually defined as receptors and molecules which bind specifically to the receptor are defined as ligands. Upon binding, most of these receptor-ligand complexes will be internalized along with the cell membrane through either a clathrin-dependent or a clathrin-independent pathway. In the clathrin-dependent pathway, receptor-ligand complexes are concentrated in clathrin-coated pit regions of the plasma membrane which contribute to about 2% of cell surface area of fibroblasts. The coated pits then invaginate and turn into coated vesicles by pinching off from the cell membrane. After their formation, the coated vesicles lose their coats rapidly, and as a result, smooth membrane vesicles and tubules (endosome) are formed. These endosome-entrapped receptor-ligand complexes are then subjected to a sequence of intracellular processing and sorting events. Most ligands, especially those with important physiological functions such as LDL, Tf, insulin, polymeric IgA, and EGF, enter cells through this pathway. Opportunistic ligands, such as influenza virus and diphtheria toxin, may also exploit this mechanism to enter cells.
• The formation of coated pits during receptor-mediated endocytosis has been proposed to occur by two different models. In the first model, it is assumed that coated pits are preformed by assembly of clathrin in the plasma membrane, and in this model, receptors or receptor-ligand complexes are randomly distributed to the pre-existing pits, sequestered within the pits, and endocytosed. In the second model, it is proposed that receptors or receptor-ligand complexes aggregate to form microdomains (or patches) in the plasma membrane, which then trigger the attachment of clathrin to the cytoplasmic tails of receptors. To date, there is insufficient data to determine whether either of these models, or some entirely different mechanism, is in fact operable under normal physiological conditions.
• In the clathrin-independent pathway, the invagination of non-coated plasma membrane-derived vesicles is responsible for the endocytosis of receptor-ligand complexes. In general, this type of endocytosis occurs to a much lesser extent of the receptor population and in a less efficient manner as compared to that of clathrin-dependent endocytosis. The exact internalization mechanism of this pathway is still largely unknown. A limited number of ligands, including IgG-ferritin, cholera toxins, ricin, and class I histocompatibility molecules, have been shown to enter cells by this pathway.
• Internalization of the membrane receptor can occur with or without the binding of ligands. For example, LDL and transferrin receptors can be concentrated in coated pits and be endocytosed in the absence of bound ligands. On the other hand, insulin receptors will concentrate in coated-pits and be endocytosed only upon binding of an insulin ligand. Different types of ligands can be entrapped in the same coated pits and be internalized in the same vesicle; the divergence of different receptor-ligand complexes occurs at the late stage of the endosome. For example, Tf and EGF have been shown to be taken up in the same vesicles by human epithelial cells and then diverge; EGF goes to the lysosome and Tf is recycled.
• In some instances, endocytosis of receptor-ligand complexes is not necessarily required for certain ligands to exert their physiological functions. For example, the binding of EGF and insulin to their receptors alone is sufficient to initiate a physiological response. The endocytosis of receptor-ligand complexes in this case is only responsible for the control of receptor number and the removal of receptor-bound ligands. On the other hand, endocytosis of other complexes, such as the Tf-TfR complex, is required for the physiological function of transferrin (i.e., delivery of ferric ions to the cytosol).
• Under physiological conditions, as the cell membrane undergoes constant invagination and turnover, a limited degree of non-receptor mediated endocytosis also occurs. The mechanisms involved in this constant internalization of plasma membranes are still not clear. Solutes in the extracellular fluid or macromolecules non-specifically bound to the cell membrane may be introduced into the cells by this type of endocytosis without the involvement of specific membrane receptors. This type of endocytosis is usually unsaturable and occurs along both clathrin-dependent and clathrin-independent pathways. The internalization of solutes containing fluid is defined as fluid phase endocytosis, and that of the membrane-bound molecules as non-specific adsorptive endocytosis. In fluid-phase endocytosis, a minute droplet of liquid can be randomly enclosed by an invaginated membrane vesicle and then internalized, carrying in whatever solutes happen to be in the droplet. This is an important route for the cell to nonspecifically import extracellular fluids in which nutrients and other important molecules are contained. As fluid-phase endocytosis does not require a membrane receptor, the uptake is dependent only on the concentration of the macromolecules in the extracellular fluid and the rate of membrane internalization. Because most of the endocytosed contents are usually targeted to and degraded in lysosomes, this route may not be useful for drug transport across the cells. In adsorptive endocytosis, non-specific cell membrane-bound molecules will be internalized via membrane invagination, the majority of which will eventually also be targeted to and degraded in lysosomes. This pathway is non-saturable at low concentrations, but it may be saturable at high concentrations due to limits on membrane-binding sites and on the rate of membrane internalization. As a consequence, endocytosis does not appear to provide a viable mechanism for generalized transport of active agents across the walls of endothelial or epithelial cells.
• Transcytosis is the transcellular transport of internalized vesicles from their sites of entry to the opposite surface of the cell. The contents enclosed in the vesicles, (i.e., solutes in the fluid phase or specifically or non-specifically membrane-bound molecules) are then released undegraded into the extracellular fluid. The initial cell membrane internalization processes of transcytotic vesicles usually share identical mechanisms with that of endocytotic vesicles; however, the intracellular sorting events differentiate the ultimate destination of transcytotic and endocytotic vesicles.
• Transcytosis occurs in many type of cells, such as placental syncytiotrophoblasts, enterocytes of the small intestine, colostrum cells, liver epithelial cells and endothelial cells. The main functions of transcytosis are believed to be the distribution of membrane-impermeable molecules (especially of macromolecules between separated biological compartments) and maintenance of a distinct cell membrane polarity. Transcytosis can be receptor-mediated or non-receptor mediated; for example, the transcytosis of albumin, cationized albumin, transferrin, IgG, thyroglobulin, nerve growth factor, EGF, ricin, and insulin have been shown to be mediated by receptors, whereas the transcytosis of HRP, Ferritin, dextran and lectin wheat germ agglutinin proceed via a non-specific mechanism. Transcytosis in polarized epithelial cells occurs in a vectorial fashion. For example, transcytosis of IgG, thyroglobulin, nerve growth factor, EGP and ricin occur in the apical-to-basal direction; the transcytosis of IgA and Tf usually occur in the basal-to-apical direction. Transcytosis via a non-specific mechanism usually occurs at a very low rate.
• A sequence of intracellular sorting and processing events determines the ultimate fate of plasma membrane-derived vesicles. Different cells may process membrane vesicles containing the same receptors in a different manner, and even a single cell may process the same vesicles in different ways at different times. The underlying mechanisms of sorting and processing of these internalized membrane vesicles as well as the intracellular and extracellular conditions which can affect the sorting events are still largely unknown.
• Endosomes, the Golgi apparatus, and possibly the ER have been considered the sorting compartments for internalized vesicles. Lysosomes, on the other hand, have been considered the end-points where the vesicles (as well as their contents) are degraded. Although the detailed sorting events and the associated mechanisms that determine the intracellular routing of the internalized vesicles is not yet known, available data indicate that the signals in the cytoplasmic tail of membrane receptors and the extracellular environments may play a role.
• Although the exact intracellular sorting and processing mechanisms remain to be elucidated, the apparent routing of the vesicles can be characterized in several ways. One involves receptor recycling and ligand degradation; in this route, dissociation of ligands from their receptors occurs within acidic endosomes. The released ligands are then delivered to a lysosomal compartment, where they are degraded; the membrane-bound receptors leave the endosome by budding off as a membrane vesicle and recycling back to the cell surface to bind new ligands from the extracellular fluid. Many ligand-receptor complexes, such as LDL, asialoglycoproteins, and transcobalamine II undergo this intracellular routing.
• In accordance with another mechanism, both receptor and ligand are degraded. The EGF receptor is an example for this route. Although EGF dissociates from its receptor in the endosome due to the acidic pH, the receptor does not segregate into a separate vesicle for recycling back to the cell surface. Instead, both EGF and its receptor eventually reach a lysosome where both are degraded. Other ligands such as multivalent IgG, interferons and their receptors also follow this pathway.
• Pursuant to yet another mechanism, both receptor and ligand are transported. This route is usually utilized in the transcellular transport of macromolecules across endothelial and epithelial cells. A typical example of this route is the transcellular transport of maternal IgG across the intestinal lining of epithelial monolayer in neonates. Once the IgG binds to its receptor on the cell, the complex is internalized; instead of reaching lysosomes or recycling back to the cell surface, the complex is transported to the other side of the cell where the IgG is released. Secretory IgA (sIgA) and its receptor also follow this route, but in an opposite direction; the receptor is partially degraded during the transcellular process and released together with IgA as a secretory component.
• In a final type of routing mechanism, both receptor and ligand recycle to the cell surface. Transferrin, a major serum glycoprotein which transports iron into cells, is a typical example for this route. After entry into an endosome, TfR-bound diferric Tf releases iron due to the low-pH environment of the endosomes. The resultant apotransferrin remains bound to its receptor, due to its high affinity for the receptor under acidic pH. Thereafter, apo-Tf-receptor complexes are segregated into a vesicle and recycled back to the cell surface. Other ligands, such as intrinsic factor and class I and II MHC molecules, may also follow this pathway.
• Three main factors limiting the penetration of active agents, and in particular peptide and protein drugs, across cells: the physicochemical properties of the drugs; the environment in the vicinity of the cells; and the membrane structure and the biological function of the cells. Many approaches have been designed and developed to minimize the above factors and therefore to increase drug penetration across the cells. Incompatibility with the lipid molecules in the cell membrane (hydrophilicity and hydrogen-bonding capacity), large molecular size and lability to proteolytic and hydrolytic degradation are unique properties associated with peptide- and protein-based drugs. Chemical modifications (prodrug approaches) and chemical systheses of analogues (analogue approaches) have been explored; to date, both types of approach have been of only limited applicability.
• Another category of approach involves altering the environment in the vicinity of the epithelial or endothelial cells. There are two major routes for large macromolecules (e.g., peptide and protein drugs) to pass through an epithelial or endothelial cell monolayer: the paracellular pathway, which is normally sealed by the tight junctions that exist between adjacent cells; and the transcellular pathway, which is normally obscured by the hydrophobic lipid bilayer of the cell membrane. Various types of enhancers, which act either by relaxing the tight junction or by increasing the fluidity (and hence, the permeability) of the membrane, have been developed to increase drug transport across the cells. Although some degree of success has been achieved, these approaches suffer from non-specificity and toxicity. When the tight junction is opened up by selected enhancers, substances other than the intended agents may also pass through the open gap between the cells, causing undesirable effects. Moreover, the perturbation of the cell membrane by enhancers can be harmful and toxic to the cells; irritation and inflammation of local tissues after the use of enhancers has been reported.
• In eukaryotic cells, there is an interconnected membrane traffic network responsible for a constant cellular membrane internalization. The half-life of membrane turnover in fibroblasts has been shown to be 2 hours and in macrophages, 0.5 hour. In epithelial cells, constant renewal of the brush-border membrane and membrane-associated proteins/glycoproteins has also been reported. The formation of membrane vesicles was shown to occur at a rate of 3000 vesicles/cell/min, or 5-50 times the cell-surface area per hour in liver epithelial cells. These membrane vesicles are naturally used to ingest macromolecules by endocytosis, to discharge their secretory products by exocytosis, or to transport macromolecules from one side of cell to the other by transcytosis.
• One of the most notable cellular barriers which drug delivery formulations attempt to circumvent is the blood-brain barrier (BBB), which is composed of 95% brain capillary endothelium. Clinical observations indicate that drug delivery across the BBB is a major factor in the management of the many central nervous system (CNS) diseases, such as primary or metastatic tumors in the brain, primary or secondary CNS infection in AIDS, neurological degenerations in Parkinson's disease, multiple sclerosis, and genetic enzyme deficiencies.
• Several approaches have been considered in an attempt to increase the transport of impermeable drugs across the BBB. For an approach to be clinically applicable, such a method must fulfill at least the following three criteria: (1) reversibility--the increase in permeability of the BBB must be transient and controllable, and should not cause permanent changes in transendothelial resistance; (2) nontoxicity--the increase in permeability of the BBB should not damage the endothelial cells, and more importantly should not cause any neurotoxicity; and (3) selectivity--the increase in permeability should be effective only for the transport of the therapeutic drug to the target tissue, but not induce transport of other potentially-harmful molecules or cause deposition into non-targeted tissues.
• Unfortunately, among the currently available penetration enhancers for epithelial and endothelial drug absorption, none can adequately fulfill these criteria. Their use in therapeutics is generally unreliable and sometimes quite dangerous; hence, the use of these techniques should be considered with extensive caution. In fact, to date the only clinically-acceptable method for increasing the BBB permeability is the osmotic disruption of endothelial junctions by hypertonic treatment. Although this method is indeed reversible, it can cause severe neurotoxicity due to its lack of selectivity. Hypertonic treatment extensively loosens BBB tight junctions and indiscriminately increases the paracellular transport of many impermeable molecules from the blood, which may include many pathogens and toxins. As reported in a clinical study, 5% of the patients who received a single osmotic disruption treatment developed a stroke, and 14% experienced various other types of seizures [Neuwelt, E. A. and Dahlborg, S. A. (1989) in Implications of the Blood-Brain Barrier and Its Manipulation, Plenum Publishing Co., New York, Vol. 2, pp. 195-261]. Other complications associated with this treatment include brain edema and ocular toxicity. Despite their inherent risk of side effects, osmotic disruption procedures, such as arterial infusion of hypertonic mannitol solution [Neuwelt, E. A., Barnett, P. A., Hellstrom, I., Hellstrom, K. E., Beaumier, P., McCormick, C. I. and Weigel, R. A. (1988) Cancer Res. 48, 4725-4729; Neuwelt, E. A., Frenkel, E., Gumerlock, M. K., Braziel, R., Dana, B. and Hill, S. A. (1986) Cancer 58, 1609-1620], are still being used to increase the transport of antibodies and drugs to the brain of the patient for the diagnosis or therapeutic treatment of brain tumors. Therefore, it can be anticipated that if a safe procedure could be achieved for the selective increase of specific drugs or proteins transport across the BBB, the treatment of many CNS-associated diseases would be markedly improved.
• One potentially promising route to achieve selective delivery of a drug or protein across epithelial or endothelial cells is to use receptors as markers and receptor-building ligands as vehicles for their transcellular transport [Rodman, J. S., Mercer, R. W. and Stahl, P. D. (1989) Current Opinion in Cell Biology 2, 664-672]. This type of carrier-mediated transport is known as receptor-mediated transcytosis, which has been demonstrated in epithelial and endothelial cells for the transport of hormones [King, G. L. and Johnson, S. M. (1985) Science 227, 1583-1586], albumin [Ghitescu, L., Fixman, A., Simionescu, M. and Simionescu, N. (1986) J. Cell Biol. 102, 1304-1311], and immunoglobulins [Rodewald, R. (1980) J. Cell Biol. 85, 18-32; Underdown, B. J. (1989) Immunol. Invest. 18, 287-297]. Drug delivery via receptor-mediated transcytosis is highly specific because it enhances only the transport of molecules that are conjugated to receptor-binding ligands. In addition, drug delivery via transcytosis is also specific for receptor-bearing cells, and therefore, a targeted delivery system can be developed [Gregoriadis, G., Poste, G., Senior, J. and Trouet, A. (eds.), Receptor-Mediated Targeting of Drugs, Plenum Press, New York (1984)].
• One type of receptor which has been suggested to be a potential vehicle for transcytotic transport across the BBB is the transferrin (Tf) receptor (TfR). Tf is a natural transport protein for iron [Aisen, P. and Listowsky, I. (1980) Ann. Rev. Biochem. 49, 357-393], and TfR's are expressed abundantly on the luminal surface of the brain capillary endothelium [Jefferies, A. E., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. and Mason, D. Y. (1984) Nature 312, 162-163]. Unlike the binding of hormones or growth factors to their receptors, the binding of Tf to its receptor will not alter any metabolic or physiologic pathway in the endothelial wall. In addition, the intracellular accumulation of internalized Tf or TfR is negligible due to the efficient recycling of the TfR to the cell surface [Fuller, S. D. and Simons, K. (1986) J. Cell. Biol. 103, 1767-1779]; therefore, less drug toxicity would result for the epithelial or endothelial cells.
• For example, TfR's have been found to reside predominantly on the basal membrane of MDCK epithelial cells. Because of this polarized distribution, basal membrane-associated TfR's recycle efficiently to their original membrane domain. Consequently, less than 0.16% of basal membrane-bound Tf can be transcytosed to the apical surface [Fuller et al., supra]. When an anti-TfR antibody was used as a transport carrier for methotrexate, it was found that only 0.27% of the injected dose of the drug reached the brain parenchyma in 24 hr following i.v. injection [Friden, P. M., Walus, L. R., Musso, G. F., Taylor, M. A., Malfroy, B. and Starzyk, R. M. (1991) Proc.Natl.Acad.Sci. USA 88, 4771-4775]. Similar results have also been reported in the case of acetylated low-density lipoprotein, where a high affinity of this protein to the luminal surface receptor of the brain capillary endothelium resulted in only a negligible absorption by the brain parenchyma [Triguero, D., Buciak, J. and Pardridge, W. (1990) J. Neurochem. 54, 1882-1888 (1990)]. This discrepancy between the high receptor density on the capillary surface and the low ligand transport to the brain parenchyma indicates that the translocation of ligand-receptor complexes across endothelial cells may be the rate limiting step in the receptor mediated trans-BBB drug delivery by macromolecular carriers. Nonetheless, present knowledge regarding the control and regulation of transcytosis in polarized cells especially in brain capillary endothelium, is still very limited.
• Receptors-mediated transcytosis in epithelial and endothelial cells is one of the major pathways by which many hormones, growth factors, and other proteins are distributed within mammalian tissues and is a highly regulated process in membrane-polarized cells [Mostov, K. E., and Simister, N. E. (1985) Cell 43, 389-390; Simionescu, M. (1989) in Endothelial Cell Biology in Health and Diseases, (Simionescu, N., and Simionescu, M., eds.) pp. 69-104, Plenum Press, New York]. It has been shown that although both receptors of epidermal growth factor (EGF) and transferrin (Tf) are localized on the basolateral membrane of filter-grown Madin-Darby canine kidney (MDCK) epithelial cells [Maratos-Flier, E., Yang Kao, C.-Y., Verdin, E. M., and King, G. L. (1987) J. Cell Biol. 105, 1595-1601; Fuller, S. D., and Simons, K. (1986) J. Cell Biol. 103, 1767-1779], only EGF can be transcytosed across the cell monolayers [Maratos-Flier et al., supra] while Tf remains exclusively in the recycling pathway of the basolateral membrane [Fuller et al., supra]. Factors that accurately control vectorial transcytosis of membrane-associated receptors and their bound ligands in polarized cells have been a subject of active investigation in recent years [Rodriguez-Boulan, E., and Salas, P. J. I. (1989) Annu. Rev. Physiol. 51, 741-754.; Breitfeld, P. P., Casanova, J. E., Simister, N. E., Ross, S. A., Mckinno, W. C. and Mostov, K. E. (1989) Current Opinion in Cell Biol. 1, 617-623; Rodman, J. S. , Mercer, R. W. and Stahl, P. D. (1990) Current Opinion in Cell Biol. 2, 664-672 [. It has been shown that amino acid sequence, phosphorylation of the cytoplasmic tail [Davis, C. G., van Driel, I. R., Russell, D. W., Brown, M. S., and Goldstein, J. L.(1987) J. Biol. Chem. 263, 4075-5082 ; Glenney, J. R. (1988) Cell 52, 675] of receptors, and cytosolic factors [Balch, W. E. (1989) J. Biol. Chem. 264, 16956-16968] can affect the internationization and intracellular trafficking of membrane receptors.
• It is an object of the present invention to enhance the carrier-mediated transcytosis of active agents, such as monomeric organic molecules and proteins, in endothelial and epithelial cells.
• In accordance with the present invention, transepithelial and transendothelial delivery of active agents, in particular peptides and proteins, is substantially enhanced through the use of the following combinations: (1) a conjugate carrier for the active agent comprising a ligand for a particular cell-surface receptor which undergoes endocytosis and/or transcytosis; and (2) and accelerator for the transcytosis of the particular cell-surface receptor. For example, certain agents (such as monensin and brefeldin A) can reversibly disrupt the polarized distribution of TfR's and markedly increase the rate of receptor-mediated transcytosis of Tf. Because this increase in transcytosis is specific to the cell-surface receptors (e.g., TfR), no other molecules will be co-transported with the receptor/ligned combination unless they have been conjugated to the ligand.
• Pursuant to a preferred embodiment of the present invention, there is provided a drug-delivery system 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's.
• Despite these promising characteristics of receptor-ligand interaction, receptor-mediated transcytosis has so far failed to demonstrate to be an effective means for increasing transepithelial or trasendothelial drug transport. One of the major drawbacks of drug delivery via transcytosis is that the rate of transport by this mechanism is usually very low due to the polarized distribution of receptors on the apical and basal plasma membranes. This membrane polarity renders surface receptors or markers difficult to traverse across epithelial or endothelial cells.