Drug Delivery
The therapeutic efficacy of pharmaceutical or therapeutic agents relies on the delivery of adequate doses of a pharmaceutical agent to the site of action. Many modes of delivery have been developed, including, for example, enteral (oral), parenteral (intramuscular, intravenous, subcutaneous), and topical administration. In most instances the administration system is chosen for reliable dosage delivery and convenience.
Typically, parenteral administration is the most reliable means of delivering a pharmaceutical to a patient. See, Goodman et al., Goodman and Gilman's Pharmacological Basis of Therapeutics, Pergamon Press, Ehnsford, N.Y. (1990) and Pratt et al. Principles of Drug Action: The Basis of Pharmacology, Churchill Livingstone, New York, N.Y. (1990). Each parenteral mechanism insures that a prescribed dosage of the pharmaceutical agent is inserted into the fluid compartment of the body where it can be transported. The disadvantage of these modes of delivery is that they require an invasive procedure. The invasive nature of administration is inconvenient, painful and subject to infectious contamination.
Enteral and topical administration are more convenient, generally non-painful, and do not predispose to infection, however both have limited utility. The gastrointestinal and dermal surfaces present formidable barriers to transport and therefore, some pharmaceutical agents are not absorbed across these surfaces. Another drawback to patient directed modes of administration (enteral, topical and subcutaneous) is compliance. Pharmaceutical agents that have a short half-life require multiple daily doses. As the number of doses increases, patient compliance and therapeutic efficacy decrease. Simplified and/or less frequent administration schedules can aid in optimizing patient compliance. Wilson et al. (1991) Harrison's Principles of Internal Medicine, 12th Ed., McGraw-Hill, Inc., New York, N.Y.
The skin is an efficient barrier to the penetration of water soluble substances, and the rate of transdermal pharmaceutical agent absorption is primarily determined by the agent's lipid solubility, water solubility, and polarity. Highly polar or water soluble pharmaceutical agents are effectively blocked by the skin. Even very lipophilic pharmaceutical agents penetrate the dermis very slowly compared with the rate of penetration across cell membranes. See Pratt et al. supra.
Efforts to develop more effective and convenient modes of pharmaceutical administration have led to the development of transdermal delivery systems. Many current transdermal pharmaceutical agent delivery systems rely upon pharmaceutical agents that are absorbed when admixed with inert carriers. See Cooper et al. (1987) “Penetration Enhancers”, in Transdermal Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRC Press, Boca Raton, Fla. Few pharmaceutical agents fit this profile and those which do are not always predictably absorbed. Various forms of chemical enhancers, such as those enhancing lipophilicity, have been developed to improve transdermal transport when physically mixed with certain therapeutic agents and provide more predictable absorption. See for example, U.S. Pat. Nos. 4,645,502; 4,788,062; 4,816,258; 4,900,555; 3,472,931; 4,006,218; and 5,053,227. Carriers have also been coupled to pharmaceutical agents to enhance intracellular transport. See Ames et al. (1973) Proc. Natl. Acad. Sci. USA, 70:456-458 and (1988) Proc. Int. Symp. Cont. Rel. Bioact. Mater., 15:142.
Fusogenic Proteins and Polypeptides of the Saposin Family
Saposins, a family of small (˜80 amino acids) heat stable glycoproteins, are essential for the in vivo hydrolytic activity of several lysosomal enzymes in the catabolic pathway of glycosphingolipids (see Grabowski, G. A., Gatt, S., and Horowitz, M. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 385-414; Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16; Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J. Lipid. Res. 33, 1255-1267). Four members of the saposin family, A, B, C, and D, are proteolytically hydrolyzed from a single precursor protein, prosaposin (see Fujibayashi, S., Kao, F. T., Hones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101; Rorman, E. G., and Grabowski, G. A. (1989) Genomics 5, 486-492; Nakano, T., Sandhoff, K., Stumper, J., Christomanou, H., and Suzuki, K. (1989) J. Biochem. (Tokyo) 105, 152-154; Reiner, O., Dagan, O., and Horowitz, M. (1989) J. Mol. Neurosci. 1, 225-233). The complete amino acid sequences for saposins A, B, C and D have been reported as well as the genomic organization and cDNA sequence of prosaposin (see Fujibayashi, S., Kao, F. T., Jones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101; Rorman, E. G., and Grabowski, G. A. (1989) Genomics 5, 486-492). A complete deficiency of prosaposin with mutation in the initiation codon causes the storage of multiple glycosphingolipid substrates resembling a combined lysosomal hydrolase deficiency (see Schnabel, D., Schroder, M., Furst, W., Klien, A., Hurwitz, R., Zenk, T., Weber, J., Harzer, K., Paton, B. C., Poulos, A., Suzuki, K., and Sandhoff, K. (1992) J. Biol. Chem. 267, 3312-3315).
Saposins are defined as sphingolipid activator proteins or coenzymes. Structurally, saposins A, B, C, and D have approximately 50-60% similarity including six strictly conserved cysteine residues (see Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16) that form three intradomain disulfide bridges whose placements are identical (see Vaccaro, A. M., Salvioli, R., Barca, A., Tatti, M., Ciaffoni, F., Maras, B., Siciliano, R., Zappacosta, F., Amoresano, A., and Pucci, P. (1995) J. Biol. Chem. 270, 9953-9960). All saposins contain one glycosylation site with conserved placement in the N-terminal sequence half, but glycosylation is not essential to their activities (see Qi. X., and Grabowski, G. A. (1998) Biochemistry 37, 11544-11554; Vaccaro, A. M., Ciaffoni, F., Tatti, M., Salvioli, R., Barca, A., Tognozzi, D., and Scerch, C. (1995) J. Biol. Chem. 270, 30576-30580). In addition, saposin A has a second glycosylation site in C-terminal half.
All saposins and saposin-like proteins and domains contain a “saposin fold” when in solution. This fold is a multiple α-helical bundle motif, characterized by a three conserved disulfide structure and several amphipathic polypeptides. Despite this shared saposin-fold structure in solution, saposins and saposin-like proteins have diverse in vivo biological functions in the enhancement of lysosomal sphingolipid (SL) and glycosphingolipid (GSL) degradation by specific hydrolases. Because of these roles, the saposins occupy a central position in the control of lysosomal sphingolipid and glycosphingolipid metabolisms (see Kishimoto, Y., Kiraiwa, M., and O'Brien, J. S. (1992) J. Lipid. Res. 33, 1255-1267; Fujibayashi, S., Kao, F. T., Hones, C., Morse, H., Law, M., and Wenger, D. A. (1985) Am. J. Hum. Genet. 37, 741-748; O'Brien, J. S., Kretz, K. A., Dewji, N., Wenger, D. A., Esch, F., and Fluharty, A. L. (1988) Science 241, 1098-1101).
The structural characteristic of these saposins is of great importance to the diverse mechanisms of activation. Since all of these proteins have high sequence similarity, but different mechanisms of action with lipid membranes, one can speculate that the specific biological functions of saposins and saposin-like proteins are the result of the differential interactions with the biological membrane environments. In vitro, saposin A enhances acid β-glucosidase activity at μM concentration, but saposin C deficiency leads to glucosylceramide storage and a “Gaucher disease-like” phenotype (see Schnable, D., Schroder, M., and Sandhoff, K. (1991) FEBS Lett. 284, 57-59; Rafi. M. A., deGala, G., Zhang, X. L., and Wenger, D. A. (1993) Somat. Cell Mol. Genet. 19, 1-7). Activation of saposin B takes place through solubilizing and presenting glycosphingolipid substrates to lysosomal enzymes (see Furst, W., and Sandhoff, K., (1992) Biochim. Biophys. Acta 1126, 1-16).
Saposin C promotes acid β-glucosidase activity by inducing in the enzyme conformational change at acidic pH (see Berent, S. L., and Radin, N. S. (1981) Biochim. Biophys. Acta 664, 572-582; Greenberg, P., Merrill, A. H., Liotta, D. C., and Grabowski, G. A. (1990) Biochim. Biophys. Acta 1039, 12-20; Qi. X., and Grabowski, G. A. (1998) Biochemistry 37, 11544-11554). This interaction of saposin C with the enzyme occurs on negatively charged phospholipid surfaces. In vitro and ex vivo saposins A and D function to enhance the degradation of galactosylceramide and ceramide/sphingomyelin, respectively (see Harzer, K., Paton, B. C., Christomanou, H., Chatelut, M., Levade, T., Hiraiwa, M. and O'Brien, J. S. (1997) FEBS Lett. 417, 270-274; Klien, A., Henseler, M., Klein, C., Suzuki, K., Harzer, K., and Sandhoff, K. (1994) Biochem. Biophys. Res. Commun, 200, 1440-1448). Patients lacking the individual saposins B and C showed a variant form of metachromatic leukodystrophy and Gaucher disease, respectively. (see Wenger, D. A., DeGala, G., Williams, C., Taylor, H. A., Stevenson, R. E., Pruitt, J. R., Miller, J., Garen, P. D., and Balentine, J. D. (1989) Am. J. Med. Genet. 33, 255-265) (see Christomanou, H., Aignesberger, A., and Linke, R. P. (1986) Biol. Chem. Hoppe-Seyler 367, 879-890).
Membrane fusion is a major event in biological systems driving secretion, endocytosis, excocytosis, intracellular transport, fertilization, and muscle development (see Christomanou, H., Chabas, A., Pampols, T., and Guardiola, A. (1989) Klin, Wochenschr. 67, 999-1003). Recent experimental evidence generated by this inventor has indicated that saposin-lipid membrane interactions play a critical role in saposin-mediated membrane fusion of lipids thereby facilitating transport of active agents across these biological membranes.
Accordingly, there exists a significant need for nontoxic agents which can improve the delivery or transport of pharmaceutical agents across or through biological membranes. The present invention fulfills these needs.