Ligand-Directed Uptake of Nonionic Anticoders The cellular uptake process of nucleic acid and its analogs, oligopeptides and oligosaccharides with molecular weights between 5- 10,000 is not ell understood. However, these compounds may possess great potential a effective therapeutic agents. These molecules are designed for their interactions with their intracellular targets. Therefore, a thorough understanding of ht cellular uptake mechanism(s) for these substances is necessary for development of these compounds as molecular biological tools and potentially as effective drugs. Our paper on oligonucleoside methylphosphonates (ONMP, 8-mer) indicates that cellular entrance of ONMPs is unlikely to occur via passive diffusion or receptor-mediated endocytosis but most likely through an adsorptive endocytotic process and fluid-phase pinocytosis. Our recent manuscript reports that the neoglycopeptide [YEE(ah-GalNAc)3]-directed, receptor-mediated endocytosis is highly effective (50 times higher) in inducing the cellular uptake of ONMP into human liver cells in culture. The enhancement of uptake is tissue- specific, ligand-specific and is dependent on a covalent linkage between the ONMP and the neoglycopeptide. The biological activities of ONMP taken up by liver cells are currently being assayed for their antiviral activities against HSV-1 in the nucleus (anti-splicing) and in the cytoplasm (anti-mRNA in translation). The preliminary results indicated that the anti-splicing effectiveness of ONMP in the nucleus has been increased by 20-25 fold upon linking with the neoglycopeptide. The positive biological activity of the ONMP conjugate indicates that an effective system for a tissue-specific ONMP delivery system can now be constructed for human liver cells. Similarly, our preliminary study indicates that folate can serve as a ligand, enhancing the cellular uptake of ONMP to cell lines (such as Igrov-1 and kB cells) possessing a high expression of folate receptors. Thus, the research program of the revised application is to build a """"""""delivery assembly"""""""" specifically for various tissues and organs; the """"""""delivery assembly"""""""" consists of ligand-Scaffold- Pro-drug. The following variations in the construction of a """"""""delivery assembly"""""""" are: 1) Ligands - sugars and folate; 2) Scaffold - neoglycopeptides, bovine serum albumin/human serum albumin oligopeptides; 3) Pro-drug - ONMP, oligonucleotides, polypeptides/protein and chemotherapeutic agents. The cellular assay system for the effectiveness of the """"""""delivery assembly"""""""" to constructed will be based on measurement of radioactively labeled or fluorescently labeled pro-drug for both uptake rate and exit rate, microscopic examination of intracellular location, and anti-HSV activity, particularly of ONMP or ONM{P-oligonucleotides chimera possessing known anti-HSV activities. We will evaluate and develop effective """"""""delivery assemblies' for the cellular entrance to lymphocytes, kidney and prostate cells in addition to liver cells. Subsequently, the biodistribution and pharmacokinetics in animals and tumor xenograft-nude mice will be studied by Dr. Michael Colvin in Duke as collaborator. The real time biodistribution and pharmacokinetics in animals will also be investigated by 123I-labeled delivery assembly via Single-Photon Emission Computer Tomography (SPECT) in collaboration with Dr. J. James Frost of the Radiology Department of The Johns Hopkins University. Various covalent linkages among the ligand-scaffold-pro-drug will be evaluated for their effectiveness. The studies will provide new understanding about how the pro-drug may escape from endosomes to become biologically active and how membrane receptors may recycle back for more action. The research will develop principles and practices of tissue-specific drug delivery systems from basic science to clinical application.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052984-03
Application #
2685064
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Project Start
1996-04-01
Project End
2000-03-31
Budget Start
1998-04-01
Budget End
2000-03-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218