Engineering Polymers For Gene Therapy Of Head Cancer
Peter W Swaan, Associate Professor
Pharmaceutics And Pharmaceutl Chemuniversity Of Utah
Grant 7R01CA107621-04 from National Cancer Institute, IRG: BTSS
Abstract: The long-term goal of this research is to engineer polymeric delivery systems that improve the efficacy and reduce the toxicity of head and neck cancer gene therapy. The purpose of this project is to engineer silk-elastinlike polymeric (SELP) matrices for minimally invasive controlled delivery of adenovirus constructs carrying the RB94 tumor suppressor gene (Ad-RB94). The rationale is that by genetic engineering of SELPs, it is possible to tailor-make delivery systems that are liquid at room temperature, mixed under mild conditions with adenoviral tumor suppressor gene therapy vectors, form vector-laden hydrogels at body temperature after a single intratumoral injection, release viable vectors at the site of tumor over a desired period of time, and kill the tumor cells with minimum systemic toxicity and maximum efficacy. The following Specific Aims will be addressed 1) To synthesize and characterize SELP hydrogels for localized and controlled adenoviral gene delivery. Linear SELP copolymer analogs containing silk-like and elastin-like repeating units with various sequences and lengths will be biosynthesized and characterized using recombinant techniques. Hydrogels will be formed from the linear polymers. The degree of swelling of the hydrogels will be examined as a function of polymer structure and initial polymer concentration at physiological temperature, pH, and ionic strength. 2) To examine the influence of polymer and adenoviral composition on the degree of swelling, adenoviral particle release and bioactivity in vitro. Model adenoviruses will be incorporated in the hydrogels. The degree of swelling will be evaluated in the presence of adenoviral particles. The amount released will be evaluated over time as a function of polymer structure and concentration. Release will be correlated with the bioactivity of the particles in relevant in vitro models. From the results of model adenoviral release and in vitro gene transfer, appropriate polymer and Ad-RB94 compositions will be used to examine the bioactivity of the released therapeutic tumor suppressor gene. 3) To evaluate the influence of polymer composition on transduction efficiency, duration of transgene expression, biodistribution, therapeutic efficacy, and toxicity of adenoviral-containing SELP hydrogels in vivo. A nude mouse tumor xenograft model of head and neck cancer will be used to evaluate these parameters by intratumoral administration of SELP hydrogels containing Ad-GFP (Adenoviruses containing green fluorescent protein gene as markers of gene transfer) and Ad-RB94. In the future phases, the proposed matrix-mediated adenoviral .gene therapy approach using genetically engineered polymers can be used for the development of clinically acceptable systems for gene therapy of head and neck cancer
Keywords: biomaterial development /preparation, gel, gene delivery system, gene therapy, head /neck neoplasm chemical structure function, genetic transduction, neoplasm /cancer therapy, transfection /expression vector athymic mouse, biotechnology, cell line, neoplasm /cancer transplantation
Project start date: 2005-05-01
Project end date: 2009-04-30
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Grants awarded to Peter W Swaan
High-Throughput Assay For The Intestinal Peptide Transporter
Peter W Swaan, Associate Professor
University Of Maryland Baltimore 660 W Redwood St, Rm 021 Baltimore, Md 21201
Grant 1R03DK075157-01A1 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZNS1
Abstract: This proposal aims to further develop a bioassay for the rapid determination of transporter affinity. We focus our proposed research on a pharmacologically relevant transport system, the small intestinal peptide transporter (PepT1). PepT1 plays a key role in intestinal absorption of di- and tripeptides, and mediates the absorption of a wide range of therapeutic compounds, such as aminopenicillins, cephalosporins, and angiotensin converting enzyme (ACE) inhibitors. We have developed novel, selective, peptidomimetic fluorescent compounds with affinity for PEPT1 that can be used in high-throughput bioassays. An epithelial cell line stably expressing human PepT1 will be used to validate and optimize the assay. Successful implementation of this assay will significantly increase our understanding of the structural interactions that drive intestinal peptide transport and further our structural knowledge of SLC proteins in general. Additionally, it may aid future development of strategies to optimize the oral bioavailability of drugs by targeting specifically to PepT1 and the rational development of chemotherapeutics that are targeted to tumors overexpressing PepT1, such as pancreatic adenocarcinoma. Ou specific aims are 1. To synthesize and evaluate novel fluorescent peptidomimetic substrates for PepT1In this aim we will design and synthesize novel fluorescent PepT1 substrates and select those that display a combination of high relative fluorescence quantum yield, metabolic stability in cell culture system, and high hPepTI affinity for further testing in a high-throughput setting. 2. Assay validation and optimization. In this specific aim we will validate our bioassay by determining assay accuracy, precision, limit of detection as well as limit of quantitation for known standards, specificity, linearity and range, and robustness. The long-term goal of this study is to develop a reliable, cost efficient, high-throughput bioassay for the peptide transporter system. This will aid in our understanding of the structural features that govern peptide and drug transport via this system and will significantly support studies aiming to target this transporter.
Keywords: bioassay, drug delivery system, gastrointestinal absorption /transport, high throughput technology, protein transport, technology /technique development, biomimetics, drug design /synthesis /production, drug screening /evaluation, fluorescence, gene expression, peptide, protein structure function, small intestine, cell line, tissue /cell culture
Project start date: 2005-09-30
Project end date: 2007-08-31
1R03DK075157-01A1 (2005): $74250
Engineering Polymers For Gene Therapy Of Head Cancer
Peter W Swaan, Associate Professor
Pharmaceutics And Pharmaceutl Chemuniversity Of Utah
Grant 7R01CA107621-04 from National Cancer Institute, IRG: BTSS
Abstract: The long-term goal of this research is to engineer polymeric delivery systems that improve the efficacy and reduce the toxicity of head and neck cancer gene therapy. The purpose of this project is to engineer silk-elastinlike polymeric (SELP) matrices for minimally invasive controlled delivery of adenovirus constructs carrying the RB94 tumor suppressor gene (Ad-RB94). The rationale is that by genetic engineering of SELPs, it is possible to tailor-make delivery systems that are liquid at room temperature, mixed under mild conditions with adenoviral tumor suppressor gene therapy vectors, form vector-laden hydrogels at body temperature after a single intratumoral injection, release viable vectors at the site of tumor over a desired period of time, and kill the tumor cells with minimum systemic toxicity and maximum efficacy. The following Specific Aims will be addressed 1) To synthesize and characterize SELP hydrogels for localized and controlled adenoviral gene delivery. Linear SELP copolymer analogs containing silk-like and elastin-like repeating units with various sequences and lengths will be biosynthesized and characterized using recombinant techniques. Hydrogels will be formed from the linear polymers. The degree of swelling of the hydrogels will be examined as a function of polymer structure and initial polymer concentration at physiological temperature, pH, and ionic strength. 2) To examine the influence of polymer and adenoviral composition on the degree of swelling, adenoviral particle release and bioactivity in vitro. Model adenoviruses will be incorporated in the hydrogels. The degree of swelling will be evaluated in the presence of adenoviral particles. The amount released will be evaluated over time as a function of polymer structure and concentration. Release will be correlated with the bioactivity of the particles in relevant in vitro models. From the results of model adenoviral release and in vitro gene transfer, appropriate polymer and Ad-RB94 compositions will be used to examine the bioactivity of the released therapeutic tumor suppressor gene. 3) To evaluate the influence of polymer composition on transduction efficiency, duration of transgene expression, biodistribution, therapeutic efficacy, and toxicity of adenoviral-containing SELP hydrogels in vivo. A nude mouse tumor xenograft model of head and neck cancer will be used to evaluate these parameters by intratumoral administration of SELP hydrogels containing Ad-GFP (Adenoviruses containing green fluorescent protein gene as markers of gene transfer) and Ad-RB94. In the future phases, the proposed matrix-mediated adenoviral .gene therapy approach using genetically engineered polymers can be used for the development of clinically acceptable systems for gene therapy of head and neck cancer
Keywords: biomaterial development /preparation, gel, gene delivery system, gene therapy, head /neck neoplasm chemical structure function, genetic transduction, neoplasm /cancer therapy, transfection /expression vector athymic mouse, biotechnology, cell line, neoplasm /cancer transplantation
Project start date: 2005-05-01
Project end date: 2009-04-30
Structural Biology Of The Apical Bile Acid Transporter
Peter W Swaan, Associate Professor
University Of Maryland Baltimore 660 W Redwood St, Rm 021 Baltimore, Md 21201
Grant 5R01DK061425-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: PHRA
Abstract: The apical sodium-dependent bile acid transporter (ASBT) plays a key role in the enterohepatic recycling of bile salts, cholesterol homeostasis, and serves as a molecular target for hypercholesterolemic agents. Although the transporter sequence is known, there is controversy about its membrane topology and very little is known about ASBT structure-function and ligand binding domains. The proposed research will focus on the structural biology of ASBT. Using a novel approach that combines molecular and computational biology our long-term goal is to delineate the three-dimensional structure, ligand-binding domains, and cellular transport mechanism of ASBT. The following specific aims will be addressed (1) define the membrane topology of ASBT using a series of topology scanning approaches; (2) Construct a comprehensive structural and predictive model of ASBT that can correlate structural point mutations to changes in ligand affinity and transport; (3) Define the functional regions of ASBT by site-directed mutagenesis; we have developed a computer-assisted site-directed mutagenesis approach to probe ASBT protein for amino acid residues implicated in ligand and sodium interactions; (4) Determine the ligand binding domains of ASBT by mass spectrometry; we will employ selective photoaffinity labels to determine ligand-binding peptide sequences. Information gained by these studies will significantly increase our understanding of the structural interactions that drive bile acid transport and further our structural knowledge of solute carrier proteins in general. Additionally, it may aid future development of specific therapeutic strategies against hypercholesterolemia and related cardiovascular diseases.
Keywords: biological transport, cholanate compound, membrane transport protein, molecular biology information system, protein structure function, structural biology, cholesterol, computational biology, hypercholesterolemia, ligand, SDS polyacrylamide gel electrophoresis, computer assisted sequence analysis, confocal scanning microscopy, high performance liquid chromatography, mass spectrometry, site directed mutagenesis, tissue /cell culture
Project start date: 2003-05-01
Project end date: 2008-02-29
5R01DK061425-05 (2007): $231624
5R01DK061425-04 (2006): $238541
5R01DK061425-03 (2005): $244283
5R01DK061425-02 (2004): $244283
1R01DK061425-01A1 (2003): $244283
Epithelial Transport And Function Of Riboflavin
Peter W Swaan, Associate Professor
Ohio State University 1960 Kenny Road Columbus, Oh 43210
Grant 5R01DK056631-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: PHRA
Abstract: Riboflavin (RF), a water-soluble vitamin, is essential for normal cellular functions and growth. During periods of dietary deprivation or physiological and pathological stress humans are vulnerable to developing RF deficiency. This results in a variety of clinical abnormalities, including growth retardation, anemia, skin lesions and degenerative changes in the nervous system. Humans cannot biosynthesize RF and, thus, must obtain the vitamin from the diet through absorption in the small intestine. Although many studies have focused on the mechanism of RF uptake, its definitive transepithelial absorption mechanism is controversial and remains to be defined in detail. Intracellular processes in RF absorption, such as cellular homeostasis, and RF function and regulation are also poorly understood. The current proposal aims to close these gaps in our knowledge of intestinal RF absorption. Our long-term objectives aim to identify, isolate, clone, and characterize the protein(s) involved in te epithelial translocation of this important vitamin. Our specific aims are 1) Is riboflavin transported into enterocytes by a receptor- mediated and/or a carrier-mediated pathway? 2) How is RF trafficked within the cell and what is the subcellular localization of its storage compartment? 3) Is a soluble plasma RF-binding protein involved in RF translocation? Our multidisciplinary approach is designed to provide new and integrated information at the functional, cellular and molecular levels of RF transport. These studies will yield new and important information regarding a complex epithelial transport mechanism and provide important new insights in its structural specificity and function.
Keywords: binding protein, biological transport, chemical structure function, epithelium, gastrointestinal absorption /transport, intermolecular interaction, membrane transport protein, riboflavin, cell component structure /function, cell membrane, chemical kinetics, glycosylphosphatidylinositol, growth /development, model design /development, receptor binding, receptor mediated endocytosis, structural model, chemical synthesis, confocal scanning microscopy, fluorescence microscopy, histochemistry /cytochemistry, laboratory mouse, morphometry, nutrition related tag, radiotracer, video microscopy
Project start date: 2001-06-01
Project end date: 2003-08-31
5R01DK056631-03 (2003): $199125
1R01DK056631-01A2 (2001): $198450
Porous Silicon Particles For Oral Drug Delivery
Peter W Swaan, Associate Professor
University Of Maryland Baltimore 660 W Redwood St, Rm 021 Baltimore, Md 21201
Grant 5R01EB002687-03 from National Institute Of Biomedical Imaging And Bioengineering, IRG: ZRG1
Abstract: Oral delivery remains the preferred route for drug administration. However, therapeutic macromolecular drugs currently under development suffer from poor oral bioavailability. Microfabrication technology may offer potential advantages over conventional drug delivery stratagems. This technology, combined with appropriate surface chemistry, can permit highly localized delivery of drugs and permeation enhancers. In this proposal, we investigate microfabrication strategies to create reservoir-containing microdevices and a surface chemistry protocol that can be used to bind muco- or cytoadhesives to these platforms. The long-term objective of this proposal is to develop a biomedical microsystem for oral delivery of pharmacologically active macromolecules into the systemic circulation via the creation of a robust hybrid organic/inorganic delivery system. It is expected that the proposed drug delivery system will enable directional release at the lumen-enterocyte interface resulting in elevated local concentrations. The central hypothesis to our proposed research is that the penetration potential of poorly permeable drugs is significantly enhanced by increasing both the epithelial residence time and local concentration. We propose the following specific aims 1. To asymmetrically conjugate bioadhesive ligands to microdevices 2. To determine the in vitro release mechanism of drug-loaded microsystems 3. To compare the permeation enhancement effects of bioadhesive microdevices against established oral drug delivery standards 4. Test microdevices in vivo for their ability to deliver therapeutically relevant amounts of drug. We expect to find that microdevices can exhibit enhanced biocompatibility and bioadhesion due to our ability to control device architecture in terms of bulk material, shape, size, and surface chemistry. This may allow for the delivery of multiple therapeutic macromolecules in a more controlled and targeted manner to the GI tract.
Keywords: biomedical equipment development, consumable /disposable biomedical equipment, drug delivery system, oral administration, particle, pharmacokinetics, silicon, adhesion, blood glucose, cellular immunity, cytotoxicity, gastrointestinal drug absorption, gastrointestinal epithelium, immune response, lactate dehydrogenase, lectin, polymethacrylate, resin, streptozotocin, atomic force microscopy, cell line, confocal scanning microscopy, fluorescent dye /probe, laboratory rat, radiotracer, whole body imaging /scanning
Project start date: 2005-03-01
Project end date: 2009-02-28
5R01EB002687-03 (2007): $242896
5R01EB002687-02 (2006): $251377
1R01EB002687-01A1 (2005): $270771
Related Publications
Intracellular processing of riboflavin in human breast cancer cells. Mol Pharm. 2008 Sep-Oct; 5( 5): 839-48. Epub 2008 Aug 29. PMID: 18754669 
Machine learning methods and docking for predicting human pregnane X receptor activation. Chem Res Toxicol. 2008 Jul; 21( 7): 1457-67. Epub 2008 Jun 12. PMID: 18547065 Conserved aspartic acid residues lining the extracellular loop 1 of sodium-coupled bile acid transporter ASBT Interact with Na+ and 7alpha-OH moieties on the ligand cholestane skeleton. J Biol Chem. 2008 Jul 25; 283( 30): 20653-63. Epub 2008 May 28. PMID: 18508772 Bacterial peptide recognition and immune activation facilitated by human peptide transporter PEPT2. Am J Respir Cell Mol Biol. 2008 Nov; 39( 5): 536-42. Epub 2008 May 12. PMID: 18474668 Design of high-affinity peptide conjugates with optimized fluorescence quantum yield as markers for small peptide transporter PEPT1 (SLC15A1). Bioorg Med Chem Lett. 2008 Apr 15; 18( 8): 2555-7. Epub 2008 Mar 20. PMID: 18374571 Cytosolic half of transmembrane domain IV of the human bile acid transporter hASBT (SLC10A2) forms part of the substrate translocation pathway. Biochemistry. 2008 Mar 25; 47( 12): 3606-14. Epub 2008 Mar 1. PMID: 18311924 Design, synthesis, cytoselective toxicity, structure-activity relationships, and pharmacophore of thiazolidinone derivatives targeting drug-resistant lung cancer cells. J Med Chem. 2008 Mar 13; 51( 5): 1242-51. Epub 2008 Feb 8. PMID: 18257542 Multi-level analysis of organic anion transporters 1, 3, and 6 reveals major differences in structural determinants of antiviral discrimination. J Biol Chem. 2008 Mar 28; 283( 13): 8654-63. Epub 2008 Jan 3. PMID: 18174163 Endocytosis inhibitors prevent poly(amidoamine) dendrimer internalization and permeability across Caco-2 cells. Mol Pharm. 2008 Mar-Apr; 5( 2): 364-9. Epub 2008 Jan 4. PMID: 18173246 Evaluation of the effect of ethanol's toxic metabolite acetaldehyde on the gastrointestinal oligopeptide transporter, PEPT1: in vitro and in vivo studies. Alcohol Clin Exp Res. 2008 Jan; 32( 1): 162-70. Epub 2007 Nov 20. PMID: 18028524 Electrostatic and potential cation-pi forces may guide the interaction of extracellular loop III with Na+ and bile acids for human apical Na+-dependent bile acid transporter. Biochem J. 2008 Mar 1; 410( 2): 391-400. PMID: 18028035 Conformational flexibility of helix VI is essential for substrate permeation of the human apical sodium-dependent bile acid transporter. Mol Pharmacol. 2008 Feb; 73( 2): 305-13. Epub 2007 Oct 30. PMID: 17971420 Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability. Bioconjug Chem. 2007 Nov-Dec; 18( 6): 2054-60. Epub 2007 Oct 26. PMID: 17960872 Computational models to assign biopharmaceutics drug disposition classification from molecular structure. Pharm Res. 2007 Dec; 24( 12): 2249-62. Epub 2007 Sep 11. PMID: 17846869 Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells. Pharm Res. 2007 Nov; 24( 11): 2138-45. Epub 2007 Aug 15. PMID: 17701324 Endocytic mechanisms for targeted drug delivery. Adv Drug Deliv Rev. 2007 Aug 10; 59( 8): 748-58. Epub 2007 Jun 28. Review. PMID: 17659804 Human pregnane X receptor antagonists and agonists define molecular requirements for different binding sites. Mol Pharmacol. 2007 Sep; 72( 3): 592-603. Epub 2007 Jun 18. PMID: 17576789 
Dynamin 2 regulates riboflavin endocytosis in human placental trophoblasts. Mol Pharmacol. 2007 Sep; 72( 3): 553-62. Epub 2007 Jun 12. PMID: 17565002 Structural variation governs substrate specificity for organic anion transporter (OAT) homologs. Potential remote sensing by OAT family members. J Biol Chem. 2007 Aug 17; 282( 33): 23841-53. Epub 2007 Jun 5. PMID: 17553798 Analogs of methyllycaconitine as novel noncompetitive inhibitors of nicotinic receptors: pharmacological characterization, computational modeling, and pharmacophore development. Mol Pharmacol. 2007 May; 71( 5): 1288-97. Epub 2007 Feb 16. PMID: 17308033