Jeoung Soo Lee

Synthesis and In Vitro/In Vivo Evaluation of 5‐Aminosalicyl‐Glycine as a Colon‐Specific Prodrug of 5‐Aminosalicylic Acid

A simple synthetic route for the preparation of amino acid conjugate of 5-aminosalicylic acid (5-ASA) was exploited and prepared 5-aminosalicyl-glycine (5-ASA-Gly) in good yield. In vitro and in vivo properties of 5-ASA-Gly as a colon-specific prodrug of 5-ASA were investigated using rats as the test animal. Incubation of 5-ASA-Gly with cecal or colonic contents at 37 degrees C released 5-ASA in 65 or 27% of the dose in 8 h, respectively. No 5-ASA was detected from the incubation of 5-ASA-Gly with the homogenates of stomach or small intestine. Plasma concentration of 5-ASA-Gly decreased rapidly after intravenous administration of 5-ASA-Gly, and no 5-ASA was detected in the blood, which indicated 5-ASA-Gly was not degraded in the plasma. After oral administration of 5-ASA-Gly, about 50% of the administered dose was recovered as 5-ASA and N-acetyl-ASA and 3% as 5-ASA-Gly from feces and 14% as 5-ASA-Gly and 28% as 5-ASA and N-acetyl-ASA from urine in 24 h. These results suggested that a large fraction of 5-ASA-Gly was delivered to the large intestine and activated to liberate 5-ASA. For comparison, total recovery of 5-ASA and N-acetyl-5-ASA from feces after oral administration of 5-ASA-Gly was greater than that from sulfasalazine, which is one of the most commonly prescribed prodrugs of 5-ASA.

Synthesis and properties of dextran-5-aminosalicylic acid ester as a potential colon-specific prodrug of 5-aminosalicylic acid

Dextran-5-aminosalicylic acid ester (dextran-5-ASA) was synthesized as a colon-specific prodrug of 5-aminosalicylic acid (5-ASA) which is active against inflammatory bowel diseases. Chemical stability of dextran-5-ASA in the bath of pH 1.2 or 6.8 was investigated at 37°C for 6 hrs, and 5-ASA was not released on such conditions. Depolymerization (%) of dextran-5-ASA by dextranase with the degree of substitution (DS) of 18, 23, or 30 was 92, 62 or 45 in 8 hrs respectively, but was not affected by the MW of dextran (9,000, 40,600, 80,200 or 580,000). Distribution of 5-ASA in dextran, determined by gel filtration chromatography, appeared to be relatively uniform. Incubation of dextran-5-ASA (DS 18) in cecal contents of rats released 20% (28 g) and 35% (49 g) of 5-ASA in 8 hrs and 24 hrs, respectively, but no 5-ASA was liberated from small intestinal contents.

Formulation and Characterization of Poloxamine-based Hydrogels as Tissue Sealants

In situ cross linkable polyethylene glycol (PEG)-based polymers play an increasing role in surgical practice as sealants that provide a barrier to fluid/gas leakage and adhesion formation. This study investigated the gelation behavior and physical properties of hydrogels formed from homogeneous and blended solutions of two acrylated poloxamines (Tetronics® T1107 and T904) of various molecular weights and hydrophilic/lipophilic balances relative to a PEG control. Hydrogels were formed by reverse thermal gelation at physiological temperature (T1107-containing formulations) and covalent crosslinking by Michael-type addition with dithiothreitol. All poloxamine-based hydrogels exhibited thermosensitive behavior and achieved significantly reduced swelling, increased tensile properties and increased tissue bond strength relative to the PEG hydrogel at physiological temperature. Swelling and tensile properties of all poloxamine-based hydrogels were significantly greater at 37°C relative to 4°C, suggesting that their improved physical properties derive from cooperative crosslinking by both noncovalent and covalent mechanisms. Poloxamine-based hydrogels were cytocompatible and underwent hydrolytic degradation over 2-5weeks, depending on their T1107/T904 composition. In conclusion, select poloxamine-based hydrogels possess a number of properties potentially beneficial to tissue sealant applications, including a substantial increase in viscosity between room/physiological temperatures, resistance to cell adhesion and maintenance of a stable volume during equilibration.

Synthesis and properties of dextran-nalidixic acid ester as a colon-specific prodrug of nalidixic acid.

Dextran–nalidixic acid ester (dextran-NA) with a varied degree of substitution (DS) was synthesized as a colon-specific prodrug of nalidixic acid (NA). Solubility in water (mg/ml) of dextran-NA with DS (mg NA/100 mg dextran-NA) of 7, 19, or 32 was 57.57 (equivalent to 4.00 mg NA/ml), 0.53 (equivalent to 0.10 mg NA/ml), or 0.03 (equivalent to 0.01 mg NA/ml), respectively, and that for NA was 0.03 at 25°C. To ensure the chemical stability of dextran-NA at conditions similar to those of the stomach and small intestine, dextran-NA was placed in a solution of pH 1.2 hydrochloric acid buffer or pH 6.8 phosphate buffer and incubated at 37°C; no NA was detected during the 6 h of the incubation period, which indicated that dextran-NA might be chemically stable during the transit through the gastrointestinal tract. Degree of depolymerization (%) by dextranase determined by the 2,4-dinitrosalicylic acid (DNS) method at 37°C for dextran-NA with DS of 7, 19, or 32 was 81, 68, or 8, respectively, in 8 h, and that for dextran was 91. When dextran-NA (equivalent to 50 μg of NA) with a DS of 7 or 17 was incubated with cecal contents (100 mg) of rats at 37°C, the extent of NA released in 24 h was 41% or 32% of the dose, respectively. NA was not liberated from the incubation of dextran-NA with the homogenate of tissue and contents of the small intestine.

A novel synthetic route for the preparation of hydrolytically degradable synthetic hydrogels

A variety of approaches have been described for the modification of synthetic, water soluble polymers with hydrolytically degradable bonds and terminal vinyl groups that can be crosslinked in situ by photo- or redox-initiated free radical polymerization. However, changes in macromer concentration, functionality, and molecular weight commonly used to achieve variable degradation rates simultaneously alter hydrogel mechanical properties. Herein, we describe a novel, two-step synthetic route for the preparation of hydrolytically degradable, crosslinkable PEG-based macromers based on chemical intermediaries that form ester linkages with variable alkyl chain length. Changes in the concentration of a single macromer were shown to provide effective variation of degradation, but with corresponding significant changes in tensile properties. Through variation in the alkyl chain length of the chemical intermediary, variable degradation times ranging from weeks to months are achieved, without significantly affecting initial gelation efficiency, swelling, or tensile properties. When modified with adhesive ligands, hydrogels supported viability of encapsulated and adherent cells. Controlled release of a model protein (Immunoglobulin G) was attained as a function of hydrogel degradation rate. Independent control of hydrogel degradation and mechanical properties will offer improved flexibility for studying the effect of these material characteristics on cellular function and may be useful in the design of matrices for tissue engineering and controlled release of bioactive molecules.

Poly(ethylene glycol) diacrylate/hyaluronic acid semi-interpenetrating network compositions for 3-D cell spreading and migration.

To serve as artificial matrices for therapeutic cell transplantation, synthetic hydrogels must incorporate mechanisms enabling localized, cell-mediated degradation that allows cell spreading and migration. Previously, we have shown that hybrid semi-interpenetrating polymer networks (semi-IPNs) composed of hydrolytically degradable poly(ethylene glycol) diacrylates (PEGdA), acrylate-PEG-GRGDS and native hyaluronic acid (HA) support increased cell spreading relative to fully synthetic networks that is dependent on cellular hyaluronidase activity. This study systematically investigated the effects of PEGdA/HA semi-IPN network composition on 3-D spreading of encapsulated fibroblasts, the underlying changes in gel structure responsible for this activity, and the ability of optimized gel formulations to support long-term cell survival and migration. Fibroblast spreading exhibited a biphasic response to HA concentration, required a minimum HA molecular weight, decreased with increasing PEGdA concentration and was independent of hydrolytic degradation at early time points. Increased gel turbidity was observed in semi-IPNs, but not in copolymerized hydrogels containing methacrylated HA, which did not support cell spreading. This suggests that there is an underlying mechanism of polymerization-induced phase separation that results in HA-enriched defects within the network structure. PEGdA/HA semi-IPNs were also able to support cell spreading at relatively high levels of mechanical properties (∼10kPa elastic modulus) compared to alternative hybrid hydrogels. In order to support long-term cellular remodeling, the degradation rate of the PEGdA component was optimized by preparing blends of three different PEGdA macromers with varying susceptibility to hydrolytic degradation. Optimized semi-IPN formulations supported long-term survival of encapsulated fibroblasts and sustained migration in a gel-within-gel encapsulation model. These results demonstrate that PEGdA/HA semi-IPNs provide dynamic microenvironments that can support 3-D cell survival, spreading and migration for a variety of cell therapy applications.

Synthesis and evaluation of 5-aminosalicyl-glycine as a potential colon-specific prodrug of 5-aminosalicylic acid

As a new colon-specific prodrug of 5-aminosalicylic acid (5-ASA), 5-aminosalicyl-glycine (5-ASA-Gly) was prepared by a simple synthetic route in good yield. Apparent partition coefficients of 5-ASA-Gly were lower than those of 5-ASA, which determined in CHCl3/pH 6.8 buffer or n-octanol/pH 6.8 buffer system. Stability of 5-ASA-Gly by peptidases was investigated by incubation of 5-ASA-Gly with the homogenates of tissue and contents of stomach, proximal small intestine or distal small intestine of rats at 37°C. 5-ASA was not detected, indicating that the prodrug was stable in the upper intestine. The amount of 5-ASA liberated from incubation of the prodrug in cecal or colonic contents of rats was about 65% or 27% in 8 hrs, respectively, which indicated that the prodrug activation took place more readily in the rat cecum whose bacterial counts are high like human colon. Results fromin vitro experiments suggested 5-ASA-Gly as a promising candidate of a colon-specific prodrug of 5-ASA.

RhoA knockdown by cationic amphiphilic copolymer/siRhoA polyplexes enhances axonal regeneration in rat spinal cord injury model

Spinal cord injury (SCI) results in permanent loss of motor and sensory function due to developmentally-related and injured-induced changes in the extrinsic microenvironment and intrinsic neuronal biochemistry that limit plasticity and axonal regeneration. Our long term goal is to develop cationic, amphiphilic copolymers (poly (lactide-co-glycolide)-g-polyethylenimine, PgP) for combinatorial delivery of therapeutic nucleic acids (TNAs) and drugs targeting these different barriers. In this study, we evaluated the ability of PgP to deliver siRNA targeting RhoA, a critical signaling pathway activated by multiple extracellular inhibitors of axonal regeneration. After generation of rat compression SCI model, PgP/siRhoA polyplexes were locally injected into the lesion site. Relative to untreated injury only, PgP/siRhoA polyplexes significantly reduced RhoA mRNA and protein expression for up to 4 weeks post-injury. Histological analysis at 4 weeks post-injury showed that RhoA knockdown was accompanied by reduced apoptosis, cavity size, and astrogliosis and increased axonal regeneration within the lesion site. These studies demonstrate that PgP is an efficient non-viral delivery carrier for therapeutic siRhoA to the injured spinal cord and may be a promising platform for the development of combinatorial TNA/drug therapy.

Prednisolone 21-sulfate sodium: a colon-specific pro-drug of prednisolone.

Prednisolone 21‐sulfate sodium (PDS) was synthesized as a colon‐specific pro‐drug of prednisolone with the expectation that it would be stable and non‐absorbable in the upper intestine and release prednisolone by the action of sulfatase once it was delivered to the colon. In‐vitro/in‐vivo properties were investigated using rats as test animals. PDS was chemically stable at pH 1.2, 4.5, 6.8 and 8.0, and the apparent partition coefficient was 0.11 in 1‐octanol/pH 6.8 buffer solution at 37°C. PDS was stable on incubation with the contents of the stomach or small intestine. When PDS (0.1 mg equiv. of prednisolone) was incubated with the caecal contents (0.05g), prednisolone was produced to a maximum 54% of the dose in 6 h and decreased thereafter, which suggested that reduction of the A ring took place in addition to the hydrolysis by sulfatase. After oral administration of PDS, a small portion of prednisolone was recovered from the cecal contents but not from the small intestine. Neither PDS nor prednisolone was detected in the plasma, suggesting that absorption of PDS is limited. The data demonstrate that the sulfate ester can serve as a novel colon‐specific promoiety by limiting the absorption of the pro‐drug in the upper intestine and releasing the active compound by the action of microbial sulfatase in the colon.

Cationic, amphiphilic copolymer micelles as nucleic acid carriers for enhanced transfection in rat spinal cord

UNLABELLED Spinal cord injury commonly leads to permanent motor and sensory deficits due to the limited regenerative capacity of the adult central nervous system (CNS). Nucleic acid-based therapy is a promising strategy to deliver bioactive molecules capable of promoting axonal regeneration. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to its cytotoxicity and low transfection efficiency in the presence of serum proteins. In this study, we synthesized cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), by grafting low molecular weight PLGA (4kDa) to bPEI (25kDa) at approximately a 3:1 ratio as an efficient nonviral vector. We show that PgP micelle is capable of efficiently transfecting plasmid DNA (pDNA) and siRNA in the presence of 10% serum in neuroglioma (C6) cells, neuroblastoma (B35) cells, and primary E8 chick forebrain neurons (CFN) with pDNA transfection efficiencies of 58.8%, 75.1%, and 8.1%, respectively. We also show that PgP provides high-level transgene expression in the rat spinal cord in vivo that is substantially greater than that attained with bPEI. The combination of improved transfection and reduced cytotoxicity in vitro in the presence of serum and in vivo transfection of neural cells relative to conventional bPEI suggests that PgP may be a promising nonviral vector for therapeutic nucleic acid delivery for neural regeneration. STATEMENT OF SIGNIFICANCE Gene therapy is a promising strategy to overcome barriers to axonal regeneration in the injured central nervous system. Branched polyethylenimine (bPEI: 25kDa) is one of the most widely studied nonviral vectors, but its clinical application has been limited due to cytotoxicity and low transfection efficiency in the presence of serum proteins. Here, we report cationic amphiphilic copolymers, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) that are capable of efficiently transfecting reporter genes and siRNA both in the presence of 10% serum in vitro and in the rat spinal cord in vivo. The combination of improved transfection and reduced cytotoxicity in the presence of serum as well as transfection of neural cells in vivo suggests PgP may be a promising nucleic acid carrier for CNS gene delivery.