Development and characterization of cross-linked poly(malate) microspheres with dipyridamole.

Biodegradable cross-linked microspheres containing up to 63 wt.% of the active substance were obtained in a polycondensation process between D,L-malic acid and the tetrahydroxy compound dipyridamole. The in vitro release mechanism from biodegradable cross-linked microspheres has been studied. It was found that dipyridamole was released due to two-step hydrolysis of the ester bonds of the network. Initially, the only product of the hydrolytic degradation was found to be an oligomeric ester fraction with M(w)=1000 Da. The release of the free drug started after 8 days due to a further hydrolysis of the oligomers in solution. It was found that blood plasma enzymes in rats did not affect the hydrolytic processes. Biodegradable poly(malate) microspheres containing an anti-aggregating agent dipyridamole can be considered as a novel drug delivery system for a prolonged period of time implying a future parenteral application.

Synthesis and biological activity of polyethylene glycol-mouse nerve growth factor conjugate.

Conjugation of poly(ethylene glycol) derivatives with therapeutic proteins is a promising approach for enhancing protein stability and, therefore, effectiveness. An N-hydroxysuccinimidyl ester of fluorescein-PEG 2000 was used for chemical modification of mouse nerve growth factor (mNGF), a dimeric protein with therapeutic potential for Alzheimer's disease. The mNGF-PEG2000-fluorescein conjugate was characterized by RP-HPLC, spectrofluorometry, and SDS-PAGE and was biologically active, as determined by two independent NGF-specific assays (enhancement of ChAT activity in fetal neurons and neurite outgrowth in PC12 cells). The conjugate was not detectable by a standard NGF ELISA, suggesting a fortuitous reduction in protein recognition by antibodies.

Controlled DNA delivery systems.

PURPOSE: Genes are of increasing interest as pharmaceuticals, but current methods for long-term gene delivery are inadequate. Controlled release systems using biocompatible and/or biodegradable polymers offer many advantages over conventional gene delivery approaches. We have characterized systems for controlled delivery of DNA from implantable polymer matrices (EVAc: poly (ethylene-co-vinyl acetate)) and injectable microspheres (PLGA and PLA: poly (D, L-lactide-co-glycolide) copolymer and poly (L-lactide), respectively). METHODS: Herring sperm DNA and bacteria phage lambda DNA were encapsulated as a model system. Released DNA concentration was determined by fluoroassays. Agarose electrophoresis was used to determine the dependence of release rate on DNA size. The Green Fluorescent Protein (GFP) gene was used to determine the integrity and functionality of released DNA. RESULTS: Both small and large DNA molecules (herring sperm DNA, 0.1-0.6 kb; GFP, 1.9 kb; lambda DNA, 48.5 kb) were successfully encapsulated and released from EVAc matrices, and PLGA or PLA microspheres. The release from DNA-EVAc systems was diffusion-controlled. When co-encapsulated in the same matrix, the larger lambda DNA was released more slowly than herring sperm; the rate of release scaled with the DNA diffusion coefficient in water. The chemical and biological integrity of released DNA was not changed. CONCLUSIONS: These low cost, and adjustable, controlled DNA delivery systems, using FDA-approved biocompatible/biodegradable and implantable/injectable materials, could be useful for in vivo gene delivery, such as DNA vaccination and gene therapy.

Synthesis and characterization of polymer-(multi)-peptide conjugates for control of specific cell aggregation.

A new synthetic approach has been applied to obtain novel di-, tetra-, and (multi)-peptide containing polymer conjugates in quantitative yields with a high degree of conjugation. Bis-(N-hydroxysuccinimidyl) esters of PEG (Mw = 200, 600, 1400, 2000, and 3400) were synthesized and studied in a condensation reaction with synthetic peptides: glycine-glycine-tyrosine-arginine (GGYR), a model peptide, and glycine-arginine-glycine-aspartic acid-tyrosine (GRGDY), a sequence known to promote cell adhesion and aggregation. Tetra-substituted derivatives of PEG-based conjugates were synthesized by coupling L-aspartic acid and L-aspartyl-L-phenylalanine through a condensation procedure in organic media. Poly(acrylic acid) and co-polymers (Mw = 2000 and 5000) were studied as a model of multifunctional linear polymers in the reaction with L-tryptophan and GGYR. Alternative polymer-(multi)-peptide conjugates were successfully synthesized using Starburst dendrimer PAMAM (G = 3), 'short' and 'long'-chain PEG-based active esters and GRGDY. The structure of the intermediate precursors and peptide-conjugates was confirmed by spectral (UV-Vis, FTIR, H-NMR) and chromatographic (RP-HPLC and SEC) methods. By varying the properties of the interconnecting polymer--such as hydrophobicity, molecular weight, and functionality--a set of polymer-GRGDY conjugates was synthesized.

Synthesis, toxicological, pharmacological, and bronchodilating activity in vitro of some xanthineacetic acid derivatives.

The possibility of the preparation of some ester derivatives of dimethylxanthines from 1-theobromine- and 7-theophylline acetic acids and 7-(2-hydroxyethyl)-theophylline by DCC/DMAP-mediated esterification under mild conditions was studied. The structures of the compounds synthesized and by products isolated were demonstrated by microanalyses, UV-, IR-, and 1H NMR data. Acute toxicity assessment of the compounds on mice showed that compounds 4, 5, 6, and 7 are less toxic than aminophylline. A pharmacological study of the in vitro broncholytic effect (IC50 and pD2 values) of the derivatives and aminophylline showed that the new compound 4 (1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purine-7-acetic acid 2-(1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purin-7-yl)ethyl ester) has a strong bronchodilating effect on serotonine- and acetylcholine-induced spasm in guinea pig trachea. The same compound does not influence barbiturate-induced hypnosis and locomotor activity, unlike to the effect of the aminophylline, used as a reference substance.

Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis--I. Activities of phosphatases and composition and amount of lipids.

Maintenance of Mizuhopecten yessoensis in water with 0.5 mg Cd/l for 60 days increased Cd concentration in the mantle and ovaries about 200 times, in gills 70 times and hepatopancreas 20 times. Alkaline phosphatase activity was inhibited in all organs except the hepatopancreas after a 1-month exposure to Cd and became equal to the control in all the organs after 2 months exposure. Molluscs transferred to flowing water showed a considerable decrease in the activity of alkaline and acid phosphatases. The activity of both acid phosphatase and Mg2+-ATPase decreased in all the organs of experimental molluscs after exposure to Cd. The amount of phospholipids and cholesterol in the organs of control and experimental molluscs did not essentially differ.

Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis--II. Subcellular distribution of metals and metal-binding proteins.

Cd accumulation in the hepatopancreas and gills of the scallop resulted in an increase of metal concentration in the cytoplasm, while in control animals the general amount of Cd in the hepatopancreas was concentrated in the microsomes and cytoplasm. Cd appears to be equally distributed among high molecular weight proteins and metallothioneins in control animals. It was shown that during the experiment the metal was bound mainly to high molecular weight proteins. After 30 days of exposure of scallops to flowing water Cd was redistributed to metallothionein (MT)-like proteins. Cd concentration in the lipids of the hepatopancreas of control and experimental scallops was equal.