Preparation and characterization of monopegylated human granulocyte-macrophage colony-stimulating factor.

ABSTRACT Conjugates of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) attached to polyethylene glycol (PEG) chains were prepared using amine-reactive chemistry. Molecular masses of the PEGs were 20, 30, and 40 kDa. The monopegylated forms were isolated by anion-exchange chromatography and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography, mass spectrometry, reverse-phase high-performance liquid chromatography (HPLC), peptide mapping, in vitro cell proliferation bioassays, and rat pharmacokinetic studies. The pegylation site of the purified monopegylated products was identified as the N-terminus of the protein. All forms of pegylated GM-CSF were able to stimulate TF-1 cell proliferation in a colorimetric bioassay at concentrations equal to or lower than that of GM-CSF. Pharmacokinetic studies in rats demonstrated 32-fold, 27-fold, and 40-fold extensions in elimination half-lives for 20, 30, and 40 kDa PEG-GM-CSF, respectively, as compared with nonmodified GM-CSF.

Correlation of photosensitizer delivery to lipoproteins and efficacy in tumor and arthritis mouse models; comparison of lipid-based and Pluronic P123 formulations.

PURPOSE: The purpose of this study was the use of animal models to demonstrate the importance of drug delivery (verteporfin) to plasma lipoproteins in order to attain efficacy of photodynamic therapy (PDT) in vivo. METHODS: Photosensitizers appropriately formulated in various vehicles such as pluronics and lipid-based systems were compared to delivery of the drug in DMSO in two in vivo systems. The first was a tumor model using male DBA/2 mice inoculated intradermally with M1 rhabdomyosarcoma cells and in the second, arthritis in the MRL -lpr mouse strain was enhanced by two intradermal injections of complete Freunds adjunct. RESULTS: Those formulations in which the drug was in a monomeric form were better able to transfer drug to lipoproteins, which in turn led to superior PDT in vivo. CONCLUSIONS: The ability to introduce drug in monomeric form into the circulation correlates well with efficacy of photosensitizer formulations in mouse arthritis and tumor models.

Formulation of benzoporphyrin derivatives in Pluronics.

This study investigates the potential of Pluronics for the formulation of tetrapyrrole-based photosensitizers, with a particular focus on B-ring benzoporphyrin derivatives. The B-ring derivatives have a high tendency to aggregate in aqueous solutions, and this poses a significant formulation problem. Pluronics are ABA-type triblock copolymers composed of a central hydrophobic polypropylene oxide section with two hydrophilic polyethylene oxide sections of equal length at either end. Out of a range of different commercially available block copolymers studied, it was found that the longer the hydrophobic block, the better the stabilization of tetrapyrrolic drugs in monomeric form in aqueous suspensions. Of these the best performance was observed in the micelle-forming Pluronic P123. Micelle size determination by laser light scattering confirmed that particle size in stable Pluronic formulations was around 20 nm. Pluronics such as L122 formed emulsions spontaneously without the need for emulsion stabilizers; emulsions were highly stable at ambient temperatures over several days and also highly effective as potential drug delivery agents.