PEGylated bioactive molecules in biodegradable polymer microparticles.

Injectable peptide and oligonucleotide biotherapeutics offer great promise for treatment of serious chronic diseases but almost always need further formulation work to increase stability and circulation lifetimes. Covalent attachment of poly(ethylene glycol) (PEG) will increase circulation lifetimes up to a week or so and decrease degradation in favorable cases. Encapsulation in biodegradable polymer microparticles has been highly successful, mostly for peptides to provide sustained release up to several months after injection. Although products are on the market using these technologies, PEGylation and microparticle encapsulation each have drawbacks that prevent more widespread use. When they are combined, the limitations of one technology may be resolved by the other. Work in several laboratories on encapsulation of PEGylated bioactive molecules has revealed a synergy. Activity reduction and restricted circulation lifetimes for PEGylated bioactive agents is addressed by microencapsulation and using a lower PEG molecular weight. Chemical degradation, excessive burst release and limited drug content are typical problems for microparticles that are ameliorated by using PEGylated actives. The case for synergy between PEGylation and microencapsulation is illustrated in this review by work with several proteins and peptides including insulin, and the oligonucleotide therapeutic, pegaptanib.

Tumor targeting by an aptamer.

UNLABELLED: Aptamers are small oligonucleotides that are selected to bind tightly and specifically to a target molecule. We sought to determine whether aptamers have potential for in vivo delivery of radioisotopes or cytotoxic agents. METHODS: TTA1, an aptamer to the extracellular matrix protein tenascin-C, was prepared in fluorescent and radiolabeled forms. After in vivo administration, uptake and tumor distribution of Rhodamine Red-X-labeled aptamer was studied by fluorescence microscopy. In glioblastoma (U251) and breast cancer (MDA-MB-435) tumor xenografts, biodistribution and imaging studies were performed using TTA1 radiolabeled with (99m)Tc. Tenascin-C levels and tumor uptake were studied in a variety of additional human tumor xenografts. To assess the effect of radiometal chelate on biodistribution, mercapto-acetyl diglycine (MAG(2)) was compared with diethylenetriaminepentaacetic acid and with MAG(2)-3,400-molecular-weight PEG (PEG(3,400)). RESULTS: Intravenous injection of fluorescent aptamer TTA1 produced bright perivascular fluorescence in a xenografted human tumor within 10 min. In the ensuing 3 h, fluorescence diffused throughout the tumor. Labeled with (99m)Tc, TTA1 displayed rapid blood clearance, a half-life of less than 2 min, and rapid tumor penetration: 6% injected dose (%ID)/g at 10 min. Tumor retention was durable, with 2.7 %ID/g at 60 min and a long-lived phase that stabilized at 1 %ID/g. Rapid tumor uptake and blood clearance yielded a tumor-to-blood ratio of 50 within 3 h. Both renal and hepatic clearance pathways were observed. Using the (99m)Tc-labeled aptamer, images of glioblastoma and breast tumors were obtained by planar scintigraphy. Aptamer uptake, seen in several different human tumors, required the presence of the target protein, human tenascin-C. Modification of the MAG(2) radiometal chelator dramatically altered the uptake and clearance patterns. CONCLUSION: TTA1 is taken up by a variety of solid tumors including breast, glioblastoma, lung, and colon. Rapid uptake by tumors and rapid clearance from the blood and other nontarget tissues enables clear tumor imaging. As synthetic molecules, aptamers are readily modified in a site-specific manner. A variety of aptamer conjugates accumulate in tumors, suggesting imaging and potentially therapeutic applications.

PEGylated insulin in PLGA microparticles. In vivo and in vitro analysis.

A novel controlled release formulation has been developed with PEGylated human insulin encapsulated in PLGA microspheres that produces multi-day release in vivo. The insulin is specifically PEGylated at the amino terminus of the B chain with a relatively low molecular weight PEG (5000 Da). Insulin with this modification retains full biological activity, but has a limited serum half-life, making encapsulation necessary for sustained release beyond a few hours. PEGylated insulin can be co-dissolved with PLGA in methylene chloride and microspheres made by a single o/w emulsion process. Insulin conformation and biological activity are preserved after PEGylation and PLGA encapsulation. The monolithic microspheres have inherently low burst release, an important safety feature for an extended release injectable insulin product. In PBS at 37 degrees C, formulations with a drug content of approximately 14% show very low (< 1%) initial release of insulin over one day and near zero order drug release after a lag of 3-4 days. In animal studies, PEG-insulin microspheres administered subcutaneously as a single injection produced < 1% release of insulin in the first day but then lowered the serum glucose levels of diabetic rats to values < 200 mg/dL for approximately 9 days. When doses were given at 7-day intervals, steady state drug levels were achieved after only 2 doses. PEG-insulin PLGA microparticles show promise as a once-weekly dosed, sustained release basal insulin formulation.

Long-term delivery of ivermectin by use of poly(D,L-lactic-co-glycolic)acid microparticles in dogs.

OBJECTIVE: To evaluate the potential utility of poly(D,L-lactic-co-glycolic)acid (PLGA) as a long-acting biodegradable drug delivery matrix for ivermectin used in the prevention of heartworm disease in dogs. ANIMALS: 30 adult female dogs. PROCEDURE: Microparticle formulations containing 25 weight percent (wt%), 35 wt%, and 50 wt% ivermectin were prepared by an oil-in-water emulsion technique with solvent extraction into excess water. A fourth formulation, consisting of a mixture of 15 wt% and 50 wt% ivermectin microparticles, was blended in a 1:1 ratio to result in a 32.5 wt% ivermectin formulation. Formulations were administered once on Day 0 to groups of 6 dogs at a dose of 0.5 mg of ivermectin/kg, s.c. Half of the dogs in each treatment group and 3 untreated control dogs were infected with Dirofilaria immitis larvae 121 and 170 days after treatment. Six months after infection, dogs were euthanatized and necropsies were performed. Pharmacokinetics and efficacy were investigated. RESULTS: Analysis of pharmacokinetic data revealed sustained release of ivermectin during at least 287 days in 3 distinct phases: a small initial peak, followed by release of drug through diffusion, and polymer degradation. Untreated control dogs were all infected with heartworms. Heartworms were not found in any of the dogs in the ivermectin-PLGA treated groups. Adverse clinical signs were not observed. CONCLUSIONS AND CLINICAL RELEVANCE: All formulations were 100% effective in preventing development of adult heartworms. Results indicate that PLGA microparticles are a promising drug delivery matrix for use with ivermectin for the prevention of heartworm disease for at least 6 months after treatment.