Patrick P. DeLuca

Emerging PEGylated drugs

PEGylation is a pharmaceutical technology that involves the covalent attachment of polyethylene glycol (PEG) to a drug to improve its pharmacokinetic, pharmacodynamic, and immunological profiles, and thus, enhance its therapeutic effect. Currently, PEGylation is used to modify proteins, peptides, oligonucleotides, antibody fragments, and small organic molecules. Research groups are striving to improve the consistencies of PEGylated drugs and to PEGylate commercialized proteins and small organic molecules. Furthermore, the PEGylations of novel medications, like oligonucleotides and antibody fragments, are being pursued to improve their bioavailabilities. This active research in the PEGylation field and the continued growth of the biopharmaceutical market predicts that PEGylated drugs have a bright future.

Methods to assess in vitro drug release from injectable polymeric particulate systems.

This review provides a compilation of the methods used to study real-time (37°C) drug release from parenteral microparticulate drug delivery systems administered via the subcutaneous or intramuscular route. Current methods fall into three broad categories, viz., sample and separate, flow-through cell, and dialysis techniques. The principle of the specific method employed along with the advantages and disadvantages are described. With the “sample and separate” technique, drug-loaded microparticles are introduced into a vessel, and release is monitored over time by analysis of supernatant or drug remaining in the microspheres. In the “flow-through cell” technique, media is continuously circulated through a column containing drug-loaded microparticles followed by analysis of the eluent. The “dialysis” method achieves a physical separation of the drug-loaded microparticles from the release media by use of a membrane, which allows for sampling without interference of the microspheres. With all these methods, the setup and sampling techniques seem to influence in vitro release; the results are discussed in detail, and criteria to aid in selection of a method are stated. Attempts to establish in vitro–in vivo correlation for these injectable dosage forms are also discussed. It would be prudent to have an in vitro test method for microparticles that satisfies compendial and regulatory requirements, is user friendly, robust, and reproducible, and can be used for quality-control purposes at real-time and elevated temperatures.

Effect of solvent removal technique on the matrix characteristics of polylactide/glycolide microspheres for peptide delivery

Abstract Porous salmon calcitonin (sCT)-loaded PLGA microspheres (size range 35–140μm) of varying matrix characteristics were prepared by an aqueous emulsification process using either a temperature gradient (Tmp) or dilution (Dil) of the continuous phase (CP). The Tmp technique resulted in microspheres with a hollow internal core and a porous wall. The core size and thickness of the porous wall were dependent on the temperature gradient used. A rapid ramp of temperature from 15 to 40°C resulted in a large core and a thin wall, while a gradual temperature rise resulted in a smaller core. The Dil technique produced microspheres with a uniform, honeycomb like pore structure without a core, pore size being dependent on the dilution volume used. The specific surface area was higher and bulk density lower for microspheres prepared by the Tmp technique while there was no significant difference in the peptide load (3.2–4.5%) between both techniques. A rapid removal of CH2C12 was observed in case of the Tmp technique while the Dil technique facilitated a slower and gradual CH2C12 removal. Residual CH2Cl2 was approximately

Peptide containing microspheres from low molecular weight and hydrophilic poly(d,l-lactide-co-glycolide)

Abstract Biodegradable poly(d,l-lactide-co-glycolide) (PLGA) polymers of molecular weight lower than 30 000 Da and free terminal carboxylic acid end groups were evaluated for incorporation of a peptide. These low molecular weight hydrophilic polymers require characterization for carboxylic acid content and fraction of water soluble polymer, both of which significantly affected the structure and peptide incorporation. The polymers from different suppliers have different properties due to their methods of synthesis. The glass transition temperature increased with increase in the molecular weight, where as the carboxylic acid content and the bulk density of the microspheres decreased with an increase in the molecular weight. The particle size of the microspheres increased with molecular weight. The total yield of microspheres decreased with an increase in the water soluble fraction of the polymer. The 75:25 lactide/glycolide polymers showed a decrease in peptide incorporation with molecular weight but the 50:50 did not. The peptide not incorporated into the microsphere was extracted by the aqueous continuous phase during the microsphere preparation. Microspheres with higher molecular weight show lower initial peptide release. The peptide incorporation in low molecular weight polymers can be controlled by modification in the microsphere preparation process. The results suggest that peptide microspheres can be prepared from low molecular weight hydrophilic lactide/glycolide polymers.

Porous Biodegradable Microspheres for Controlled Drug Delivery. I. Assessment of Processing Conditions and Solvent Removal Techniques

Microspheres containing methylene blue and prednisolone acetate were prepared by one of three methods: freeze-drying, evaporation, and solvent-extraction-precipitation. An extremely porous structure was obtained by the freeze-dry and solvent-extraction-precipitation procedures. The specific surface area of 6.33-µm particles was 20.6 m2/g, or 35 times that of a particle devoid of pores, and the void space was 59–61%. The sphericity, size, and yields of the microspheres were influenced by the preparation procedure, surfactant type and concentration, temperature of the continuous phase, polymer concentration in the dispersed phase, and ratio of marker to polymer. The most suitable processing conditions were a polymer concentration of 5–10%, a marker loading of 10%, 0.1% sorbitan sesquioleate as the surfactant, and temperature adjustment of the continuous phase from 15 to 50°C following the addition of the dispersed phase. Complete release of the highly water soluble methylene blue occurred within 72 hr, while the less soluble prednisolone acetate released much more slowly, i.e., 90% after 7 days. The microspheres remained relatively intact during the in vitro release of methylene blue, confirming that the incorporated agent was confined to the walls of the porous network. Collapse of the polymer structure was evident after 7 days. The release therefore was believed to be governed principally by the solubility of the drug and the porosity of the matrix.

Preparation and characterization of poly (D,L-lactide-co-glycolide) microspheres for controlled release of poly(L-lysine) complexed plasmid DNA.

AbstractPurpose. To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). Methods. Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. Results. The microspheres were found to be spherical with average particle size of 3.1-3.5 μm. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. Conclusions. The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.

Assessment of protein release kinetics, stability and protein polymer interaction of lysozyme encapsulated poly(d,l-lactide-co-glycolide) microspheres

Using lysozyme as a model protein, this study investigated protein stability, protein--polymer interaction in different release media and their influence on protein release profile and in vitro--in vivo correlation. Lysozyme was microencapsulated into PLGA 50:50 by a double emulsion--solvent extraction/evaporation method. Protein stability, protein--PLGA adsorption and protein in vitro release were studied in various test media. Differential scanning calorimetry analysis showed lysozyme to be most conformationally stable in pH 4.0 acetate buffer with highest T(m) at 77.2 degree C and DeltaH(cal) 83.1 kcal/mol. Lysozyme exhibited good stability in pH 2.5 glycine buffer with T(m) at 63.8 degree C and DeltaH(cal) 69.9 kcal/mol. In pH 7.4 phosphate-buffered saline (PBS), lysozyme showed a trend toward aggregation when the temperature was elevated. When PLGA polymer was incubated with lysozyme in the various buffers, adsorption was found to occur in PBS only. The adsorption severely limited the amount of lysozyme available for release from microspheres, resulting in slow and incomplete release in PBS. In contrast, the release of the microspheres in acetate and glycine buffers was complete within 40 and 70 days, respectively. Radiolabeled lysozyme blood levels in rats from the microspheres correlated qualitatively well with in vitro release in glycine buffer as a release medium. This study suggests that protein stability and adsorption are critical factors controlling protein release kinetics and in vitro--in vivo correlation of PLGA microspheres.

Biodegradable microspheres as depot system for patenteral delivery of peptide drugs

Abstract Synthetic biodegradable polymers of lactic and glycolic acid have been extensively investigated for sustained drug delivery. Drug release from lactide/glycolide polymers can be either diffusion controlled or matrix erosion controlled. Polymer degradation was dependent on the molecular weight of the polymer and matrix structure of the delivery system. High surface area, lower particle size and lower bulk density increased the rate of degradation. Gamma irradiation of the delivery system also increased the rate of matrix erosion by reduction of molecular weight. Microspheres containing peptide, salmon calcitonin (sCT), were prepared by solvent extraction techniques using temperature or dilution. sCT was also incorporated into pre-formed microspheres by adsorption. sCT has a strong hydrophobic region and hence can bind to the polymer by a combination of hydrophobic and ionic forces, resulting in high incorporation from an aqueous medium. Adsorption appeared to result in multiple layers of adsorbed peptide on the polymer surface. The microspheres with entrapped sCT exhibited in vivo release of sCT between days 5 and 9, whereas microspheres with adsorbed sCT showed low but detectable in vivo release for 3–4 days. Combination of microspheres with different release properties can be used to achieve various in vivo serum profiles.

Influence of formulation parameters on the characteristics of poly(D, L-lactide-co-glycolide) microspheres containing poly(L-lysine) complexed plasmid DNA.

This study describes the influence of polymer type, surfactant type/concentration, and target drug loading on the particle size, plasmid DNA (pDNA) structure, drug loading efficiency, in vitro release, and protection from DNase I degradation of poly(D, L-lactide-co-glycolide) (PLGA) microspheres containing poly(L-lysine) (PLL) complexed pDNA. PLGA microspheres containing pDNA-PLL were prepared using the water-in-oil-in-water (w-o-w) technique with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) as surfactants in the external aqueous phase. A complex ratio of 1:0.33 (pDNA-PLL, w/w) enhanced the stability of pDNA during microsphere preparation. Higher pDNA-PLL loading efficiency (46.2%) and supercoiled structure (64.9%) of pDNA were obtained from hydrophobic PLGA (M(w) 31000) microspheres compared with hydrophilic PLGA or low-molecular-weight PLGA microspheres. The particle size decreased from 6.6 to 2.2 microm when the concentration of PVA was increased from 1 to 7%. At the same concentration of surfactant, PVA stabilized microspheres showed higher pDNA-PLL loading efficiency (46.2%) than PVP stabilized microspheres (24.1%). Encapsulated pDNA in PLGA microspheres was protected from enzymatic degradation and maintained in the supercoiled form. The pDNA-PLL microspheres showed in vitro release of 95.9 and 84.9% within 38 days from the low-molecular-weight PLGA and hydrophilic PLGA microspheres, respectively, compared to 54.2% release from the hydrophobic, higher-molecular-weight PLGA microspheres. The results suggest loading and release of pDNA-PLL complex can be influenced by surfactant concentration and polymer type.

A short term (accelerated release) approach to evaluate peptide release from PLGA depot-formulations.

An accelerated method to evaluate peptide release from poly(dl-lactide-co-glycolide) (PLGA) depot formulations in short time is described. Peptide-loaded microspheres were made from hydrophilic 50∶50 PLGA by a dispersionsolyent extraction technique, and peptide release was studied at 37°C and at higher temperatures in various media. For all accelerated conditions, release was faster at temperatures above the glass transition, Tg, of the host polymer. Complete release of peptide from 8600 MW PLGA was achieved in 35 hours at 50°C in buffered and nonbuffered media containing 0.5% polyvinyl alcohol (PVA). Type of release media and concentration of PVA influenced the release profiles. A PVA concentration of 0.1 to 0.5% was found to prevent aggregation of microspheres at higher temperatures, with an increase in release at the higher PVA concentration. Peptide release was associated with a reduction of pH of the releasing media and increased mass loss. Complete peptide release at pH 4 from 8.6 kd and 28 kd PLGA at 50 and 60°C occurred within 30–40 hours and correlated well with the real-time release at 37°C and pH 7.0. At the higher molecular weight, a slightly longer accelerated release time and higher temperature were required to correlate with the real-time release. The data suggest that by optimization of release conditions such as temperature, surfactant concentration, buffer component, and pH, an accelerated study could be employed to evaluate depot formulations for a given polymer type.