Eva Horn Møller

Delivery of dermatan sulfate from polyelectrolyte complex-containing alginate composite microspheres for tissue regeneration.

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) with a great potential as a new therapeutic agent in tissue engineering. The aim of the present study was to investigate the formation of polyelectrolyte complexes (PECs) between chitosan and dermatan sulfate (CS/DS) and delivery of DS from PEC-containing alginate/chitosan/dermatan sulfate (Alg/CS/DS) microspheres for application in tissue regeneration. The CS/DS complexes were initially formed at different conditions including varying CS/DS ratio (positive/negative charge ratio), buffer, and pH. The obtained CS/DS complexes exhibited stronger electrostatic interaction, smaller complex size, and more stable colloidal structure when chitosan was in large excess (CS/DS 3:1) and prepared at pH 3.5 as compared to pH 5 using acetate buffer. The CS/DS complexes were subsequently incorporated into an alginate matrix by spray drying to form Alg/CS/DS composite microspheres with a DS encapsulation efficiency of 90-95%. The excessive CS induced a higher level of sustained DS release into Tris buffer (pH 7.4) from the microspheres formulated at pH 3.5; however, the amount of CS did not have a significant effect on the release from the microspheres formulated at pH 5. Significant cell proliferation was stimulated by the DS released from the microspheres in vitro. The present results provide a promising drug delivery strategy using PECs for sustained release of DS from microspheres intended for site-specific drug delivery and ultimately for use in tissue engineering.

Insulin diffusion and self-association characterized by real-time UV imaging and Taylor dispersion analysis

Assessment of release kinetics of subcutaneously administered protein therapeutics remains a complex challenge. In vitro methods capable of visualizing and characterizing drug transport properties, in the formulation as well as surrounding subcutaneous tissue environment, are desirable in drug development. Diffusion is a key process in drug release and transport. Thus, our objective was to develop a UV imaging in vitro method for direct visualization and characterization of insulin diffusivity and self-association behavior. Agarose hydrogels were used for mimicking subcutaneous tissue. Diffusivity, self-association, and apparent size of insulin were further characterized by Taylor dispersion analysis, size exclusion chromatography, and dynamic light scattering. At low insulin concentrations and pH 3.0, the hydrodynamic radius of insulin was determined by Taylor dispersion analysis to 1.5±0.1nm, corresponding to the size of insulin monomer. Increasing concentration and pH to 1mM and pH 7.4, respectively, favoring insulin hexamers, increased the insulin hydrodynamic radius to 3.0±0.1nm. The UV imaging method developed was adequately sensitive to identify and characterize, in terms of diffusion coefficients, the changes in insulin transport in hydrogel due to pH and concentration changes. In conclusion, UV imaging allowed insulin diffusion in hydrogel matrixes to be studied in real-time, and showed that insulin self-association properties were reflected in the diffusion behavior. UV imaging is a useful tool for characterization of the influence of environmental conditions on protein mass transport. Hydrogels combined with UV imaging may be of utility for in vitro testing of protein therapeutics.

In vitro release studies of insulin from lipid implants in solution and in a hydrogel matrix mimicking the subcutis

Widely accepted in vitro methodologies for sustained release parenteral drug formulations remain to be established. Hydrogels have been proposed as a release matrix more closely resembling the in vivo conditions for formulations intended for subcutaneous administration. The perspective of the current work was to investigate the feasibility of developing UV imaging-based in vitro methods enabling visualization and characterization of drug release and transport of protein therapeutics intended for subcutaneous administration. Specifically, the objectives were to prepare lipid implants providing sustained release of the model protein insulin and investigate the release into 0.5% (w/v) agarose hydrogels, pH7.40, using UV imaging- and a gel sampling-based release testing method. These results were compared to insulin release into well agitated buffer solution. Irrespective of the applied in vitro release method, the insulin release from Sterotex implants with a drug load of 20% (w/w) was faster as compared to the release from implants with a load of 10% (w/w), most likely due to the higher porosity of the implants with increasing drug load. Insulin release from 10% (w/w) implants into agitated solution was faster as compared to release into agarose hydrogel. This was ascribed to the additional mass transfer resistance provided by the agarose hydrogel. Interestingly, the release profiles of insulin from implants with an initial drug load of 20% (w/w) obtained by the three in vitro methods were relatively similar. The gel-based methods, in particular UV imaging, enable monitoring local drug concentrations in the vicinity of the implant over time thereby facilitating assessment of, e.g., sink conditions. The study highlights that the selection of the in vitro release method should take into account various factors including mass transport, drug stability, data analysis and simplicity of the methodology.

Design and characterization of core-shell mPEG-PLGA composite microparticles for development of cell-scaffold constructs.

Appropriate scaffolds capable of providing suitable biological and structural guidance are of great importance to generate cell-scaffold constructs for cell-based tissue engineering. The aim of the present study was to develop composite microparticles with a structure to provide functionality as a combined drug delivery/scaffold system. Composite microparticles were produced by incorporating either alginate/dermatan sulfate (Alg/DS) or alginate/chitosan/dermatan sulfate (Alg/CS/DS) particles in mPEG-PLGA microparticles using coaxial ultrasonic atomization. The encapsulation and distribution of Alg/DS or Alg/CS/DS particles in the mPEG-PLGA microparticles were significantly dependent on the operating conditions, including the flow rate ratio (Qout/Qin) and the viscosity of the polymer solutions (Vout, Vin) between the outer and the inner feeding channels. The core-shell composite microparticles containing the Alg/DS particles or the Alg/CS/DS particles displayed 40% and 65% DS release in 10 days, respectively, as compared to the DS directly loaded microparticles showing 90% DS release during the same time interval. The release profiles of DS correlate with the cell proliferation of fibroblasts, i.e. more sustainable cell growth was induced by the DS released from the core-shell composite microparticles comprising Alg/CS/DS particles. After seeding fibroblasts onto the composite microparticles, excellent cell adhesion was observed, and a successful assembly of the cell-scaffold constructs was induced within 7 days. Therefore, the present study demonstrates a novel strategy for fabrication of core-shell composite microparticles comprising additional particulate drug carriers in the core, which provides controlled delivery of DS and favorable cell biocompatibility; an approach to potentially achieve cell-based tissue regeneration.

Formulation technologies to overcome unfavorable properties of peptides and proteins for pulmonary delivery

The pulmonary delivery of peptides and proteins has been considered as a promising alternative administration route to injections. Several advanced formulation technologies have been developed to deliver peptide and protein drugs to and through the lungs by overcoming the unfavorable properties of these macromolecular drugs. Some of them have shown the great potential; however, none of them is flawless. The future innovation in pulmonary delivery for biopharmaceuticals may lie in better understanding of interplay between the lung biology and the advanced formulation technologies.

Soft hydrogels interpenetrating silicone—A polymer network for drug‐releasing medical devices

Materials for the next generation of medical devices will require not only the mechanical stability of current devices, but must also possess other properties such as sustained release of drugs in a controlled manner over a prolonged period of time. This work focuses on creating such a sophisticated material by forming an interpenetrating polymer network (IPN) material through modification of silicone elastomers with a poly(2-hydroxyethyl methacrylate) (PHEMA)-based hydrogel. IPN materials with a PHEMA content in the range of 13%-38% (w/w) were synthesized by using carbon dioxide-based solvent mixtures under high pressure. These IPNs were characterized with regard to microstructure as well as ability of the hydrogel to form a surface-connected hydrophilic carrier network inside the silicone. A critical limit for hydrogel connectivity was found both via simulation and by visualization of water uptake in approximately 25% (w/w) PHEMA, indicating that entrapment of gel occurs at low gel concentrations. The optimized IPN material was loaded with the antibiotic ciprofloxacin, and the resulting drug release was shown to inhibit bacterial growth when placed on agar, thus demonstrating the potential of this IPN material for future applications in drug-releasing medical devices.

Real-time UV imaging identifies the role of pH in insulin dissolution behavior in hydrogel-based subcutaneous tissue surrogate

For parenteral biopharmaceuticals, subcutaneous diffusion and, in the case of solid implants or suspensions, dissolution may govern the clinical profile of the drug product. Insight into the dissolution and diffusion processes of biopharmaceuticals after parenteral administration is fundamental in the development of new protein drug formulations. Using insulin as a model compound, the aim of this work was to develop a UV imaging-based method to study the real-time dissolution and diffusion behavior of solid protein drugs under stagnant conditions in a hydrogel matrix mimicking the subcutaneous tissue. Dissolution of proteins and peptides is a complex phenomenon as it may be coupled to the complicated acid base properties of these substances. UV imaging allowed the real-time dissolution and diffusion processes of insulin at different pH values and of different insulins to be studied. Dissolution rates were obtained, and the quantitative performance of the developed UV imaging method was verified. It was shown that the UV imaging dissolution method was able to differentiate between the behavior of different insulins and that human insulin dissolution was highly dependent on pH. pH effects in the microenvironment of the human insulin compacts at pH 7.40 and 3.00 were observed by UV-Vis imaging, explaining the different dissolution kinetics of human insulin at pH 7.40 and 3.00 as compared to pH 5.40. In conclusion, UV-Vis imaging may be a useful tool for studying dissolution, diffusion and pH effects in the vicinity of solid protein drug in a hydrogel matrix with the aim of achieving a better understanding of in vivo dissolution processes.

Immunogenicity of Biopharmaceuticals

Immune Reactions Towards Biopharmaceuticals - a General, Mechanistic Overview.- Clinical Aspects of Immunogenicity to Biopharmaceuticals.- Assessment of Unwanted Immunogenicity.- Models for Prediction of Immunogenicity.- Immunogenicity of Biopharmaceuticals: Causes, Methods to Reduce Immunogenicity, and Biosimilars.- Case Study: Immunogenicity of rhEPO.- Case Study: Immunogenicity of Interferon-Beta.- Case Study: Immunogenicity of Insulin.- Case Study: Immunogenicity of Factor VIII.- Case Study: Immunogenicity of Natalizumab.- Case Study: Immunogenicity of Anti-TNF Antibodies.- Heparin-Induced Thrombocytopenia.- Presenting an Immunogenicity Risk Assessment to Regulatory Agencies.

Immunogenicity of Biopharmaceuticals: Causes, Methods to Reduce Immunogenicity, and Biosimilars

The immune response to biopharmaceuticals is still an elusive process, governed by a large number of factors. In this chapter an overview is given of several of the factors implicated in the development of an immune response, and the potential strategies to reduce immunogenicity are discussed. Finally, the implications of the complex immune response to the development of biosimilars are described in Section 5.4. The factors most commonly associated with immunogenicity of biopharmaceuticals are listed in Table 5.1 (Hermeling et al. 2004, Schellekens 2002a, Schellekens 2002b, Schellekens and Casadevall 2004). Those related to the analytical aspects of immunoassays will not be discussed here, and the reader is referred to Chapter 3 for a more in-depth discussion on these aspects. The other factors can be subdivided into three subheadings: structural characteristics of the biopharmaceutical, product-related factors, and patient-related factors. Although most of the discussion will be focused around protein-based biopharmaceuticals, several of the aspects discussed are also of relevance to other biopharmaceuticals. Wherever possible, we have tried to include some references to those other biopharmaceuticals.

Optimization of infrared microscopy to assess secondary structure of insulin molecules within individual subvisible particles in aqueous formulations

The analysis of subvisible particles is currently challenging but pivotal to the understanding and control of the quality of protein therapeutics. While a range of characterization methods is available for subvisible particles, information on the protein conformation in a particle-considered a possible parameter in eliciting unwanted immunogenicity of protein therapeutics-is especially challenging in the lower micrometer range using existing analytical technologies. Using 6 different protein particle populations, we show that transmission Fourier transform infrared (FTIR) microscopy can determine protein secondary structure in single particles down to 10 μm. The analytical setup presented here is able to immobilize protein particles and obtain transmission FTIR spectra on individual protein particles in their intact aqueous environment. Spectra of dried particles, on the other hand, were found to occasionally differ from spectra of particles in aqueous environment. In summary, using the analytical setup described in this study, transmission FTIR microscopy uniquely provides information on single protein particles in particle populations in their aqueous environment without interference from the background protein solution.