Ruud Verrijk

Shifting paradigms: biopharmaceuticals versus low molecular weight drugs

Biopharmaceuticals are pharmaceutical products consisting of (glyco)proteins. Nowadays a substantial part of the FDA-approved drugs belong to this class of drugs. Biopharmaceuticals deserve special attention as they have a number of characteristics that set them aside from low molecular weight drugs. Their activity depends on their complicated shape based on secondary, tertiary and (sometimes) quaternary structures. These structures cannot be fully defined with our present set of analytical techniques and approaches for potency testing. They often are the same as (or closely resemble) endogenous proteins. This means that in safety testing and clinical test programs questions have to be addressed regarding species specific responses, selection of dosing schedules and route of administration, and the possible occurrence of immunogenicity. As the conformational structure of a protein is easily disturbed, formulation and handling of biopharmaceuticals needs special attention in order to optimize the therapeutic effect and minimize adverse reaction, among which immune responses. The issue of biogenerics is gaining more and more interest and different critical elements in the development of biogenerics are touched upon. In conclusion, biopharmaceuticals cannot be characterized fully in terms of their structure like low molecular weight drugs. The performance of biopharmaceuticals relies on strict production protocols and close monitoring of their activity in the clinical situation.

The effect of formulation parameters on the size of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes

The aim of this study was to gain insight into the formulation parameters affecting the size of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes (polyplexes). Experimental designs were applied to screen and optimize several variables, which may influence the complex size. In a screening design, it was demonstrated that at a fixed concentration of plasmid (40 micrograms/ml) after incubation with polymer, the size of the resulting polyplexes was highly dependent on the polymer/plasmid ratio as well as on the pH, viscosity (i.e. sucrose concentration) and ionic strength of the aqueous solution. However, the temperature, PEG 600 (up to 5% (v/v)) and Tween 80 (up to 0.2%) had a marginal effect on the size of the polyplexes. In an optimization design, the effect of the pH, polymer/plasmid ratio and Tween on the size of the polymer/plasmid complexes prepared at relatively high concentration of plasmid (50-200 micrograms/ml) was evaluated. Based on the results of the optimization design, a mathematical model was derived, which describes the relationship between the size of the polyplexes and the different formulation parameters. This model shows that even at high plasmid concentration (200 micrograms/ml), small sized polyplexes were formed at low pH and ionic strength, especially when the solution contains 20% (w/v) sucrose. This concentrated polyplex dispersion (polymer/plasmid ratio > 3/1 (w/w), 200 micrograms plasmid/ml) can be diluted down to 5 micrograms/ml plasmid without significant changes in particle size and transfection potential. At lower ratios, a growth in particle size was observed upon dilution of the complexes, which might also explain the low transfection efficiency of these polyplexes in vitro.

Single ocular injection of a sustained-release anti-VEGF delivers 6 months pharmacokinetics and efficacy in a primate laser CNV model

A potent anti-vascular endothelial growth factor (VEGF) biologic and a compatible delivery system were co-evaluated for protection against wet age-related macular degeneration (AMD) over a 6month period following a single intravitreal (IVT) injection. The anti-VEGF molecule is dimeric, containing two different anti-VEGF domain antibodies (dAb) attached to a human IgG1 Fc region: a dual dAb. The delivery system is based on microparticles of PolyActive™ hydrogel co-polymer. The molecule was evaluated both in vitro for potency against VEGF and in ocular VEGF-driven efficacy modelsin vivo. The dual dAb is highly potent, showing a lower IC50 than aflibercept in VEGF receptor binding assays (RBAs) and retaining activity upon release from microparticles over 12 months in vitro. Microparticles released functional dual dAb in rabbit and primate eyes over 6 months at sufficient levels to protect Cynomolgus against laser-induced grade IV choroidal neovascularisation (CNV). This demonstrates proof of concept for delivery of an anti-VEGF molecule within a sustained-release system, showing protection in a pre-clinical primate model of wet AMD over 6 months. Polymer breakdown and movement of microparticles in the eye may limit development of particle-based approaches for sustained release after IVT injection.

Determination of the Porosity of PLGA Microparticles by Tracking Their Sedimentation Velocity Using a Flow Imaging Microscope (FlowCAM)

PurposeTo investigate whether particle sedimentation velocity tracking using a flow imaging microscope (FlowCAM) can be used to determine microparticle porosity.MethodsTwo different methods were explored. In the first method the sedimentation rate of microparticles was tracked in suspending media with different densities. The porosity was calculated from the average apparent density of the particles derived by inter- or extrapolation to the density of a suspending medium in which the sedimentation velocity was zero. In the second method, the microparticle size and sedimentation velocity in one suspending fluid were used to calculate the density and porosity of individual particles by using the Stokes’ law of sedimentation.ResultsPolystyrene beads of different sizes were used for the development, optimization and validation of the methods. For both methods we found porosity values that were in excellent agreement with the expected values. Both methods were applied to determine the porosity of three PLGA microparticle batches with different porosities (between about 4 and 52%). With both methods we obtained microparticle porosity values similar to those obtained by mercury intrusion porosimetry.ConclusionsWe developed two methods to determine average microparticle density and porosity by sedimentation velocity tracking, using only a few milligrams of powder.

VEGF165A microsphere therapy for myocardial infarction suppresses acute cytokine release and increases microvascular density but does not improve cardiac function

Angiogenesis induced by growth factor-releasing microspheres can be an off-the-shelf and immediate alternative to stem cell therapy for acute myocardial infarction (AMI), independent of stem cell yield and comorbidity-induced dysfunction. Reliable and prolonged local delivery of intact proteins such as VEGF is, however, notoriously difficult. Our objective was to create a platform for local angiogenesis in human-sized hearts, using polyethylene-glycol/polybutylene-terephthalate (PEG-PBT) microsphere-based VEGF165A delivery. PEG-PBT microspheres were biocompatible, distribution was size dependent, and a regimen of 10 × 10(6) 15-μm microspheres at 0.5 × 10(6)/min did not induce cardiac necrosis. Efficacy, studied in a porcine model of AMI with reperfusion rather than chronic ischemia used for most reported VEGF studies, shows that microspheres were retained for at least 35 days. Acute VEGF165A release attenuated early cytokine release upon reperfusion and produced a dose-dependent increase in microvascular density at 5 wk following AMI. However, it did not improve major variables for global cardiac function, left ventricular dimensions, infarct size, or scar composition (collagen and myocyte content). Taken together, controlled VEGF165A delivery is safe, attenuates early cytokine release, and leads to a dose-dependent increase in microvascular density in the infarct zone but does not translate into changes in global or regional cardiac function and scar composition.

Micro-Flow Imaging as a quantitative tool to assess size and agglomeration of PLGA microparticles

Graphical abstract Figure. No caption available. ABSTRACT The purpose of this study was to explore the potential of flow imaging microscopy to measure particle size and agglomeration of poly(lactic‐co‐glycolic acid) (PLGA) microparticles. The particle size distribution of pharmaceutical PLGA microparticle products is routinely determined with laser diffraction. In our study, we performed a unique side‐by‐side comparison between MFI 5100 (flow imaging microscopy) and Mastersizer 2000 (laser diffraction) for the particle size analysis of two commercial PLGA microparticle products, i.e., Risperdal Consta and Sandostatin LAR. Both techniques gave similar results regarding the number and volume percentage of the main particle population (28–220 &mgr;m for Risperdal Consta; 16–124 &mgr;m for Sandostatin LAR). MFI additionally detected a ‘fines’ population (<28 &mgr;m for Risperdal Consta; <16 &mgr;m for Sandostatin LAR), which was overlooked by Mastersizer. Moreover, MFI was able to split the main population into ‘monospheres’ and ‘agglomerates’ based on particle morphology, and count the number of particles in each sub‐population. Finally, we presented how MFI can be applied in process development of risperidone PLGA microparticles and to monitor the physical stability of Sandostatin LAR. These case studies showed that MFI provides insight into the effect of different process steps on the number, size and morphology of fines, monospheres and agglomerates as well as the extent of microparticle agglomeration after reconstitution. This can be particularly important for the suspendability, injectability and release kinetics of PLGA microparticles.