Jesper Østergaard

Intra-Articular Depot Formulation Principles: Role in the Management of Postoperative Pain and Arthritic Disorders

The joint cavity constitutes a discrete anatomical compartment that allows for local drug action after intra-articular injection. Drug delivery systems providing local prolonged drug action are warranted in the management of postoperative pain and not least arthritic disorders such as osteoarthritis. The present review surveys various themes related to the accomplishment of the correct timing of the events leading to optimal drug action in the joint space over a desired time period. This includes a brief account on (patho)physiological conditions and novel potential drug targets (and their location within the synovial space). Particular emphasis is paid to (i) the potential feasibility of various depot formulation principles for the intra-articular route of administration including their manufacture, drug release characteristics and in vivo fate, and (ii) how release, mass transfer and equilibrium processes may affect the intra-articular residence time and concentration of the active species at the ultimate receptor site.

Protein adsorption at charged surfaces: the role of electrostatic interactions and interfacial charge regulation.

The understanding of protein adsorption at charged surfaces is important for a wide range of scientific disciplines including surface engineering, separation sciences and pharmaceutical sciences. Compared to chemical entities having a permanent charge, the adsorption of small ampholytes and proteins is more complicated as the pH near a charged surface can be significantly different from the value in bulk solution. In this work, we have developed a phenomenological adsorption model which takes into account the combined role of interfacial ion distribution, interfacial charge regulation of amino acids in the proximity of the surface, electroneutrality, and mass balance. The model is straightforward to apply to a given set of experimental conditions as most model parameters are obtained from bulk properties and therefore easy to estimate or are directly measurable. The model provides a detailed understanding of the importance of surface charge on adsorption and in particular of how changes in surface charge, concentration, and surface area may affect adsorption behavior. The model is successfully used to explain the experimental adsorption behavior of the two model proteins lysozyme and α-lactalbumin. It is demonstrated that it is possible to predict the pH and surface charge dependent adsorption behavior from experimental or theoretical estimates of a preferred orientation of a protein at a solid charged interface.

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions

Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug‐human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV‐detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.

Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides

Cu(II) ions are implicated in the pathogenesis of Alzheimer disease by influencing the aggregation of the amyloid-β (Aβ) peptide. Elucidating the underlying Cu(II)-induced Aβ aggregation is paramount for understanding the role of Cu(II) in the pathology of Alzheimer disease. The aim of this study was to characterize the qualitative and quantitative influence of Cu(II) on the extracellular aggregation mechanism and aggregate morphology of Aβ1–40 using spectroscopic, microelectrophoretic, mass spectrometric, and ultrastructural techniques. We found that the Cu(II):Aβ ratio in solution has a major influence on (i) the aggregation kinetics/mechanism of Aβ, because three different kinetic scenarios were observed depending on the Cu(II):Aβ ratio, (ii) the metal:peptide stoichiometry in the aggregates, which increased to 1.4 at supra-equimolar Cu(II):Aβ ratio; and (iii) the morphology of the aggregates, which shifted from fibrillar to non-fibrillar at increasing Cu(II):Aβ ratios. We observed dynamic morphological changes of the aggregates, and that the formation of spherical aggregates appeared to be a common morphological end point independent on the Cu(II) concentration. Experiments with Aβ1–42 were compatible with the conclusions for Aβ1–40 even though the low solubility of Aβ1–42 precluded examination under the same conditions as for the Aβ1–40. Experiments with Aβ1–16 and Aβ1–28 showed that other parts than the Cu(II)-binding His residues were important for Cu(II)-induced Aβ aggregation. Based on this study we propose three mechanistic models for the Cu(II)-induced aggregation of Aβ1–40 depending on the Cu(II):Aβ ratio, and identify key reaction steps that may be feasible targets for preventing Cu(II)-associated aggregation or toxicity in Alzheimer disease.

Simultaneous Evaluation of Ligand Binding Properties and Protein Size by Electrophoresis and Taylor Dispersion in Capillaries

The interplay between biophysical characteristics such as protein size and shape and protein function is difficult to ascertain using simple methods. Here, we present an approach for characterizing both protein-ligand binding as well as protein hydrodynamic radius in one operation combining electrophoresis and size measurement by dispersion using capillaries. The methodology is based on the integration of Taylor dispersion analysis and capillary electrophoresis and is here demonstrated using commercially available capillary electrophoresis instrumentation modified with a pixel sensor UV area imager, allowing two detection points along the capillary. Analytes are the human serum proteins alpha(1)-acid glycoprotein and albumin interacting with the drug propranolol in a frontal analysis mode. Upon introduction of the propranolol-protein sample, voltage is initially applied to facilitate electrophoretically mediated separation of ligand and protein and frontal analysis. Then a pressure mobilization step is used whereby Taylor dispersion can be characterized online based on the signal from the UV area imager. Estimates of ligand binding and values for hydrodynamic radii agree with values obtained by independent methods.

Insights into the Early Dissolution Events of Amlodipine Using UV Imaging and Raman Spectroscopy

Traditional dissolution testing determines drug release to the bulk, but does not enable an understanding of the events happening close to the surface of a solid or a tablet. UV imaging is a new imaging approach that can be used to study the dissolution behavior of chemical compounds. The UV imaging instrumentation offers recording of absorbance maps with a high spatial and temporal resolution which facilitates the abundant collection of information regarding the evolving solution concentrations. In this study, UV imaging was used to visualize the dissolution behavior of amlodipine besylate (amorphous and dihydrate forms) and amlodipine free base. The dissolution of amlodipine besylate was faster from the amorphous form than from the crystalline forms. The UV imaging investigations suggested that a solvent mediated phase transformation occurred for the amorphous amlodipine besylate and the amlodipine free base samples. Raman spectroscopy was used to confirm and probe the changes at the solid surface occurring upon contact with the dissolution media and verified the recrystallization of the amorphous form to the monohydrate. The combination of UV imaging and Raman spectroscopy is an efficient tool to obtain a deeper insight into the early events of the dissolution process.

Oral bioavailability of cinnarizine in dogs : relation to SNEDDS droplet size, drug solubility and in vitro precipitation

The in vivo performance of self-nanoemulsifying drug delivery systems (SNEDDSs) with different in vitro physicochemical properties were determined with the purpose of elucidating the parameters determining the in vivo performance of SNEDDSs. The in vitro characterisation included the use of pulsed field gradient NMR and the dynamic lipolysis model. In vivo characterisation was carried out in dogs with elevated gastric pH. Four SNEDDSs containing cinnarizine were dosed orally, and the obtained PK profiles were related to in vitro characterisation data. The SNEDDSs with the lowest solubility of cinnarizine in the preconcentrates and the smallest droplet size had the highest AUC values after oral administration. No difference in C(max) and t(max) was observed between the SNEDDSs. Despite of precipitation occurring during in vitro lipolysis of one of the SNEDDS this SNEDDS performed as well in vivo as another SNEDDS that did not show any precipitation. The area under the colloidal dispersion curves as well as under the lipolysis curves could be used to rank order the in vivo performance of the SNEDDSs. Selection of in vitro optimisation parameters for SNEDDSs should be done carefully. It may not always be best to aim for the highest solubility in the preconcentrate and to avoid precipitation during in vitro lipolysis.

Characterization of bupivacaine-loaded formulations based on liquid crystalline phases and microemulsions: the effect of lipid composition.

This report details the structural characterization and the in vitro drug-release properties of different local anesthetic bupivacaine (BUP)-loaded inverted-type liquid crystalline phases and microemulsions. The effects of variations in the lipid composition and/or BUP concentration on the self-assembled nanostructures were investigated in the presence of the commercial distilled glycerol monooleate Myverol 18-99K (GMO) and medium-chain triglycerides (MCT). Synchrotron small-angle X-ray scattering (SAXS) and rotating dialysis cell model were used to characterize the BUP formulations and to investigate the in vitro BUP release profiles, respectively. The evaluation of SAXS data for the BUP-loaded GMO/MCT formulations indicates the structural transition of inverted-type bicontinuous cubic phase of the symmetry Pn3m → inverted-type hexagonal (H(2)) phase → inverted-type microemulsion (L(2)) with increasing MCT content (0-40 wt %). In the absence of MCT, the solubilization of BUP induces the transition of Pn3m → H(2) at pH 7.4; whereas a transition of Pn3m → (Pn3m + H(2)) is detected as the hydration is achieved at pH 6.0. To mimic the drug release and transport from in situ formed self-assembled systems after subcutaneous administration, the release experiments were performed by injecting low viscous stimulus-responsive precursors to a buffer in the dialysis cell leaving the surface area between the self-assembled system and the release medium variable. Our results suggest that the pH-dependent variations in the lipidic partition coefficient, K(l/w), between the liquid crystalline nanostructures and the surrounding buffer solution are significantly affecting BUP release rates. Thus, a first step toward understanding of the drug-release mechanism of this drug-delivery class has been undertaken tackling the influence of drug ionization as well as the type of the self-assembled nanostructure and its release kinetics under pharmaceutically relevant conditions.

Role of in vitro release models in formulation development and quality control of parenteral depots.

This review article provides an assessment of advantages/limitations of the use of current in vitro release models to predict in vivo performance of parenteral sustained release products (injectable depots). As highlighted, key characteristics influencing the in vivo drug fate may vary with the route of administration and the type of sustained release formulation. To this end, an account is given on three representative injection sites (intramuscular, subcutaneous and intra-articular) as well as on in vitro release mechanism(s) of drugs from five commonly investigated depot principles (suspensions, microspheres, hydrogels, lipophilic solutions, and liposomes/other nano-size formulations). Current in vitro release models are, to a different extent, able to mimic the rate, transport and equilibrium processes that the drug substance may experience in the environment of the administration site. Their utility for the purpose of quality control including in vitro–in vivo correlations and formulation design is discussed.

Rapid Formation of a Preoligomeric Peptide–Metal–Peptide Complex Following Copper(II) Binding to Amyloid β Peptides

Copper(II)-induced extracellular aggregation of amyloid b peptides (Ab) is implicated in the pathogenesis of Alzheimer s disease (AD). The exact role of the Cu–Ab interaction is not fully understood but it has been demonstrated that Cu–Ab oligomeric complexes may catalyze the formation of neurotoxic reactive oxygen species. Cu also induces the rapid formation of insoluble Ab oligomers that dissolve if Cu is removed. Low-order oligomers both with and without Cu are key to the neurodegeneration associated with AD. In the brain, Ab are primarily found as 40-residue (Ab1–40) and 42-residue (Ab1–42) peptides. It is known that Cu II binds to the N-terminal part of Ab and the 16-residue fragment Ab1–16 is well established as a nonfibrillating model for the Cu–Ab complex. Ab1–16 coordinates Cu II with the side chains of the amino acid residues D1, H6, H13, and H14 and presumably the N-terminal amino group; two different pHdependent coordination modes exist. The importance of the elucidation of the mechanism of Cu binding to Ab and its specific role in the aggregation process is emphasized by the fact that the Cu concentration is elevated in the amyloid plaques in brains from AD patients, and that Cu concentration transiently can reach micromolar concentrations in the extracellular space. However, detailed knowledge of the dynamics of the initial events in Cu binding to Ab and its relation to the formation of higher-order oligomers and aggregates is lacking. This information could be essential for the development of therapeutic strategies that could convert a toxic oligomerization pathway into a nontoxic pathway. Herein we address the kinetic mechanism of Cu binding to both Ab1–16 and Ab1–40, and copper(II)-induced Ab1–40 oligomerization by a combination of stopped-flow spectroscopy (fluorescence spectroscopy and light scattering), NMR relaxation, and dynamic simulations. Global fitting and dynamic simulations resulted in a unifying model for the initial steps of Cu binding to Ab, which includes a transient peptide–metal– peptide (Ab–Cu–Ab) complex that does not participate in the subsequent aggregation. Binding of Cu to Ab1–16 induces a large change in the environment of Y10 in Ab1–16 that allows characterization of the binding kinetics by stopped-flow fluorescence spectroscopy. Ab1–16 (40 mm) was mixed with solutions of Cu II with varying concentrations in HEPES buffer (0–40 mm), thus resulting in biphasic time traces (Figure 1a). The fast phase (< 30 ms) that exhibits a large decrease in signal intensity shows observed rates from approximately 35 to 1.7 10 s 1