Simon Bjerregaard

Quality by design – Spray drying of insulin intended for inhalation

Quality by design (QBD) refers to a holistic approach towards drug development. Important parts of QBD include definition of final product performance and understanding of formulation and process parameters. Inhalation of proteins for systemic distribution requires specific product characteristics and a manufacturing process which produces the desired product. The objective of this study was to understand the spray drying process of insulin intended for pulmonary administration. In particular, the effects of process and formulation parameters on particle characteristics and insulin integrity were investigated. Design of experiments (DOE) and multivariate data analysis were used to identify important process parameters and correlations between particle characteristics. The independent parameters included the process parameters nozzle, feed, and drying air flow rate and drying air temperature along with the insulin concentration as a formulation parameter. The dependent variables included droplet size, geometric particle size, aerodynamic particle size, yield, density, tap density, moisture content, outlet temperature, morphology, and physical and chemical integrity. Principal component analysis was performed to find correlations between dependent and independent variables. Prediction equations were obtained for all dependent variables including both interaction and quadratic terms. Overall, the insulin concentration was found to be the most important parameter, followed by inlet drying air temperature and the nozzle gas flow rate. The insulin concentration mainly affected the particle size, yield and tap density, while the inlet drying air temperature mainly affected the moisture content. No change was observed in physical and chemical integrity of the insulin molecule.

Secondary structure alterations in insulin and growth hormone water-in-oil emulsions

Water-in-oil (w/o) emulsions have shown a promising release profile of small drug molecules and proteins. However, the major concerns are the structural stability, the retention of the activity and to avoid unwanted immunological reactions caused by the changes in protein structure. In the present study, the secondary structure of insulin and growth hormone is investigated after manufacture of w/o emulsions, using Fourier transform infrared (FTIR) spectroscopy. Initial investigations indicate an altered distribution in the secondary structure elements, e.g. alpha-helix and beta-sheet, measured by area overlap calculations. The changes are more pronounced for growth hormone than for insulin. The overlapping area is 0.93 +/- 0.01 for the emulsion containing insulin manufactured at 0 degrees C and homogenised for 3 min, the corresponding value for growth hormone is 0.83 +/- 0.01. The droplet size changes from 0.27 +/- 0.04 microm in the blank w/o emulsion to 0.79 +/- 0.13 and 0.66 +/- 0.21 microm when insulin or growth hormone is incorporated into the w/o emulsions, respectively.

Formulation and evaluation of release and swelling mechanism of a water-in-oil emulsion using factorial design

Water-in-oil emulsions have a potential as a parenteral prolonged release system for hydrophilic drugs. A consistent challenge when developing an emulsion drug delivery system is to obtain a proper release characteristic of the entrapped drug. The aim of the present study was to study the release mechanism from water-in-oil emulsions. Secondly, to study the effects of droplet size, phase ratio and osmotic pressure on the release rate of glucose from water-in-oil emulsions in a factorial experimental design. The release mechanism of glucose was deduced from the release kinetics of two coentrapped marker molecules, glucose and inulin, with a molecule weight of 180 and 5000 g/mol, respectively. The results indicate that release of glucose was dominated by diffusion through the oily barrier as opposed to membrane rupture. Using statistical methodology, the release rate of glucose could be varied 8 fold in a controlled manner with osmotic pressure as the most important parameter. The osmotic behaviour of the emulsions was further studied in a dynamic swelling study. These results show that the release of entrapped hydrophilic drug can be controlled within certain limits using pharmaceutical formulation principles.

Cyclodextrin inclusion complexes of antimycotics intended to act in the oral cavity--drug supersaturation, toxicity on TR146 cells and release from a delivery system.

The dissolution rate, the toxicity and the release from chewing gum of miconazole and econazole cyclodextrin products and complexes were investigated. The dissolution rate studies showed that an amorphous miconazole hydroxypropyl-beta-cyclodextrin product gave drug supersaturation, whereas drug supersaturation was not present during dissolution rate testing of an econazole hydroxypropyl-beta-cyclodextrin product. The miconazole hydroxypropyl-beta-cyclodextrin product and genuine cyclodextrin inclusion complexes of miconazole, econazole and clotrimazole were toxic on a human TR146 buccal cell culture model. The toxicity was probably due to drug supersaturation, thereby increasing the bioavailability of the antimycotics. The econazole hydroxypropyl-beta-cyclodextrin product and physical mixtures of miconazole or econazole and beta-cyclodextrin did not give supersaturation and were not as toxic as the above-mentioned compounds. Neat econazole and miconazole, a genuine econazole beta-cyclodextrin complex and the miconazole hydroxypropyl-beta-cyclodextrin product were incorporated in chewing gum. The miconazole hydroxypropyl-beta-cyclodextrin gum had a much higher drug release in vitro than the neat miconazole gum. The genuine econazole beta-cyclodextrin complex only increased the drug release moderately when compared with the release from the neat econazole gum. The release studies were performed on a mastication device.

Sustained elevated plasma aprotinin concentration in mice following intraperitoneal injections of w / o emulsions incorporating aprotinin

This study was initiated to test the feasibility of w/o emulsions as a sustained release system for aprotinin following intraperitoneal injection in mice. The emulsion was well tolerated in mice and sustained release was observed over a period of 96 h. The time for maximum plasma concentration of aprotinin was 10 min and 12 h after injection of a control solution and the emulsion dosage form, respectively. Furthermore, the hemolytic activity of the emulsion constituents was low indicating a low acute toxicological potential of the emulsion. The present study also showed that the lipolytic activity in peritoneal exudate from mice is important for the clearance of oily vehicles from the peritoneal cavity with lipolytic rate constants ranging from 50 to 130 nmol free fatty acid released/min/mg exudate protein at 37 degrees C, pH 8.5. It was concluded that the w/o emulsion was well suited to provide sustained elevated plasma aprotinin concentrations in mice.

Parenteral water/oil emulsions containing hydrophilic compounds with enhanced in vivo retention: formulation, rheological characterisation and study of in vivo fate using whole body gamma-scintigraphy

The preparation and characterization of parenteral water-in-oil (w/o) emulsions with a potential for sustained release of hydrophilic drugs was described with emphasis on rheological behaviour and spreading phenomenon after intramuscular (i.m.) injection in rabbit thigh muscle. Both steady state and dynamic rheological parameters were investigated showing Newtonian behaviour at low fraction of disperse phase ratio as opposed to viscoelastic and pseudoplastic behaviour at high fraction of disperse phase. Disappearance and spreading behaviour of hydrophilic radioactive markers, aprotinin (6512 g/mol) and pertechnetate (193 g/mol) entrapped in w/o emulsions from an i.m. injection site was studied by whole body gamma-scintigraphy. The retention of entrapped aprotinin 24 h postinjection was 83 +/- 5% for a low spreading emulsion and 76 +/- 6% for a high spreading emulsion. The corresponding values for pertechnetate were 50 +/- 11 and 23 +/- 2%, respectively. The relatively long retention times were suggested to be related to the good physical stability properties of the present emulsions. It was concluded that the presented w/o emulsions are promising vehicles for sustained release of hydrophilic drugs from an i.m. injection site.

A genuine clotrimazole γ-cyclodextrin inclusion complex-isolation, antimycotic activity, toxicity and an unusual dissolution rate

A crystalline clotrimazole γ-cyclodextrin inclusion complex, molar ratio 1:1.0, was isolated from phosphate buffer 0.05 M, pH 7.1. Due to the low water solubility of the inclusion complex, the clotrimazole dissolution rate from the complex was very low. However, application of a new method to disclose supersaturation phenomena showed that the complex gave rise to a profound clotrimazole supersaturation during the dissolution rate test. Probably, the clotrimazole supersaturation was the reason why the inclusion complex had higher antimycotic activity than both clotrimazole and a physical mixture of clotrimazole and γ-cyclodextrin, molar ratio 1:1.0. In addition, the inclusion complex was the most toxic on human erythrocytes and on non-differentiated monolayers of epithelial like human TR146 cells. On the other hand, no difference in the toxicity of the inclusion complex, the physical mixture and clotrimazole was observable when robust multilayers of differentiated TR146 cells were exposed to the compounds.

An econazole β-cyclodextrin inclusion complex : An unusual dissolution rate, supersaturation, and biological efficacy example

Genuine cyclodextrin inclusion complexes of the antimycotic econazole and β-cyclodextrin had higher antimycotic activity than a physical mixture of econazole and β-cyclodextrin. Surprisingly, the econazole dissolution rate from the physical mixture was higher than the dissolution rate from the inclusion complex. The improved antimycotic activity of the inclusion complex might be due to the superior ability of the complex to cause econazole supersaturation. A new procedure was applied to disclose the drug supersaturation. The genuine inclusion complex molar ratio econazole:β-cyclodextrin 2:3 gave rise to more hemolysis than the corresponding physical mixture. Toxicity testing on a human buccal epithelium in vitro model — based on TR146 cells — showed that the physical mixture was more toxic than the inclusion complex when TR146 cell mortality was evaluated. Neither measurement of the transepithelial electrical resistance of TR146 cell layers exposed to either the physical mixture or the inclusion complex nor analysis of the protein liberation from the TR146 cells during exposure revealed any differences between the two compositions.

Analysis of insulin allostery in solution and solid state with FTIR

The insulin hexamer acts as an allosteric unit mediated by homotropic and heterotropic effects shifting the equilibrium between three distinct conformational states (T(6), R(3)T(3) and R(6)). The homotropic ligand phenol stabilises the R(6) state by binding to hydrophobic pockets only present in the R(6) state and shifts the equilibrium towards the R(6) state. The structural difference between the T(6) and R(6) state is primarily a change in the B1-B8 residues from extended conformation (T(6)) to alpha-helix (R(6)). The aim of this study was to investigate FTIR as an alternative method to monitor the T-R transition in the insulin hexamer upon phenol binding, and in addition to explore the advantage of infrared spectroscopy to measure solid state samples, and support the ability to maintain an allosteric state upon drying. The FTIR spectra of insulin in solution showed an increase in alpha-helix upon phenol binding and correlated well with the transition measured by CD yielding similar dissociation constants. Furthermore it was possible to maintain the increase in alpha-helix upon phenol binding after lyophilisation. The overall structure of the FTIR spectra changed upon lyophilisation but an increase in alpha-helix content was retained. Reconstitution of lyophilised insulin resulted in a change in structure resembling the structure of insulin prior to lyophilisation. Principal component analysis of all spectra was computed resulting in distinct clusters, and most variation in the data set could be explained by PC1 corresponding to a change in alpha-helix.

The effect of controlled osmotic stress on release and swelling properties of a water-in-oil emulsion.

The purpose of this study was to investigate the effect of osmotic gradients in a water-in-oil (w/o) emulsion on release properties in order to control the release of hydrophilic drugs. The magnitude and direction of the osmotic gradient was shown to have a pronounced effect on the apparent permeability of the hydrophilic marker, [3H]glucose. The apparent permeability coefficient of glucose could be varied between 1.0x10(-5) and 5.0x10(-8) cm s-1 using osmotic gradients. The release rate of glucose was related to the swelling properties. The larger the degree of swelling, the lower the release rate. Furthermore the present w/o emulsion has a low viscosity and a long-term physical stability. This makes the emulsion a promising parenteral drug delivery system in which the release of hydrophilic drugs such as peptides, can be controlled.