Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

<p>The purpose of this thesis was to develop methods suitable for the incorporation of drug substancessuch as proteins into microparticles intended for controlled release. In particular a novel techniquefor the preparation of microparticles using supercritical fluids was investigated.</p><p>Supercritical fluids offer a considerable promise as extraction media for the formation ofmicroparticles of drugs and pharmaceutical excipients. There are two main reasons for using this technique. Firstly, the selective solvating power of supercritical fluids makes it possible to separatea particular component from a multi-component mixture. Secondly, the favourable mass transfer properties and high solubility of solvent in supercritical fluid make the formation of the microparticles rapid and efficient. </p><p>The Solution Enhanced Dispersion by Supercritical fluids process (SEDS) was used for the production of microparticles from several different biodegradable polymers. Briefly, particles were formed by the extraction of solvent from a solution which was sprayed into a supercritical fluid. </p><p>The use of a combination of supercritical N₂ and CO₂ in the SEDS process, improved the dispersion of polymer solutions, as compared with CO₂ alone. This resulted in reduction of the particle size of discrete microparticles produced from amorphous biodegradable polymers. Proteins (lysozyme and urease) were successfully incorporated into the poly(d,l-lactide-co-glycolide): copolymer composition 50:50 (DL-PLG) microparticles. The particles showed high entrapment efficiencies and the incorporated proteins retained a high degree of biological activity. Compared with conventional technologies for the preparation of such drug delivery systems, e.g. solvent-evaporation emulsion techniques, this new technique is environmentally superior, and suitable for up-scaling. Moreover the higher degree of control as indicated by the high reproducibility, makes validation of the process feasible. In conclusion, the SEDS process is an attractive way of incorporating proteins and peptides into biodegradable microparticles for controlled release.</p>

<p>In this thesis local anaesthetic formulations based on PEO-PPO-PEO block copolymers (PEO and PPO being poly(ethylene oxide) and poly(propylene oxide), respectively) or nonionic cellulose ethers undergoing temperature- or dilution-induced thickening were investigated. The aim of the work was to develop formulations which can be easily administered to the periodontal pocket, stay at the application site, give a fast onset of anaesthesia and have a duration sufficient to perform periodontal scaling procedures. </p><p>Emulsions, (mixed) micellar solutions and microemulsions fulfilling the requirements stated above were achieved by combining the active ingredients lidocaine and prilocaine with the nonionic block copolymers Lutrol^® F127 and Lutrol^® F68. The critical micellisation and gelation temperatures of the systems were found to be interconnected and influenced by the total polymer concentration and the polymer mixture composition, as well as the presence of cosolutes and pH. </p><p>A low-viscous isotropic phase that turns into a high-viscous hexagonal phase as the water content increases was found by combining Lutrol^® F68, water, a eutectic mixture of lidocaine and prilocaine and Akoline MCM. The system has a slower release rate compared to the microemulsion formulation which might make it suitable for indications where a longer duration is needed. </p><p>Finally, a temperature-induced gelling system was achieved by adding lidocaine and prilocaine to mixtures of ethyl(hydroxyethyl)cellulose (EHEC) and sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB) or myristoylcholine bromide systems at, or just below, the surfactant concentration found to give a maximum viscosity increase at room temperature. In particular, the myristoylcholine bromide system may be interesting considering its antibacterial properties and biodegradability.</p>

<p>Less than 2% of the lead compounds generated by the pharmaceutical industry enter the exploratory drug-development phase, from which point they stand only a 10% chance of becoming a commercial medicine. A large proportion of the compounds fail due to poor biopharmaceutical properties, such as permeability and solubility. The main theme of this thesis is, therefore, the development of better experimental and computational methods for modelling and predicting the biopharmaceutical properties of drug candidates. Immobilised liposome chromatography (ILC) was used for studying drug-membrane interactions and for the prediction of passive drug transport. For the drugs studied in this thesis, ILC and octanol/water partitioning showed a similar performance with regard to the prediction of passive drug transport.</p><p>The theoretical work was directed at the modelling and prediction of drag transport processes using calculated molecular properties and PLS analysis. In the initial studies, the molecular properties were calculated with an advanced computational tool (MolSurf) that takes the three-dimensional structural information and electronic properties of the compound into consideration. Statistical models well suited to the prediction of drug transport processes such as Caco-2 cell permeability, intestinal absorption and CNS penetration were derived.</p><p>This approach was also successfully applied to the modelling of the interaction of drugs with P-glycoprotein. Subsequently, rapidly calculated descriptors based on two-dimensional structural information and PLS analysis were also found to give good predictive models of drug transport properties. The preferred use of the latter models is for screening compound collections and virtual libraries. It can be concluded that calculated molecular properties in conjunction with PLS analysis are well suited to the modelling and prediction of drug transport processes and to identifying compounds with potential biopharmaceutical problems at an early stage of the drug-development process.</p>

<p>Lead compounds generated in high-throughput drug discovery programs often have unfavorable biopharmaceutical properties, resulting in a low success rate for such drug candidates in clinical development. Efficient and reliable methods that predict biopharmaceutical properties, such as intestinal permeability and solubility are therefore required in order to reduce the attrition rate during development of these compounds.</p><p>One aim of this thesis was to identify molecular properties that are important for intestinal drug permeability using a wide range of drugs and model compounds. A second aim was to develop computational models for predicting intestinal drug permeability based on these properties.</p><p>The calculated molecular descriptors ranged from the simple counting of atoms and fragments to more complex descriptors derived from molecular mechanics and quantum mechanics calculations. Particular attention was given to descriptors associated with molecular surface areas. Descriptors calculated by the various methods were used to establish structure-permeability relationships for conventional drugs, peptide derivatives and large, lipophilic compounds generated by high-throughput pharmacological screening. Caco-2 cell monolayer permeabilities were determined for a structurally diverse set of compounds and were used to predict human intestinal membrane permeability and to develop computational models.</p><p>From these investigations, several new models for the computational prediction of intestinal membrane permeability were developed. Models were developed that are suitable for the prediction of membrane permeability to specific types of drugs, as well as models that are more generally applicable. One of these general models is based on partitioned total molecular surface areas, and this model can be used to predict intestinal membrane permeability to structurally diverse compounds. It was also demonstrated how these models can be applied in a manner that increases both the accuracy of the prediction and the throughput. In addition, a simplified protocol based on Caco-2 cells for the experimental prediction of intestinal permeability was developed. These improvements can be used to construct highly effective experimental and computational filters for use in drug discovery and development.</p>

<p>This thesis describes certain critical aspects of the development of semisolid topical anesthetic formulations requiring a fast onset of action. Furthermore, local anesthetics were investigated regarding their phase interaction with membrane lipids.</p><p>A new long acting and topically effective local anesthetic/analgesic agent, isopropyl-methyl-[2-(3-propoxyphenoxy)-ethyl]-amine (amino diether, AD), was used as the model compound. The nonionized form of AD is liquid oil at room temperature with low water solubility. A submicron o/w emulsion with Newtonian flow property was prepared with AD as the disperse phase. The kinetic stability of this emulsion was increased to prevent Ostwald ripening by addition of small amounts of a hydrophobic excipient to the disperse phase. The emulsion allowed a high <i>in vitro</i> release and permeation rate of AD as well as a sufficient <i>in vivo</i> efficacy.</p><p>To achieve a plastic property, hydrophilic polymers were added to the o/w emulsion resulting in a significant reduction of the release and permeation rate of AD. In order to avoid the addition of these polymers, a semisolid w/o emulsion was evaluated with AD as the continuous phase. The inherent plastic property of this formulation allows sufficient skin adhesion. Furthermore, the release and permeation rate of AD from this formulation is comparable to that of the Newtonian submicron o/w emulsion. A close correlation between the in vitro permeation studies and the <i>in vivo</i> human plasma profiles was observed using the convolution/deconvolution</p><p>method.</p><p>Furthermore, x-ray and calorimetric data indicated that local anesthetics are able to interact with skin lipids both by increasing the chain fluidity of the crystalline lipids and by probably producing phase inversions in the grain borders of the stratum corneum lipid multilayers. It was also shown that this lipid interaction was not directly correlated with the different levels of skin permeation and/or topical efficacy of the investigated compounds.</p>

<p>Drug delivery to nasal or ocular mucosa for either local or systemic action faces many obstacles – these routes are protected by effective mechanisms. Gel formulations with suitable rheological and mucoadhesive properties increase the contact time at the site of absorption. However, drug release from the gel must be sustained if benefits are to be gained from the prolonged contact time.</p><p>The work presented here is the characterization of gels and the determination of the mucoadhesive properties of polymers using rheology. Gelrite gels were formed in simulated tear fluid at concentrations of polymer as low as 0.1%, and it was shown that sodium was the most important gel-promoting ion <i>in vivo</i>. Rheology, although it may be a questionable technique for evaluating mucoadhesive properties of polymers, showed that interactions between mucin and polymers were most likely to be seen with weak gels.</p><p>It was possible to control the release of uncharged drug substances by including surfactants that form micelles in the gel. This release depended on lipophilic interactions between the drug and the polymer and/or the micelles. Controlled-release formulations of charged drugs could be designed by mixing the drugs with oppositely charged surfactants in certain ratios. In this way, vesicles in which the drug and surfactant constituted the bilayer formed spontaneously. The vesicle formation was affected by the presence of polymer, and very small vesicles that gave a slow release rate were formed when a lipophilically modified polymer was used.</p><p>The gels were also evaluated in the Ussing chamber using porcine nasal mucosa. The rate of transport of drugs through the mucosa could be controlled by the rate of release from the formulation. Furthermore, the Ussing chamber could be used to evaluate the potential toxicity of formulations.</p>

<p>Recent advances in recombinant DNA technology have resulted in a great number of proteins with a potential to enter pharmaceutical formulations. The most commonly used method for preparing protein pharmaceuticals is by freeze-drying. Freezing is an important step in this process and therefore a deeper investigation of the freeze-thawing process is qualified.</p><p>The aim of the thesis was to investigate the protection of protein during freeze-thawing. The effects on the recovered activity of the protein by different protective additives and different temperature history were evaluated, together with the protection mechanism on a molecular level.</p><p>Lactate dehydrogenase (LDH) was used as a model protein. The systems were examined by differential scanning calorimetry (DSC and MTDSC), IR-, NMR- and fluorescence spectroscopy as well as surface tension measurements.</p><p>The additives Tween 80 and Brij 35 are non-ionic surfactants and both protected LDH during freeze-thawing in concentrations far below cmc. The non-surface active polymer PEG 6000 had a protecting ability in very low concentrations. The protection was strongly affected by the temperature history; an increased freezing rate decreased the recovered activity. The optimum protecting concentration of Tween was also dependent on the cmc. During freezing below -20ºC no liquid water or amorphous ice was detected, all water was crystallized to polycrystalline ice. The relative degree of crystallinity could be determined by MTDSC at melting but not during crystallization, since it is a very fast process.</p><p>An interaction between protein and additive is not necessarily required for protection at these low concentrations of additives. An interaction was observed between LDH and PEG but very weak or no interaction at all between LDH and the non-ionic surfactants. The protein was in all cases in the native state.</p><p>The protective mechanisms are quite complex, but the amount of ice surface created during freezing is crucial for the protection. The non-ionic surfactants are able to hinder the protein from destructive interactions with the ice crystals by competing for adsorption at the ice surface. PEG can prevent LDH from denaturation at the ice surface by adsorption of a PEG hydrate that is formed only with certain temperature history.</p>

<p>The results from this thesis leads to the following conclusions; HUM is a useful tool that fills a gap in the in vitro methods previously available to study nasal drug delivery. Using HUM, the pig respiratory nasal mucosa can obtain acceptable viability and retain it longer than the period of time needed for a transport experiment. HUM has proven to be an appropriate tool for the study of liquids in low volume, gels, both unmodified and with controlled release properties, and particle suspensions. The potential local toxicity of formulations such as controlled release gels and surfactants could be evaluated and graded using HUM. The estimation of the apparent permeability can be corrected on a mathematical basis, for substances that bind to the chamber material. As seen using HUM, unmodified gels from Carbopol 934 (C934) are well tolerated by the nasal mucosa and may consequently be suitable for nasal administration. The release rate of testostenone, dihydroalprenolol and hydrocortisone from C934 gels can be successfully sustained. Protein-conjugated starch microparticles, intended to function as a vaccine carrier system, were taken up by non-ciliated epithelial cells of the pig respiratory nasal mucosa after incubation using HUM. The concentration-dependent effects on permeability and transepithelial electrical resistance on Caco-2 cells, of a series of nonionic polyoxyethylene surfactants, correlated with surfactant structure. Similar effects were seen on pig nasal mucosa using HUM, but the nasal mucosa appeared to be more tolerant to the surfactants than the intestinal cell model.</p><p>The nasal route has advantages for several classes of drugs e.g. involved in migrain treatment, nicotine substitution therapy and mucosal vaccination. The increased development of a variety of substances, in a variety of formulation types, has increased the demand for suitable investigational tools. It is in this context that the horizontal Ussing chamber method (HUM) was developed. Using HUM, the studied formulation can be applied on the mucosa without additional buffer, giving an in vivo-like situation and the possibility to study solid and semi-solid formulations. Furthermore, the influence of gravity will not result in uneven distribution of the formulation. </p>