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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. 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. 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. 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. 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. 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.

A fast reversed phase capillary electrochromatographic method was developed for the analysis of parabens preservatives in drugs and cosmetics in the presence of their main metabolite and/or impurity 4-hydroxybenzoic acid. The separation was optimised in 75 mm i.d. capillary, fully packed with 5 mm C18 stationary phase in our laboratory with a very well experienced methodology, studying the effects of mobile phase pH and composition (buffer type and organic solvent content). The mobile phase 5 mM ammonium formate pH 3.0 containing 65% of acetonitrile allowed us to obtain the baseline separation of methyl-, ethyl-, propyl-, butyl- and benzyl- parabens in mixture in less than 2.5 min with repeatability and linearity using the short end injection method (separation capillary effective length: 8 cm). Under the optimum experimental conditions the method provided high separation efficiency for parabens, in the range of 129,312-140,325 number of theoretical plates per meter, and analytes quantitation limits (LOQs) in the range 1.25-2.50 mg/mL. The method was successfully applied to the quantitative analysis of paraben preservatives in pharmaceutical and cosmetic industrial samples with direct injection or after a much reduced sample pretreatment.

Although, pulmonary drug delivery is a well established means for targeting of drugs to the lungs for the treatment of respiratory diseases as well as for the systemic delivery of volatile anesthetic agents, drug absorption kinetics in the lung have not been subjected to extensive research. The main objective of this thesis was to investigate drug absorption characteristics of the lung barrier, using the isolated and perfused rat lung model and in vivo pharmacokinetic studies in rats. Physicochemically diverse drugs (i.e. atenolol, budesonide, cromolyn, cyanocobalamin, enalapril, enalaprilate, formoterol, imipramine, losartan, metoprolol, propranolol, talinolol, terbutaline, and the tetrapeptide TArPP) were used as model compounds. In connection to these investigations, a nebulization catheter device was successfully adapted and evaluated as a new technique for delivery of defined aerosol doses to the rat lung. In addition, a physicochemical profile of the inhaled drugs on the market worldwide during 2001 was made. The pulmonary first-order absorption rate constant and bioavailability were found to correlate to the drug lipophilicity, the molecular polar surface area, and the apparent permeability of Caco-2 cell monolayers. In contrast to the intestinal mucosa and the blood-brain barrier, the pulmonary epithelium was highly permeable to drugs with a high molecular polar surface area. Accordingly, a small hydrophilic tetrapeptide (oral bioavailability ~0.5%) showed a complete bioavailability after pulmonary delivery to rats in vivo. Regional differences in bioavailability, absorption rate, and first-pass metabolism of the peptide was demonstrated after targeted delivery to different regions of the respiratory tract in rats in vivo. The high pulmonary bioavailability of the efflux transporter substrates losartan and talinolol provides functional evidence for an insignificant role of efflux transporters such as P-glycoprotein in limiting the absorption of these drugs from the rat lung. The results of this thesis demonstrate that the lung efficiently absorbs drugs with a wide range of lipophilicity. The pulmonary route should thus be regarded as a potential alternative for administration of drugs with low oral bioavailability. In addition, drug inhalation present an opportunity to attain a more rapid onset of drug action than can be attained by the oral route.

Nasal delivery is a feasible alternative to oral or parenteral administration for some drugs because of the high permeability of the nasal epithelium, rapid drug absorption across this membrane and avoidance of hepatic first-pass metabolism. The main objective of this thesis was to investigate factors influencing the permeability of the nasal mucosa to various compounds and to evaluate implications for drug absorption via the nasal route. Porcine nasal mucosa mounted in an Ussing chamber system was established as an in vitro model, and glucose, insulin, lidocaine, mannitol, melagatran, nicotine, PEG 4000, propranolol, sumatriptan, verapamil, vinblastine and an aminodiether were used as model compounds. The pharmacokinetics of melagatran and propiomazine were investigated in absorption studies in rats, and the influence of the enhancers SDS and EDTA on melagatran absorption was evaluated and compared with in vitro permeability data. The expression of P-glycoprotein in porcine nasal mucosa was investigated and compared with that in human nasal epithelial biopsies using the Western Blot technique. The results demonstrated that the Ussing chamber model using porcine nasal mucosa has potential as a tool for evaluating mechanisms of nasal absorption and predicting the in vivo effects of absorption enhancers. Moreover, porcine nasal mucosa is comparable to human nasal mucosa in its morphology and P-glycoprotein expression. The in vitro permeability data were found to weakly correlate with literature data on human absorption after nasal administration of the corresponding compounds. In vivo absorption studies of the sedative propiomazine demonstrated that nasal administration of this drug offers an interesting alternative to the oral formulation currently on the market, since the absorption was rapid and the bioavailability was promising. The bioavailability of melagatran in rats was moderate but variable, and responded to the addition of enhancers. Finally, the establishment and characterisation of an in vitro method for prediction of nasal drug absorption, and the investigation of factors influencing nasal membrane permeability and absorption offer substantial contributions for nasal drug delivery.

Similar policies may be found in different countries. This, in turn, is an indication that policies travel. But why are certain policies imitated while others are not? The author seeks to further our understanding of policy diffusion and its dynamics by investigating how foreign ideas are integrated and translated into new contexts, and how these imported ideas influence national policymaking and implementation. The National Drug Policies in Laos and Vietnam are studied in order to identify factors facilitating or restraining policy processes—and to discuss the effects of diffusion. In the 1980s reforms were initiated leading to far-reaching economic liberalisation in Laos and Vietnam. These reforms have affected all segments of society, including the health sector. The pharmaceutical sector was privatised, drugs became widely available, and self-medication as well as fake and sub-standard drugs proliferated. This was the setting when the Lao and Vietnamese governments had to consider the development of a National Drug Policy, as part of the development assistance from Swedish Sida to their health sectors. In 1993 the National Drug Policy was endorsed in Laos, and in 1996 the policy was adopted in Vietnam. Hence, the policy processes were initiated within the framework of bilateral cooperation between donors and the two Ministries of Health. In other words, there had been a spread of external ideas, or policy diffusion. Later on these ideas and NDP had to be translated into, and implemented in, the national context. Thus, the study covers the time from the development of the policies until present the time, 2001. Drawing on research in policy studies, constructivism, new institutionalism, and globalisation, the study aims to bridge the international/national divide in policymaking both theoretically and empirically. In an era of escalating globalisation and ever increasing flows of ideas, the author argues for the contextualisation of policymaking and problematisation of the often taken for granted rational actor approach inherent in much of the policy literature. The main findings indicate that although the formulation of the policies may be relatively trouble-free, it does not automatically mean that the translation of the policy into practice will follow as anticipated. In order to understand why new ideas are accepted and integrated into a new context and how this affects the implementation, one must look at the way new policies have been spread and translated into a new context. This, in turn, can provide insights as to why some policies are more successful than others, and why the policy process in Laos appears to have been more straightforward than in Vietnam. Hur kommer det sig att liknande reformprogram återfinns i olika länder? Varför väljs vissa idéer framför andra? Avhandlingen syftar till att öka förståelsen av så kallad policydiffusion, spridning av olika typer av policy, och den dynamik som omgärdar spridningen. Detta görs genom att undersöka hur externa, eller utländska, idéer integreras och översätts i en ny omgivning. På vilket sätt påverkar de nya idéerna det nationella beslutsfattandet och påföljande policyimplementering? Två läkemedelspolicies, en i Laos och en i Vietnam, studeras mer i detalj för att på så sätt identifiera faktorer som antingen underlättar eller försvårar policyprocessen. Även effekterna av policydiffusion på ett mer generellt plan diskuteras. Bakgrunden till att Laos och Vietnam antog nya läkemedelspolicies var bland annat konsekvenserna av den ekonomiska liberalisering som initierades i mitten av 1980-talet. Även hälsosektorn inkluderades i reformerna, och framför allt läkemedelsektorn privatiserades. Detta ledde i sin tur till att tillgången på läkemedel ökade samtidigt som självmedicinering och undermåliga läkemedel blev allt vanligare. Situationen var följaktligen närmast kaotisk när de laotiska och vietnamesiska regeringarna fick ta ställning till om man skulle utveckla ett handlingsprogram inom läkemedelssektorn som en del av det svenska biståndet. År 1993 antogs den laotiska läkemedelspolicyn och 1996 den vietnamesiska. Studien omfattar med andra ord en tioårsperiod fram till år 2001. Det intressanta i sammanhanget är att det tycks som om implementeringen har fungerat smidigare i Laos än i Vietnam. Detta trots att Vietnam har bättre förutsättningar eftersom hälsoindikatorer och utbildningsnivå är högre än i Laos. I debatten om läkemedel, och sjuk- och hälsovård ställs ofta kostnadseffektiva aspekter i fokus på bekostnad av de politiska. Man glömmer bort politiska processer och politiska möjligheter att implementera de riktlinjer man beslutar om. Situationen blir extra känslig då internationella överenskommelser och aktörer kommer med i bilden. Tanken är med andra ord att koppla samman internationella beslutsprocesser med nationella genom att se hur idéer och reformprogram sprids och hur dessa program förverkligas. Tidigare studier har en tendens att antingen fokusera på relationer mellan stater eller på hur reformprogram genomförs i enskilda länder. Detta leder i sin tur till att man förbiser viktiga förklaringsfaktorer till varför vissa program i utvecklingsländer är framgångsrika medan andra inte är det. Idag sträcker sig kontakter och samarbeten över sakområden, hierarkier och maktsfärer lika mycket som över nationella gränser. Stater, organisationer och individer ingår detta samarbete, som dessutom leder till en snabbare spridning av idéer och reformer. För att studera ovan nämnda problematik används ett teoretiskt ramverk baserat på forskning inom policystudier, konstruktivism, nyinstitutionalism samt globalisering. Syftet är att studien genom detta ramverk ska kunna överbrygga åtskillnaden mellan den internationella och den nationella sfären såväl teoretiskt som empiriskt. Detta är särskilt viktigt i en tid av ökande globalisering där idéer i allt högre grad och hastighet sprids över världen. I studien understryks vikten av att kontextualisera policyprocessen och att problematisera den målmedel rationalitet som så ofta präglar policylitteraturen. Resultaten pekar på att trots att det kan vara relativt smärtfritt att utveckla och anta en ny policy så bör man vara försiktig med att anta att ett framgångsrikt genomförande automatiskt blir följden. Anledningen till detta går inte endast att finna i resursbrist eller dålig planering. Även hur policyn i fråga har spridits och översatts till en ny omgivning spelar avgörande roll för hur väl den kommer att integreras och faktiskt göra någon skillnad i praktiken. Studien är ett resultat av ett samarbete mellan Statsvetenskapliga institutionen vid Lunds universitet och IHCAR ("Division of International Health"), Karolinska institutet, i Stockholm.

To assess whether molecular oxygen and oxidative stress contribute to chloroquine activity, we cultivated strains of Plasmodium falciparum in erythrocytes with carboxyhemoglobin and an atmosphere containing 2% CO, 5% CO2 and 93% N2. Results indicate that, contrary to common belief, oxygen is not involved in the activity of chloroquine. Reactive radicals formation is suggested.

Aims: 1) To assess if drug sales data indicate differences in morbidity, mortality, socio-economic conditions and deviating prescribing habits among physicians; 2) to identify areas for educational interventions and to analyse changes in prescribing after educational activities. In addition, an effort was made to estimate differences between pharmacy sales and purchases by the population, and the amount and value of unused drugs returned to pharmacies. Method: Information on drug sales was gathered from three Swedish and one Nordic drug registers, namely, Sales Statistics on Drugs, Nordic Statistics on Medicines, The National Prescription Survey and The Diagnosis-Prescription Survey, as well as from local prescription studies, copies of prescriptions processed at pharmacies, copies of issued prescriptions and computerised patient records at a health care centre. Results: There was a significant correlation between sales of tranquillizers and hypnotics/sedatives, on the one hand, and mortality, suicides and UnderPrivileged Area score, on the other, in the 33 municipalities of Skåne County. Benzodiazepine sales in the city of Helsingborg were the highest in the country and higher than the national average to all age groups and both genders. A minority of the physicians, Det finns behov att studera läkemedelsförskrivning och läkemedelsanvändning och dessas samband med och effekter på hälsa, sjuklighet och överdödlighet. Det är också önskvärt att kunna använda individdata. I Sverige är det i allmänhet inte tillåtet att använda individdata avseende läkemedelsanvändning. Det finns däremot god tillgång på befolknings- och områdesbaserade försäljningsdata av god kvalitet. Syftet med denna studie har varit att undersöka om sådana aggregerade data samvarierar med sjuklighet i befolkningen, att identifiera områden där det finns informations- och utbildningsbehov och att bedöma effekterna av informations- och utbildningsinsatser. Avsikten har också varit att mäta skillnader mellan de lokala apotekens försäljning och den lokala befolkningens inköp av läkemedel mot recept samt att undersöka hur mycket oanvända läkemedel som återlämnas till apoteken för destruktion. Uppgifterna om den lokala läkemedelsförsäljningen har hämtats från apotekens inleveransstatistik, Receptundersökningen samt lokala förskrivningsstudier. Kompletterande information har inhämtats från kopior av expedierade recept på apotek, kopior av utfärdade recept på vårdcentraler samt ur journaldata på vårdcentral. Dessutom har nationella data om läkemedelsförsäljningen hämtats från apotekens inleveransstatistik och Receptundersökningen samt uppgifter för de nordiska länderna från Nordisk läkemedelsstatistik. Nationella data om förskrivningen har hämtats från Diagnos-Recept-undersökningen. En undersökning har dessutom genomförts om hur mycket läkemedel som under två veckor återlämnades till apoteken i Skåne för destruktion. Den lokala läkemedelsstatistiken visar stora skillnader i försäljningen av läkemedel mellan kommuner inom samma län. Resultaten från studierna visar att läkemedelsförsäljningen lokalt samvarierar med och indikerar skillnader mellan kommunerna i socioekonomisk nivå, sjuklighet och överdödlighet. Informationen kan också användas för att identifiera områden där det kan vara motiverat med informations- och utbildningsinsatser för att främja en rationell läkemedelsförskrivning. Lokala data om läkemedelsförsäljningen lämpar sig väl för att bedöma effekterna av informations- och utbildningsinsatser. Apotekens försäljning av läkemedel lokalt motsvarar dock inte helt den lokala befolkningens läkemedelsinköp eller användning och inte heller receptförskrivningen lokalt. Resultaten i de studier om läkemedelsanvändning som baseras på de lokala apotekens försäljning (t.ex. studier som gjorts före 1997) kan vara missvisande och kan därför behöva revideras. Försäljningsdata i nuvarande form räcker inte heller för att identifiera vilka målgrupper som informations- och utbildningsaktiviteterna skall riktas till, utan behöver kompletteras med andra informationskällor om receptutfärdare och arbetsplats. Receptundersökningens material kan emellertid komma att få en sådan komplettering, om förslaget i regeringens proposition 2001/02:63; §6 genomförs, dvs. att arbetsplatskod krävs på recepten för att läkemedlet skall ingå i läkemedelsförmånen. Lokala försäljningsdata, med ökad kvalitet och tillgänglighet, är i många avseenden ett användbart och kostnadseffektivt instrument för att, i kombination med andra tillgängliga data, analysera förskrivning och användning av läkemedel.

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. 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.

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. 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. 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. 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.

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. 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 in vivo. 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. 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. 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.

The reversible nature of the direct esterification of acids with alcohols limits the use of this process in the biocatalitic kinetic resolution of chiral acids. A new irreversible procedure, using orthoesters, have been developed.

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. 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 in vitro release and permeation rate of AD as well as a sufficient in vivo efficacy. 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 in vivo human plasma profiles was observed using the convolution/deconvolution method. 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.

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. 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. 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.

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. 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. 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. 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.

The nasal administration of drugs offers advantages over administration by intravenous injection. Drugs can be rapidly absorbed through the nasal mucosa, resulting in a rapid onset of action, and also avoiding degradation in the gastrointestinal tract and first-pass metabolism in the liver. The olfactory receptor cells, which are in direct contact with both the environment and the central nervous system (CNS), are potential routes for drugs into the CNS. The olfactory pathway thus circumvents the blood brain barrier (BBB) which prevents many systemically administered drugs from entering the brain. The studies used compounds active in the CNS and the experiments were performed in rodents. The nasal bioavailability of (S)-UH-301, NXX-066 and [3H]-dopamine was investigated in a rat model; uptake into the cerebrospinal fluid (CSF) was compared after nasal and intravenous administration. The concentrations of S-UH-301 and NXX-066 in plasma and CSF were measured with high performance liquid chromatography. The possible transfer of dopamine and neurotensin along the olfactory pathway after nasal administration to mice was studied using brain tissue sampling and autoradiography. The radioactivity content in blood, CSF and dissected brain tissue samples after administration of [3H]-dopamine and [3H]-neurotensin was assessed using liquid scintillation, and thin layer chromatography (TLC) was used to investigate the metabolic fate of [3H]-dopamine. The results of this thesis suggest that nasal administration of CNS-active compounds with low oral bioavailability is an interesting and workable alternative to intravenous injection. The small lipophilic compounds (S)-UH-301 and NXX-066 were rapidly and completely absorbed after nasal administration, although hard evidence of direct transfer from the nose remains elusive. Radioactivity measurements in the olfactory bulb following nasal administration of [3H]-dopamine and [3H]-neurotensin indicate that transfer occurred. The TLC results showed the presence of unchanged dopamine in the olfactory bulb but it is less clear from initial results with neurotensin, which radioactive products of this molecule reached the olfactory bulb, and further studies are required.

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. 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. 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. 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.

In this thesis, the strength-enhancing mechanisms of dry binders in direct compression were studied. The systems investigated were binary mixtures containing various compounds and binders. Among the binders used were a series of different molecular weights of polyethylene glycol. The proposed simplified tablet model describing the fracture path in a tablet during strength testing offers an explanation for the increase in tablet strength caused by the binder. The model and results in this thesis indicate that fractures will usually propagate around the tablet particles and through the interparticulate voids during tablet strength testing. One important characteristic of the binder is its ability to be effectively and evenly distributed through the interparticulate voids in a compound tablet. Characteristics such as high plasticity, low elasticity and a small particle size were associated with a more even distribution and a consequent pronounced effect on pore structure and marked improvement in tablet strength. The strength of tablets containing less plastic binders was governed more by the compactibility of the binder. The tablet porosity, bonding mechanisms and volume reduction mechanisms of the compound also influenced the effect of the binder. For example, the plasticity and particle size of the binder had the most significant effects on tablet strength when the tablet porosity of the com-pound was relatively low. A combination of the plasticity and the compactibility of the binder determined the strength of tablets when the tablet of a compound was more porous. The positive effect of a binder on pore structure and tablet strength resulted in an increase in the disintegration time. Although addition of a superdisintegrant generally improved the disintegration time, the effect was decreased when the formulation included more deformable binders. The choice of a suitable binder for a tablet formulation requires extensive knowledge of the relative importance of binder properties for enhancing the strength of the tablet and also of the interactions between the various materials constituting a tablet. Thus, the increased knowledge of the functionality of a binder obtained in this thesis enables a more rational approach to tablet formulation.

In this thesis, factors important in tableting operations and for tablet properties have been studied and characterised by different spectroscopic techniques as well as by some more conventionally used particle characterisation techniques. The spectroscopic techniques solid-state NMR, FT-IR and NIR spectroscopy, proved to be valuable tools in the estimation of particle and tablet properties, offering both specificity and sensitivity in the measurements. Because of the large amount of information obtained in a spectrum, multivariate data analysis was in some cases used in the processing of the spectral data. Correlations between the solid state structure measured by spectroscopy and the particle and tablet properties could be obtained including useful prediction models. The surface area obtained using different principles has in this thesis been shown to reflect different properties and tableting behaviour of a collection of pharmaceutical materials. The particle shape and the external surface area of the powders measured by permeametry, were found to be important factors for the tensile strength of tablets made of hydroxypropyl methylcellulose. Furthermore, the external surface area could be used to access dominating interparticulate bonding mechanisms in compacts of different materials by normalising the tablet tensile strength for the tablet surface area. It was also shown that for materials prone to develop solid bridges, the actual surface area participating in the bonding was more important than the average interparticulate distance. When studying the properties of microcrystalline cellulose and cellulose powder from the alga Cladophora sp., the cellulose fibril surface area estimated by solid-state NMR resulted in better correlations to the tableting behaviour and to tablet disintegration than the external permeametric surface area did. It was suggested that the difference in fibril surface area of the two celluloses was the primary factor responsible for properties like the crystallinity and the disintegration of the tablets.

The aim of this investigation has been to study mechanisms by which glucocorticoids inhibit inflammatory processes in vitro. Type IV cytosolic 85 kDa phospholipase A2 (cPLA2) is an important modulator of inflammatory responses that is present in macrophages as well as in other cell types. In macrophages, the synthetic glucocorticoid dexamethasone (dex) was found to reduce the expression of cPLA2 and further to inhibit the activation of cPLA2. Stimulation with phorbol 12-myristate 13-acetate (PMA), zymosan or the protein phosphatase inhibitor okadaic acid increased the cPLA2 activity. After treatment with dex, okadaic acid, but not PMA or zymosan, was able to up-regulate cPLA2 activity. These results indicate that the effect of okadaic acid was exerted at, or downstream of, the dex-sensitive step(s). Phosphorylation of cPLA2 occurred on multiple sites. Upon stimulation with PMA or bacteria, the most C-terminal fragment (residues 698-749) of cPLA2 was the most heavily phosphorylated. We did not find any evidence for down-regulation of protein kinase C isoforms after dex treatment. Neither was the expression, nor the activation of, the mitogen-activated protein kinases (MAP kinases), extracellular signal- regulated kinase-2 (ERK-2) or p38, affected to the same extent as the cPLA2 activity. Although a minor inhibition of the zymosan-induced activation of the MAP kinases was observed, our results suggest that dex-mediated inhibition of cPLA2 activation is exerted down-stream of the MAP kinases. The effect of dex on the formation of two other inflammatory mediators in macrophages, the cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha) was also investigated. Treatment with dex inhibited bacteria- induced IL-1beta expression primarily at the transcriptional level whereas TNF-alpha expression was only partially inhibited at the level of transcription but further inhibited at the translational level. The translational inhibition after dex treatment was overcome by okadaic acid. Also the post-translational processing of TNF-alpha was modified by okadaic acid. TNF-alpha was found both in the cleaved and uncleaved form after okadaic acid treatment. The different TNF-alpha forms exhibited different sensitivity to dex treatment. Based on these data, we suggest that the effect of dex on TNF-alpha translation is mediated by an okadaic acid-sensitive protein phosphatase. In conclusion, dex inhibits the synthesis of cPLA2. Studies with okadaic acid revealed protein phosphatases to be likely targets for dex both in the inhibition of cPLA2 activation and in inhibition of TNF-alpha translation. En studie av hur glokokortikoider hämmar inflammation Glukokortikosteroider, varav kortison är den mest kända, är hormoner som reglerar många funktioner i kroppen och även används som läkemedel för att dämpa inflammatoriska tillstånd som t.ex. astma och reumatiska sjukdomar. Glukokortikosteroider är mycket potenta läkemedel och används flitigt med mycket goda effekter. Vid långvarigt systemiskt bruk av höga doser förekommer tyvärr biverkningar. Det är därför av stort intresse att klargöra glukokortikosteroidernas verkningsmekanismer för att utifrån dessa fakta kunna framställa mer specifika läkemedel med färre biverkningar. I denna studie har vi använt dexametason som är en syntetisk glukokortikosteroid. Effekterna av dexametason har studerats på celler som kallas makrofager. Makrofager är en sorts vita blodkroppar som deltar i kroppens försvar mot främmande organismer. Vid aktivering av makrofager är fosfolipas A2 det enzym som reglerar hastigheten med vilken olika inflammationssubstanser såsom prostaglandiner och leukotriener bildas. Cytokiner är ett annat exempel på substanser som bildas och frisätts av makrofager och medverkar vid inflammationer. I den första studien fann vi att dexametason hämmar både bildningen av proteinet fosfolipas A2 (cPLA2) och dess aktivitet. Många proteiners aktivitet regleras genom fosforylering (ett s.k. kinas kopplar på en fosfatgrupp på en aminosyra) respektive defosforylering (borttagning av fosfatgrupp som utförs av fosfatas). Vi fann att vi genom att tillsätta en substans som hämmar fosfataser (okadainsyra) kan upphäva effekten av dexametason. Detta indikerar att hämningen av cPLA2 aktiviteten eventuellt sker genom induktion av ett fosfatas. Vi har också arbetat med att kartlägga vilka aminosyror det är som fosforyleras vid aktivering av cPLA2. Tre olika kinaser har tidigare föreslagits fosforylera cPLA2. Våra data ger inte något stöd för att dexametason skulle hämma något av dessa i samma utsträckning som cPLA2 hämmas. Dex påverkar också cytokiner. Vi har valt att studera främst interleukin-1 och tumour necrosis factor vilka är två cytokiner med stor betydelse vid inflammatoriska tillstånd. Regleringen av cytokiner är viktig eftersom de behövs för att kroppen ska kunna bekämpa infektioner och även cancer, men överaktivering av cytokiner kan ha dödlig utgång. Vi fann att dexametason hämmar bildningen av dessa cytokiner. De båda cytokinerna hämmas under olika betingelser och i olika steg i processen som leder fram till det färdiga proteinet. Även för regleringen av cytokiner med dexametason fann vi att fosfataser är av stor betydelse. Sammanfattningsvis hämmas långt ifrån alla proteiner som är involverade vid inflammationer av dexametason. Dock utövade glukokortikosteroiderna en kraftfullt hämmande effekt på flera olika viktiga proteiner varav ett är cPLA2. En sådan komplex verkan är med all sannolikhet av stor betydelse för finjusteringen av balansen i kroppens immunsvar.