uu.seUppsala University Publications
Change search
Refine search result
1 - 26 of 26
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Rows per page
  • 5
  • 10
  • 20
  • 50
  • 100
  • 250
Sort
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
  • Standard (Relevance)
  • Author A-Ö
  • Author Ö-A
  • Title A-Ö
  • Title Ö-A
  • Publication type A-Ö
  • Publication type Ö-A
  • Issued (Oldest first)
  • Issued (Newest first)
  • Created (Oldest first)
  • Created (Newest first)
  • Last updated (Oldest first)
  • Last updated (Newest first)
  • Disputation date (earliest first)
  • Disputation date (latest first)
Select
The maximal number of hits you can export is 250. When you want to export more records please use the Create feeds function.
  • 1.
    Alterman, Mathias
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Design and synthesis of HIV-1 protease inhibitors2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immune Deficiency Syndrome (AIDS). The C2-symmetric HIV-1 protease is one of the prime targets for chemotherapy in the treatment of the HIV infection. Inhibition of HIV-1 protease leads to immature and non-infectious viral particles. Design and synthesis of a number of C2-symmetrical C-terminal duplicated HIV-1 protease inhibitors and subsequent biological evaluation is presented in this thesis.

    A versatile three step synthetic route has been developed using a carbohydrate as an inexpensive chiral starting material thus allowing inhibitors with the desired stereochemistry to be obtained. By this efficient method a series of tailor-made P2/P2' modified inhibitors were synthesized, and these were evaluated on purified HIV-1 protease and in HIV-1 infected cell assays. Highly active HIV-1 protease inhibitors were identified among the tested compounds. Analyses of the X-ray crystal structures of two of the most active compounds, as complexes with the protease, guided the further design of P1/P1' elongated inhibitors. Substitutions in the para-position of the P1/P1' benzyl groups were promoted efficiently by microwave-irradiated of palladium-catalyzed reactions. Particular modifications in the P1/P1' region of the inhibitors resulted in a 40-fold increase of the anti-viral activity on HIV-1 infected cells. Furthermore, a fast, efficient, and general one-pot microwave enhanced synthesis protocol for transformations of organo-bromides to tetrazoles was developed and applied on the inhibitor scaffold. Attachment of linker molecules to the P1/P1' benzyl groups of one inhibitor was used to develop of sensitivity enhancer tools in surface plasmon resonance biosensor assays. These new assays enable the evaluation of low-molecular weight compounds as HIV-1 protease inhibitors.

  • 2.
    Amini, Ahmad
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Enantiomeric separation by capillary electrophoresis: With special emphasis on the partial filling technique1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Capillary electrophoresis as a powerful separation technique was employed for resolution of racemic drugs. Taurodeoxycholate (TDC), different cyclodextrins and α1- acid glycoprotein (AGP) were dissolved in the background electrolyte at low pH (3.0 to 4.0 ) as chiral selectors.

    Enantioresolutions were based on the principle of micellar electrokinetic chromatography, inclusion complexation and affinity interactions between the enantiomers and TDC, cyclodextrins and AGP, respectively.

    The influence of chiral selector concentration and type, temperature and addition of organic modifiers on enantioselectivity were studied. The temperature was found to be an important factor for regulation of the enantioresolution.

    In order to avoid UV-absorbance interferences between AGP as a UV-absorbing chiral selector as well as to reduce consumption of chiral selector solution, the partial filling technique (PFT) was employed. The technique was developed and equations describing the feature of the technique as an efficient separation mode in CE was presented. Parameters such as applied plug length (PLapp) and effective plug length (PLeff) were determined. Equations expressing the relation between the PLeff and selectivity and resolution factors were developed.

    The PFT was used for determination of association constants between AGP as chiral selector and the enantiomers of disopyramide and remoxipride. The association was strongly temperature dependent, and a maximum in binding was observed at 25°C. The PFT was found to be a very convenient approach for measurement of association constants.

    To verity the above mentioned technique for evaluation of binding constants, the association constants between methyl-β-cyclodextrin and the enantiomers of orciprenaline at different chiral selector concentrations were measured and compared with the data obtained by the conventional technique. The results illustrate the reliability of the system based on the PFT.

  • 3.
    Bergh, Margareta
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Allergenic oxidation products in ethoxylated non-ionic surfactants: Chemical characterization and studies on allergenic activity1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The susceptibility of ethoxylated non-ionic surfactants to oxidation on exposure to air was investigated. Surfactants have a complex chemical composition and are widely used in a variety of applications for to their amphiphilic properties. Ethoxylated non-ionic surfactants are regarded by the producers to be stable at normal handling. However, ethoxylated surfactants are polyethers and as such susceptible to oxidation on exposure to air, which is theoretically discussed in literature. Hand eczema is a common occupational disease of which mom than half of the cases is considered to be caused by work with surfactants and water. Surfactants are known to cause skin irritation, but cases of allergic contact dermatitis have also been reported.

    Some ethoxylated surfactants were handled at room temperature in a manner that imitated ordinary handling at work. All samples autoxidized and the structures of some of the components formed were identified with liquid chromatography (HPLC) and gas chromatography (GC). Structural analogues were synthesized and could be identified in the complex oxidation mixture using nuclear magnetic resonance, infrared and mass spectroscopy (NMR, FT-IR, MS). The allergenic potential of the identified oxidationproducts was studied in experimental sensitization studies in guinea pigs. The physicochemical behavior of individual oxidation products in pure water solutions was investigated.

    A well-known contact allergen, i.e. formaldehyde, and previously undescribed allergenic compounds were identified after air exposure of the ethoxylated surfactants. At oxidation, the content of free formaldehyde increased above the level for warning labelling within the European Union. The allergenic potential of one of the main compounds identified, an ethoxylated aldehyde, was of similar magnitude as that of formaldehyde. The oxidation led to a gradual change in the physicochemical properties.

    The observed autoxidation of commonly used ethoxylated surfactants and formation of a complex degradation mixture indicate a potential health risk that has not been considered before. Previously unknown breakdown/oxidation products were identified, some of which had allergenic properties. This oxidation can also affect the technical performance of the surfactants. The observed allergenic properties of the formed oxidation products may be of importance in occupational exposure to polyoxyethylene surfactants.

  • 4.
    Berts, Wei
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Syntheses of functionalized Phe-Gly dipeptidomimetics1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The complete series of Boc-protected C-4 mono- and difluoroallyl Phe-Gly dipeptidomimetics and their corresponding saturated derivatives has been synthesized. A mechanism that rationalizes the formation of the two isomeric allylic mono-fluorides from an allylic alcohol with diethylaminosulfur trifluoride (DAST) has been investigated. Most likely, the reaction proceeds via an aziridine intermediate. An allylic ketone isostere wasused as starting material for all the fluorination transformations. Two synthetic approaches to this ketone were developed.

    A series of vicinal diamines was synthesized by reactions of allylic cis- and transaziridines with nucleophilic amines. The nucleophiles reacted either via ring opening or 1,4-addition reactions with the α,β-unsaturated aziridines. The 1,4-addition reaction was found to be favoured in most cases. To improve the yield of ring opening products, various reaction conditions were examined. The Lewis acids Yb(OTf)3 and Zn(OTf)2 were chosen as the reaction catalysts since they are believed to coordinate differently to acylaziridines. It was observed that addition of these Lewis acids did not influence the outcome of the reaction.

    Five Boc-protected Phe-Gly and Phe-Phe vinyl isosteres were synthesized using a Julia olefination reaction. The epoxidation of the vinyl isosteres were performed with three different peracids and VO(acac)2/TBHP. The stereoselectivities observed in the peracid epoxidation may emanate from a cooperative coordination of the incoming peracid by the allylic carbamate group and more weakly coordinating allylic methyl ester, alcohol,and acetate functions. The vanadium catalyzed reactions demonstrated that the face selectivity in the epoxidation of the double bond of the Phe-Gly and Phe-Phe vinyl isosteres is only dependent of the steric interference.

  • 5.
    Elversson, Jessica
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Spray-Dried Powders for Inhalation: Particle Formation and Formulation Concepts2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Spray drying is a method with a high potential in the preparation of protein particles suitable for pulmonary delivery. However, surface induced denaturation of bio-molecules during atomization and subsequent drying can be substantial and it is therefore important to develop new formulation concept for concurrent encapsulation and stabilization of proteins during spray drying. Hence, with an overall objective to increase the knowledge of the formation of particulate systems for systemic administration of proteins by spray drying, the first part of this thesis, systematically investigated the particle formation by droplet size and particle size measurements. It was described how specific properties, such as the solubility and the crystallization propensity of the solute, can affect the product, e.g. the particle size, internal structures, and possibly particle density. A new method using atomic force microscopy (AFM) for the assessment of the effective particle density of individual spray-dried particles was demonstrated. In the second part, two different formulation concepts for encapsulation of protein during spray drying were developed. Both systems used non-ionic polymers for competitive adsorption and displacement of protein from the air/water interface during spray drying. The aqueous two-phase system (ATPS) of polyvinyl alcohol (PVA) and dextran, and the surface-active polymers, hydroxypropyl methylcellulose (HPMC) and triblock co-polymer (poloxamer 188) used for in situ coating, proved efficient in encapsulation of a model protein, bovine serum albumin (BSA). Inclusion of polymeric materials in a carbohydrate matrix also influenced several particle properties, such as the particle shape and the surface morphology, and was caused by changes in the chemical composition of the particle surface and possibly the surface rheology. In addition, powder performance of pharmaceutical relevance, such as dissolution and flowability, were affected.

    List of papers
    1. Droplet and particle size relationship and shell thickness of inhalable lactose particles during spray drying
    Open this publication in new window or tab >>Droplet and particle size relationship and shell thickness of inhalable lactose particles during spray drying
    2003 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 92, no 4, p. 900-910Article in journal (Refereed) Published
    Abstract [en]

    To find means of controlling the size and density of particles intended for inhalation the relationship between droplet and particle size during spray drying was investigated. Lactose solutions were atomized with a two-fluid nozzle and dried in a laboratory spray drier. The effects of nozzle orifice diameter, atomization airflow and feed concentration on droplet and particle size were examined. Mass median diameter of both droplets and particles were analyzed with laser diffraction. In addition, scanning electron microscopy and transmission electron microscopy were used for studies of particle shape and morphology. It was demonstrated that nozzle orifice diameter and airflow, but not feed concentration controlled the droplet size during atomization. Increasing droplet size increased particle size but the effect was also influenced by feed concentration. Particles from solutions of a low concentration (1% w/w) were smaller than those from higher concentrations (5-20% w/w). This may be partly explained by lower yields at higher feed concentrations, but may also be related to differences in drying rate. Spray-dried lactose solutions formed hollow particles, and it was suggested that the shell thickness of the particles increased with increasing feed concentration.

    Place, publisher, year, edition, pages
    Wiley, 2003
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-93362 (URN)10.1002/jps.10352 (DOI)
    Available from: 2005-09-02 Created: 2005-09-02 Last updated: 2018-01-13Bibliographically approved
    2. Particle size and density in spray drying: effects of carbohydrate properties
    Open this publication in new window or tab >>Particle size and density in spray drying: effects of carbohydrate properties
    2005 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 94, no 9, p. 2049-2060Article in journal (Refereed) Published
    Abstract [en]

    The purpose of this study was to examine some fundamental aspects of the particle formation during spray drying, related to particle size and density. Particles were prepared in a laboratory spray dryer from carbohydrates with different solubility and crystallization propensity, such as lactose, mannitol, and sucrose/dextran 4:1. The feed concentrations ranged from 1% w/w to saturated and the size of droplets and particles were measured by laser diffraction. Particles were also characterized by various microscopy techniques (i.e., scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and light microscopy), differential scanning calorimetry (DSC), gas adsorption, and gas pycnometry. As demonstrated larger particles could be obtained by either increasing the droplet size during atomization; increasing the concentration of the feed solution; or decreasing the solubility of the solute. The apparent particle density, measured by gas pycnometry, was found negatively correlated to the feed concentration. Due to the nonlinear relationship between the feed concentration and the particle size, it was concluded that higher solids load would cause an increase in the effective particle density and that the reduction in the apparent particle density was a result of a gradually less permeable particle surface. Further, the crystallization propensity of the carbohydrate influenced the particle formation and resulted in either hollow or porous particles.

    Place, publisher, year, edition, pages
    Wiley, 2005
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-93363 (URN)10.1002/jps.20418 (DOI)
    Available from: 2005-09-02 Created: 2005-09-02 Last updated: 2018-01-13Bibliographically approved
    3. An atomic force microscopy approach for assessment of particle density applied to single spray-dried carbohydrate particles
    Open this publication in new window or tab >>An atomic force microscopy approach for assessment of particle density applied to single spray-dried carbohydrate particles
    2007 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 96, no 4, p. 905-912Article in journal (Refereed) Published
    Abstract [en]

    To evaluate an atomic force microscopy (AFM) approach for effective density analysis of single spray dried carbohydrate particles in order to investigate the internal structure of the particles. In addition, the AFM method was compared to an established technique, that is gas pycnometry. Resonant frequency AFM analysis was employed for determination of the mass of individual particles of spray-dried lactose, mannitol, and a mixture of sucrose/dextran (4:1). The effective particle density was calculated using the diameter of the spherical particles obtained from light microscopy. The apparent particle density was further analyzed with gas pycnometry. It was observed by microscopy that particles appeared either solid or hollow. A solid appearance applied to an effective particle density close to the true density of the material, whereas a density around 1 g/cm3 corresponded to a hollow appearance. However, carbohydrates, which crystallized during spray drying, for example, mannitol appeared solid but the average effective particle density was 0.95 g/cm3, indicating a continuous but porous structure. AFM measurements of effective particle density corroborate the suggestion of differences in particle structure caused by the varying propensity of carbohydrates to crystallize during spray drying, resulting in mainly either amorphous hollow or crystalline porous particles.

    Keywords
    Amorphous, Atomic force microscopy (AFM), Crystallization, Gas pycnometry, Microparticles, Morphology, Particle density, Spray drying
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-93364 (URN)10.1002/jps.20795 (DOI)000245426900016 ()17094123 (PubMedID)
    Available from: 2005-09-02 Created: 2005-09-02 Last updated: 2018-01-13Bibliographically approved
    4. Aqueous two-phase systems as a formulation concept for spray-dried protein
    Open this publication in new window or tab >>Aqueous two-phase systems as a formulation concept for spray-dried protein
    2005 (English)In: International Journal of Pharmaceutics, Vol. 294, no 1-2, p. 73-87Article in journal (Refereed) Published
    Abstract [en]

    This study investigates to what extent an aqueous two-phase system (ATPS) can encapsulate and protect the secondary structure of a protein during spray drying. The ATPSs contained polyvinyl alcohol (PVA) and dextran solutions, in different proportions. A model protein, bovine serum albumin (BSA) and, in some experiments, trehalose were added to the ATPS prior to spray drying. Electron spectroscopy for chemical analysis (ESCA), differential scanning calorimetry (DSC), UV spectrophotometry, size exclusion high-performance liquid chromatography (SEC-HPLC) and Fourier transform infrared spectroscopy (FTIR) were used for analysis of solid and reconstituted samples. The anticipated function of the ATPS was to improve the stability of the protein by preventing interactions with the air–liquid interface during drying and by improving the encapsulation of the protein in the dried powder. BSA was found to preferentially partition to the dextran phase and in the absence of PVA, BSA dominated the powder surface. In samples containing PVA, the polymer mainly covered the powder surface, even though the dextran-rich phase was continuous, thus preventing protein surface interactions and providing improved encapsulation. However, PVA was found to cause partial loss of the native structure of BSA although the protein was well encapsulated during spray drying.

    Place, publisher, year, edition, pages
    Elsevier, 2005
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-93365 (URN)10.1016/j.ijpharm.2005.01.015 (DOI)
    Available from: 2005-09-02 Created: 2005-09-02 Last updated: 2018-01-13Bibliographically approved
    5. In situ coating: an approach for particle modification and encapsulation of proteins during spray-drying
    Open this publication in new window or tab >>In situ coating: an approach for particle modification and encapsulation of proteins during spray-drying
    2006 (English)In: International Journal of Pharmaceutics, ISSN 0378-5173, E-ISSN 1873-3476, Vol. 323, no 1-2, p. 52-63Article in journal (Refereed) Published
    Abstract [en]

    In this paper, we present a method for in situ coating of individual protein particles in a respirable size. The aim of the coating was to influence the particle/powder properties, and to reduce or prevent surface-induced conformational changes of the protein, during spray-drying, which was the method used for simultaneously preparing and coating particles. The investigated formulations included bovine serum albumin (BSA), trehalose and either of the two non-ionic polymers, hydroxypropyl methylcellulose (HPMC) and poly(ethylene oxide)–poly(propylene oxide) triblock co-polymer (Poloxamer 188). Complete protein coating as measured by electron spectroscopy for chemical analysis (ESCA) was achieved at a polymer concentration of approximately 1% of the total solids weight, and could be predicted from the dynamic surface tension at the air/water interface, as measured by the pendant drop method. Further, particle properties such as: size, dissolution time, powder flowability, and apparent particle density, as measured by gas pycnometry, were affected by the type and concentration of the polymer. In addition, the particle surface morphology could possibly be correlated to the surface elasticity of the droplet surface during drying. Moreover, an extensive investigation (Fourier transform infrared spectroscopy, circular dichroism and size exclusion chromatography) of the structural effects of protein encapsulated in a polymeric coating suggested that in situ coating provide particulate formulations with preserved native conformation and with a high stability during rehydration.

    Keywords
    polymeric particle coating, spray-drying, competitive surface adsorption, dynamic surface tension, surface morphology
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-93366 (URN)10.1016/j.ijpharm.2006.05.066 (DOI)000241346400007 ()16887302 (PubMedID)
    Available from: 2005-09-02 Created: 2005-09-02 Last updated: 2018-01-13Bibliographically approved
  • 6.
    Evertsson, Hans
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Molecular dynamics and aggregation behaviour in aqueous polymer-drug model systems1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pharmaceutical formulations often contain polymers used as binders, swelling agents, etc., to give the formulation mechanical stability or specific drug release features. Many drug substances, as well as polymers, are amphiphilic which will give an associative polymer-drug interaction, affecting the dissolution rate and drug release. This thesis focuses on the molecular dynamics of model nonionic polymer-anionic drug complexesformed in aqueous solution. Two low molecular weight amphiphiles with different surface activities are used; the surfactant sodium dodecyl sulfate (SDS) and the sodium salt of ibuprofen. The polymers studied are a set of cellulose ethers of different hydrophobicity, with special emphasis on ethyl(hydroxyethyl)cellulose (EHEC).

    Fluorescence probe techniques and nuclear magnetic resonance (NMR) are the main methods used. The molecular dynamics of the EHEC/SDS system is strongly composition dependent. Mixed EHEC/SDS micelles with low SDS aggregation numbers and a high relative content of EHEC is more than three times as rigid as mixed micelles with a high relative content of SDS, as monitored by the fluorescent probe 1,3-di(1-pyrenyl)propane(P3P) and 1H NMR transverse relaxation.

    The microviscosities as monitored by P3P of a set of ten cellulose ethers with different hydrophobicity, interacting with SDS, are compared. A correlation between the microviscosity and the polymer-SDS interaction energy, as qualitatively estimated from the SDS concentration at onset of polymer-SDS association, is found. This correlation is further described by principal component analysis for a subset of six EHEC samples. The microviscosity of mixed polymer-SDS micelles as monitored by P3P is found to be a valuable parameter for the characterization of polymer samples, and can be used to predict the surface tension of the polymer.

    The interaction between EHEC and the sodium salt of ibuprofen resembles the normally accepted model for polymer-surfactant interaction, but is more complex. Fluorescence probe- and pulsed gradient spin echo self diffusion NMR- (PGSE-NMR) measurements indicate that premicellar aggregates of ibuprofen adsorb onto EHEC below the onset of cooperative adsorption with respect to the ibuprofen concentration. The thesis shows theimportance of detailed knowledge of the molecular dynamics for design of controlled release preparations.

  • 7.
    Hedeland, Mikael
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Cellobiohydrolase I as a chiral sector1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The protein cellobiohydrolase I (CBH I) immobilized on silica particles has been used as a chiral selector in liquid chromatography (LC) and the free enzyme has served as a chiral complexing agent in the background electrolyte in capillary electrophoresis (CE) using the partial tilling technique.

    The CE systems were much more efficient and more solutes could be enantioseparated than in the LC systems. Due to the high enantioselectivity, a few substances could be completely enantioresolved in an injected CBH I zone that was shorter than the sample zone. The affinity for the amino alcohol propranolol was very high at neutral pH and in order to displace this solute for detection, a plug of the disaccharide cellobiose was injected after the sample zone. The high affinity of propranolol combined with the displacing ability of cellobiose made it possible to preconcentrate the solute prior to enantioseparation by compression of long dilute sample zones.

    The mechanisms of retention and enantioselectivity in LC have been studied using charged and uncharged additives in the mobile phase. Site-specific mutation and covalent modification of the protein were also used to elucidate retention mechanisms. The enantioselective sites of amino alcohols and the acid warfarin overlap in the vicinity of the catalytically active site of CBH I. The main binding mechanism for both amines and acids was electrostatic attraction. Interestingly, both the retention and the enantioselectivity increased upon changing from potassium to sodium as mobile phase cation.

    The thermodynamics of the system was studied by microcalorimetry and LC. Both enantiomers of the amino alcohol alprenolol bind to CBH I with an endothermic enthalpy change. Hence, the complexation is entropy driven.

    Molecular mechanics calculations were carried out on the complex between propranolol and CBH I. The bound conformation of (S)-piopranolol differs both in geometry and energy compared to the preferred conformation in water solution. The ability of different parameter setups to reproduce the experimental structure of the (S)-propranolol - CBH I complex was investigated. One setup was chosen to suggest possible conformations of the (R)-enantiomer. The water structure in the bindning site seems to be an important factor in the complexation mechanism.

  • 8.
    Henriksson, Hongbin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Munoz, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Isaksson, Roland
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBHI (T.r. Cel 7A) in enantioseparations2000In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 898, no 1, p. 63-74Article in journal (Refereed)
    Abstract [en]

    Cellobiohydrolase 58 (EC 3.2.1.91, pc. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta -adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, To the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.

  • 9.
    Hugerth, Andreas M.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Effects of polyelectrolyte conformation, charge density and ion specificity in polyelectrolyte and polyelectrolyte-drug interaction2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The central theme is how the structural characteristics of sulphated polyelectrolytes and theirinteraction with counter- and coions determine the degree of polyelectrolyte self-association (gelformation), interaction with oppositely charged polyelectrolyte and polyelectrolyte-amphiphilic druginteraction in protolytic solvent medium. Such knowledge is essential for understanding and regulatingthe interaction between polyelectrolytes as well as between polyelectrolytes and a drug substance in apharmaceutical formulation and for its fate in the human organism.

    The gel formation of the sulphated polyelectrolyte ê-carrageenan was found to be dependent on theextent of inter-chain helical association and thus the nature of the polyelectrolyte counterions. This wasshown from the relationship between the gel-sol melting temperature of the hydrophobic microdomains(T0), the fraction of polymer in helical conformation at T0, and the storage modulus of the samples inthe gel-state. Interaction between the oppositely charged polyelectrolytes carrageenan and chitosanresulted in the formation of polyelectrolyte complexes with a charge ratio of unity. However, in casesof inter helical association of carrageenan chitosan acted as a bridging element producing complexeswith a charge ratio below unity. This mechanism may be used to control the charge ratio ofpolyelectrolyte complexes.

    The solvation characteristics of the polyelectrolyte counterions also affected the polyelectrolyte-amphiphilic drug interaction (amitriptyline). The binding isotherm was shifted to a higher concentrationof free amphiphile according to the counterion sequence Li+ < Na+ < K+ < Rb+ ≈Cs+. The change inGibbs free energy per monomer amphiphile originating from exchanging the counterions (Li+ for Cs+),was of the order of kT. This is of the same order of magnitude as that obtained by significantlychanging the hydrophobicity of the amphiphilic drug molecule. Increasing the polyelectrolyte chargedensity decreased the critical aggregation concentration, increased the degree of cooperativity andincreased the magnitude of ion specific effects. Furthermore, the dependence of the. critical aggregationconcentration on ionic strength was linear and the polymer flexibility affected the efficiency of thepolycounterion properties of the polyelectrolyte. The binding isotherms, micropolarity, microviscosityand surface tension indicated the amitriptyline-polyelectrolyte interaction to be qualitatively andquantitatively similar to that observed for "typical" cationic surfactants.

  • 10.
    Hultén, Johan
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Cyclic sulfamides as HIV-1 protease inhibitors: Synthesis, X-ray structure analysis and structure-activity relationship1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immune Deficiency Syndrome (AIDS). Inhibition of HIV-1 protease leads to immature and non-infectious viral particles. The synthesis of a number of cyclic HIV-1 protease inhibitors and the subsequent evaluation in an enzyme assay is presented in this thesis. A centrally positioned water molecule (W301), unique to retroviral proteases, guided the design ofthe inhibitors.

    A synthetic procedure utilising carbohydrates as chiral starting materials has been used to gain control of the stereochemistry of the target compounds. Synthesis of four C2-symmetric cyclic urea inhibitors revealed the importance of correct stereochemistry in rigid cyclic structures for activity. According to the X-ray crystal structures of the cyclic inhibitors, in complex with the protease, a displacement of the water molecule W301 by aurea carbonyl, or alternatively by a sulfamide group was accomplished.

    Changing the water-mimicking group from urea to sulfamide resulted in an unexpected non-symmetric binding mode as deduced from comparison of the X-ray crystal structure of a urea and a sulfamide inhibitor in complex with the protease. A small X-ray structure of a sulfamide inhibitor in absence of the protease established that thenon-symmetric conformation of the inhibitor was an inherent feature of the sulfamide scaffold and not induced by the protease.

    In an attempt to establish the structure activity relationship (SAR) of the sulfamide class of inhibitors, symmetric and non-symmetrically substituted sulfamide inhibitors were prepared. A comparative molecular field analysis (CoMFA) was performed to rationalise the SAR and give a model of predictive value. The cyclic inhibitors wereevaluated against a series of mutant forms of the protease and found to have a considerably lower affinity towards I84V and V82A than towards the wild type enzyme.

  • 11.
    Huynh, Ngoc Hang
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Enantioseparation of drugs and related compounds using chiral mobile phase additives1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Neutral and anionic chiral mobile phase additives have been employed to separate enantiomers of drugs and related compounds using aqueous buffer solution and hydro-organic solvent mixtures as mobile phases.

    Ester derivatives of tartaric acid, e. g. (2R,3R)-dicyclohexyl tartrate, was adsorbed on an achiral solid phase of porous graphitic carbon (PGC) and acted as a chiral stationary phase for the enantiomer separation of neutral, acidic and basic compounds (e.g. atropine, mandelic acid, ephedrine) The antipode, (2S,3S)-dicyclohexyl tartrate was applied for the determination of the enantiomeric impurity of a pharmaceutical preparation of (S)-atropine.

    (2R,3R)-(-)-Dibenzoyl-tartaric acid mono (dimethylamide), a tartaric acid amide derivative having a free carboxylic group was also adsorbed on PGC and simultaneously acted as a chiral counter ion to several racemic drugs. Complete chiral resolution was obtained for several β-blocking agents (e.g. alprenolol, metoprolol and propranolol) and antiulcer agents (e.g. omeprazole and lansoprazole)

    In contrast to the tartaric acid derivatives, N-protected di-and tripeptides, e.g. N-benzyloxycarbonyl-glycyl-L-proline (L-ZGP), had low affinity to PGC when dissolved in mobile phase of methanol. Nevertheless they gave high enantiomeric resolution for several racemic drugs of pharmacological interest (e.g. terbutalin, alprenolol, trimipramine and promethazine) by acting as a counter ion to the solute enantiomers. This chiral counter ion enabled the determination of the enantiomeric composition of mefloquine at micro-molar levels in biological fluids.

    When charged selectors were used, solute retention conformed with ion-pair retention mechanism. Counter ion concentration and pH were the main parameters utilized to control both retention and enantioselectivity. Other achiral additives e.g. organic modifiers, co-ions etc might also influence the enantioselective retetion. Thus many factors could be used to regulate the chiral separation by ion-pair chromatography. The chemometric approach, using experimental design and multivariate data analysis, was shown to be an effective tool for optimization of the separation of mefloquine. (1S,3S,4S)-(+)-3-bromocamphor-10-sulfonic acid was used as a counter ion.

  • 12.
    Högberg, Marita
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Synthesis and structure activity relationship studies of non-nucleoside HIV-1 reverse transcriptase inhibitors1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    HIV-1 reverse transcriptase (RT) is a target for anti-HIV chemotherapy. A series of PETT PhenylEthylThiazoleThiourea) non-nucleoside HIV-1 RT inhibitors were synthesised. All compounds were tested in an HIV-1 RT enzyme assay, and in cell culture using MT-4 cells and wild-type virus. Many compounds were also tested in HIV-1 RT enzyme assays with three constructed mutants, Ile100, Cys181 and Asn103, and in cell culture using virus containing a mutation in residue 100, 181 or 103.

    The lead PETT compound, N-(2-phenethyl)-N'-(2-thiazolyl)thiourea, has an IC50 of 0.9 µM in an HIV-1 RT enzyme assay and an ED50 of 1.3 µM in an HIV-1 cell culture assay. The optimisation of this compound resulted in a clinical candidate, trovirdine, N-[2-(2-pyridylethyl)]-N'-[2-(5-bromopyridyl)]thiourea, which inhibits HIV-1 RT with an IC50 of 15 nM and has an ED50 of 20 nM in cell culture. Further optimisation produced more potent thiourea compounds comprising 2, 3, 6-trisubstituted phenethyl 5-chloro, 5-bromo or 5-cyano-2-pyridyl thiourea analogs. The IC50 and ED50 values of the most active of these analogs were 1 to 5 nM. Bioisosteric substitution of the thiourea moiety of PETT compounds with cyanoguanidine, guanidine and sulfonyldiamide is studied. Finally, the synthesis and structure activity relationship studies of highly potent, conformationally restricted, racemic and enantiomeric cyclopropyl urea analogs are described with enhanced antiviral activities even on mutant form of HIV-1 virus in the nanomolar range.

    The three-dimensional structures of complexes between HIV-1 RT and an enantiomeric pair of cyclopropyl compounds have been determined. The structures show similar binding in the NNI binding pocket. The cyclopropyl moiety of both enantiomers has close-packing interactions with leucine residue 100, valine residue 179 and tyrosine residue 181, but in addition to these interactions the cyclopropyl moiety of the (+)-enantiomer has extensive hydrophobic close-packing interactions with glutamine residue 1138.

  • 13.
    Karami, Kiomars
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Electrolyte structures and ionization conditions for iontophoretic drug formulation of local anesthetics1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Iontophoresis is a modern method of drug delivery by which charged bioactive molecules (drugs) are transferred from an electrolytic reservoir into and through a tissue, normally skin, by means of a weak electric current. The drug is placed in a conductive medium, usually an aqueous solution or a hydrogel, between the "active" electrode and the skin. The circuit is completed by a second "passive" electrode and the skin. Although iontophoresis is a well-known method for transdermal delivery of drugs, basic physicochemical knowledge of this phenomenon is still lacking.

    This thesis aimed to obtain a wide understanding of electrolyte structures and ionization conditions in iontophoresis, and a firm physicochemical basis for improvement of iontophoretic drug formulation of local anesthetics. The precision conductometric technique was used mainly. Lidocaine hydrochloride (LidHCl) was selected as a model local anesthetic substance. The conductance theory of Fuoss, Hsia and Fernandez-Prini was used to interpret the experimental data, with respect to ionization and mobility of LidHCl in different solvent media. LidHCl was studied in the following solvents:, water, propylene glycol (a transdermal enhancer),aqueous propylene glycol (20 weight-% PG) and 1-octanol. To compare the ionization properties of prilocaine hydrochloride (PrilHCl) with those of LidHCl, PrilHCl was studied in the same pharmaceutical aqueous-enhancer medium. Furthermore, the molecular diffusive transport properties of the local anesthetics lidocaine-, prilocaine-,bupivacaine-, etidocaine-, mepivacaine- and ropivacaine hydrochloride in an agarose hydrogel (1 weight-% agarose) were studied.

    The results indicate that, for the solvents studied, LidHCl has the highest ionic mobility in aqueous propylene glycol; u(LidH+) = 1.35 mm2 V-1 min-1. No measurable difference in ionization properties and ionic mobility between LidHCl and PrilHCl in aqueous propylene glycol was observed. Within the concentration range investigated in this solvent, more than 98% of both LidHCl and PrilHCl are in ionized form (LidH+ and PrilH+). Of the six local anesthetics studied by diffusion measurements in agarose hydrogel (1 weight-% agarose) as medium, LidHCl and PrilHCl have the highest diffusion coefficients; D(lido) = 7.79 · 10-6 and D(prilo) = 7.76  · 10-6 cm2/s.

    From a pharmaceutical point of view, it might be interesting to study the possibility of combining lidocaine- and prilocaine hydrochloride in an aqueous-enhancer medium as an iontophoretic drug formulation of local anesthetics.

  • 14.
    Lindman, Susanna
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Design and synthesis of -turn peptidomimetics: Applications to angiotensin II2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This study addresses the issue of how to convert peptides into drug-like non-peptides while retaining the biological activity at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide.

    Small bioactive peptides are in most cases conformationally flexible molecules. Rigidified peptide analogues or peptidomimetic scaffolds can be introduced into the peptide, to enforce a particular backbone conformation, and thereby locate the side-chains at defined positions in space. The conformationally constrained analogues are of considerable value in determining biologically active conformation(s) of the studied peptide. The strategy applied in this thesis includes identification of non-pharmacophoric amino acid residues, rigidification, conformational analysis and incorporation of turn mimicking scaffolds in

    Ang II. Several side-chain cyclized (disulfide and methylendithioether) Ang II analogues have been synthesized. The binding studies of the rigidified analogues demonstrated that the compounds designed for the AT1-receptor had affinity for both receptor subtypes, while the compounds designed for the AT2-receptor displayed high selectivity only for this receptor subtype. Conformational evaluation revealed that several of the cyclized Ang II analogues most probably adopt a γ-turn like conformation around Tyr-4 while interacting with the

    Ang II receptor. Based on this hypothesis, three different γ-turn mimetics replacing amino acid residues 3-5 were designed, synthesized and incorporated into Ang II. One of the synthesized pseudopeptides, incorporating an azepine-containing γ-turn mimetic, exerted high binding affinity and agonistic activity. These results strongly support the theory that Ang II adopts a γ-turn like conformation when activating the AT1 receptor. The other Ang II analogues, incorporating bicyclic and aromatic γ-turn mimetics, did not display any binding to the AT1 receptor.

  • 15.
    Linnanen, Tero
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Serotonergic aporphine derivatives: Synthesis and structure-activity relationships2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Novel series of well-characterized and stereochemically pure 11-substituted (R)-aporphine derivatives have been prepared and their interaction with serotonin 5-HT7 and 5-HT1A receptors and dopamine D2A and D1 receptors have been studied. Efficient palladium catalyzed reactions were utilized for the diastereoselective synthesis of atropisomers as well as for the synthesis of several of the other (R)-aporphine derivatives. A number of 11-arylated (R)-aporphines displayed high affinity for 5-HT7 and 5-HT1A receptors and derivatives with selectivity for either 5-HT7 or 5-HT1A receptors were obtained by chosing the proper substituents on the 11-phenyl ring. Simultaneous substitution of the 2' and 6' positions of (R)-11-phenylaporphine afforded derivatives, including some atropisomers, with increased 5-HT7 selectivity and several of these analogues were characterized as selective 5-HT7 receptor antagonists. Introduction of substituents in the other positions of the 11-phenyl group resulted in derivatives with selectivity for 5-HT1A receptors.

    In addition, new series of novel pentacyclic (R)-aporphines were synthesized either by the introduction of a methylene group between carbon atoms C1 and C11 in the (R)-aporphine skeleton or by ring expansion reactions of (R)-1,11-carbonylaporphine. These series of derivatives afforded the possibility to introduce substituents/functional groups below, above and in the plane of the ring system thereby allowing for structural extensions into previously unexplored areas of receptor binding sites. Compounds with various affinities for the serotonin and dopamine receptors were obtained and novel 5-HT7 receptor antagonists were identified.

    The identification of selective serotonin 5-HT7 receptor antagonists is of particular interest since only a few putatively selective 5-HT7 receptor antagonistic ligands have been reported.

  • 16.
    Olofsson, Kristofer
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Regiocontrol in the Heck-reaction and fast fluorous chemistry2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The palladium-catalysed Heck-reaction has been utilised in organic synthesis, where the introduction of aryl groups at the internal, β-carbon of different allylic substrates has been achieved with high regioselectivity.

    The β-stabilising effect of silicon enhances the regiocontrol in the internal arylation of allyltrimethylsilane, while a coordination between palladium and nitrogen induces very high regioselectivities in the arylation of N,N-dialkylallylamines and the Boc-protected allylamine, producing β-arylated arylethylamines, which are of interest for applications in medicinal chemistry. Phthalimido-protected allylamines are arylated with poor to moderate regioselectivity.

    Single-mode microwave heating can reduce the reaction times of Heck-, Stille- and radical mediated reactions drastically from approximately 20 hours to a few minutes with, in the majority of cases, retained, high regioselectivity.

    The use of heavily fluorinated tin reagents, which proved to be unreactive under thermal heating, is shown to be applicable with microwave-heating and the high fluorous content of the products is utilised with the aim of improving and simplifying the work-up procedure.

  • 17.
    Persson, Bengt
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Drug-polymer interaction in relation to surface tension and polymer properties1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Polymers are important in a variety of pharmaceutical applications. Many of them are derived from cellulose but there are several biological sources of importance for their production. Polymers display a very wide diversity of properties which makes a detailed characterization necessary. The investigated polymers are more or less surface active due to the presence of hydrophilic and hydrophobic parts. They show surface activity at very low concentrations which makes them useful as stabilizers in suspensions and emulsions. The surface tension measurements were performed by the pendant drop method.

    The surface activity and the cloud point (CP) are related to each other. These parameters depend on the hydrophilic/hydrophobic balance of the polymer. The time dependence of surface tension is mainly related to the concentration in the dynamic region (2-10 ppm). The half-life of the surface tension (t1/2) at these concentrations was found to be inversely correlated to the diffusion coefficient and to the square of the bulk concentration, which is expected for a diffusion process. Furthermore, t1/2 was related to the intrinsic viscosity of the polymers.

    The in situ colonic effective permeability of fluvastatin in the rat was found to be inversely proportional to the surface tension of fluvastatin.

    The interaction between charged amphiphiles and oppositely charged polymers was investigated by surface tension measurements since surface activity is an important property of pharmaceutical formulation. The adsorption of charged amphiphiles to oppositely charged polyelectrolytes involve charge to charge interaction at low amphiphile concentration. An increase in amphiphile concentration is followed by a cooperative increase in adsorption at a certain concentration due to hydrophobic interaction between already adsorbed amphiphiles. The strength of the interaction is related to the hydrophobicity of the amphiphile. Also the charge density isimportant since the distance between the charges influence the concentration where the hydrophobic interaction starts.

  • 18.
    Saenger-van de Griend, Carl
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Majors, Ronald E.
    Revival of Capillary Electrophoresis Techniques in the Pharmaceutical Industry2012In: LC GC North America, ISSN 1527-5949, E-ISSN 1939-1889, Vol. 30, no 11, p. 954-+Article in journal (Other academic)
  • 19.
    Sjöberg, Hans
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Release and transport of drugs in some complex and interacting systems2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Drug formulations come in many forms, some intended for direct and immediate action and others aiming at a controlled manner of drug delivery. Viewed in a more general way drug transport often involves charged units, it takes place in a medium which in many respects must be considered complex - to some degree even reactive. Drug transport may also be competitive because of thermo- and hydrodynamic interactions, These three aspects of drug transport in general is discussed and exemplified in specific studies: 1) A conductivity study as a fundamental investigation for iontophoretic drug delivery; 2) a methodological development and diffusivity investigation of a steroidal hormone transport in silica containing silicone; 3) an investigation of competitive diffusion transport of small amphiphilic molecules in a polyelectrolyte containing semi-solid system.

    Iontophoresis - drug administration by electric current - normally requires charged drug molecules, hence their mobility and state of dissociation are essential parameters. The medium in which the drug is situated regulates the electrolytic drug content (e.g. dissociation equilibrium) and thus the iontophoretic efficiency. The conductivity of the local anesthetic, IidocaineHCl was investigated by high precision conductometry in aqueous and octanol solutions. From conductivity data and applied advanced electrolyte theory drug mobility parameters as well as, dissociation and association constants were obtained. The drug ion mobility was found, to be a factor 50 lower in octanol. The equilibrium constants obtained show that increasing drug concentration favors ion formation in water while favoring ion pair association in octanol. Hence, such a drug behaves very differently in different media, with obvious clinical implications.

    The dynamic diffusion in the complex system silicone/silica/estradiol was studied by means of a new equipment design which has considerably improved the possibility to perform precise quantitative dynamic diffusion measurements in such systems, The method avoids all disturbing interfaces between different media and allows for a precise spatial resolution of concentration of the diffusing drug. Data showed the method to be dependable and accurate. The effect on estradiol diffusivity due to varied silica content in the silicone is found to be paramount, increasing [SiO2] from 17 to 30% decreased the diffusivity by 68%. Some mechanisms are discussed.

    The diffusion of amphiphiles within and the release from agarose hydrogels containing the polyelectrolyte carrageenan was investigated. All drugs exhibit same apparent diffusivity in pure agarose gel. The diffusivity decreased when carrageenan was introduced in the gel. The order of the decrease follows the order of increased hydrophobic character of the drug. Competing diffusion was studied for amitriptyline and chlorpromazine, the former released significantly faster than chlorpromazine when simultaneously released from a hydrogel slab. Data indicate the possibility to adjust the simultaneous release rates individually for the two drugs by means of polyion charge density.

  • 20.
    Ståhlberg, Jerry
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Henriksson, Hongbin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Divne, Christina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Isaksson, Roland
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Alwyn, Jones T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 angstrom resolution.2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 305, no 1, p. 79-93Article in journal (Refereed)
    Abstract [en]

    Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta -blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers.

    In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta -blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1.9 Angstrom resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/ + 1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O-epsilon1 and to Gln175 N-epsilon2, whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q.

    Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 muM and 44 muM, respectively, corresponding to binding free energies of 20 kJ/ mol and 24 kJ/mol, respectively. The K-i value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed.

    By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non-selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP

  • 21.
    Svensson, Lars A.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Chiral packed capillary chromatography using an in-column immobilized selector: With special emphasis on vancomycin1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Packed fused silica capillary columns have been used for enantioseparation of drugs and related compounds. The technique to prepare the chiral stationary phase (CSP) involved a reductive amination of aldehyde functionalized silica with a chiral selector. The immobilization was performed in the packed column, thereby minimizing the use of selector. An α-chymotrypsin CSP was used to investigate the influence of the pore size on the chromatographic performance. Both selectivity and efficiency were better on a 1000 Å pore size material.

    The retention mechanism of some arylpropionic acids on a vancomycin CSP was studied. A two-site retention model was sufficient to qualitatively describe the effects of different mobile phase compositions, including buffer, modifier and counter ion concentration as well as pH. A closer investigation of the immobilization reaction was also conducted for the vancomycin selector with the aim of elucidating if the linkage to the silica affected the enantioselective retention. To accomplish this one of the two amino groups was temporarily protected while the other was linked to the silica. However, no significant difference in enantioresolution between the two defined CSPs formed was obtained in comparison with the one resulting from the immobilization of native vancomycin.

    Different types of interactions seemed to be important in different modes of chromatography. Reversed phase LC primarily separated acidic compounds while polar organic phase LC (POPLC) was better suited to resolve basic compounds. Supercritical fluid chromatography (SFC) and normal phase LC could separate both acidic and basic as well as neutral compounds, but SFC had superior chromatographic performance resulting in both higher efficiency and selectivity.

    Acid and base interactions between analyte and selector seems to be the key process for chiral discrimination. It has previously been shown that the N-terminal of vancomycin was important for acidic compounds. With the use of an analogous selector, ristocetin A, it is here indicated that the C-terminal is equally important for resolution of bases.

  • 22.
    Sänger-van de Griend, Cari Elise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Enantiomeric separations by capillary electrophoresis in pharmaceutical analysis1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The chiral capillary electrophoresis mechanism is studied by evaluating the enantiomeric separations of a series of local anaesthetic analogues, the sixteen different optical isomers of a tetrapeptide and a series of glycyl, alanyl and leucyl dipeptides. The usefulness of mobility difference plots is appraised and important method parameters, such as the selection of the chiral selector, the chiral selector concentration, the chiral selector purity, the pH and the composition of the electrophoresis solution, are identified and discussed, bearing method robustness in mind. The evaluation of the mobility difference plots obtained for the eight enantiomer pairs of the tetrapeptide resulted in an extension of the mobility difference model. In the extended model the formation of enantiomer-chiral selector complexes with a stoichiometry higher than 1:1 is taken into account. With this model it is demonstrated that complex formation of one tetrapeptide molecule with more than one cyclodextrin molecule is a possible explanation for the experimentally obtained data.

    Cyclodextrins were evaluated as chiral selectors for dipeptides and have been shown to be applicable to the enantiomeric separation of aromatic and non-polar aliphatic dipeptides. By developing and validating an enantiomeric purity determination method for ropivacaine substance and products, it is shown that chiral capillary electrophoresis is a feasible technique for pharmaceutical analysis, which can offer an alternative and/or complement to chiral liquid chromatography.

  • 23.
    Thorsteinsdóttir, Margrét
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Separation of peptides by capillary electrophoresis: Application of chemometrics for evaluation of separation performance in micellar electrokinetic chromatography1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The separation of enkephalin-related peptides and protein kinase A peptide substrates werestudied in micellar electrokinetic chromatography and compared with capillary zoneelectrophoresis. Special interest was focused on systems where the electroosmosis wasreversed in combination with neutral micellar agents. Such systems involve the unique combination of minimizing surface interactions by electrostatic effects and increasing themigration time window. This was accomplished either by addition of cationic monomericamines to the background electrolyte or by using fused silica capillaries modified byimmobilizing an amine to the surface.

    The selectivities were enhanced by adding anionic taurodeoxycholate micelles to the BGE.The efficiencies obtained were highly dependent on the extent of distribution of the peptides tothe micellar phase. The effect of experimental factors that cause band broadening duringseparation were evaluated by fractional factorial design and response surface modelling. Partialleast square (PLS) regression analysis revealed that very high efficiencies were obtained forpeptides with low distribution to the micelles, while the efficiencies drastically decreased forpeptides strongly associated to the micelles, probably due to slow sorption-desorption kinetics.

    The separation of enkephalin-related peptides in a system with anionic sodium dodecylmicelles (SDS) was optimized by utilizing experimental design and response surfacemodelling. Such strategies have the advantages of greatly reducing the number of experimentsand allowing identification of interaction effects between the variables. The effect ofacetonitrile was studied, in four different concentration domains, together with SDSconcentration, temperature and the ionic strength of the buffer via central composite design,and related to resolution, migration factor and migration time window utilizing PLS-regression. The results revealed a complex system with very different influences of theexperimental variables in respective domain. The effect of acetonitrile is highly non-linear insystems of this kind and dependent on the temperature used. A change in the thermodynamicsof the distribution behaviour was observed at increasing acetonitrile concentrations. Further,the relationships between different parameters that influence the resolution were investigatedby principal component analysis.

  • 24.
    Torstensson, Richard
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Functional regulation of the dopaminergic system in vivo: Experimental and clinical studies with positron emission tomography1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Positron emission tomography (PET) is a non-invasive imaging technique used to quantitate the kinetics of a radiotracer in such physiological processes as receptor binding and enzyme activity. Radiolabelled L-DOPA has previously been used as a tracer for the dopaminergic system. The focus of the present study was to investigate the validity and feasibility of the 11C-labelled L-DOPA to measure aromatic amino acid decarboxylase (AADC) activity as an indicator of the functional tone of the presynaptic dopaminergic system in vivo. The regulation of the presynaptic dopaminergic system was studied in rhesus monkeys, as well as in humans in the healthy and diseased states.

    The intra- and inter-individual variations of the striatal influx rate constant for L-[β-11C]DOPA, i.e. Ki, measured with PET were found to be 10 and 15% between days, respectively. Furthermore, in rhesus monkeys, the Ki value was shown to decrease slightly (1 to 2% per year) as a function of time. Radiolabelled L-DOPA and its 6-fluoroanalogue were compared as PET tracers. Differences in the peripheral metabolism of L-DOPA and the fluorinated analogue were confirmed. The modulating effects of 6R-BH4 on L-[β-11C]DOPA Ki were only found for the L-6-fluorodopa tracer when animals had been pretreated with a COMT inhibitor.

    Pharmacological treatments induced changes in the L-[β-11C]DOPA Ki. The dopamine receptor agonist, apomorphine, decreased the striatal Ki value and L-DOPA as well as 6R-BH4, a co-factor for tyrosine hydroxylase, increased the Ki values. Moreover, the effects of these drugs on the striatal Ki value showed significant baseline dopaminergic state-dependent effects. The regulating effect on the L-[β-11C]DOPA Ki induced by different pharmacological treatments was shown to correlate with changes in AADC activity measured in rat brain homogenates.

    An intriguing new compound, (-)-OSU6162, was investigated with respect to its functional effects in the regulation of the presynaptic dopaminergic system. The compound induced a normalising effect on striatal L-[β-11C]DOPA Ki values, i.e. (-)-OSU6162 increased the striatal Ki value in monkeys with initial low values and decreased the Ki values in monkeys with initial high values.

    In patients with Parkinson's disease (PD), marked differences in the functional regulation of the presynaptic dopaminergic tone were found in patients with early and advanced disease. After therapeutic levodopa infusions, patients with advanced PD showed an increase in striatal L-[β-11C]DOPA Ki values; in patients with early PD, levodopa induced a decrease in striatal Ki values. A diminished presynaptic autoreceptor function in advanced PD was proposed and demonstrated in a clinical study using a therapeutic apomorphine challenge.

    In conclusion, the radiotracer L-[β-11C]DOPA was shown not only to measure the AADC integrity in the presynaptic dopaminergic system but also to reflect the functional tone of this neurotransmitter system in vivo.

  • 25.
    Wulff, Marie
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Studies on structure and mechanism at formation of solid solutions and solid dispersions of hydrophobic drugs in polymers1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The aim of the studies was to investigate the structures formed and mechanisms involved, when a hydrophobic drug is dispersed in a polymer carrier. The effect of different additives, like surfactants and cyclodextrins, was evaluated. The dispersions were prepared by the melting method. The phase composition and structure of the samples were studied by X-ray powder diffraction (XRD) and solid-state NMR. The thermal analysis was made by differential scanning calorimetry (DSC) and temperature-modulated DSC (MTDSC).

    The structure of the systems formed upon addition of surfactants was found to depend on several factors. The critical surfactant concentration for formation of solid solutions with different counterions correlated very well to the relative size of the counterions. The ion with the highest charge-to-radius ratio, Li+, formed bonds that were different from those formed with the Na+ and K+ ions. This could explain the observed increased crystallinity and the deviation in the thermal behavior of the Li+ compounds, compared to that obtained for the Na+ and K+ systems.

    The critical surfactant concentrations in different PEGs were correlated to the structure of the PEGs. The polymers contained varying amounts of folded and extended chains, which influences the amorphous parts of the polymer structure, where the surfactant-drug aggregates are likely to dissolve. The solid solubility of griseofulvin was found to be much higher in PEG 6000 than in PEG 3000 or PEG 20000 with surfactant added, implying that a certain balance between the amount of folded chains and surfactant concentration is crucial for the creation of the solid solution. The prerequisites of the formation of a solid solution were given already in the melt.

    The melting method was introduced as a new method for producing cyclodextrin (CD) inclusion complexes of hydrophobic drugs. The structures formed when cyclodextrins were added to the solid dispersions depended on the character of the CD. The results demonstrated the different competition between the polymer, the drug and the CDs when forming bonds to each other. Since the interaction between α-CD and PEG is stronger than between β-CD and the polymer, β-CD can form a stronger bond to indomethacin (IM) than α-CD. In the γ-CD case, a new phase with tetragonal structure was formed where the indomethacin molecule was bound to the CD in an inclusion complex. The interactions resulted in a delayed release of indomethacin from PEG 6000 in the order α-CD < γ-CD <=CD.

    The specific information on the relative crystallinity obtained by the NMR, XRD, DSC and MTDSC techniques was emphasized for the γ-CD system.

    The selection of operational parameters for proper measurements by MTDSC is crucial. A dimensionless quantity, degree of oscillation, was introduced to be able to predict the reproducibility of the heat of fusion determinations. A degree of oscillation ~1-2 gave reliable results for a variety of combinations for the systems examined. With proper operational settings MTDSC can be used for determining the relative crystallinity of certain compounds. It is recommended to test a number of variations of the parameters whenever examining a new type of sample.

  • 26.
    Yu, Zuoxiang
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Direct injection of plasma samples into restricted-access media precolumns for drug analysis in column-switching systems1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis focuses on the theoretical and practical aspects of using column-switching systems for direct injection of plasma samples to assay drugs at ng/ml levels. The systems utilized a restricted-access media (RAM) precolumn for the sample clean-up and trace enrichment. The separation of analytes of interest was accomplished on a reversed-phase analytical column via a switching device in the back-flush mode.

    Retention principles for the functional permission of direct injection of biological samples on RAMs were proposed. In the long course of plasma injections, RAM packings behaved distinctly different from conventional reversed-phase materials. Changes of retention, peak performance and column resistance after the injection of 10-20 ml of plasma were mainly resulted from the blockage of sealings or the adsorption of proteins on the hydrophilic layer of the packings.

    The clean-up effect, the stability of the system and the choice of UV wavelength depended on the character of the RAM packings. However, the composition of the mobile phase serving for the RAM columns could affect the plasma elution profile, the recovery of plasma matrix and column lifetime.

    Based on the developed systems, a detection limit of 10 ng/ml was obtained for the determination of weakly UV absorbing local anaesthetics with UV at 210 nm in conjunction with large volumes of plasma injection (500 _l). Analysis of methotrexate and its main metabolite in plasma was achieved with ion-pair conditions using on-line post-column photochemical reaction and fluorimetric detection. The method gave a limit of quantitation of methotrexate at 3.6 ng/ml.

1 - 26 of 26
CiteExportLink to result list
Permanent link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf