Sigridur Jonsdottir

Stabilisation of ionic drugs through complexation with non-ionic and ionic cyclodextrins

The effects of negatively charged (i.e. carboxymethyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin), positively charged (i.e. trimethylamoniumpropyl-β-cyclodextrin) and neutral cyclodextrins (i.e. hydroxypropyl-β-cyclodextrin, acetyl-β-cyclodextrin and randomly methylated β-cyclodextrin) on the chemical stability of various drugs were investigated. The degradation rate of each drug in aqueous cyclodextrin solutions was determined and the stability constant (Kc) of the drug–cyclodextrin complex and the degradation rate of the drug within the complex (kc) was obtained by non-linear fitting of the data. Compared to drug complexes with neutral cyclodextrins, the values of Kc were from 20 to 1600% larger when the drug and cyclodextrin molecules carried opposite charges, but from 50 to 80% smaller when the molecules carried the same type of charge. The values of kc were not affected by the charge of the cyclodextrin molecule. NMR studies of chlorambucil complexes indicated that the structure of the cyclodextrin complex was at least in some cases affected by the charge on the cyclodextrin molecules.

Conformational Properties of 1-Fluoro-1-silacyclohexane, C5H10SiHF: Gas Electron Diffraction, Low-Temperature NMR, Temperature-Dependent Raman Spectroscopy, and Quantum Chemical Calculations†

The molecular structures of axial and equatorial conformers of 1-fluorosilacyclohexane, C5H10SiHF, as well as the thermodynamic equilibrium between these species were investigated by means of gas electron diffraction (GED), dynamic nuclear magnetic resonance, temperature-dependent Raman spectroscopy, and quantum chemical calculations (MP2, DFT, and composite methods). According to GED, the compound exists in the gas phase as a mixture of two conformers possessing the chair conformation of the six-membered ring and Cs symmetry and differing in the axial or equatorial position of the Si-F bond (axial ) 63(8) mol %/equatorial ) 37(8) mol %) at T ) 293 K, corresponding to an A value of –0.31(20) kcal mol. Density functional theory (DFT) calculations were employed to obtain the minimal energy path of the conformational inversion. The MP2, G3B3, and CBS-QB3 methods were also employed to calculate the equilibrium geometries and energies of the local minima in the gas phase and in solution. The gas-phase results are in good agreement with the experiment, whereas a combined PCM/IPCM(B3LYP/6-311G(d)) approach overestimates the stabilization of the axial conformer by 0.3-0.4 kcal mol in solution at 112 K. Temperature-dependent Raman spectroscopy in the temperature ranges of 210–300 K (neat liquid), 120–300 K (pentane solution), and 200–293 K (dichloromethane solution) also indicates that the axial conformer is favored over the equatorial one by 0.25(5), 0.22(5), and 0.28(5) kcal mol (∆H values), respectively.

Tetraphenylporphyrin tethered chitosan based carriers for photochemical transfection.

Water-soluble amphiphilic chitosan nanocarriers tethered with the photosensitizer meso-tetraphenylporphyrin were synthesized in a seven-step procedure, starting from 3,6-di-O-tert-butyldimethylsilyl-chitosan and 5-(p-aminophenyl)-10,15,20-triphenylporphyrin. The lipophilic photosensitizer could be introduced in a quantitative and reproducible reaction to give either 0.1 or 0.25 degrees of substitution per glucosamine monomer. Fluorescence and NMR investigations revealed the dynamic structures of the carriers, which formed nanoparticles in aqueous solution with a core of π-stacked photosensitizers. These carriers can then unfold in the lipophilic environment, and the photosensitizer moiety can thus be inserted into the cell membrane. The efficacy of the carriers for photochemical internalization (PCI) mediated gene delivery was evaluated in vitro using the HCT116/LUC human colon carcinoma cell line. The efficacy of transfection was comparable to what could be achieved by the reference compound and current clinical candidate TPCS(2a).

Inositols and methylinositols in sea buckthorn (Hippophaë rhamnoides) berries.

Sea buckthorn (Hippophaë rhamnoides L.) berries, especially of ssp. sinensis, contain significant quantities of an unknown, water-soluble compound, evidently a cyclitol derivative. The compound was isolated by HPLC and analyzed by GC-MS [trimethylsilyl (TMS) derivative, selected ion monitoring (SIM) and total ion chromatogram (TIC) analyses], by (1)H and (13)C NMR and by optical activity measurements. The results together with analyses of reference compound verified the unambiguous structure (-)-2-O-methyl-L-chiro-inositol (L-quebrachitol). In addition, chiro-inositol and myo-inositol existing in trace amounts were identified based on reference compounds, chromatographic data and mass spectra of the TMS derivatives. Methyl-myo-inositol was tentatively identified based on chromatography and mass spectrometry. Inositols and methyl inositols are bioactive compounds essential for regulating physiological processes of plants and humans. To our knowledge, this is the first report on the presence of chiro-inositol and myo-inositol in sea buckthorn and L-quebrachitol in edible berries. The identification of the inositols and l-quebrachitol in sea buckthorn may bring new insights into the sensory properties and also mechanisms behind the health effects of the berry.

Examination of 19F-NMR as a Tool for Investigation of Drug-Cyclodextrin Complexes

Abstract Fluorine nuclear magnetic-resonance spectroscopy (19F-NMR) was used to measure complexation of three fluorine-containing drugs—dexamthasone, fluoxetine hydrochloride, and diflunisal sodium—with 2-hydroxypropyl-β-cyclodextrin (HPβCD). Poor aqueous solubility inhibited investigation of dexamethasone complexes with this method. Complexation caused separation of the fluorine peaks that could be assigned to the two enantiomers of fluoxetine hydrochloride. The trifluoromethyl group of the drug was not included, or only partially included, in the cyclodextrin cavity and the shift changes resulting from complexation were small (0.04 and −0.05 ppm). The NMR method, therefore, could not be used to determine complex stoichiometry and complex stability constants, as chemical-shift changes were influenced by changes in the composition of the solvent medium. The difluorophenyl group of diflunisal sodium was fully included in the cyclodextrin cavity and the chemical-shift changes were large, 2.0 and 1.4 ppm, for C2′ and C4′ fluorine atoms, respectively. Using the continuous variation method, a 1:1 stoichiometry was determined for the complex. The chemical shift changes could also be used to determine the stability constant (Kc) for complex formation. The value obtained for the fluorine that enters deeper into the cavity was 2000 M−1. The data shows that, given that the drug has sufficient solubility, one-dimensional 19F-NMR can be a fast and convenient method to investigate drug-cyclodextrin complexes. However, when the results are interpreted it must be taken into account that the solvent medium can affect the chemical shifts of the fluorine peaks.

Developing a preventive immunization approach against insect bite hypersensitivity using recombinant allergens: A pilot study.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of midges (Culicoides spp.). IgE-mediated reactions are often involved in the pathogenesis of this disease. IBH does not occur in Iceland due to the absence of Culicoides, but it occurs with a high frequency in Icelandic horses exported to mainland Europe, where Culicoides are present. We hypothesize that immunization with the Culicoides allergens before export could reduce the incidence of IBH in exported Icelandic horses. The aim of the present study was therefore to compare intradermal and intralymphatic vaccination using four purified recombinant allergens, in combination with a Th1 focusing adjuvant. Twelve horses were vaccinated three times with 10μg of each of the four recombinant Culicoides nubeculosus allergens. Six horses were injected intralymphatically, three with and three without IC31(®), and six were injected intradermally, in the presence or absence of IC31(®). Antibody responses were measured by immunoblots and ELISA, potential sensitization in a sulfidoleukotriene release test and an intradermal test, cytokine and FoxP3 expression with real time PCR following in vitro stimulation of PBMC. Immunization with the r-allergens induced a significant increase in levels of r-allergen-specific IgG1, IgG1/3, IgG4/7, IgG5 and IgG(T). Application of the r-allergens in IC31(®) adjuvant resulted in a significantly higher IgG1, IgG1/3, IgG4/7 allergen-specific response. Intralymphatic injection was slightly more efficient than intradermal injection, but the difference did not reach significance. Testing of the blocking activity of the sera from the horses immunized intralymphatically with IC31(®) showed that the generated IgG antibodies were able to partly block binding of serum IgE from an IBH-affected horse to these r-allergens. Furthermore, IgG antibodies bound to protein bands on blots of C. nubeculosus salivary gland extract. No allergen-specific IgE was induced and there was no indication of induction of IgE-mediated reactions, as horses neither responded to Culicoides extract stimulation in a sulfidoleukotriene release test, nor developed a relevant immediate hypersensitivity reaction to the recombinant allergens in skin test. IL-4 expression was significantly higher in horses vaccinated intralymphatically without IC31(®), as compared to horses intradermally vaccinated with IC31(®). Both routes gave higher IL-10 expression with IC31(®). Both intralymphatic and intradermal vaccination of horses with recombinant allergens in IC31(®) adjuvant induced an immune response without adverse effects and without IgE production. The horses were not sensitized and produced IgG that could inhibit allergen-specific IgE binding. We therefore conclude that both the injection routes and the IC31(®) adjuvant are strong candidates for further development of immunoprophylaxis and therapy in horses.

A preventive immunization approach against insect bite hypersensitivity: Intralymphatic injection with recombinant allergens in Alum or Alum and monophosphoryl lipid A

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides insects, not indigenous to Iceland. Horses born in Iceland and exported to Culicoides-rich areas are frequently affected with IBH. The aims of the study were to compare immunization with recombinant allergens using the adjuvant aluminum hydroxide (Alum) alone or combined with monophosphoryl lipid A (MPLA) for development of a preventive immunization against IBH. Twelve healthy Icelandic horses were vaccinated intralymphatically three times with 10 μg each of four recombinant Culicoides nubeculosus allergens in Alum or in Alum/MPLA. Injection with allergens in both Alum and Alum/MPLA resulted in significant increase in specific IgG subclasses and IgA against all r-allergens with no significant differences between the adjuvant groups. The induced antibodies from both groups could block binding of allergen specific IgE from IBH affected horses to a similar extent. No IgE-mediated reactions were induced. Allergen-stimulated PBMC from Alum/MPLA horses but not from Alum only horses produced significantly more IFNγ and IL-10 than PBMC from non-vaccinated control horses. In conclusion, intralymphatic administration of small amounts of pure allergens in Alum/MPLA induces high IgG antibody levels and Th1/Treg immune response and is a promising approach for immunoprophylaxis and immunotherapy against IBH.

A preventive immunization approach against insect bite hypersensitivity: Intralymphatic injection with recombinant allergens in Alum or Alum and monophosphoryl lipid A. Vet Immunol Immunopathol

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides insects, not indigenous to Iceland. Horses born in Iceland and exported to Culicoides-rich areas are frequently affected with IBH. The aims of the study were to compare immunization with recombinant allergens using the adjuvant aluminum hydroxide (Alum) alone or combined with monophosphoryl lipid A (MPLA) for development of a preventive immunization against IBH. Twelve healthy Icelandic horses were vaccinated intralymphatically three times with 10 μg each of four recombinant Culicoides nubeculosus allergens in Alum or in Alum/MPLA. Injection with allergens in both Alum and Alum/MPLA resulted in significant increase in specific IgG subclasses and IgA against all r-allergens with no significant differences between the adjuvant groups. The induced antibodies from both groups could block binding of allergen specific IgE from IBH affected horses to a similar extent. No IgE-mediated reactions were induced. Allergen-stimulated PBMC from Alum/MPLA horses but not from Alum only horses produced significantly more IFNγ and IL-10 than PBMC from non-vaccinated control horses. In conclusion, intralymphatic administration of small amounts of pure allergens in Alum/MPLA induces high IgG antibody levels and Th1/Treg immune response and is a promising approach for immunoprophylaxis and immunotherapy against IBH.

Tuneable regioselectivity during the mono-etherification of the 2,3-diol of a mannose derivative

The paper reports selective mono-etherification of the 2-, and 3-hydroxyl groups of methyl 4,6-O-isopropylidene-α-d-mannopyranoside using tin(II) chloride catalysed reactions of diaryldiazomethanes. By the use of different diazo compounds and the variation of the tin(II) chloride concentration the ether formation can be shifted from over 90% 3-selectivity to over 90% 2-selectivity.

Generation of equine TSLP-specific antibodies and their use for detection of TSLP produced by equine keratinocytes and leukocytes

Allergic horses react to innocuous environmental substances by activation of Th2 cells and production of allergen-specific IgE antibodies. The mechanisms leading to Th2 differentiation are not well understood. In humans and mice, epithelial cell-derived thymic stromal lymphopoietin (TSLP) plays a central role in this process. Little is known about equine TSLP (eqTSLP) and its role in allergic diseases and our current knowledge is limited to the assessment of TSLP mRNA expression. In order to be able to study eqTSLP at the protein level, the aim of the present study was to produce recombinant eqTSLP protein and generate TSLP-specific antibodies. EqTSLP was cloned from a skin biopsy sample from a horse with chronic urticaria and eqTSLP protein was expressed in E.coli and in mammalian cells. Recombinant proteins were designed to include C-terminal Histag, which allowed subsequent purification and detection by Histag-specific Ab. Polyclonal and monoclonal eqTSLP-specific Ab were generated after immunization of mice with E.coli-expressed TSLP. Their specificity was tested by western blotting and ELISA. In addition, a commercially available polyclonal human TSLP-specific antibody was tested for cross-reactivity with eqTSLP. Expression of TSLP protein was confirmed by western blotting using Histag-specific Ab. E.coli-expressed TSLP appears as a band of ∼13 kDa, whereas mammalian cell-expressed TSLP showed several bands at 20-25 kDa, probably representing several glycosylation forms. Polyclonal and monoclonal antibodies generated in this study, as well as commercially available human TSLP-specific Ab reacted with both E.coli- and mammalian cell-expressed TSLP in western blotting and ELISA. A capture ELISA was established to quantitate TSLP in cell supernatants and validated using supernatants from primary equine keratinocytes and peripheral blood leukocytes (PBL). Increased TSLP concentrations were found after stimulation of keratinocytes with PMA+ionomycine and with Culicoides extract. Similarly, increased TSLP concentrations were detected in PBL after stimulation with ConA, Culicoides extract, or IgE cross-linking. In conclusion, recombinant TSLP proteins and TSLP-specific antibodies produced in this study will allow further studies of the role of TSLP in equine allergic diseases.