The fate of islets in clinical transplantation is unclear. To elude on this positron emission tomography combined with computed tomography (PET/CT) was performed for 60 min during islet transplantation in five patients receiving six transplants. A fraction of the islets (23%) were labeled with 18F-fluorodeoxyglucose ([(18)F]FDG) and carefully mixed with unlabeled islets just prior to intraportal transplantation. The peak radioactivity concentration in the liver was found at 19 min after start of islet infusion and corresponded to only 75% of what was expected, indicating that islets are lost during the transplantation procedure. No accumulation of radioactivity was found in the lungs. A nonphysiological peak of C-peptide was found in plasma during and immediately after transplantation in all subjects. Distribution in the liver was heterogeneous with wide variations in location and concentration. Islets found in areas with concentrations of >400 IEQ/cc liver tissue varied between 1% and 32% of the graft in different subjects. No side effects attributed to the PET/CT procedure were found. Clinical outcome in all patients was comparable to that previously observed indicating that the [(18)F]FDG labeling procedure did not harm the islets. The technique has potential to be used to assess approaches to enhance islet survival and engraftment in clinical transplantation.

Introduction. Occupancy-over-time was determined for two dopamine transporter (DAT) inhibitors through modeling of their ability to displace the PET ligand [C-11]CIT. The tracer was held at a pseudo steady state in a reference tissue by target controlled infusion. Methods. Rhesus monkeys (n = 5) were given [C-11]CIT and studied with a PET scanner. Tracer uptake in the reference tissue cerebellum was held at a pseudo steady state by use of target controlled infusion. The pharmacokinetics/pharmacodynamics (PK/PD) of [C-11]CIT was assessed through the simplified reference tissue model (SRTM). Bupropion (n = 2) and GBR-12909 (n = 2) receptor occupancies were estimated through modeling of their effects on [C-11]CIT displacement. Results. There was a high uptake of [C-11]CIT in striatum, which contains a high DAT density. The reference tissue cerebellum had a comparatively low uptake. The modeling of [C-11]CIT PK/PD properties in striatum showed high binding potential (BP = 5.34 +/- 0.78). Both DAT inhibitors caused immediate displacement of [C-11]CIT after administration. The occupancy-over-time was modeled as a mono-exponential function, describing initial maximal occupancy (Occ(0)) and rate of ligand-receptor dissociation (k(off)). GBR-12909 showed irreversible binding (k(off) = 0) after an initial occupancy of 76.1%. Bupropion had a higher initial occupancy (84.5%) followed by a release half-life of 33 minutes (k(off) = 0.021). Conclusions. The proposed model can be used for assessment of in-vivo occupancy-over-time of DAT ligands by use of target controlled infusion of [C-11]CIT. The concept of assessing drug-receptor interactions by studying perturbations of a PET tracer from a pseudo steady state can be transferred to other CNS systems.

BACKGROUND:

A computer controlled infusion pump (UIPump) for regulation of target tissue concentration of radioactive compounds was developed for use in biological research and tracer development for PET.

METHODS:

Based on observed tissue or plasma kinetics after a bolus injection of the tracer an algorithm calculates the infusion needed to obtain a specified target kinetic curve. A computer feeds this infusion scheme into an infusion pump connected to an animal via a venous catheter. The concept was validated using [¹¹C]Flumazenil administrated to Sprague-Dawley rats where the whole brain distribution and kinetic of the tracer was measured over time using a microPET-scanner. The accuracy and precision of the system was assessed by producing steady-state levels of the tracer and by mimicking kinetics after oral administration.

RESULTS:

Various kinetic profiles could be generated, including rapid achievement of constant levels, or step-wise increased levels. The resulting tissue curves had low deviation from the target curves according to the specified criteria: AUC (%): 4.2 +/- 2.8, Maximal deviation (%): 13.6 +/- 5.0 and R²: 0.95 +/- 0.02.

CONCLUSION:

The UIPump-system is suitable for use in PET-research for assessment of PK/PD properties by simulation of different tracer tissue kinetics in vivo.

Triorganylsulfonium, -selenonium and -telluronium salts were reduced by carbon dioxide radical anions/solvated electrons produced in aqueous solution by radiolysis. The radical expulsion accompanying reduction occurred with the expected leaving group propensities (benzyl > secondary alkyl> primary alkyl> methy> phenyl), although greater than expected loss of the phenyl group was often observed. Diorganyl chalcogenides formed in the reductions were conveniently isolated by extraction with an organic solvent. Product yields based on the amount of reducing radicals obtained from the y-source were often higher than stoichiometric (up to 1800%) in the reduction of selenonium an dtelluronium compounds; it is likely that this result can be accounted for in terms of a chain reaction with carbon-centred radicals/formate serving as the chain transfer agent. The product distribution was essentially independent of the reducing species for diphenyl alkyl telluronium salts, whereas significant variations were seen for some of the corresponding selenonium salts. This would suggest the intermediacy of telluranyl radicals in the one-electron reduction of telluronium salts. However, pulse radiolysis experiments indicated that the lifetimes of such a species (the triphenyltelluranyl radical) would have to be less than 1 us.

Tripeptidyl-peptidase II (TPP II) is a cytosolic peptidase that has been implicated in fat formation and cancer, apparently independent of the enzymatic activity. In search for alternative functional regions, conserved motifs were identified and eleven signatures were constructed. Seven of the signatures covered previously investigated residues, whereas the functional importance of the other motifs is unknown. This provides directions for future investigations of alternative activities of TPP II. The obtained signatures provide an efficient bioinformatic tool for the identification of TPP II homologues. Hence, a TPP II sequence homologue from fission yeast, Schizosaccharomyces pombe, was identified and demonstrated to encode the TPP II-like protein previously reported as multicorn. Furthermore, an homologous protein was found in the prokaryote Blastopirellula marina, albeit the TPP II function was apparently not conserved. This gene is probably the result of a rare gene transfer from eukaryote to prokaryote.

This thesis deals with the synthesis and development of ¹⁸F-labelled alkyl etomidate and vorozole analogues, and their use as positron emission tomography (PET) tracers for the imaging of the steroid enzymes 11β-hydroxylase and aromatase. Two synthetic ¹⁸F-labelling approaches to the etomidate and vorozole analogues were developed, and the analogues were evaluated in some biological assays.

The two-step labelling method was used to synthesise many compounds for biological evaluation. In the first step, a ¹⁸F-labelled intermediate based on a ditosylate or a halogenated diethyl ether was synthesised and used directly in the next alkylation step. The decay-corrected (d.c.) radiochemical yield was higher compared to other known two-step labelling methods.

Once an appropriate candidate has been chosen for clinical evaluation, a one-step labelling method will be more suitable. We therefore developed a method based on precursors that had leaving groups at the end of their alkyl chains, and used these directly in the ¹⁸F-labelling synthesis. The one-step ¹⁸F-labelling synthesis required less reaction time and produced higher specific radioactivity and d.c. radiochemical yield than our two-step synthesis. With microwave heating, the reaction time was reduced to seconds and the d.c. radiochemical yield was better than that obtained with conventional heating. The one-step synthesis simplified the technical handling by allowing the tracer syntheses to be automated on the TRACERLab FX_FN.

By replacing the alkyl chain in a metomidate ester with F-18-labelled   di- or tri(ethylene glycol) chains, two F-18-labelled PET tracers, i.e.  2-(2-[F-18]fluoroethoxy)ethyl 1-[(1R)-1-phenylethyll-1   H-imidazole-5-carboxylate (1) and   2-[2-(2-[F-18]fluoroethoxy)-ethoxylethyl   1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (2), were   synthesized. Two precursors, 2-(2-bromoethoxy)ethyl  1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate and   2-[2-(2-chloroethoxy)ethoxylethyl  1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate, were prepared and   used in one-step nucleophilic [F-18]fluorination reactions using   conventional and microwave heating. Organ distribution, frozen section   autoradiography and metabolite analysis were performed. The   decay-corrected radiochemical yields of 1 and 2 were 26 +/- 8 and 23   +/- 8%, respectively, when they were prepared using conventional   heating. By performing microwave heating, the reaction time could be   decreased and the yields of analogues 1 and 2 could be increased to 57   +/- 12 and 51 +/- 18%, respectively. Organ distribution studies in the   rat showed considerable uptake in the lungs, adrenals and liver. Both   compounds bound with low nonspecific binding (1: approx. 20-30%; 2:   2.9% or lower) to tissue from pig and human normal and pathologic   adrenals. Metabolite analyses were performed in rats after 5 and 30 min   for tracer 1 (20 +/- 6 and 2 +/- 1 %) and tracer 2 (27 +/- 5 and 6 +/-   4%). Both compounds are interesting candidates for the detection of   different types of adrenal disorders.

Introduction: Two- and one-step syntheses of ¹⁸F-labelled analogues of Metomidate, such as 2-[¹⁸F]fluoroethyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (1), 2-[¹⁸F]fluoroethyl 1-[(1R)-1-(4-chlorophenyl)ethyl]-1H-imidazole-5-carboxylate (2), 2-[¹⁸F]fluoroethyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate (3), 3-[¹⁸F]fluoropropyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate (4) and 3-[¹⁸F]fluoropropyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate (5) are presented.

Methods: Analogues 1-5 were prepared by a two-step reaction sequence that started with the synthesis of either 2-[¹⁸F]fluoroethyl 4-methylbenzenesulfonate or 3-[¹⁸F]fluoropropyl 4-methylbenzenesulfonate. These were used as ¹⁸F-alkylating agents in the second step, in which they reacted with the ammonium salt of a 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylic acid. One-step-labelling syntheses of 1, 2 and 5 were also explored. Analogues 1-4 were biological validated by frozen-section autoradiography and organ distribution. Metabolite analysis was performed for 2 and 3.

Results: The radiochemical yield of the two-step synthesis was in the range of 10-29%, and thatof the one-step synthesis was 25-37%. Using microwave irradiation in the one-step synthesis of 1 and 2 increased the radiochemical yield to 46 ± 3 and 79 ± 30%, respectively.

Conclusion: Both the frozen-section autoradiography and organ distribution results indicated that analogue 2 has a potential as an adrenocortical imaging agent, having the highest degree of specific adrenal binding and best ratio of adrenal to organ uptake among the compounds studied.

One- and two-step syntheses for the F-18-labelling of 6-[(S)-(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-(2-[F-18]fluoroe thyl)-1H-benzotriazole, [F-18]FVOZ, 1 and 6-[(S)-(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1-[2-(2-[18F]fluor oethoxy)ethyl]-1H-benzotriazole, [F-18]FVOO, 2 were developed. In the two-step synthesis, the nucleophilic fluorination step was performed by reacting (S)-6-[(4-chlorophenyl)-(1H-1,2,4-triazol-1-yl)methyl]-1H-benzotriazole (VOZ) with either the F-18-labelled ethane-1,2-diyl bis(4-methylbenzenesulfonate) or the oxydiethane-2,1-diyl bis(4-methylbenzenesulfonate). The radiochemical yields were in the range of 9-13% after the 110-120 min total syntheses and the specific radioactivities were 175 +/- 7 GBq/mu mol and 56 GBq/mu mol for compounds 1 and 2, respectively. In the one-step synthesis, the precursor 2-{6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-1,2,3-benzotriaz ol-1-yl}ethyl 4-methylbenzenesulfonate (7) or 1-[2-(2-bromoethoxy)ethyl]-6-[(4-chlorophenyl)(1H-1,2,4-triazol-1-yl)met hyl]-1H-benzotriazole (8) was directly labelled via, an 18F nucleophilic substitution to give the corresponding tracer. The labelled compounds were obtained in 36-99% radioichemical yield after 75-min syntheses. The specific radioactivities are 100 GBq/mu mol for compound 1 and 80 GBq/pmol for compound 2. In vitro autoradiography using frozen rat brains illustrated specific binding in the medial amygdala, the bed nucleus of stria terminalis and the preoptic area, all of which corresponded well to the result of C-11-labelled vorozole.

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide, and antagonism of NPY Y1 and NPY Y5 receptors (NPYxR) is considered a potentially important anti-obesity drug target. We tested the hypothesis that blockade of the NPY5R will lead to weight loss in humans using MIK-0557, a potent, highly selective, orally active NPY5R antagonist. The initial series of experiments reported herein, including a multiple-dose positron-emission tomography study and a 12 week proof-of concept/dose-ranging study, suggested an optimal MK-0557 dose of 1 mg/day. The hypothesis was then tested in a 52 week, multicenter, randomized, double-blind, placebo-controlled trial involving 1661 overweight and obese patients. Although statistically significant at 52 weeks, the magnitude of induced weight loss was not clinically meaningful. These observations provide the first clinical insight into the human NPY-energy homeostatic pathway and suggest that solely targeting the NPY5R in future drug development programs is unlikely to produce therapeutic efficacy.

Subtle changes in the second coordination sphere of [Cl(2)bdtFe(2)-(CO)(4)(Ph2P-CH2-X-CH2-PPh2)] (bdt = benzene-1,2-dithiolate, X = NCH3, NCH2CF3, CH2) that do not influence the electronic character of the Fe-2 center can however direct protonation to three different sites: the N in the bis-phosphane, the Fe-Fe bond or the bdt-S.

The secondary a- and B-deuterium, the a-carbon, the nucleophile carbon, the nucleophile nitrogen, and the chlorine leaving group kinetic isotope effects forthe SN2 reaction between cyanide ion an dethyl chloride were determined in the very slightly polar solvent THF at 30 C. A comparison of these KIEs with those reported earlier for the same reaction in the polar solvent DMSO shows that the transition state in THF is only sligthly tighter with very slightly shorter NC-Ca-CI bonds. This minor change in transition state structure does not account for the different transition structures that were earlier suggested by interpreting the experimental KIEs and the gas-phase calculations, respectively. It therefore seems unlikely that the different transition states suggested by the two methods are due to the lack of appropriate solvent modeling in the theoretical calculations. Previously it was predicted that the transition state of SN2 reactions where the nucleophile and the leaving group have the same charge would be unaffected by a charge in solvent. The experimental KIEs support this view.

Miniaturization of analytical separation methods offers several advantages, including short run times, high resolution, and high recovery of the sample constituents. To optimize these parameters, the reversible adsorption (to minimize loss in resolution), as well as the irreversible adsorption (to minimize loss of analytes) must be quantified. However, no useful equation is available for the calculation of the variance of reversible adsorption. Therefore, we have taken another approach to quantify the reversible interaction. The method is unique and important since no equation for calculation of this variance is required. Instead, two experiments are required, which are run under such conditions that the variance of a certain parameter has the same numerical value in the two experiments (one with and without EOF), except for the variance of reversible adsorption. The approach is universal in the sense that it can be used for many Is different mathematical concepts and be modified to also cover certain functions other than a sum of parameters. We have also introduced a simple expression for the irreversible adsorption, which shows that the hydrophobic interaction from only two methyl groups in the coating gives rise to as much as 40-50% loss of protein, and the width of the zones in the capillary with this coating was 8-15% larger compared to the zone width in the polyacrylamide-coated capillaries. The reproducibility in migration time, peak area, and peak width in two consecutive runs in capillaries with two methyl groups in the coating was very low, but in EOF-free polyacrylamide-coated capillaries extremely high, indicating that the reversible and irreversible adsorption of proteins to this coating is negligible. The scanning detector, frequently used in free zone electrophoresis in the 1960s-1970s, gives true separation parameters and is, therefore, much preferable to the stationary detector used in most CE experiments, because this detector gives apparent separation parameters.

Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. This thesis contributes to knowledge about glutathione transferases from Sus scrofa (pig). The study is needed for better understanding of biochemical processes in this species and is desirable for drug development, for food industry research and in medicine.

A primary role of GSTs is detoxication of electrophilic compounds. Our study presents porcine GST A1-1 as a detoxication enzyme expressed in many tissues, in particular adipose tissue, liver and pituitary gland. Based on comparison of activity and expression profiles, this enzyme can be expected to function in vivo similarly to human GST A2-2 (Paper II).

In addition to its protective function, human GST A3-3 is an efficient steroid isomerase and contributes to the biosynthesis of steroid hormones in vivo. We characterized a porcine enzyme, pGST A2-2, displaying high steroid-isomerase activity and resembling hGST A3-3 in other properties as well. High levels of pGST A2-2 expression were found in ovary, testis and liver. The properties of porcine enzyme strengthen the notion that particular GSTs play an important role in steroidogenesis (Paper I).

Combination of time-dependent and enzyme concentration-dependent losses of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurements of GSTs. Enzyme adsorption to surfaces was found to be the main explanation of high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Several approaches to improved functional comparison of highly active GSTs were proposed (Paper III).

Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets. We have purified catalytically active human PHD3 after heterologous expression in Escherichia coli. Histidine-tagged enzyme was isolated as monomer by immobilized Ni-affinity chromatography followed by gel filtration. Overexpression of bacterial chaperonins GroEL/ES at 30 °C substantially increased the yield of soluble PHD3. High concentrations of salt and reducing agent during purification prevented protein aggregation. The enzyme activity with peptide derived from HIF-1α was inhibited by Zn²⁺, desferrioxamine and imidazole. The hydroxylation activity was verified by mass spectrometry, and Pro567 in HIF-1α was discovered as a new site of hydroxylation.

Glutathione transferases (GSTs, EC 2.5.1.18) possess multiple functions and have potential applications in biotechnology. Direct evidence of underestimation of activity of human GST A3-3 and porcine GST A2-2 measured at submicromolar enzyme concentrations is reported here for the first time. Combination of time-dependent and enzyme concentration-dependent loss of activity as well as the choice of the organic solvent for substrates were found to cause irreproducibility of activity measurementsof GSTs. These effects contribute to high variability of activity values of porcine GST A2-2 and human Alpha-class GSTs reported in the literature. Adsorption of GSTs to surfaces was found to be the main explanation of the observed phenomena. Several approaches to improved functional comparison of highly active GSTs are proposed.

An Alpha-class glutathione transferase (GST) has been cloned from pig gonads. In addition to two conservative point mutations our nucleotide sequence presents a frame shift resulting from a missing A as compared to a previously published porcine GST A1-1 sequence. The deduced C-terminal amino-acid segment of the protein differs between the two variants. Repeated sequencing of cDNA isolated from different tissuesand animals ruled out the possibility of a cloning artifact, and the deduced amino acid sequence ofour clone showed higher similarity to related mammalian GST sequences. Hereafter, we refer to ourcloned enzyme as GST A1-1 and to the previously published enzyme as GST A1-1*. The study of the tissue distribution of the GSTA1 mRNA revealed high expression levels in many organs, in particular adipose tissue, liver, and pituitary gland. Porcine GST A1-1 was expressed in Escherichia coli and its kinetic properties were determined using alternative substrates. The catalytic activity in steroid isomerization reactionswas at least 10-fold lower than the corresponding values for porcine GST A2-2, whereas the activity with 1-chloro-2,4-dinitrobenzene was approximately 8-fold higher. Differences in the H-site residues of mammalian Alpha-class GSTs may explain the catalytic divergence.

A primary role of GSTs (glutathione transferases) is detoxication of electrophilic compounds. In addition to this protective function, hGST (human GST) A3-3, a member of the Alpha class of soluble GSTs, has prominent steroid double-bond isomerase activity. The isomerase reaction is an obligatory step in the biosynthesis of steroid hormones, indicating a special role of hGST A3-3 in steroidogenic tissues. An analogous GST with high steroid isomerase activity has so far not been found in any other biological species. In the present study, we characterized a Sus scrofa (pig) enzyme, pGST A2-2, displaying high steroid isomerase activity. High levels of pGST A2-2 expression were found in ovary, testis and liver. In its functional properties, other than steroid isomerization, pGST A2-2 was most similar to hGST A3-3. The properties of the novel porcine enzyme lend support to the notion that particular GSTs play an important role in steroidogenesis.

Glutathione S-transferases (GSTs) are a group of multifunctional enzymes that are found in animals, plants and microorganisms. Their primary function is to remove toxins derived from exogenous sources or the products of metabolism from the cell. Mammalian GSTs have been extensively studied, in contrast to bacterial GSTs which have received relatively scant attention. A new class of GSTs called Chi has recently been identified in cyanobacteria. Chi GSTs exhibit a high glutathionylation activity towards isothiocyanates, compounds that are normally found in plants. Here, the crystallization of two GSTs are presented: TeGST produced by Thermosynechococcus elongates BP-1 and SeGST from Synechococcus elongates PCC 6301. Both enzymes formed crystals that diffracted to high resolution and appeared to be suitable for further X-ray diffraction studies. The structures of these GSTs may shed further light on the evolution of GST catalytic activity and in particular why these enzymes possess catalytic activity towards plant antimicrobial compounds.

Carbohydrate binding modules (CBMs) are noncatalytic substrate binding domains of many enzymes involved in carbohydrate metabolism. Here we used fluorescent labeled recombinant CBMs specific for crystalline cellulose (CBM1_HjCel7A) and mannans (CBM27_TmMan5 and CBM35_CjMan5C) to analyze the complex surfaces of wood tissues and pulp fibers. The crystalline cellulose CBM1_HjCel7A was found as a reliable marker of both bacterially produced and plant G-layer cellulose, and labeling of spruce pulp fibers with CBM1_HjCel7A revealed a signal that increased with degree of fiber damage. The mannan-specific CBM27_TmMan5 and CBM35_CjMan5C CBMs were found to be more specific reagents than a monoclonal antibody specific for (1→4)-β-mannan/galacto-(1→4)-β-mannan for mapping carbohydrates on native substrates. We have developed a quantitative fluorometric method for analysis of crystalline cellulose accumulation on fiber surfaces and shown a quantitative difference in crystalline cellulose binding sites in differently processed pulp fibers. Our results indicated that CBMs provide useful, novel tools for monitoring changes in carbohydrate content of nonuniform substrate surfaces, for example, during wood or pulping processes and possibly fiber biosynthesis.

The geometries of 35 conformers of Me(SiMe2) nMe (n= 4, 1; n = 5, 2; n = 6, 3; n = 7, 4) were optimized at the MP2/VTDZ level, and CCSD(T) single-point calculations were done at three MP2/VTDZ conformer geometries of 1. The relative ground-state energies of the conformers of 1-4 in the gas phase were obtained from the MP2/VTDZ electronic energy, zero- point vibrational energy, and thermal corrections at 0, 77, and 298 K. Relative energies in an alkane solvent at 77 and 298 K were obtained by the addition of solvation energies, obtained from the SM5.42R model. The calculated energies of 26 of the conformers (n = 4-6) have been least-squares fitted to a set of 15 additive increments associated with each Si-Si bond conformation and each pair of adjacent bond conformations, with mean deviations of 0.06-0.20 kcal/mol. An even better fit for the energies of 24 conformers (mean deviations, 0.01-0.09 kcal/mol) has been obtained with a larger set of 19 increments, which also contained contributions from selected combinations of conformations of three adjacent bonds. The utility of the additive increments for the prediction of relative conformer energies in the gas phase and in solution has been tested on the remaining nine conformers (n = 6, 7). With the improved increment set, the average deviation from the SM5.42R//MP2 results for solvated conformers at 298 K was 0.18 kcal/mol, and the maximum error was 0.98 kcal/mol.

PURPOSE: For the treatment of cancer, the therapeutic potential of short-range, low-energy Auger-electron emitters, such as (125)I, is getting progressively wider recognition. The potency of Auger-electron emitters is strongly dependent on their location in close vicinity to DNA. We have developed a new two-step targeting strategy to transport (125)I into cancer-cell nuclei using PEG-stabilized tumour-cell targeting liposomes named "Nuclisome-particles". METHODS: In the present study, epidermal growth factor (EGF) was used as a tumour-cell-specific agent to target the EGF-receptor (EGFR) and the liposomes were loaded with (125)I-Comp1, a recently synthesized daunorubicin derivative. RESULTS: As analysed with cryo-TEM, the derivative precipitates inside liposomes at a drug-to-lipid molar ratio of 0.05:1. Receptor-specific uptake in cultured U-343MGaCl2:6 tumour cells of EGFR-targeting liposomes increased with time while non-specific and receptor-blocked uptake remained low. Nuclisome-particles were able to target single U-343MGaCl2:6 cells circulating in human blood during 4 h, with low uptake in white blood cells, as demonstrated in an ex vivo system using a Chandler loop. Autoradiography of targeted cells indicates that the grains from the radiolabelled drug are mainly co-localized with the cell nuclei. The successful targeting of the nucleus is shown to provide high-potency cell killing of cultured U-343MGaCl2:6 cells. At the concentration used, Nuclisome-particles were up to five orders of magnitude more effective in cell killing than EGFR-targeting liposomes loaded with doxorubicin. CONCLUSION: The results thus provide encouraging evidence that our two-step targeting strategy for tumour cell DNA has the potential to become an effective therapy against metastasizing cancer cells in the bloodstream.

Background:

New in vivo amyloid PET imaging tracers, such as 11C-PIB, provide possibilities to deeper understand the underlying pathological processes in Alzheimers disease (AD). In this study we investigated how 11C-PIB retention is related to cerebral glucose metabolism, episodic memory and CSF biomarkers.

Method:

Thirty-seven patients with mild AD and 21 patients with mild cognitive impairment (MCI) underwent PET examinations with the amyloid tracer 11C-PIB, 18F-FDG for measurement of regional cerebral metabolic rate of glucose (rCMRglc), assessment of episodic memory and assay of cerebral spinal fluid (CSF) levels of amyloid-ß (Aβ1-42), total tau and phosphorylated tau respectively. Analyses were performed using Statistical Parametric Mapping (SPM) and regions of interest (ROIs).

Results:

Pooled data from AD and MCI patients showed strong correlations between 11C-PIB retention, levels of CSF biomarkers (especially Aß1-42), rCMRglc and episodic memory. Analysis of the MCI group alone revealed significant correlations between 11C-PIB retention and CSF biomarkers and between CSF biomarkers and episodic memory respectively. A strong correlation was observed in the AD group between rCMRglc and episodic memory as well as a significant correlation between 11C-PIB retention and rCMRglc in some cortical regions. Regional differences were observed as sign for changes in temporal patterns across brain regions.

Conclusions:

A complex pattern was observed between pathological and functional markers with respect to disease stage (MCI versus AD) and brain regions. Regional differences over time were evident during disease progression. 11C-PIB PET and CSF Aß1-42 allowed detection of prodromal stages of AD. Amyloid imaging is useful for early diagnosis and evaluation of new therapeutic interventions in AD.

mild cognitive impairment, converters, amyloid, PET, PIB, FDG, CSF biomarkers/k It is of great clinical value to identify Subjects at a high risk of developing AD. We previously found that the amyloid positron emission tomography (PET) tracer PIB showed a robust difference in retention in the brain between AD patients and healthy controls (HC). Twenty-one patients diagnosed with MCI (mean age 63.3 +/- 7.8 (S.D.) years) Underwent PET Studies with C-11-PIB, and F-18-fluoro-deoxy-glucose (FDG) to measure cerebral glucose metabolism, its well as assessment of cognitive function and CSF sampling. Reference group data from 27 AD patients and 6 healthy controls, respectively. were Used for comparison. The mean cortical PIB retention for the MCI patients was intermediate compared to HC and AD. Seven MCI patients that later Lit clinical follow-up converted to AD (8.1 +/- 6.0 (S.D.) months) showed significant higher PI B retention compared to non-converting MCI patients and HC, respcctively (ps < , 0.01). The PIB retention in MCI converters was comparable to AD patients (p > , 0.01). Correlations were observed in the MCI patients between PI B retention and CSF A beta(1-42). total Tau and episodic memory, respectively.

Innovation in basic and applied science has brought radiotracers to fruition as diagnostics. Non-invasive, longitudinal, and quantifiable molecular imaging is the key to diagnosing and monitoring numerous illnesses, with more to come from characterization of the clinical relevance of findings from genomics research. Radiotracers enable real-time in vivo studies of the effects of drug candidates on receptors, pathways, pharmacodynamics, and clinically relevant endpoints, thereby providing both early detection of pathophysiology to enable early intervention, and then monitoring of treatment responses to enable individualization of treatment regimens. We review developments which have translated imaging from bench to bedside, or biomarkers to diagnostics. Notable developments include (1) synthesis methods for rapid ¹¹C labeling of biomolecules to high specific radioactivity; (2) ligand-binding assays for screening molecular imaging agents rather than drugs; (3) in vivo imaging of radiotracers in animals; (4) discovering the imaging advantages of ^99mTc, ¹¹C, and ¹⁸F; (5) co-registration and automated quantitative assessment of high spatial resolution CT and MR images with molecular images from PET for longitudinal studies of treatment effect.

The preparation of cis-1α-acetoxy-7-methoxy-1,2,3,4,4a,10a-hexahydro-9(10H)- phenanthrenone 5 was accomplished starting from 6-methoxy-1-tetralone. Reduction of 7-methoxy-1,2,3, 4,9,10-hexahydro-1-oxo-phenanthrene 8, acetylation and subsequent oxidation delivered 5. Application of an analogus procedure to the preparation of cis-1β-acetoxy-5-methoxy-1,2,3,4,,4a,10a-hexahydro-9(10H)- phenanthrenone 6 was not feasible. A more elaborate route was developed for the synthesis of compound 6, where an epoxide-arene reaction involving a 1,2-alkyl shift rearrangement, constituted a highly selective key transformation.

The compounds 5 and 6 were prepared. A route was developed for the synthesis of compound 6, where an epoxide-arene reaction involving a 1,2-alkyl shift rearrangement, constituted a highly selective key transformation.

An SPR biosensor-based assay for studies of the interactions with full-length NS3 protease from hepatitis C virus (HCV) has been developed. It was used to characterize the interaction kinetics for a series of NS3 protease inhibitors. Moreover, the interaction between NS3 and the NS4A cofactor could be studied.

The K_D of the NS3-NS4A interaction was 30 nM in the standard buffer. It was reduced 600-fold by increasing the ionic strength to 300 mM NaCl. By using surfaces with only NS3 or with NS3 and co-immobilised NS4A, the effect of this protein cofactor on the interaction with several protease inhibitors was investigated. NS4A increased the affinity for all compounds, between 2 to 40 times, indicating that the NS3-NS4A complex binds inhibitors better than only NS3. The obtained interaction data was also compared with inhibition data, revealing a very good correlation between k_off or K_D with K_i (r = 0.92 and r = 0.90 respectively) over a broad range of affinities and potencies, showing that this biosensor based assay is a good and powerful tool for detailed studies of NS3 protease inhibitors which can serve as a future cure for HCV infection.

The mechanism and kinetics of the interactions between ligands and immobilized full-length hepatitis C virus (HCV) genotype 1a NS3 have been characterized by SPR biosensor technology. The NS3 interactions for a series of NS3 protease inhibitors as well as for the NS4A cofactor, represented by a peptide corresponding to the sequence interacting with the enzyme, were found to be heterogeneous. It may represent interactions with two stable conformations of the protein. The NS3-NS4A interaction consisted of a high-affinity (K(D) = 50 nM) and a low-affinity (K(D) = 2 µM) interaction, contributing equally to the overall binding. By immobilizing NS3 alone or together with NS4A it was shown that all inhibitors had a higher affinity for NS3 in the presence of NS4A. NS4A thus has a direct effect on the binding of inhibitors to NS3 and not only on catalysis. As predicted, the mechanism-based inhibitor VX 950 exhibited a time-dependent interaction with a slow formation of a stable complex. BILN 2061 or ITMN-191 showed no signs of time-dependent interactions, but ITMN-191 had the highest affinity of the tested compounds, with both the slowest dissociation (k(off)) and fastest association rate, closely followed by BILN 2061. The k(off) for the inhibitors correlated strongly with their NS3 protease inhibitory effect as well as with their effect on replication of viral proteins in replicon cell cultures, confirming the relevance of the kinetic data. This approach for obtaining kinetic and mechanistic data for NS3 protease inhibitor and cofactor interactions is expected to be of importance for understanding the characteristics of HCV NS3 functionality as well as for anti-HCV lead discovery and optimization.

Kinetic, mechanistic and chemodynamic aspects of the interaction between five non-nucleoside inhibitors and the HCV NS5B polymerase (genotype 2a) were assessed using SPR biosensor technology. The compounds were selected to represent different structural classes (benzothiadiazine, , α,γ-diketo acid, benzimidazole, thiophene carbocyclic acid and benzofuran), each known to interact with different binding sites. The viral polymerase interacted with the compounds with different kinetics and surprisingly also with different capacities. Cooperativity between the different allosteric inhibitor binding sites and the active site binding diketoacid was observed, but no cooperativity was seen between the allosteric sites. The interaction with diketoacid was stronger in phosphate buffer as compared to Tris buffer, indicating a phosphate ion-mediated interaction mechanism. The enzyme generally had reduced affinity for the inhibitors in the presence of RNA. Interaction parameters determined for human serum albumin revealed the propensity of the compounds to be distributed by HSA. This study provides important information for the design of optimized NS5B inhibitors and illustrates the complementarity of a biosensor-based analysis with inhibition studies, in particular for allosteric compounds with complex interaction mechanisms or when the target contains multiple ligand binding sites.

The thermodynamics of the interaction between mutant HIV-1 reverse transcriptase (K103N and Y181C) and a nonnucleoside reverse transcriptase inhibitor (NNRTI), the phenylethylthiazolylurea compound MIV-150, was obtained by determining the temperature dependence of the kinetic rate constants. Large entropic changes in the forward and backward steps of the isomerization between a non-binding competent and a binding competent conformation of the enzyme, as well as in the binding steps, implied the involvement of major structural rearrangements upon interaction with the inhibitor. Despite of the entropic character of the overall interaction, the equilibrium for the binding of inhibitor was found to be predominantly enthalpy-driven. The high affinity and the low affinity interactions of the heterogeneously interacting inhibitor showed different energetics in the analysis, revealing an expectedly higher enthalpic component for the high-affinity interaction. The thermodynamic profiles of the two enzyme variants displayed significant differences, which could not be derived from their kinetics at a single temperature.

A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).

The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein, and acetylcholine, nicotine, epibatidine and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model and a structural interpretation of the interaction was obtained by molecular modelling involving induced-fit and loop flexibility simulations. The data suggests that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.

Details of the interaction between HIV-1 reverse transcriptase and non-nucleoside inhibitors (NNRTIs) have been elucidated using a biosensor-based approach. This initial study was performed with HIV-1 reverse transcriptase mutant K103N, the phenethylthioazolylthiourea compound (PETT) MIV-150, and the three NNRTIs licensed for clinical use: nevirapine, delavirdine, and efavirenz. Mathematical evaluation of the experimental data with several interaction models revealed that the four inhibitors interacted with HIV-1 RT with varying degrees of complexity. The simplest adequate model accounted for two different conformations of the free enzyme, of which only one can bind the inhibitor, consistent with a previously hypothesized population-shift model including a preformation of the NNRTI binding site. In addition, a heterogeneous binding was observed for delavirdine, efavirenz, and MIV-150, indicating that two noncompetitive and kinetically distinct enzyme-inhibitor complexes could be formed. Furthermore, for these compounds, there were indications for ligand-induced conformational changes.

Two 1,3,5-trisubstituted aromatic scaffolds intended to serve as γ-turn mimetics have been synthesized and incorporated in five pseudopeptide analogues of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), replacing Val-Tyr-Ile, Val-Tyr, or Tyr-Ile. All the tested compounds exhibited nanomolar affinity for the AT₂ receptor with the best compound (3) having a K_i of 1.85 nM. Four pseudopeptides were AT₂ selective, while one (5) also exhibited good affinity for the AT₁ receptor (K_i = 30.3 nM). This pseudopeptide exerted full agonistic activity in an AT₂ receptor induced neurite outgrowth assay but displayed no agonistic effect in an AT₁ receptor functional assay. Molecular modeling, using the program DISCOtech, showed that the high-affinity ligands could interact similarly with the AT₂ receptor as other ligands with high affinity for this receptor. A tentative agonist model is proposed for AT₂ receptor activation by angiotensin II analogues. We conclude that the 1,3,5-trisubstituted benzene rings can be conveniently prepared and are suitable as γ-turn mimics.

High selectivity of a biomarker is a basic requirement when it is used for diagnosis, prognosis and treatment of a disease. The artificial gel antibodies, which we synthesise by a molecular imprinting method, have this property not only for proteins, but also for bioparticles, such as viruses and bacteria. However, diagnosis of a disease requires not only that the biomarker can be "fished out" from a body fluid with high selectivity, but also that its concentration in the sample can rapidly be determined and preferably by a simple technique. This paper deals primarily with the development of a spectrophotometric method, which is so simple and fast that it can be used with advantage in a Doctor's Office. The development of this method was not straight-forward. However, by modifications of the performance of these measurements we can now design standard curves in the form of a straight line, when we plot the true (not the recorded "apparent" absorption) against known protein concentrations. In an additional publication (see the following paper in this issue of JSS) we show an application of such a plot: determination of the concentration of albumin in serum and cerebrospinal fluid from patients with neurological disorders to investigate whether albumin is a biomarker for these diseases.

We have previously used the molecular-imprinting method for the synthesis of artificial gel antibodies, highly selective for various proteins. In the present work, we have synthesized artificial gel antibodies against human albumin with the aim to develop a simple and rapid procedure to measure the concentration of this protein in samples of clinical interest. The procedure, based on the design of a standard curve (see the preceding paper), was applied on a quantitative analysis of albumin in human plasma and cerebrospinal fluid (CSF). We found that our technique permitted detection of albumin in these body fluids with high precision and that the concentration of this protein was significantly enhanced in CSF from patients with amyotrophic lateral sclerosis (ALS), compared to control samples. This finding is in agreement with results from earlier studies, which confirms the validity of our analysis technique and suggests that the barrier permeability may be affected in ALS, perhaps also for other proteins. No enhancement in plasma levels of albumin was seen in patients with ALS, but rather a decrease. The results further indicate that our approach might also apply well to other biomarkers for the actual neurological disease and other disorders.

Although protein biomarkers have a great potential as biomarkers for diagnosis of diseases, they are seldom used in hospitals. There are many reasons for this, for instance, the difficulties to (i) find a biomarker for which the concentration in body fluids clearly differs between patients and healthy subjects, (ii) attain purification of the biomarker close to 100%, which is required for production of conventional protein antibodies as well as artificial gel antibodies for selective capture of a biomarker, (iii) design a standard curve for rapid and accurate determination of the concentration of the biomarker in the body fluid because of adsorption of the biomarker onto vials, pipettes, etc., (iv) determine accurately the sample volume delivered by a pipette, (v) avoid polymerization of the biomarker upon storage and to decide whether it is in the form not only of monomers, but also of dimers, trimers, etc., in the native state, (vi) determine the degree of possible glycosylation and amidation of the biomarker and (vii) decide whether glycosylation and amidation positively or negatively affects the possibility to use the protein as a biomarker. In this article, we discuss in quantitative terms the difficulties (iii-vii) and how to overcome them, which also may help to overcome the difficulty (ti), which in turn minimizes difficulty (i).

A method for assignment of the relative stereochemistry in acyclic pi-allyl)palladium complexes by H-1 NMR H-H coupling constants has been developed. It is based on the introduction of nitrogen chelating ligands of the bipyridyl type into the complexes. The analytical suitability of several other types of nitrogen chelating ligands has also been investigated. A model for rationalization of the observed relation between stereochemistry and spectral parameters is proposed. Introduction of the chelating ligand also affects the syn,anti equilibrium of the complexes. Isomer ratios depend upon the relative stereochemistry of the side chain as well as on the chelating ligand.

A series of (σ-π)palladium complexes derived from cyclooctadiene were investigated by ¹H, ¹³C and ¹⁹F NMR. The stereochemical assignment was based on intramolecular NOEs in conjunction with molecular modelling and semi-empirical methods, and confirmed by interligand NOEs in nitrogen chelate complexes derived from the title compounds. The nitrogen chelating ligands are involved in a rotation with respect to the ligand-palladium axis.

A series of 3,7-diazabicyclo[3.3.1]nonane (bispidine) derivatives have been synthesized, and their properties as bidentate nitrogen ligands for (pi-allyl)palladium complexes have been investigated. Complexes of these ligands and of N,N'-diphenylpiperazin

2-Oxopurine reacted with benzyl bromide and ethanol to give the covalent adduct 1,3,7-tribenzyl-6-ethoxy-2-oxopurine, as well as dibenzylated products. Carbon-carbon bond formation was observed in the reaction between 2-oxopurine, dry silica gel, and benzyl bromide, giving rise to 6-hydroxy-1,3,8-tribenzyl-2-oxopurine.