Liquid Chromatography ICP-MS to Assess the Stability of 175Lu- and natGa-Based Tumor-Targeting Agents towards the Development of 177Lu- and 68Ga-Labeled Radiopharmaceuticals.

In recent years, nuclear medicine has gained great interest, partly due to the success story of [177Lu]Lu-PSMA-617 (PluvictoTM). Still, in-depth preclinical characterization of radiopharmaceuticals mainly happens at centers that allow working with radioactive material. To support the development of novel radiopharmaceuticals, alternative non-radioactive characterization assays are highly desirable. The aim of this study was to demonstrate that inductively coupled plasma mass spectrometry (ICP-MS) associated with a chromatographic system can serve as a surrogate for the classical high-performance liquid chromatography (HPLC)-radiodetector combination for preclinical in vitro characterization of non-radioactive metal-labeled analogs of radiopharmaceuticals. In this proof-of-concept study, we demonstrate the applicability of HPLC-ICP-MS by assessing the stability of 175Lu- and natGa-labeled prostate-specific membrane antigen (PSMA)-targeting peptidomimetics, single domain antibody (sdAb) conjugates, and monoclonal antibody (mAb) conjugates. 175Lu-labeled DOTAGA-conjugated and natGa-labeled NODAGA-conjugated sdAbs and mAbs showed the highest stability with >90% still intact after 24 h. The peptidomime-tics [175Lu]Lu-PSMA-617 and [natGa]Ga-PSMA-11 showed identical in vitro serum stability as it was reported for their corresponding radioligands with >99% intact species after 24 h incubation in mouse serum, demonstrating the reliability of the method. Hence, the established HPLC-ICP-MS methods can support the development of novel radiopharmaceuticals in a classical pharmaceutical setting.

Inductively Coupled Plasma Mass Spectrometry─A Valid Method for the Characterization of Metal Conjugates in View of the Development of Radiopharmaceuticals.

This study addresses the question whether inductively coupled plasma mass spectrometry (ICP-MS) can be used as a method for the in vitro and in vivo characterization of non-radioactive metal conjugates to predict the properties of analogous radiopharmaceuticals. In a "proof-of-concept" study, the prostate-specific membrane antigen (PSMA)-targeting [175Lu]Lu-PSMA-617 and [159Tb]Tb-PSMA-617 were compared with their respective radiolabeled analogues, [177Lu]Lu-PSMA-617 (PLUVICTO, Novartis) and [161Tb]Tb-PSMA-617. ICP-MS and conventional γ-counting of the cell samples revealed almost identical results (<6% absolute difference between the two technologies) for the in vitro uptake and internalization of the (radio)metal conjugates, irrespective of the employed methodology. In vivo, an equal uptake in PSMA-positive PC-3 PIP tumor xenografts was determined 1 h after the injection of [175Lu]Lu-/[177Lu]Lu-PSMA-617 (41 ± 6% ID/g and 44 ± 12% IA/g, respectively) and [159Tb]Tb-/[161Tb]Tb-PSMA-617 (44 ± 5% ID/g and 44 ± 5% IA/g, respectively). It was further revealed that it is crucial to use the same ratios of the (radio)metal-labeled and unlabeled ligands for both methodologies to obtain equal data in organs in which receptor saturation was reached such as the kidneys (12 ± 2% ID/g vs 10 ± 1% IA/g, 1 h after injection). The data of this study demonstrate that the use of high-sensitivity ICP-MS allows reliable and predictive quantification of compounds labeled with stable metal isotopes in cell and tissue samples obtained in preclinical studies. It can, hence, be employed as a valid alternative to the state-of-the-art γ-counting methodology to detect radioactive ligands.

Distribution and efficacy of ofatumumab and ocrelizumab in humanized CD20 mice following subcutaneous or intravenous administration.

Approval of B-cell-depleting therapies signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the administration route of anti-CD20 monoclonal antibodies (mAbs) alters tissue distribution patterns and subsequent downstream effects. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT)/CT 72 h after SC/IV administration of ofatumumab or SC/IV administration of ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC/IV ofatumumab or SC/IV ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, albeit with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, where no differences between the route of administration or mAb were observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was mostly achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain.

Efficient Distribution of a Novel Zirconium-89 Labeled Anti-cd20 Antibody Following Subcutaneous and Intravenous Administration in Control and Experimental Autoimmune Encephalomyelitis-Variant Mice.

Objective: To investigate the imaging and biodistribution of a novel zirconium-89 (89Zr)-labeled mouse anti-cd20 monoclonal antibody (mAb) in control and experimental autoimmune encephalomyelitis (EAE) mice following subcutaneous (s. c.) and intravenous (i.v.) administration. Background: Anti-cd20-mediated B-cell depletion using mAbs is a promising therapy for multiple sclerosis. Recombinant human myelin oligodendrocyte glycoprotein (rhMOG)-induced EAE involves B-cell-mediated inflammation and demyelination in mice. Design/Methods: C57BL/6J mice (n = 39) were EAE-induced using rhMOG. On Day 14 post EAE induction, 89Zr-labeled-anti-cd20 mAb was injected in control and EAE mice in the right lower flank (s.c.) or tail vein (i.v.). Positron emission tomography/computed tomography (PET/CT) imaging and gamma counting (ex vivo) were performed on Days 1, 3, and 7 to quantify tracer accumulation in the major organs, lymphatics, and central nervous system (CNS). A preliminary study was conducted in healthy mice to elucidate full and early kinetics of the tracer that were subsequently applied in the EAE and control mice study. Results:89Zr-labeled anti-cd20 mAb was effectively absorbed from s.c. and i.v. injection sites and distributed to all major organs in the EAE and control mice. There was a good correlation between in vivo PET/CT data and ex vivo quantification of biodistribution of the tracer. From gamma counting studies, initial tracer uptake within the lymphatic system was found to be higher in the draining lymph nodes (inguinal or subiliac and sciatic) following s.c. vs. i.v. administration; within the CNS a significantly higher tracer uptake was observed at 24 h in the cerebellum, cerebrum, and thoracic spinal cord (p < 0.05 for all) following s.c. vs. i.v. administration. Conclusions: The preclinical data suggest that initial tracer uptake was significantly higher in the draining lymph nodes (subiliac and sciatic) and parts of CNS (the cerebellum and cerebrum) when administered s.c. compared with i.v in EAE mice.

Imaging Mass Cytometry and Single-Cell Genomics Reveal Differential Depletion and Repletion of B-Cell Populations Following Ofatumumab Treatment in Cynomolgus Monkeys.

Ofatumumab is the first, fully human, anti-CD20 monoclonal antibody in Phase 3 development for multiple sclerosis (MS). The study focused on changes in lymphocyte subsets in blood and lymphoid tissues and on potential novel biomarkers as a result of anti-CD20 antibody action in Cynomolgus monkeys treated with human equivalent doses of subcutaneous (s.c.) ofatumumab on Days 0, 7, and 14. Axillary lymph nodes (LNs) and blood samples were collected at various time points until Day 90. Lymphocyte subsets were quantified by flow cytometry, while morphological and immune cell changes were assessed by imaging mass cytometry (IMC), immunohistochemistry (IHC), in situ hybridization (ISH), and transcriptome analyses using single-cell methodology. Ofatumumab treatment resulted in a potent and rapid reduction of B cells along with a simultaneous drop in CD20+ T cell counts. At Day 21, IHC revealed B-cell depletion in the perifollicular and interfollicular area of axillary LNs, while only the core of the germinal center was depleted of CD20+CD21+ cells. By Day 62, the perifollicular and interfollicular areas were abundantly infiltrated by CD21+ B cells and this distribution returned to the baseline cytoarchitecture by Day 90. By IMC CD20+CD3+CD8+ cells could be identified at the margin of the follicles, with a similar pattern of distribution at Day 21 and 90. Single-cell transcriptomics analysis showed that ofatumumab induced reversible changes in t-distributed stochastic neighbor embedding (t-SNE) defined B-cell subsets that may serve as biomarkers for drug action. In summary, low dose s.c. ofatumumab potently depletes both B cells and CD20+ T cells but apparently spares marginal zone (MZ) B cells in the spleen and LN. These findings add to our molecular and tissue-architectural understanding of ofatumumab treatment effects on B-cell subsets.

Modular synthesis and modification of novel bifunctional dendrons.

Herein, we report the design and synthesis of two novel bifunctional dendrons bearing multiple amine termini at the periphery and an azide at the focal point. Copper-catalyzed alkyne-azide cycloaddition enabled modular dendritic scaffold assembly resulting in a first generation dendron carrying six amines and a second generation dendron carrying eighteen amines. Peripheral amines were labeled with multiple copies of a metal isotope, whereas the azide functionality at the focal point was employed in conjugation to a single anti-human CD4 antibody. We demonstrated that the highly monomeric first generation dendron-antibody conjugate selectively detected CD4+ T cells in the PMBC culture.

GPR91 senses extracellular succinate released from inflammatory macrophages and exacerbates rheumatoid arthritis.

When SUCNR1/GPR91-expressing macrophages are activated by inflammatory signals, they change their metabolism and accumulate succinate. In this study, we show that during this activation, macrophages release succinate into the extracellular milieu. They simultaneously up-regulate GPR91, which functions as an autocrine and paracrine sensor for extracellular succinate to enhance IL-1ß production. GPR91-deficient mice lack this metabolic sensor and show reduced macrophage activation and production of IL-1ß during antigen-induced arthritis. Succinate is abundant in synovial fluids from rheumatoid arthritis (RA) patients, and these fluids elicit IL-1ß release from macrophages in a GPR91-dependent manner. Together, we reveal a GPR91/succinate-dependent feed-forward loop of macrophage activation and propose GPR91 antagonists as novel therapeutic principles to treat RA.

SOM230: a new therapeutic modality for Cushing's disease.

A rational drug design approach involving transposition of functional groups from SRIF into a reduced size cyclohexapeptide template has led to the discovery of SOM230, a novel, stable cyclohexapeptide somatostatin mimic which exhibits unique high affinity binding to human somatostatin receptors (sst1-5). This unique receptor subtype binding profile, in particular the exceptional high affinity binding to sst5, led to SOM230 being approved by EMEA and FDA in 2012 as the first effective pituitary directed therapeutic modality for Cushing's disease.

Annulated and bridged tetrahydrofurans from alkenoxyl radical cyclization.

4-Pentenoxyl radicals sharing two or more carbon atoms with a cycloalkane cyclize in a predictable manner stereoselectively and regioselectively to afford in solutions of bromotrichloromethane cycloalkyl-fused or -bridged 2-bromomethyltetrahydrofurans in up to 95% yield. Stereoselectivity in alkenoxyl radical ring closures arises from cumulative steric effects. The substituent positioned the closest to the alkene carbon, which is being attacked by the oxygen radical, exerts the strongest stereodirecting effect. This principal inductor guides 5-exo-cyclization 2,3-trans- or 2,4-cis-selectively. The substituent located further from the attacked π-bond is the secondary inductor. A secondary inductor in the relative trans-configuration enhances stereodifferentiation by the primary inductor; a cis-configured secondary inductor decreases this effect. A secondary inductor is not able to overrule the guiding effect of a similar sized primary inductor. Intramolecular 4-pentenoxyl radical additions to a cyclohexene-bound exo-methylene group or to endocyclic double bonds proceed cis-specifically, as exemplified by synthesis of a diastereomerically pure bromobicyclo[2.2.1]heptyl-annulated tetrahydrofuran from the verbenylethyloxyl radical. According to theory, the experimental 2,3-cis-specificity in alkoxyl radical cyclization to an endocyclic π-bond arises from strain associated with the 2,3-trans-ring closure.

In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

BACKGROUND AND AIMS: Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. METHODS: Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent "smart" probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. RESULTS: We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. CONCLUSIONS: We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.

Optical and magnetic resonance imaging as complementary modalities in drug discovery.

Imaging has the ability to study various biological and chemical processes noninvasively in living subjects in a longitudinal way. For this reason, imaging technologies have become an integral part of the drug-discovery and development program and are commonly used in following disease processes and drug action in both preclinical and clinical stages. As the domain of imaging sciences transitions from anatomical/functional to molecular applications, the development of molecular probes becomes crucial for the advancement of the field. This review summarizes the role of two complementary techniques, magnetic resonance and fluorescence optical imaging, in drug discovery. While the first approach exploits intrinsic tissue characteristics as the source of image contrast, the second necessitates the use of appropriate probes for signal generation. The anatomical, functional, metabolic and molecular information that becomes accessible through imaging can provide invaluable insights into disease mechanisms and mechanisms of drug action.

Fluorescent nanoprobes as a biomarker for increased vascular permeability: implications in diagnosis and treatment of cancer and inflammation.

This article describes the use of a fluorescent nanoprobe as a functional biomarker for the identification of increased vascular permeability in cancer/arthritis disease models. Synthesis of the fluorescent nanoprobe was achieved by passive loading of a fluorophore inside the nanoparticle using thin film hydration method. The outer layer of the nanoprobe was decorated with poly(ethylene glycol) arms to increase the bioavailability of the fluorophore. Stability studies of the nanoprobe showed that the particles were stable up to 70 days. The uptake and internalization of the fluorescent nanoprobe inside target cells was confirmed by fluorescence microscopy studies. Co-localization of the probe with the target tissue in vivo was unambiguously identified using intravital microscopy. Results from in vivo imaging studies showed that the particles had a long half-life in the circulation and passively targeted tumor or arthritic tissue. The increased and specific uptake of the fluorescent nanoprobe in tumor/arthritic tissue is attributed to an enhanced permeation and retention (EPR) effect. Use of an optical method to validate anti-inflammatory drugs in an arthritis disease model is demonstrated in this study. In general, this methodology could be used for detection of leaky vasculature in different pathological states.

In vivo assessments of mucus dynamics in the rat lung using a Gd-Cy5.5-bilabeled contrast agent for magnetic resonance and optical imaging.

Dysfunctions in mucociliary clearance are associated with the accelerated loss of lung function in several respiratory diseases. Approaches enabling the in vivo visualization of mucus dynamics in rodents at high resolution and sensitivity would be beneficial for experimental lung research. We describe the synthesis and characterization of two bilabeled amino dextran-based probes binding specifically to mucin. Labeling of secreted mucus and of mucin in goblet cells in the lungs of lipopolysaccharide-challenged rats has been demonstrated in vivo with near-infrared fluorescence and MRI and confirmed by histology. The effects of uridine triphosphate were then studied in lipopolysaccharide-challenged rats by simultaneously administering the imaging probe and the compound. The data suggest that uridine triphosphate increased the mucociliary clearance, but at the same time induced a release of mucin from goblet cells, thus not contributing to the overall reduction of mucus in the lung. The approach outlined here enables one to derive information on mucus clearance, as well as secretion. Such a global view on mucus dynamics may prove invaluable when testing new pharmacological agents aimed at improving mucociliary clearance.

In vivo visualization of macrophage infiltration and activity in inflammation using magnetic resonance imaging.

Because macrophages play a key role on host defense, visualization of the migration of these cells is of high relevance for both diagnostic purposes and the evaluation of therapeutic interventions. The present article addresses the use of iron oxide and gadolinium-based particles for the noninvasive in vivo detection of macrophage infiltration into inflamed areas by magnetic resonance imaging (MRI). A general introduction on the functions and general characteristics of macrophages is followed by a discussion of some of the agents and acquisition schemes currently used to track the cells in vivo. Attention is then devoted to preclinical and clinical applications in the following disease areas: atherosclerosis and myocardial infarction, stroke, multiple sclerosis, rheumatoid arthritis, and kidney transplantation.

Investigating pharmacology in vivo using magnetic resonance and optical imaging.

The better and earlier a disease can be diagnosed and characterized, the greater the chance of being able to intervene in this process with a chemical entity. This is the rationale for the use of in vivo imaging techniques in the drug discovery and development process. In this article we address the value of two imaging modalities in this area, i.e. magnetic resonance imaging (MRI) and optical imaging. The multiparametric nature of MRI enables anatomical, functional, metabolic and, to a certain extent, also cellular and target-related information to be obtained noninvasively at high spatial resolution. This favours characterization of a disease state and the corresponding drug intervention. The noninvasiveness of MRI strengthens the link between preclinical and clinical pharmaceutical research. The high sensitivity of optical techniques enables molecular information to be obtained in vivo. Within pharmacological research, the main applications of optical techniques relate to the early drug discovery process and acquisition of target-related information. However, potential clinical applications of optical imaging are also emerging. The complementary character of both imaging modalities renders them useful in various portions of the drug discovery process, from early target selection and validation to clinical studies.

In vivo monitoring the fate of Cy5.5-Tat labeled T lymphocytes by quantitative near-infrared fluorescence imaging during acute brain inflammation in a rat model of experimental autoimmune encephalomyelitis.

T cells and macrophages directed against myelin proteins orchestrate the inflammation process in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). So far, assessment of macrophages infiltration or structural alterations has been achieved by in vivo imaging. In this work, we show the infiltration of Cy5.5-labeled T lymphocytes into the brains of EAE rats by reflectance near-infrared fluorescence imaging. T lymphocytes were labeled with Cy5.5-Tat and administered intravenously to naïve or EAE animals. The highest fluorescence signal was observed for EAE animals, which received myelin-activated T cells during the acute phase of the disease. The temporal profile of fluorescence in this group paralleled the pattern of neurological impairment during the acute phase, the remittance and first relapses of EAE. No disease specific fluorescence pattern was observed for EAE animals, which received naïve T cells. However, uptake of Cy5.5-Tat by scavenger cells (e.g. macrophages) following death of labeled T cells in vivo prevents prolonged longitudinal studies. Our work demonstrates that Cy5.5-Tat labeling of T cells is suitable for in vivo fluorescence imaging of inflammation initiation in the EAE model. This approach may particularly be useful for evaluation of novel anti-inflammatory therapies.

In vivo mouse imaging and spectroscopy in drug discovery.

Imaging modalities such as micro-computed tomography (micro-CT), micro-positron emission tomography (micro-PET), high-resolution MRI, optical imaging, and high-resolution ultrasound have become invaluable tools in preclinical pharmaceutical research. They can be used to non-invasively investigate, in vivo, rodent biology and metabolism, disease models, and pharmacokinetics and pharmacodynamics of drugs. The advantages and limitations of each approach usually determine its application, and therefore a small-rodent imaging laboratory in a pharmaceutical environment should ideally provide access to several techniques. In this paper we aim to illustrate how these techniques may be used to obtain meaningful information for the phenotyping of transgenic mice and for the analysis of compounds in murine models of disease.

Physicochemical and MRI characterization of Gd3+-loaded polyamidoamine and hyperbranched dendrimers.

Generation 4 polyamidoamine (PAMAM) and, for the first time, hyperbranched poly(ethylene imine) or polyglycerol dendrimers have been loaded with Gd3+ chelates, and the macromolecular adducts have been studied in vitro and in vivo with regard to MRI contrast agent applications. The Gd3+ chelator was either a tetraazatetracarboxylate DOTA-pBn4- or a tetraazatricarboxylate monoamide DO3A-MA3- unit. The water exchange rate was determined from a 17O NMR and 1H Nuclear Magnetic Relaxation Dispersion study for the corresponding monomer analogues [Gd(DO3A-AEM)(H2O)] and [Gd(DOTA-pBn-NH2)(H2O)]- (kex298=3.4 and 6.6x10(6) s-1, respectively), where H3DO3A-AEM is {4-[(2-acetylaminoethylcarbamoyl)methyl]-7,10-bis(carboxymethyl-1,4,7,10-tetraazacyclododec-1-yl)}-acetic acid and H4DOTA-pBn-NH2 is 2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. For the macromolecular complexes, variable-field proton relaxivities have been measured and analyzed in terms of local and global motional dynamics by using the Lipari-Szabo approach. At frequencies below 100 MHz, the proton relaxivities are twice as high for the dendrimers loaded with the negatively charged Gd(DOTA-pBn)- in comparison with the analogous molecule bearing the neutral Gd(DO3A-MA). We explained this difference by the different rotational dynamics: the much slower motion of Gd(DOTA-pBn)--loaded dendrimers is likely related to the negative charge of the chelate which creates more rigidity and increases the overall size of the macromolecule compared with dendrimers loaded with the neutral Gd(DO3A-MA). Attachment of poly(ethylene glycol) chains to the dendrimers does not influence relaxivity. Both hyperbranched structures were found to be as good scaffolds as regular PAMAM dendrimers in terms of the proton relaxivity of the Gd3+ complexes. The in vivo MRI studies on tumor-bearing mice at 4.7 T proved that all dendrimeric complexes are suitable for angiography and for the study of vasculature parameters like blood volume and permeability of tumor vessels.

In vivo detection of amyloid-beta deposits by near-infrared imaging using an oxazine-derivative probe.

As Alzheimer's disease pathogenesis is associated with the formation of insoluble aggregates of amyloid beta-peptide, approaches allowing the direct, noninvasive visualization of plaque growth in vivo would be beneficial for biomedical research. Here we describe the synthesis and characterization of the near-infrared fluorescence oxazine dye AOI987, which readily penetrates the intact blood-brain barrier and binds to amyloid plaques. Using near-infrared fluorescence imaging, we demonstrated specific interaction of AOI987 with amyloid plaques in APP23 transgenic mice in vivo, as confirmed by postmortem analysis of brain slices. Quantitative analysis revealed increasing fluorescence signal intensity with increasing plaque load of the animals, and significant binding of AOI987 was observed for APP23 transgenic mice aged 9 months and older. Thus, AOI987 is an attractive probe to noninvasively monitor disease progression in animal models of Alzheimer disease and to evaluate effects of potential Alzheimer disease drugs on the plaque load.

On the 6-endo selectivity in 4-penten-1-oxyl radical cyclizations.

Regioselectivities in cyclizations of 4-substituted 4-penten-1-oxyl radicals have been investigated in a combined experimental and computational study (density functional theory). The progressive increase of the 6-endo-trig selectivity along the series of 4-substituents H < CH(3) < C(CH(3))(3) < C(6)H(5) has been interpreted to originate from a balance between strain and FMO interactions. Torsional strain, which is associated with geometrical changes upon an approach of the reacting entities, is relevant for the 6-endo-trig but not for the 5-exo-trig reactions, as seen, for instance, in selective tetrahydrofuran formation from the 4-penten-1-oxyl radical and its 4-methyl derivative. The preference for tetrahydropyran formation in cyclizations of the 4-tert-butyl and the 4-phenyl-4-penten-1-oxyl radical has been attributed to FMO interactions between the terminal carbon atom of the pi bond and the O-radical center thus favoring the 6-endo-trig reaction on the basis of lower transition state energies.