Bridging vascular physiology to vascular medicine: an integrative laboratory class.

Vascular diseases of the legs are highly prevalent and constitute an important part of medical curricula. The understanding of these diseases relies on strongly interwoven aspects of vascular physiology and vascular medicine. We aimed to connect these within a horizontally integrated laboratory class on vascular physiology of the leg that was designed in cooperation between the departments of physiology and vascular surgery. Conceptually, we applied examination techniques of vascular medicine to visualize physiological parameters that are altered by the most frequent diseases. This facilitates integrative discussions on malfunctions, trains diagnostic skills, and bridges to vascular medicine. In four experiments, we use oscillometry and impedance venous occlusion plethysmography to address key aspects of the arterial and venous system of the legs: 1) arterial pulse wave, 2) arterial systolic blood pressure, 3) venous capacitance and venous outflow, and 4) reactive hyperemia. After the experiments, physiological vascular function, the associated diseases, their impact on the recorded parameters, and diagnostic options are discussed. To allow reproduction, we describe the course structure and the experimental setup in detail. We present the experimental data of a cohort of medical students and document learning success and student satisfaction. All experiments were feasible and provided robust data on physiologically and clinically relevant vascular functions. The activity was perceived positively by the students and led to a substantial improvement of knowledge. With this work, we offer a template for reproduction or variation of a proven concept of horizontally integrated teaching of vascular physiology of the leg.NEW & NOTEWORTHY This article presents an integrative laboratory class on vascular physiology bridging to vascular medicine. The four experiments rely on oscillometry and venous occlusion plethysmography. We describe in detail this new class regarding structure, experimental setup, and experimental procedure, and we give insight into the applied materials. Moreover, we present the experimental data of 74 students and a quantitative evaluation of the students' learning success and acceptance.

The baroreceptor reflex brought to life outside the classroom - an e-learning based asynchronous laboratory class using a non-supervised modified Active Standing Test.

BACKGROUND: E-learning based laboratory classes can replace or enhance in-classroom laboratories. They typically offer temporal flexibility, self-determined learning speed, repeatability and do not require supervision or face-to-face contact. The aim of this feasibility study was to investigate whether the established in-classroom laboratory class on the baroreceptor reflex (BRR) can be transformed into a new e-learning based asynchronous laboratory class for untrained, non-supervised students without medical equipment. The BRR is a fundamental cardiovascular process which is regularly visualized in physiology during in-classroom laboratories by a student-performed Active Standing Test (AST). During this voluntary provocation of orthostatic stress, the BRR reliably causes a solid rise in heart rate (HR) and a stabilization or even increase in blood pressure (BP). METHODS: The conventional AST was modified by omission of BP measurements which would require medical devices and was embedded into a framework of interactive digital material allowing independent student performance. With specific adaptions, this instrument was implemented to 1st and 2nd year curricula of human medicine, dental medicine, midwifery and pharmacy. An audience response system was used to collect the students' data on HR, epidemiology, technical problems, satisfaction and orthostatic symptoms. As primary outcome, we investigated the students' correct performance of the modified AST regarding textbook conformity of the HR data. Secondary outcomes included technical feasibility, the students' satisfaction and consistency of HR data within predefined subgroups (e.g., gender, curricula). Descriptive statistics are reported. RESULTS: The class was completed by 217 students (mean age: 23 ± 8 [SD], 81% female, 19% male). Mean reported rise of HR during standing was ~ 20 bpm (~ 30%) which is highly concordant to textbooks. Reported feasibility (~ 80% negated any technical issues) and students' satisfaction (4.4 on 5-point Likert-scale) were high. The HR data were consistent within the subgroups. CONCLUSION: This study demonstrates that the highly relevant BRR can be successfully addressed in an e-learning based asynchronous laboratory class implementing a non-supervised AST restricted to HR measurements embedded in digital material. The robust HR response and the adjustable complexity allow an application to different healthcare-related curricula. This class, therefore, provides a broad audience access to a fundamental concept of cardiovascular physiology.

Public support for development aid during the COVID-19 pandemic.

Global pandemics are a serious concern for developing countries, perhaps particularly when the same pandemic also affects donors of development aid. During crises at home, donors often cut aid, which would have grave ramifications for developing countries with poor public health capacity during a time of increased demand for health care. Because the major donors are democracies, whether they renege on promises would depend intimately on how donor citizens respond to the specific crisis. We conduct two survey experiments with 887 U.S. residents to examine how the 2020 COVID-19 pandemic influences their attitudes toward aid. We demonstrate that people's concern about the impact of COVID-19 on their country's financial situation reduces their support for aid. If they think that aid can help curb the next wave of the disease at home by first alleviating its impact in developing countries, people become substantially more supportive of giving aid. In contrast, merely stressing how COVID-19 might ravage developing countries barely changes their aid attitudes. Our findings have implications for what to expect from donors during global pandemics as well as how advocates may prevent aid from being cut.

18-kDa translocator protein ligand 18F-FEMPA: Biodistribution and uptake into atherosclerotic plaques in mice.

BACKGROUND: Radioligands of 18-kDa translocator protein (TSPO) expressed on activated macrophages are a potential approach for imaging of inflammation in atherosclerosis. We evaluated a novel TSPO-targeted tracer 18F-FEMPA for the detection of atherosclerotic plaque inflammation in mice. METHODS AND RESULTS: The distribution kinetics of 18F-FEMPA was evaluated by in vivo PET/CT imaging. 18F-FEMPA uptake was compared in atherosclerotic (LDLR-/-ApoB100/100, n = 10) and healthy mice (C57BL/6 N, n = 7) ex vivo at twenty minutes post-injection. Biodistribution was analyzed from harvested tissue samples, and aortas were sectioned for autoradiography. Aortas of LDLR-/-ApoB100/100 mice showed large, macrophage-rich atherosclerotic plaques. In vivo, 18F-FEMPA showed rapid blood clearance but no difference in aortic uptake between atherosclerotic and healthy mice. In the mice studied ex vivo at 20 minutes post-injection, quantification of radioactivity in the whole aorta showed 1.3-fold higher 18F-FEMPA accumulation in atherosclerotic than healthy mice (P = .028). Autoradiography showed higher tracer uptake in plaque areas with high macrophage content as compared with areas of no macrophages (count densities 190 ± 54 vs 40 ± 13 PSL/mm2, P < .001), but the uptake in the plaques was not higher than in the normal vessel wall (230 ± 78 PSL/mm2). In vitro blocking showed specific accumulation in mouse and human atherosclerotic plaques. Immunohistochemistry confirmed co-localization of TSPO and macrophages. CONCLUSIONS: 18F-FEMPA shows rapid blood clearance and uptake in the mouse aorta. Uptake in atherosclerotic plaques correlated with the amount of macrophages, but did not exceed that in the normal vessel wall.

Positron emission tomography imaging of the 18-kDa translocator protein (TSPO) with [18F]FEMPA in Alzheimer's disease patients and control subjects.

PURPOSE: Imaging of the 18-kDa translocator protein (TSPO) is a potential tool for examining microglial activation and neuroinflammation in early Alzheimer's disease (AD). [(18)F]FEMPA is a novel high-affinity second-generation TSPO radioligand that has displayed suitable pharmacokinetic properties in preclinical studies. The aims of this study were to quantify the binding of [(18)F]FEMPA to TSPO in AD patients and controls and to investigate whether higher [(18)F]FEMPA binding in AD patients than in controls could be detected in vivo. METHODS: Ten AD patients (five men, five women; age 66.9 ± 7.3 years; MMSE score 25.5 ± 2.5) and seven controls (three men, four women; age 63.7 ± 7.2 years, MMSE score 29.3 ± 1.0) were studied using [(18)F]FEMPA at Turku (13 subjects) and at Karolinska Institutet (4 subjects). The in vitro binding affinity for TSPO was assessed using PBR28 in a competition assay with [(3)H]PK11195 in seven controls and eight AD patients. Cortical and subcortical regions of interest were examined. Quantification was performed using a two-tissue compartment model (2TCM) and Logan graphical analysis (GA). The outcome measure was the total distribution volume (V T). Repeated measures analysis of variance was used to assess the effect of group and TSPO binding status on V T. RESULTS: Five AD patients and four controls were high-affinity binders (HABs). Three AD patients and three controls were mixed-affinity binders. V T estimated with Logan GA was significantly correlated with V T estimated with the 2TCM in both controls (r = 0.97) and AD patients (r = 0.98) and was selected for the final analysis. Significantly higher V T was found in the medial temporal cortex in AD patients than in controls (p = 0.044) if the TSPO binding status was entered as a covariate. If only HABs were included, significantly higher V T was found in the medial and lateral temporal cortex, posterior cingulate, caudate, putamen, thalamus and cerebellum in AD patients than in controls (p < 0.05). CONCLUSION: [(18)F]FEMPA seems to be a suitable radioligand for detecting increased TSPO binding in AD patients if their binding status is taken into account.

Which foreign vaccine should the government purchase in a pandemic? Evidence from a survey experiment in the United States.

BACKGROUND: For many countries confronting a future pandemic, the initial vaccines available will come from abroad. Public hesitancy to receive these foreign vaccines is important, as it may create an incentive for governments to forego procuring them for public use. We investigate the influence of the vaccine's country of origin on public support for government procurement during the early stages of a pandemic and examine whether endorsements from the WHO can mitigate such biases. METHODS: In the summer of 2023, we carried out a survey experiment of 1,110 U.S. residents where we asked respondents to rate their support for vaccine purchasing policies for 20 hypothetical vaccines (13,320 evaluations). We varied the vaccine's country of origin and its endorsement status from the WHO, while also randomizing other vaccine attributes. RESULTS: Compared to foreign vaccines from countries Americans see favorable (e.g., Germany, the United Kingdom), those originating from less favorable countries (e.g., China, Russia), garnered lower support for government procurement. Our causal mediation analysis indicates that this country-of-origin effect is primarily driven by participants' sentiments toward the vaccine. Surprisingly, WHO endorsement does little to mitigate the effect of the vaccine's country of origin. These findings are consistent across various sample subsets and considerations of vaccine quality. CONCLUSION: Our study advances previous work on vaccine country-of-origin effects by assessing its impact on policy preferences for procuring initial vaccines from overseas (as opposed to uptake intentions), identifying a mechanism by which vaccine favoritism occurs, and documenting that neither personal disease susceptibility nor vaccine quality fully mitigates country of origin effects. We conclude by discussing why the study of "vaccine diplomacy" ought to not only include interstate dynamics governing vaccine purchasing and availability but also consider vaccine-producing countries' more general reputations.

Discovery of YAP1/TAZ pathway inhibitors through phenotypic screening with potent anti-tumor activity via blockade of Rho-GTPase signaling.

This study describes the identification and target deconvolution of small molecule inhibitors of oncogenic Yes-associated protein (YAP1)/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen (HTS) of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies identified the geranylgeranyltransferase-I (GGTase-I) complex as the direct target of YAP1/TAZ pathway inhibitors. The small molecule inhibitors block the activation of Rho-GTPases, leading to subsequent inactivation of YAP1/TAZ and inhibition of cancer cell proliferation in vitro. Multi-parameter optimization resulted in BAY-593, an in vivo probe with favorable PK properties, which demonstrated anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.

In vivo evaluation in cynomolgus monkey brain and metabolism of [¹8F]fluorodeprenyl: a new MAO-B pet radioligand.

In this study, we evaluated the in vivo characteristics of a new monoamine oxidase type B (MAO-B) radioligand, [¹8F]fluorodeprenyl, by positron emission tomography (PET) in two cynomolgus monkeys. The brain uptake of [¹8F]fluorodeprenyl was more than 7% (600% SUV) of the total injected radioactivity and similar to that of [¹¹C]deprenyl, an established MAO-B radioligand. The highest uptake was observed in the striatum, one of the MAO-B-rich regions, with a peak at approximately 2-3 min after injection, followed by lower uptake in the thalamus and the cortex and lowest uptake in the cerebellum. Brain uptake of [¹8F]fluorodeprenyl was largely inhibited by preadministration of the MAO-B inhibitor, L-deprenyl, whereas clorgyline, a MAO Type A blocker, had no significant inhibitory effect, thus demonstrating selectivity for MAO-B. [¹8F]Fluorodeprenyl showed relatively slow metabolism with the presence of two radiometabolite peaks with similar retention time as the labeled metabolites of [¹¹C]deprenyl. These results suggest that [¹8F]fluorodeprenyl is a potential PET radioligand for visualization of MAO-B activity.

Investigating how historical legacies of militarized violence can motivate COVID-19 vaccine hesitancy: Evidence from global dyadic survey.

BACKGROUND: In almost all countries, COVID-19 vaccines available for public use are produced outside of that country. Consistent with recent social science research, we hypothesize that legacies of violent conflict from vaccine-producing against vaccine-consuming countries may motivate vaccine hesitancy among people in targeted countries that purchase vaccines produced by the erstwhile aggressor. METHODS: Our analyses draw on data from the Correlates of War project and a large, representative survey of 18,291 adults that asked respondents in 16 countries to self-report their attitudes toward COVID-19 vaccines originating from 12 potential vaccine-producing countries in December 2020 (184 country-pairs, 208,422 ratings). For the main analysis, we used random-effect linear probability models and turned to Bayesian Model Averaging to probe the robustness of the main findings. RESULTS: We demonstrate that elevated levels of historical violence between vaccine-producing and vaccine-consuming countries are associated with increased negative feelings toward a COVID-19 vaccine produced by the vaccine producer. CONCLUSION: Global vaccine hesitancy may result, at least in part, from public perceptions of historical conflict between vaccine-producing and vaccine-consuming countries. These results can help public health practitioners better preempt and adjust for cross-national vaccine resistance.

Vaccine hesitancy, state bias, and Covid-19: Evidence from a survey experiment using Phase-3 results announcement by BioNTech and Pfizer.

BACKGROUND: Past survey studies document that people strongly prefer Covid-19 vaccines developed domestically over those developed abroad. Available evidence suggests that this preference for domestic vaccines over foreign ones may stem from prejudice against foreign countries, but identifying prejudice-based vaccine preferences is difficult because people also draw inferences about the quality of vaccines based on country of origin. We exploit a unique opportunity provided by the announcement of a viable vaccine by a bi-national venture, BioNTech and Pfizer, to examine the effect of such prejudice on vaccination intentions while controlling for beliefs about the vaccine quality. METHODS: We implemented a survey experiment in Germany and the United States (n = 582, 661 respectively) a few days after the BioNTech/Pfizer announcement of a viable vaccine. We randomized the identified company (and country) responsible for the vaccine development between BioNTech (Germany) and Pfizer (U.S.) and asked respondents when they would take said vaccine. RESULTS: In either the German and U.S. samples, we find little evidence that a country of origin of the vaccine makes a difference in when respondents intend to get vaccinated. We also see no evidence that those with a general animus toward the other foreign country would be more biased against a foreign vaccine. CONCLUSION: Our findings suggest that prejudice against foreign countries may be less of a concern for vaccine hesitancy and that its effect may be highly context specific.

Chiral high-harmonic generation and spectroscopy on solid surfaces using polarization-tailored strong fields.

Strong-field methods in solids enable new strategies for ultrafast nonlinear spectroscopy and provide all-optical insights into the electronic properties of condensed matter in reciprocal and real space. Additionally, solid-state media offers unprecedented possibilities to control high-harmonic generation using modified targets or tailored excitation fields. Here we merge these important points and demonstrate circularly-polarized high-harmonic generation with polarization-matched excitation fields for spectroscopy of chiral electronic properties at surfaces. The sensitivity of our approach is demonstrated for structural helicity and termination-mediated ferromagnetic order at the surface of silicon-dioxide and magnesium oxide, respectively. Circularly polarized radiation emanating from a solid sample now allows to add basic symmetry properties as chirality to the arsenal of strong-field spectroscopy in solids. Together with its inherent temporal (femtosecond) resolution and non-resonant broadband spectrum, the polarization control of high harmonics from condensed matter can illuminate ultrafast and strong field dynamics of surfaces, buried layers or thin films.

The potent AMPK inhibitor BAY-3827 shows strong efficacy in androgen-dependent prostate cancer models.

PURPOSE: 5' adenosine monophosphate-activated kinase (AMPK) is an essential regulator of cellular energy homeostasis and has been associated with different pathologies, including cancer. Precisely defining the biological role of AMPK necessitates the availability of a potent and selective inhibitor. METHODS: High-throughput screening and chemical optimization were performed to identify a novel AMPK inhibitor. Cell proliferation and mechanistic assays, as well as gene expression analysis and chromatin immunoprecipitation were used to investigate the cellular impact as well as the crosstalk between lipid metabolism and androgen signaling in prostate cancer models. Also, fatty acid turnover was determined by examining lipid droplet formation. RESULTS: We identified BAY-3827 as a novel and potent AMPK inhibitor with additional activity against ribosomal 6 kinase (RSK) family members. It displays strong anti-proliferative effects in androgen-dependent prostate cancer cell lines. Analysis of genes involved in AMPK signaling revealed that the expression of those encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), fatty acid synthase (FASN) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), all of which are involved in lipid metabolism, was strongly upregulated by androgen in responsive models. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) analysis identified several androgen receptor (AR) binding peaks in the HMGCR and PFKFB2 genes. BAY-3827 strongly down-regulated the expression of lipase E (LIPE), cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) and serine-threonine kinase AKT3 in responsive prostate cancer cell lines. Also, the expression of members of the carnitine palmitoyl-transferase 1 (CPT1) family was inhibited by BAY-3827, and this was paralleled by impaired lipid flux. CONCLUSIONS: The availability of the potent inhibitor BAY-3827 will contribute to a better understanding of the role of AMPK signaling in cancer, especially in prostate cancer.

Localisation analysis of nerves in the mouse pancreas reveals the sites of highest nerve density and nociceptive innervation.

BACKGROUND: Neuropathy and neuro-inflammation drive the severe pain and disease progression in human chronic pancreatitis and pancreatic cancer. Mice, especially genetically induced-mouse models, have been increasingly utilized in mechanistic research on pancreatic neuropathy, but the normal "peripheral neurobiology" of the mouse pancreas has not yet been critically compared to human pancreas. METHODS: We introduced a standardized tissue-harvesting technique that preserves the anatomic orientation of the mouse pancreas and allows complete sectioning in an anterior to posterior fashion. We applied immunohistochemistry and quantitative colorimetry of all nerves from the whole organ for studying pancreatic neuro-anatomy. KEY RESULTS: Nerves in the mouse pancreas appeared as "clusters" of nerve trunks in contrast to singly distributed nerve trunks in the human pancreas. Nerve trunks in the mouse pancreas were exclusively found around intrapancreatic blood vessels, and around lymphoid structures. The majority of nerve trunks were located in the pancreatic head (0.15 ± 0.08% of tissue area) and the anterior/front surface of the corpus/body (0.17 ± 0.27%), thus significantly more than in the tail (0.02 ± 0.02%, P = .006). Nerves in the tail included a higher proportion of nociceptive fibers, but the absolute majority, ie, ca. 70%, of all nociceptive fibers, were localized in the head. Mice heterozygous for Bdnf knockout allele (Bdnf+/- ) exhibited enrichment of nitrergic nerve fibers specifically in the head and corpus. CONCLUSIONS & INFERENCES: Neuro-anatomy of the "mesenteric type" mouse pancreas is highly different from the "compact" human pancreas. Studies that aim at reproducing human pancreatic neuro-phenomena in mouse models should pay diligent attention to these anatomic differences.

Discovery of BAY-985, a Highly Selective TBK1/IKKε Inhibitor.

The serine/threonine kinase TBK1 (TANK-binding kinase 1) and its homologue IKKε are noncanonical members of the inhibitor of the nuclear factor κB (IκB) kinase family. These kinases play important roles in multiple cellular pathways and, in particular, in inflammation. Herein, we describe our investigations on a family of benzimidazoles and the identification of the potent and highly selective TBK1/IKKε inhibitor BAY-985. BAY-985 inhibits the cellular phosphorylation of interferon regulatory factor 3 and displays antiproliferative efficacy in the melanoma cell line SK-MEL-2 but showed only weak antitumor activity in the SK-MEL-2 human melanoma xenograft model.

Targeting nNOS ameliorates the severe neuropathic pain due to chronic pancreatitis.

BACKGROUND: Pain due to pancreatic cancer/PCa or chronic pancreatitis/CP, is notoriously resistant to the strongest pain medications. Here, we aimed at deciphering the specific molecular mediators of pain at surgical-stage pancreatic disease and to discover novel translational targets. METHODS: We performed a systematic, quantitative analysis of the neurotransmitter/neuroenzmye profile within intrapancreatic nerves of CP and PCa patients. Ex vivo neuronal cultures treated with human pancreatic extracts, conditional genetically engineered knockout mouse models of PCa and CP, and the cerulein-induced CP model were employed to explore the therapeutic potential of the identified targets. FINDINGS: We identified a unique enrichment of neuronal nitric-oxide-synthase (nNOS) in the pancreatic nerves of CP patients with increasing pain severity. Employment of ex vivo neuronal cultures treated with pancreatic tissue extracts of CP patients, and brain-derived-neurotrophic-factor-deficient (BDNF+/-) mice revealed neuronal enrichment of nNOS to be a consequence of BDNF loss in the progressively destroyed pancreatic tissue. Mechanistically, nNOS upregulation in sensory neurons was induced by tryptase secreted from perineural mast cells. In a head-to-head comparison of several genetically induced, painless mouse models of PCa (KPC, KC mice) or CP (Ptf1a-Cre;Atg5fl/fl) against the hypersecretion/cerulein-induced, painful CP mouse model, we show that a similar nNOS enrichment is present in the painful cerulein-CP model, but absent in painless genetic models. Consequently, mice afflicted with painful cerulein-induced CP could be significantly relieved upon treatment with the specific nNOS inhibitor NPLA. INTERPRETATION: We propose nNOS inhibition as a novel strategy to treat the unbearable pain in CP. FUND: Deutsche Forschungsgemeinschaft/DFG (DE2428/3-1 and 3-2).

In Vivo and In Vitro Characterization of a Novel MAO-B Inhibitor Radioligand, 18F-Labeled Deuterated Fluorodeprenyl.

UNLABELLED: The aim of this study was to radiolabel a novel bis-deuterium substituted l-deprenyl analog (fluorodeprenyl-D2) with (18)F and to evaluate its potential to visualize and quantify monoamine oxidase (MAO) B activity in vivo. METHODS: The precursor compound ( 5A: + 5B: ) and reference standard ( 6: ) were synthesized in multistep syntheses. Recombinant human MAO-B and MAO-A enzyme preparations were used to determine inhibitory concentrations of 50%. Radiolabeling was accomplished by a nucleophilic substitution reaction. Whole-hemisphere autoradiography was performed with (18)F-fluorodeprenyl-D2. A PET study was performed on a cynomolgus monkey. Radiometabolites were measured in monkey plasma using high-performance liquid chromatography. RESULTS: The 50% inhibitory concentration of compound 6 for MAO-B was 227 ± 36.8 nM. Radiolabeling was accomplished with high radiochemical yield, purity, and specific radioactivity. The autoradiography binding density of (18)F-fluorodeprenyl-D2 was consistent with known MAO-B expression in the human brain. In vivo, (18)F-fluorodeprenyl-D2 showed favorable kinetic properties, with relatively fast washout from the brain. Regional time-activity curves were better described by the 2-tissue-compartment model. Administration of a 1 mg/kg dose of l-deprenyl yielded 70% inhibition of MAO-B in all regions. Radiometabolite studies demonstrated 20% unchanged radioligand at 120 min after injection. (18)F-fluorodeprenyl-D2 showed less irreversibility than did previously reported MAO-B radioligands. CONCLUSION: The results suggest that (18)F-fluorodeprenyl-D2 is a suitable PET radioligand for visualization of MAO-B activity in the human brain.

Gene expression levels of matrix metalloproteinases in human atherosclerotic plaques and evaluation of radiolabeled inhibitors as imaging agents for plaque vulnerability.

INTRODUCTION: Atherosclerotic plaque rupture is the primary cause for myocardial infarction and stroke. During plaque progression macrophages and mast cells secrete matrix-degrading proteolytic enzymes, such as matrix metalloproteinases (MMPs). We studied levels of MMPs and tissue inhibitor of metalloproteinases-3 (TIMP-3) in relation to the characteristics of carotid plaques. We evaluated in vitro two radiolabeled probes targeting active MMPs towards non-invasive imaging of rupture-prone plaques. METHODS: Human carotid plaques obtained from endarterectomy were classified into stable and vulnerable by visual and histological analysis. MMP-1, MMP-2, MMP-8, MMP-9, MMP-10, MMP-12, MMP-14, TIMP-3, and CD68 levels were investigated by quantitative polymerase chain reaction. Immunohistochemistry was used to localize MMP-2 and MMP-9 with respect to CD68-expressing macrophages. Western blotting was applied to detect their active forms. A fluorine-18-labeled MMP-2/MMP-9 inhibitor and a tritiated selective MMP-9 inhibitor were evaluated by in vitro autoradiography as potential lead structures for non-invasive imaging. RESULTS: Gene expression levels of all MMPs and CD68 were elevated in plaques. MMP-1, MMP-9, MMP-12 and MMP-14 were significantly higher in vulnerable than stable plaques. TIMP-3 expression was highest in stable and low in vulnerable plaques. Immunohistochemistry revealed intensive staining of MMP-9 in vulnerable plaques. Western blotting confirmed presence of the active form in plaque lysates. In vitro autoradiography showed binding of both inhibitors to stable and vulnerable plaques. CONCLUSIONS: MMPs differed in their expression patterns among plaque phenotypes, providing possible imaging targets. The two tested MMP-2/MMP-9 and MMP-9 inhibitors may be useful to detect atherosclerotic plaques, but not the vulnerable lesions selectively.

Design, synthesis, and initial evaluation of a high affinity positron emission tomography probe for imaging matrix metalloproteinases 2 and 9.

The activity of matrix metalloproteinases (MMPs) is elevated locally under many pathological conditions. Gelatinases MMP2 and MMP9 are of particular interest because of their implication in angiogenesis, cancer cell proliferation and metastasis, and atherosclerotic plaque rupture. The aim of this study was to identify and develop a selective gelatinase inhibitor for imaging active MMP2/MMP9 in vivo. We synthesized a series of N-sulfonylamino acid derivatives with low to high nanomolar inhibitory potencies. (R)-2-(4-(4-Fluorobenzamido)phenylsulfonamido)-3-(1H-indol-3-yl)propanoic acid (7) exhibited the best in vitro binding properties: MMP2 IC50 = 1.8 nM, MMP9 IC50 = 7.2 nM. Radiolabeling of 7 with no carrier added (18)F-radioisotope was accomplished starting from iodonium salts as precursors. The radiochemical yield strongly depended on the iodonium counteranion (ClO4(-) > Br(-) > TFA(-) > tosylate). (18)F-7 was obtained in up to 20% radiochemical yield (decay corrected), high radiochemical purity, and >90 GBq/µmol specific radioactivity. The radiolabeled compound showed excellent stability in vitro and in mice in vivo.

Preclinical characterization of a novel class of 18F-labeled PET tracers for amyloid-ß.

UNLABELLED: Imaging of amyloid-ß (Aß) plaques by PET is more and more integrated into concepts for Alzheimer disease (AD) diagnosis and drug development. The objective of this study was to find novel chemical entities that can be transformed into (18)F-labeled Aß tracers with favorable brain washout kinetics and low background signal. METHODS: High-throughput screening of a large chemical library was used to identify new ligands for fibrillar aggregates of Aß(1-42) peptide. Thirty-two fluorinated derivatives were synthesized and tested for their affinity toward AD brain homogenate. Twelve ligands have been radiolabeled with (18)F. The pharmacokinetic properties of the radioligands were investigated in mouse and monkey biodistribution studies. Binding characteristics were determined by autoradiography of AD brain sections in vitro and using amyloid precursor protein transgenic mice in vivo. RESULTS: The systematic search for Aß imaging agents revealed several fluorinated derivatives with nanomolar affinity for Aß. The fluoropyridyl derivative BAY 1008472 showed a high initial brain uptake (6.45 percentage injected dose per gram at 2 min) and rapid brain washout (ratio of percentage of injected dose per gram of tissue at 2 and 30 min after injection, 9.2) in mice. PET studies of healthy rhesus monkeys confirmed the high initial brain uptake of BAY 1008472 (2.52 standardized uptake value at peak) and a fast elimination of total radioactivity from gray and white matter areas (ratio of standardized uptake value at peak uptake and 60 min 11.0). In autoradiographic analysis, BAY 1008472 selectively detected Aß deposits in human AD brain sections with high contrast and did not bind to τ- or α-synuclein pathologies. Finally, ex vivo autoradiography of brain sections from amyloid precursor protein-transgenic mice confirmed that BAY 1008472 is indeed suitable for the in vivo detection of Aß plaques. CONCLUSION: A new chemical class of Aß tracers has been identified by high-throughput screening. The fluoropyridyl derivative BAY 1008472 shows a favorable preclinical profile including low background binding in gray and white matter. These properties might qualify this new tracer, in particular, to detect subtle amounts or changes of Aß burden in presymptomatic AD and during therapy.

Synthesis of three novel fluorine-18 labeled analogues of L-deprenyl for positron emission tomography (PET) studies of monoamine oxidase B (MAO-B).

The aim in this project was to synthesize and to study fluorine-18 labeled analogues of l-deprenyl which bind selectively to the enzyme monoamine oxidase B (MAO-B). Three fluorinated l-deprenyl analogues have been generated in multistep organic syntheses. The most promising fluorine-18 compound N-[(2S)-1-[(18)F]fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c) was synthesized by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography on human brain tissue sections demonstrated specific binding for compound 4c to brain regions known to have a high content of MAO-B. In addition, the corresponding nonradioactive fluorine-19 compound (13) inhibited recombinant human MAO-B with an IC(50) of 170.5 ± 29 nM but did not inhibit recombinant human MAO-A (IC(50) > 2000 nM), demonstrating its specificity. Biodistribution of 4c in mice showed high initial brain uptake leveling at 5.2 ± 0.04%ID/g after 2 min post injection. In conclusion, compound 4c is a specific inhibitor of MAO-B with high initial brain uptake in mice and is, therefore, a candidate for further investigation in PET.