Metabolism of a Bioorthogonal PET Tracer Candidate [F-19/F-18]SiFA-Tetrazine in Mouse Liver Microsomes: Biotransformation Pathways and Defluorination Investigated by UHPLC-HRMS

Organofluorosilicon based F-18-radiolabeling is an efficient method for incorporating fluorine-18 into BF-radiopharmaceuticals for positron emission tomography (PET) by F-19/F-18 isotopic exchange (1E). The first PET radiopharmaceutical, F-18 SiFAlin-TATE, radiolabeled with a silicon-based [F-18]fluoride acceptor (SiFA), namely, a para-substituted di-tert-butyl[F-18]-fluorosilylbenzene, has entered clinical trials, and is paving the way for other potential [F-18]SiFA-labeled radiopharmaceuticals for diagnostic use. In this study, we report the in vitro metabolism of an oxime-linked SiFA tetrazine (SiFA-Tz), a new PET-radiotracer candidate, recently evaluated for pretargeted PET imaging and macromolecule labeling. Metabolism of SiFA-Tz was studied in mouse liver microsomes (MLM) for elucidating its major biotransformation pathways. Nontargeted screening by ultrahigh performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) was utilized for detection of unknown metabolites. The oxime bond between the SiFA and Tz groups forms two geometric (E/Z) isomers, which underwent the same biotransformations, but unexpectedly with different kinetics. In total, nine proposed metabolites of SiFA-Tz from phase I and II reactions were detected, five of which were defluorinated in MLMs, elucidating the metabolic pathway leading to previously reported defluorination of [F-18]SiFA-Tz in vivo. Based on the HRMS studies a biotransformation pathway is proposed: hydroxylation (+O) to tert-butyl group adjacent to the silicon, followed by oxidative defluorination (+OH/-F) cleaving the fluorine off the silicon. Interestingly, eight proposed metabolites of a reduced dihydrotetrazine analogue, SiFA-H,Tz, from phase I and II reactions were additionally detected. To the best of our knowledge, this is the first reported comprehensive investigation of enzyme mediated metabolic pathway of tetrazines and para-substituted di-tertbutylfluorosilylbenzene fluoride acceptors, providing novel structural information on the biotransformation and fragmentation patterns of radiotracers bearing these structural motifs. By investigating the metabolism preceding defluorination, structurally optimized new SiFA compounds can be designed for expanding the portfolio of efficient F-19/F-18 isotopic exchange labeling probes for PET imaging

Synthesis, in vitro and in vivo evaluation of 1,3,5-triazines as cannabinoid CB2 receptor agonists

The cannabinoid receptors type 2 (CBR2) are attractive therapeutic targets of the endocannabinoid signaling system (ECS) as they are not displaying the undesired psychotropic and cardiovascular side-effects seen with cannabinoid receptor type 1 (CB1R) agonists. In continuation of our previous work on 2,4,6-trisubstituted 1,3,5-triazines as potent CB2 agonists, we synthesized an additional series of more polar analogues (1-10), which were found to possess high CB2R agonist activity with enhanced water solubility. The most potent compound in the series was N-(adamantan-1-yl)-4-ethoxy-6-(4-(2-fluoroethyl)piperazin-1-yl)-1,3,5-triazin-2-amine (9) with EC50 value of 0.60nM. To further evaluate the biological effects of the compounds, the selected compounds were tested in vitro against four different cell lines. A human retinal pigment epithelial cell line (ARPE-19) was used to evaluate the cytotoxicity of the compounds whereas an androgen-sensitive human prostate adenocarcinoma cell line (LNCaP), a Jurkat leukemia cell line and a C8161 melanoma cell line were used to assess the antiproliferative activity of the compounds. The most interesting results were obtained for N-(adamantan-1-yl)-4-ethoxy-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (6), which induced cell viability decrease in prostate and leukemia cell lines, and diminished proliferation of C8161 melanoma cells. The results could be reversed in leukemia cells with the selective CB2R antagonist AM630, whereas in prostate cells the AM630 induced a significant cell viability decrease with a mechanism probably unlinked to CB2 cannabinoid receptor. The antiproliferative effect of 6 on the melanoma cells seemed not to be mediated via the CB1R or CB2R. No cytotoxicity was detected against ARPE-19 cell line at concentrations of 1 and 10μM for compound 6. However, at 30μM concentration the compound 6 decreased the cell viability. Finally, in order to estimate in vivo behavior of these compounds, (18)F labeled PET ligand, N-cyclopentyl-4-ethoxy-6-(4-(2-fluoro-18-ethyl)piperazin-1-yl)-1,3,5-triazin-2-amine ([(18)F]5), was synthesized and its biodistribution was determined in healthy male Sprague-Dawley rats. As a result, the tracer showed a rapid (<15min) elimination in urine accompanied by a slower excretion via the hepatobiliary route. In conclusion, we further demonstrated that 1,3,5-triazine scaffold serves as a suitable template for the design of highly potent CB2R agonists with reasonable water solubility properties. The compounds may be useful when studying the role of the endocannabinoid system in different diseases. The triazine scaffold is also a promising candidate for the development of new CB2R PET ligands

Pretargeted PET Imaging of trans-Cyclooctene-Modified Porous Silicon Nanoparticles

Pretargeted positron emission tomography (PET) imaging based on bioorthogonal chemical reactions has proven its potential in immunoimaging. It may also have great potential in nanotheranostic applications. Here, we report the first successful pretargeted PET imaging of trans-cyclooctene-modified mesoporous silicon nanoparticles, using F-18-labeled tetrazine as a tracer. The inverse electron-demand Diels-Alder cycloaddition (IEDDA) reaction was fast, resulting in high radioactivity accumulation in the expected organs within 10 min after the administration of the tracer. The highest target-to-background ratio was achieved 120 min after the tracer injection. A clear correlation between the efficiency of the in vivo IEDDA labeling reaction and the injected amount of the tracer was observed. The radioactivity accumulation decreased with the increased amount of the co-injected carrier, indicating saturation in the reaction sites. This finding was supported by the in vitro results. Our study suggests that pretargeted imaging has excellent potential in nanotheranostic PET imaging when using high-specific-activity tracers

An updated overview of clinical guidelines for the management of non-specific low back pain in primary care

The aim of this study was to present and compare the content of (inter)national clinical guidelines for the management of low back pain. To rationalise the management of low back pain, evidence-based clinical guidelines have been issued in many countries. Given that the available scientific evidence is the same, irrespective of the country, one would expect these guidelines to include more or less similar recommendations regarding diagnosis and treatment. We updated a previous review that included clinical guidelines published up to and including the year 2000. Guidelines were included that met the following criteria: the target group consisted mainly of primary health care professionals, and the guideline was published in English, German, Finnish, Spanish, Norwegian, or Dutch. Only one guideline per country was included: the one most recently published. This updated review includes national clinical guidelines from 13 countries and 2 international clinical guidelines from Europe published from 2000 until 2008. The content of the guidelines appeared to be quite similar regarding the diagnostic classification (diagnostic triage) and the use of diagnostic and therapeutic interventions. Consistent features for acute low back pain were the early and gradual activation of patients, the discouragement of prescribed bed rest and the recognition of psychosocial factors as risk factors for chronicity. For chronic low back pain, consistent features included supervised exercises, cognitive behavioural therapy and multidisciplinary treatment. However, there are some discrepancies for recommendations regarding spinal manipulation and drug treatment for acute and chronic low back pain. The comparison of international clinical guidelines for the management of low back pain showed that diagnostic and therapeutic recommendations are generally similar. There are also some differences which may be due to a lack of strong evidence regarding these topics or due to differences in local health care systems. The implementation of these clinical guidelines remains a challenge for clinical practice and research

Comparison of Ga-68-DOTA-Siglec-9 and F-18-Fluorodeoxyribose-Siglec-9: Inflammation Imaging and Radiation Dosimetry

Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a ligand of inflammation-inducible vascular adhesion protein-1 (VAP1). We compared Ga-68-DOTA-and F-18-fluorodeoxyribose-(FDR) labeled Siglec-9motif peptides for PET imaging of inflammation. Methods. Firstly, we examined Ga-68-DOTA-Siglec-9 and F-18-FDR-Siglec-9 in rats with skin/muscle inflammation. We then studied F-18-FDR-Siglec-9 for the detection of inflamed atherosclerotic plaques in mice and compared it with previous Ga-68-DOTA-Siglec-9 results. Lastly, we estimated human radiation dosimetry fromthe rat data. Results. In rats, Ga-68-DOTA-Siglec-9 (SUV, 0.88 +/- 0.087) and F-18-FDR-Siglec-9 (SUV, 0.77 +/- 0.22) showed comparable (P = 0.29) imaging of inflammation. In atherosclerotic mice, 18 FFDR- Siglec-9 detected inflamed plaques with a target-to-background ratio (1.6 1/8 0.078) similar to previously tested Ga-68-DOTASiglec- 9 (P = 0.35). Humaneffectivedose estimates for Ga-68-DOTA-Siglec-9 and (18) F-FDR-Siglec-9were 0.024 and 0.022 mSv/MBq, respectively. Conclusion. Both tracers are suitable for PET imaging of inflammation. The easier production and lower cost of (68)GaDOTA-Siglec-9 present advantages over F-18-FDR-Siglec-9, indicating it as a primary choice for clinical studies

Quantitative radiologic criteria for the diagnosis of lumbar spinal stenosis: a systematic literature review

Background: Beside symptoms and clinical signs radiological findings are crucial in the diagnosis of lumbar spinal stenosis (LSS). We investigate which quantitative radiological signs are described in the literature and which radilogical criteria are used to establish inclusion criteria in clincical studies evaluating different treatments in patients with lumbar spinal stenosis. Methods: A literature search was performed in Medline, Embase and the Cochrane library to identify papers reporting on radiological criteria to describe LSS and systematic reviews investigating the effects of different treatment modalities. Results: 25 studies reporting on radiological signs of LSS and four systematic reviews related to the evaluation of different treatments were found. Ten different parameters were identified to quantify lumbar spinal stenosis. Most often reported measures for central stenosis were antero-posterior diameter (< 10 mm) and cross-sectional area (< 70 mm2) of spinal canal. For lateral stenosis height and depth of the lateral recess, and for foraminal stenosis the foraminal diameter were typically used. Only four of 63 primary studies included in the systematic reviews reported on quantitative measures for defining inclusion criteria of patients in prognostic studies. Conclusions: There is a need for consensus on well-defined, unambiguous radiological criteria to define lumbar spinal stenosis in order to improve diagnostic accuracy and to formulate reliable inclusion criteria for clinical studies

Integrative care for the management of low back pain: use of a clinical care pathway

<p>Abstract</p> <p>Background</p> <p>For the treatment of chronic back pain, it has been theorized that integrative care plans can lead to better outcomes than those achieved by monodisciplinary care alone, especially when using a collaborative, interdisciplinary, and non-hierarchical team approach. This paper describes the use of a care pathway designed to guide treatment by an integrative group of providers within a randomized controlled trial.</p> <p>Methods</p> <p>A clinical care pathway was used by a multidisciplinary group of providers, which included acupuncturists, chiropractors, cognitive behavioral therapists, exercise therapists, massage therapists and primary care physicians. Treatment recommendations were based on an evidence-informed practice model, and reached by group consensus. Research study participants were empowered to select one of the treatment recommendations proposed by the integrative group. Common principles and benchmarks were established to guide treatment management throughout the study.</p> <p>Results</p> <p>Thirteen providers representing 5 healthcare professions collaborated to provide integrative care to study participants. On average, 3 to 4 treatment plans, each consisting of 2 to 3 modalities, were recommended to study participants. Exercise, massage, and acupuncture were both most commonly recommended by the team and selected by study participants. Changes to care commonly incorporated cognitive behavioral therapy into treatment plans.</p> <p>Conclusion</p> <p>This clinical care pathway was a useful tool for the consistent application of evidence-based care for low back pain in the context of an integrative setting.</p> <p>Trial registration</p> <p>ClinicalTrials.gov NCT00567333</p

Barriers and progress in the treatment of low back pain

Low back pain is a common and costly condition and for most people is likely to be a recurrent problem throughout their lifetime. The management of patients with low back pain has been positively influenced by the rise in high quality clinical trials and systematic reviews in recent decades, and this body of evidence, synthesized in many clinical practice guidelines, has improved our knowledge about which treatments for low back pain are useful and which are not. For the largest group of patients, those with non-specific low back pain for whom a clear diagnosis cannot be given, the reality is that the treatments we have to offer tend to produce small effects, often only in the short term and none appear to effectively change long-term prognosis. This commentary summarizes the array of treatments currently available, notes the results of recent trials and guidelines and considers alternative approaches that may prove more valuable in achieving better patient outcomes in the future

Cost-effectiveness of minimal interventional procedures for chronic mechanical low back pain: design of four randomised controlled trials with an economic evaluation

Background: Minimal interventional procedures are frequently applied in patients with mechanical low back pain which is defined as pain presumably resulting from single sources: facet, disc, sacroiliac joint or a combination of these. Usually, these minimal interventional procedures are an integral part of a multidisciplinary pain programme. A recent systematic review issued by the Dutch Health Insurance Council showed that the effectiveness of these procedures for the total group of patients with chronic low back pain is yet unclear and cost-effectiveness unknown. The aim of the study is to evaluate whether a multidisciplinary pain programme with minimal interventional procedures is cost-effective compared to the multidisciplinary pain programme alone for patients with chronic mechanical low back pain who did not respond to conservative primary care and were referred to a pain clinic. Methods. All patients with chronic low back pain who are referred to one of the 13 participating pain clinics will be asked to participate in an observational study. Patients with a suspected diagnosis of facet, disc or sacroiliac joint problems will receive a diagnostic block to confirm this diagnosis. If confirmed, they will be asked to participate in a Randomized Controlled Trial (RCT). For each single source a separate RCT will be conducted. Patients with a combination of facet, disc or sacroiliac joint problems will be invited for participation in a RCT as well. An economic evaluation from a societal perspective will be performed alongside these four RCTs. Patients will complete questionnaires at baseline, 3 and 6 weeks, 3, 6, 9 and 12 months after start of the treatment

Crucial role of calbindin-D28k in the pathogenesis of Alzheimer's disease mouse model

Calbindin-D28k (CB), one of the major calcium-binding and buffering proteins, has a critical role in preventing a neuronal death as well as maintaining calcium homeostasis. Although marked reductions of CB expression have been observed in the brains of mice and humans with Alzheimer disease (AD), it is unknown whether these changes contribute to AD-related dysfunction. To determine the pathogenic importance of CB depletions in AD models, we crossed 5 familial AD mutations (5XFAD; Tg) mice with CB knock-out (CBKO) mice and generated a novel line CBKO·5XFAD (CBKOTg) mice. We first identified the change of signaling pathways and differentially expressed proteins globally by removing CB in Tg mice using mass spectrometry and antibody microarray. Immunohistochemistry showed that CBKOTg mice had significant neuronal loss in the subiculum area without changing the magnitude (number) of amyloid β-peptide (Aβ) plaques deposition and elicited significant apoptotic features and mitochondrial dysfunction compared with Tg mice. Moreover, CBKOTg mice reduced levels of phosphorylated mitogen-activated protein kinase (extracellular signal-regulated kinase) 1/2 and cAMP response element-binding protein at Ser-133 and synaptic molecules such as N-methyl-D-aspartate receptor 1 (NMDA receptor 1), NMDA receptor 2A, PSD-95 and synaptophysin in the subiculum compared with Tg mice. Importantly, this is the first experimental evidence that removal of CB from amyloid precursor protein/presenilin transgenic mice aggravates AD pathogenesis, suggesting that CB has a critical role in AD pathogenesis