Direct Evidence for Central Sites of Action of Zolmitriptan (311C90)

Cephalalgia ◽  
1997 ◽  
Vol 17 (3) ◽  
pp. 153-158 ◽  
Author(s):  
PJ Goadsby ◽  
YE Knight
Keyword(s):  
Ex Vivo ◽  
Cervical Spinal Cord ◽  
Specific Binding ◽  
Femoral Vein ◽  
Brain Slices ◽  
Trigeminal Nucleus ◽  
Trigeminal Nucleus Caudalis ◽  
Dorsal Horns ◽  
Sites Of Action

The trigeminovascular system consists of bipolar neurons which innervate pain-sensitive intracranial structures and projecting to neurons in the superficial laminae of the caudal trigeminal nucleus and of the dorsal horns of C1 and C2. The serotonin (5HT1B/D) agonist zolmitriptan (311C90) has been shown to be effective in the treatment of acute attacks of migraine and experimental data suggest that it may have both peripheral and central sites of action. This study sought to further investigate possible central effects of zolmitriptan (311C90) by examining its distribution in the central nervous system. Specific binding of [3H]-zolmitriptan was determined both ex vivo and in vitro in the cat brain. For the ex vivo studies, cats were anaesthetized with halothane and -chloralose (60 mg/kg intraperitoneal). A femoral vein catheter was inserted for injection of the [3H]-zolmitriptan and then 1 h after injection the brain removed. For the in vitro studies fresh frozen brain slices were incubated with labelled and masking concentrations of zolmitriptan. The distribution of [3H]-zolmitriptan was determined using quantitative autoradiographic methods. The in vitro work demonstrated specific binding of [3H]-zolmitriptan in the superficial laminae of the trigeminal nucleus caudalis and dorsal horns of the C and C2 cervical spinal cord. The density of binding was 53 9 fmol/mg for the trigeminal nucleus caudalis, 47 7 fmol/mg for C1 and 50 6 fmol/mg for C2. The ex vivo work demonstrated binding in anatomically identical areas which was less dense than that seen with the in vitro method. These data confirm the existence of a population of receptors that specifically bind zolmitriptan following systemic administration. These receptors may, in part, be responsible for its clinical efficacy and reinforce the importance of central trigeminal neurons as a possible site of action of anti-migraine drugs.

scholarly journals Neuropeptide Expression in the Human Trigeminal Nucleus Caudalis and in the Cervical Spinal Cord C1 and C2

Cephalalgia ◽  
2002 ◽  
Vol 22 (2) ◽  
pp. 112-116 ◽  
Author(s):  
R Uddman ◽  
J Tajti ◽  
M Hou ◽  
F Sundler ◽  
L Edvinsson
Keyword(s):  
Spinal Cord ◽  
Cervical Spinal Cord ◽  
Primary Headaches ◽  
Trigeminal Nucleus ◽  
Trigeminal Nucleus Caudalis ◽  
Dorsal Horns ◽  
Calcitonin Gene ◽  
Pituitary Adenylate Cyclase ◽  
Vascular Component ◽  
Oxide Synthase

In migraine and other primary headaches there is a strong vascular component. Besides the trigeminovascular components some of the associated symptoms point to the involvement of brain stem regions. The central limb of the trigeminal vascular pathway is its projection to the trigeminal nucleus caudalis (TNC) and to the C1-C2 levels of the spinal cord. The aim of the present study was to demonstrate the occurrence of some neurotransmitters in these regions in man. In both the TNC and in the Rexed's laminae I and II of the dorsal horns at the C1 and C2 levels there were numerous substance P immunoreactive fibres. Fibres containing calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) were moderately dense in number. Fibres containing vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS) were not seen in the TNC or at the C1 and C2 levels of the spinal cord.

scholarly journals A New Transgenic Mouse Line for Imaging Mitochondrial Calcium Signals

Function ◽  
2021 ◽  
Vol 2 (3) ◽  
Author(s):  
Nelly Redolfi ◽  
Elisa Greotti ◽  
Giulia Zanetti ◽  
Tino Hochepied ◽  
Cristina Fasolato ◽  
...  
Keyword(s):  
Transgenic Mouse ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Brain Slices ◽  
Resonance Energy ◽  
Mouse Line ◽  
Isolated Cells ◽  
Genomic Locus ◽  
Transgenic Mouse Line

AbstractMitochondria play a key role in cellular calcium (Ca2+) homeostasis. Dysfunction in the organelle Ca2+ handling appears to be involved in several pathological conditions, ranging from neurodegenerative diseases, cardiac failure and malignant transformation. In the past years, several targeted green fluorescent protein (GFP)-based genetically encoded Ca2+ indicators (GECIs) have been developed to study Ca2+ dynamics inside mitochondria of living cells. Surprisingly, while there is a number of transgenic mice expressing different types of cytosolic GECIs, few examples are available expressing mitochondria-localized GECIs, and none of them exhibits adequate spatial resolution. Here we report the generation and characterization of a transgenic mouse line (hereafter called mt-Cam) for the controlled expression of a mitochondria-targeted, Förster resonance energy transfer (FRET)-based Cameleon, 4mtD3cpv. To achieve this goal, we engineered the mouse ROSA26 genomic locus by inserting the optimized sequence of 4mtD3cpv, preceded by a loxP-STOP-loxP sequence. The probe can be readily expressed in a tissue-specific manner upon Cre recombinase-mediated excision, obtainable with a single cross. Upon ubiquitous Cre expression, the Cameleon is specifically localized in the mitochondrial matrix of cells in all the organs and tissues analyzed, from embryos to aged animals. Ca2+ imaging experiments performed in vitro and ex vivo in brain slices confirmed the functionality of the probe in isolated cells and live tissues. This new transgenic mouse line allows the study of mitochondrial Ca2+ dynamics in different tissues with no invasive intervention (such as viral infection or electroporation), potentially allowing simple calibration of the fluorescent signals in terms of mitochondrial Ca2+ concentration ([Ca2+]).

scholarly journals Development of 18F-Labeled Radiotracers for PET Imaging of the Adenosine A2A Receptor: Synthesis, Radiolabeling and Preliminary Biological Evaluation

2021 ◽  
Vol 22 (5) ◽  
pp. 2285
Author(s):  
Thu Hang Lai ◽  
Susann Schröder ◽  
Magali Toussaint ◽  
Sladjana Dukić-Stefanović ◽  
Mathias Kranz ◽  
...  
Keyword(s):  
Specific Binding ◽  
Brain Slices ◽  
Total Activity ◽  
Biological Evaluation ◽  
Adenosine A2a Receptor ◽  
Positron Emission ◽  
A2a Receptor ◽  
Adenosine A2a

The adenosine A2A receptor (A2AR) represents a potential therapeutic target for neurodegenerative diseases. Aiming at the development of a positron emission tomography (PET) radiotracer to monitor changes of receptor density and/or occupancy during the A2AR-tailored therapy, we designed a library of fluorinated analogs based on a recently published lead compound (PPY). Among those, the highly affine 4-fluorobenzyl derivate (PPY1; Ki(hA2AR) = 5.3 nM) and the 2-fluorobenzyl derivate (PPY2; Ki(hA2AR) = 2.1 nM) were chosen for 18F-labeling via an alcohol-enhanced copper-mediated procedure starting from the corresponding boronic acid pinacol ester precursors. Investigations of the metabolic stability of [18F]PPY1 and [18F]PPY2 in CD-1 mice by radio-HPLC analysis revealed parent fractions of more than 76% of total activity in the brain. Specific binding of [18F]PPY2 on mice brain slices was demonstrated by in vitro autoradiography. In vivo PET/magnetic resonance imaging (MRI) studies in CD-1 mice revealed a reasonable high initial brain uptake for both radiotracers, followed by a fast clearance.

scholarly journals PSMA-D4 Radioligand for Targeted Therapy of Prostate Cancer: Synthesis, Characteristics and Preliminary Assessment of Biological Properties

2021 ◽  
Vol 22 (5) ◽  
pp. 2731
Author(s):  
Piotr Garnuszek ◽  
Urszula Karczmarczyk ◽  
Michał Maurin ◽  
Arkadiusz Sikora ◽  
Jolanta Zaborniak ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Targeted Therapy ◽  
Ex Vivo ◽  
Specific Binding ◽  
Biological Evaluation ◽  
In Vitro Assays ◽  
The Novel ◽  
Distribution Profile ◽  
System L

A new PSMA ligand (PSMA-D4) containing the Glu-CO-Lys pharmacophore connected with a new linker system (L-Trp-4-Amc) and chelator DOTA was developed for radiolabeling with therapeutic radionuclides. Herein we describe the synthesis, radiolabeling, and preliminary biological evaluation of the novel PSMA-D4 ligand. Synthesized PSMA-D4 was characterized using TOF-ESI-MS, NMR, and HPLC methods. The novel compound was subject to molecular modeling with GCP-II to compare its binding mode to analogous reference compounds. The radiolabeling efficiency of PSMA-D4 with 177Lu, 90Y, 47Sc, and 225Ac was chromatographically tested. In vitro studies were carried out in PSMA-positive LNCaP tumor cells membranes. The ex vivo tissue distribution profile of the radioligands and Cerenkov luminescence imaging (CLI) was studied in LNCaP tumor-bearing mice. PSMA-D4 was synthesized in 24% yield and purity >97%. The radio complexes were obtained with high yields (>97%) and molar activity ranging from 0.11 to 17.2 GBq mcmol−1, depending on the radionuclide. In vitro assays confirmed high specific binding and affinity for all radiocomplexes. Biodistribution and imaging studies revealed high accumulation in LNCaP tumor xenografts and rapid clearance of radiocomplexes from blood and non-target tissues. These render PSMA-D4 a promising ligand for targeted therapy of prostate cancer (PCa) metastases.

scholarly journals P2Y12 receptor mediates microglial activation via RhoA/ROCK pathway in the trigeminal nucleus caudalis in a mouse model of chronic migraine

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Feng Jing ◽  
Yixin Zhang ◽  
Ting Long ◽  
Wei He ◽  
Guangcheng Qin ◽  
...  
Keyword(s):  
Chronic Migraine ◽  
Microglial Activation ◽  
Morphological Changes ◽  
Critical Role ◽  
Radiant Heat ◽  
P2y12 Receptor ◽  
Trigeminal Nucleus ◽  
Double Labeling ◽  
Trigeminal Nucleus Caudalis

Abstract Background Microglial activation contributes to the development of chronic migraine (CM). The P2Y12 receptor (P2Y12R), a metabolic purinoceptor that is expressed on microglia in the central nervous system (CNS), has been indicated to play a critical role in the pathogenesis of chronic pain. However, whether it contributes to the mechanism of CM remains unknown. Thus, the present study investigated the precise details of microglial P2Y12R involvement in CM. Methods Mice subjected to recurrent nitroglycerin (NTG) treatment were used as the CM model. Hyperalgesia were assessed by mechanical withdrawal threshold to electronic von Frey and thermal withdrawal latency to radiant heat. Western blot and immunohistochemical analyses were employed to detect the expression of P2Y12R, Iba-1, RhoA, and ROCK2 in the trigeminal nucleus caudalis (TNC). To confirm the role of P2Y12R and RhoA/ROCK in CM, we systemically administered P2Y12R antagonists (MRS2395 and clopidogrel) and a ROCK2 inhibitor (fasudil) and investigated their effects on microglial activation, c-fos, and calcitonin gene-related peptide (CGRP) expression in the TNC. To further confirm the effect of P2Y12R on microglial activation, we preincubated lipopolysaccharide (LPS)-treated BV-2 microglia with MRS2395 and clopidogrel. ELISA was used to evaluate the levels of inflammatory cytokines. Results The protein levels of P2Y12R, GTP-RhoA, ROCK2, CGRP, c-fos, and inducible nitric oxide synthase (iNOS) in the TNC were increased after recurrent NTG injection. A double labeling study showed that P2Y12R was restricted to microglia in the TNC. MRS2395 and clopidogrel attenuated the development of tactile allodynia and suppressed the expression of CGRP, c-fos, and GTP-RhoA/ROCK2 in the TNC. Furthermore, fasudil also prevented hyperalgesia and suppressed the expression of CGRP in the TNC. In addition, inhibiting P2Y12R and ROCK2 activities suppressed NTG-induced microglial morphological changes (process retraction) and iNOS production in the TNC. In vitro, a double labeling study showed that P2Y12R was colocalized with BV-2 cells, and the levels of iNOS, IL-1β, and TNF-α in LPS-stimulated BV-2 microglia were reduced by P2Y12R inhibitors. Conclusions These data demonstrate that microglial P2Y12R in the TNC plays a critical role in the pathogenesis of CM by regulating microglial activation in the TNC via RhoA/ROCK pathway.

scholarly journals Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243286
Author(s):  
Mira Woitok ◽  
Elena Grieger ◽  
Olusiji A. Akinrinmade ◽  
Susanne Bethke ◽  
Anh Tuan Pham ◽  
...  
Keyword(s):  
Blood Cells ◽  
Fluorescent Dyes ◽  
Ex Vivo ◽  
Specific Binding ◽  
Coupling Reaction ◽  
Annexin V ◽  
Therapeutic Agents ◽  
Apoptotic Cells ◽  
Cell Markers

In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.

scholarly journals Small molecule control of neurotransmitter sulfonation

2020 ◽  
pp. jbc.RA120.015177
Author(s):  
Ian Cook ◽  
Mary Cacace ◽  
Ting Wang ◽  
Kristie Darrah ◽  
Alexander Deiters ◽  
...  
Keyword(s):  
Active Site ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Specific Binding ◽  
Protein A ◽  
Binding Pocket ◽  
Human Mammary Epithelial Cell ◽  
Primary Objective ◽  
Epithelial Cell Line

Controlling unmodified serotonin levels in brain synapses is a primary objective when treating major depressive disorder — a disease that afflicts ~20% of the world’s population. Roughly 60% of patients respond poorly to first-line treatments and thus new therapeutic strategies are sought. Toward this end, we have constructed isoform-specific inhibitors of the human cytosolic sulfotransferase 1A3 (SULT1A3) — the isoform responsible for sulfonating ~80% of the serotonin in extracellular brain fluid. The inhibitor design includes a core ring structure, which anchors the inhibitor into a SULT1A3-specific binding pocket located outside the active site, and a sidechain crafted to act as a latch to inhibit turnover by fastening down the SULT1A3 active-site cap. The inhibitors are allosteric, they bind with nanomolar affinity and are highly specific for the 1A3 isoform. The cap-stabilizing effects of the latch can be accurately calculated and are predicted to extend throughout the cap and into the surrounding protein. A free energy correlation demonstrates that the percent inhibition at saturating inhibitor varies linearly with cap stabilization — the correlation is linear because the rate-limiting step of the catalytic cycle, nucleotide release, scales linearly with the fraction of enzyme in the cap-open form. Inhibitor efficacy in cultured cells was studied using a human mammary epithelial cell line that expresses SULT1A3 at levels comparable to those found in neurons. The inhibitors perform similarly in ex vivo and in vitro studies; consequently, SULT1A3 turnover can now be potently suppressed in an isoform-specific manner in human cells.

scholarly journals Fully Characterized Mature Human iPS- and NMP-Derived Motor Neurons Thrive Without Neuroprotection in the Spinal Contusion Cavity

2022 ◽  
Vol 15 ◽  
Author(s):  
Zachary T. Olmsted ◽  
Cinzia Stigliano ◽  
Brandon Marzullo ◽  
Jose Cibelli ◽  
Philip J. Horner ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Progenitor Cells ◽  
Motor Neurons ◽  
Ex Vivo ◽  
Cervical Spinal Cord ◽  
Neural Cell ◽  
Oligodendrocyte Progenitor Cells ◽  
Limited Information ◽  
Cord Injury

Neural cell interventions in spinal cord injury (SCI) have focused predominantly on transplanted multipotent neural stem/progenitor cells (NSPCs) for animal research and clinical use due to limited information on survival of spinal neurons. However, transplanted NSPC fate is unpredictable and largely governed by injury-derived matrix and cytokine factors that are often gliogenic and inflammatory. Here, using a rat cervical hemicontusion model, we evaluate the survival and integration of hiPSC-derived spinal motor neurons (SMNs) and oligodendrocyte progenitor cells (OPCs). SMNs and OPCs were differentiated in vitro through a neuromesodermal progenitor stage to mimic the natural origin of the spinal cord. We demonstrate robust survival and engraftment without additional injury site modifiers or neuroprotective biomaterials. Ex vivo differentiated neurons achieve cervical spinal cord matched transcriptomic and proteomic profiles, meeting functional electrophysiology parameters prior to transplantation. These data establish an approach for ex vivo developmentally accurate neuronal fate specification and subsequent transplantation for a more streamlined and predictable outcome in neural cell-based therapies of SCI.

scholarly journals Novel cytokine–antibody fusion protein, N-820, to enhance the functions of ex vivo expanded natural killer cells against Burkitt lymphoma

2020 ◽  
Vol 8 (2) ◽  
pp. e001238
Author(s):  
Yaya Chu ◽  
Gaurav Nayyar ◽  
Nang Kham Su ◽  
Jeremy M Rosenblum ◽  
Patrick Soon-Shiong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Fusion Protein ◽  
Natural Killer ◽  
Burkitt Lymphoma ◽  
Ex Vivo ◽  
Specific Binding ◽  
Expression Profiles ◽  
Nsg Mice

BackgroundThe prognosis of patients with relapsed or progressive B cell (CD20+) non-Hodgkin’s lymphoma (B-NHL), including Burkitt lymphoma (BL), is dismal due to chemoradiotherapy resistance. Novel therapeutic strategies are urgently needed. N-820 is a fusion protein of N-803 (formerly known as ALT-803) to four single-chains of rituximab. This agent has tri-specific binding activity to CD20 and enhanced antibody-dependent cell-mediated cytotoxicity.MethodsWe investigated the anti-tumor combinatorial effects of N-820 with ex vivo expanded peripheral blood natural killer (exPBNK) cells against rituximab-sensitive and rituximab-resistant CD20+ BL in vitro using cytoxicity assays and in vivo using human BL xenografted NOD/SCID/IL2rγnull (NSG) mice. We also investigated the cytokines/chemokines/growth factors released using ELISA and multiplex assay. Gene expression changes were examined using real-time PCR arrays.ResultsN-820 significantly enhanced the expression of NK activating receptors (p<0.001) and the proliferation of exPBNK cells with enhanced Ki67 expression and Stat5 phosphorylation (p<0.001). N-820 significantly enhanced the secretion of cytokines, chemokines, and growth factors including GM-CSF, RANTES, MIP-1B (p<0.001) from exPBNK cells as compared with the combination of rituximab+N-803. Importantly, N-820 significantly enhanced in vitro cytotoxicity (p<0.001) of exPBNK with enhanced granzyme B and IFN-γ release (p<0.001) against BL. Gene expression profiles in exPBNK stimulated by N-820+Raji-2R showed enhanced transcription of CXCL9, CXCL1, CSF2, CSF3, GZMB, and IFNG. Moreover, N-820 combined with exPBNK significantly inhibited rituximab-resistant BL growth (p<0.05) and extended the survival (p<0.05) of BL xenografted NSG mice.ConclusionsOur results provide the rationale for the development of a clinical trial of N-820 alone or in combination with endogenous or ex vivo expanded NK cells in patients with CD20+ B-NHL failing prior rituximab containing chemoimmunotherapy regimens.

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