In Vitro and in Vivo Investigations of the Specific Binding of Substance P-γ-Cyclodextrin Adducts to Rat Brain Nk1 Receptors

Summary Aim: Disturbances of the D4 receptor subtype have been implicated in the genesis of a broad range of psychiatric disorders. In order to assess the suitability of a radioiodinated analogue of the D4-selective ligand FAUC 113 for tracer studies in vivo, we investigated the in-vivo stability, biodistribution and brain-uptake of 7-131I-FAUC 113 in Sprague-Dawley rats. Methods: Radiolabelling was carried out with high radiochemical yield and specific activity. After intravenous injection, blood and tissue samples, taken at designated time intervals, were collected for analysis. Analyses of metabolites were performed by radiohplc and radio-tlc. For in-vivo evaluation, sagittal cryo-sections of the rat brain were investigated by in-vitro and exvivo autoradiography on a μ-Imager system. Results: 7-131I-FAUC 113 was rapidly cleared from blood. Highest uptake was observed in kidney (0.603±0.047% ID/g, n=4) and liver (0.357±0.070% ID/g, n=4) at 10 min p.i.; 7-131I-FAUC 113 displayed rapid uptake (0.21-0.26% ID/g) and fast clearance in various brain regions consistent with the determined logP-value of 2.36±0.15 (n=4). In-vivo stability of 7-131I-FAUC 113 was confirmed in the frontal cortex (>95%). Ex-vivo autoradiography revealed a frontal cortex-to-cerebellum ratio of 1.57±0.13 at 10 min p.i. (n=6). Coinjection with L-750667 could not suppress any putative specific binding of 7-131I-FAUC 113. In-vitro autoradiography using authentic 7-iodo-FAUC 113 or L-750667 failed to cause significant displacement of the radioligand. Conclusions: Radioiodinated FAUC 113 does not allow imaging of D4 receptors in the rat brain in vivo nor in vitro. Further work should aim at the development of selective dopamine D4 radioligands with improved tracer characteristics, such as receptor affinity and subtype selectivity, specific activity or blood-brainbarrier permeability.

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Correlation between in vitro and in vivo Data of Radiolabeled Peptide for Tumor Targeting

Mini-Reviews in Medicinal Chemistry ◽

10.2174/1389557519666190304120011 ◽

2019 ◽

Vol 19 (12) ◽

pp. 950-960

Author(s):

Soghra Farzipour ◽

Seyed Jalal Hosseinimehr

Keyword(s):

Tumor Targeting ◽

Single Photon ◽

Specific Binding ◽

Specific Interaction ◽

Photon Emission ◽

Emission Computed Tomography ◽

Mixed Effect ◽

Radiolabeled Peptides

Tumor-targeting peptides have been generally developed for the overexpression of tumor specific receptors in cancer cells. The use of specific radiolabeled peptide allows tumor visualization by single photon emission computed tomography (SPECT) and positron emission tomography (PET) tools. The high affinity and specific binding of radiolabeled peptide are focusing on tumoral receptors. The character of the peptide itself, in particular, its complex molecular structure and behaviors influence on its specific interaction with receptors which are overexpressed in tumor. This review summarizes various strategies which are applied for the expansion of radiolabeled peptides for tumor targeting based on in vitro and in vivo specific tumor data and then their data were compared to find any correlation between these experiments. With a careful look at previous studies, it can be found that in vitro unblock-block ratio was unable to correlate the tumor to muscle ratio and the success of radiolabeled peptide for in vivo tumor targeting. The introduction of modifiers’ approaches, nature of peptides, and type of chelators and co-ligands have mixed effect on the in vitro and in vivo specificity of radiolabeled peptides.

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Improved in vivo PET imaging of the adenosine A2A receptor in the brain using [18F]FLUDA, a deuterated radiotracer with high metabolic stability

European Journal of Nuclear Medicine and Molecular Imaging ◽

10.1007/s00259-020-05164-4 ◽

2021 ◽

Author(s):

Thu Hang Lai ◽

Magali Toussaint ◽

Rodrigo Teodoro ◽

Sladjana Dukić-Stefanović ◽

Daniel Gündel ◽

...

Keyword(s):

Specific Binding ◽

Radiochemical Yield ◽

Toxicity Study ◽

In Vivo Evaluation ◽

One Pot ◽

Specific Binding Ratio ◽

Mri Studies ◽

The Brain

Abstract Purpose The adenosine A2A receptor has emerged as a therapeutic target for multiple diseases, and thus the non-invasive imaging of the expression or occupancy of the A2A receptor has potential to contribute to diagnosis and drug development. We aimed at the development of a metabolically stable A2A receptor radiotracer and report herein the preclinical evaluation of [18F]FLUDA, a deuterated isotopologue of [18F]FESCH. Methods [18F]FLUDA was synthesized by a two-step one-pot approach and evaluated in vitro by autoradiographic studies as well as in vivo by metabolism and dynamic PET/MRI studies in mice and piglets under baseline and blocking conditions. A single-dose toxicity study was performed in rats. Results [18F]FLUDA was obtained with a radiochemical yield of 19% and molar activities of 72–180 GBq/μmol. Autoradiography proved A2A receptor–specific accumulation of [18F]FLUDA in the striatum of a mouse and pig brain. In vivo evaluation in mice revealed improved stability of [18F]FLUDA compared to that of [18F]FESCH, resulting in the absence of brain-penetrant radiometabolites. Furthermore, the radiometabolites detected in piglets are expected to have a low tendency for brain penetration. PET/MRI studies confirmed high specific binding of [18F]FLUDA towards striatal A2A receptor with a maximum specific-to-non-specific binding ratio in mice of 8.3. The toxicity study revealed no adverse effects of FLUDA up to 30 μg/kg, ~ 4000-fold the dose applied in human PET studies using [18F]FLUDA. Conclusions The new radiotracer [18F]FLUDA is suitable to detect the availability of the A2A receptor in the brain with high target specificity. It is regarded ready for human application.

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In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

Scientific Reports ◽

10.1038/s41598-021-84622-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Merricka C. Livingstone ◽

Alexis A. Bitzer ◽

Alish Giri ◽

Kun Luo ◽

Rajeshwer S. Sankhala ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Monoclonal Antibodies ◽

Disease Burden ◽

Vaccine Candidate ◽

Specific Binding ◽

Circumsporozoite Protein ◽

Target Epitope ◽

Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

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Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽

10.1038/s41375-021-01282-6 ◽

2021 ◽

Author(s):

Christos Georgiadis ◽

Jane Rasaiyaah ◽

Soragia Athina Gkazi ◽

Roland Preece ◽

Aniekan Etuk ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Specific Binding ◽

Base Editing ◽

Car T Cells ◽

Dna And Rna ◽

Car T ◽

T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22052285 ◽

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.

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Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

Biochemical Journal ◽

10.1042/bj20020123 ◽

2002 ◽

Vol 364 (2) ◽

pp. 343-347 ◽

Cited By ~ 51

Author(s):

Gareth J.O. EVANS ◽

Alan MORGAN

Keyword(s):

Rat Brain ◽

Direct Interaction ◽

Secretory Vesicle ◽

Brain Membrane ◽

Synaptotagmin I ◽

Phosphorylated Form ◽

Order Of Magnitude ◽

And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

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Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00253.2005 ◽

2007 ◽

Vol 292 (4) ◽

pp. L915-L923 ◽

Cited By ~ 10

Author(s):

Jaime Chávez ◽

Patricia Segura ◽

Mario H. Vargas ◽

José Luis Arreola ◽

Edgar Flores-Soto ◽

...

Keyword(s):

Substance P ◽

Guinea Pigs ◽

Smooth Muscle Contraction ◽

In Vivo Experiments ◽

Intracellular Ca ◽

Neurokinin 1 ◽

Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

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