Chemical assays--new opportunities.

1987 ◽  
Vol 33 (9) ◽  
pp. 1574-1578 ◽  
Author(s):  
A E Burkhardt
Keyword(s):  
Quality Control ◽  
Solid Phase ◽  
Clinical Chemistry ◽  
Test Format ◽  
Home Testing

Abstract The acceptance of the solid-phase format in various areas of clinical chemistry is the consequence of the advantages of this test format, which include stability of the reagents, unitized packaging, convenient and small instruments, and minimal preparations by users before testing. Overall, these advantages provide very convenient tests. Future successful uses of solid-phase reagents depend upon how well these features meet the needs of the users. Needs for systems to be used in the decentralized laboratory include even less cost, even more convenience, and improved quality control. Needs for home testing include convenient tests with clinically useful accuracy, improved quality control, and improved recording systems to overcome user bias in recording results. New solid-phase technologies being developed include noncolorimetric systems suitable for use with miniature probes, for in vitro or in vivo use, and spectrophotometric systems for determinations of analytes directly in capillaries of the skin without invasive sampling.

68Ga-labeled HBED-CC Variant of uPAR Targeting Peptide AE105 Compared with 68Ga-NODAGA-AE105

2019 ◽  
Vol 18 (9) ◽  
pp. 1289-1294 ◽  
Author(s):  
Kusum Vats ◽  
Rohit Sharma ◽  
Haladhar D. Sarma ◽  
Drishty Satpati ◽  
Ashutosh Dash
Keyword(s):  
Solid Phase ◽  
Evaluation Studies ◽  
Radiochemical Yield ◽  
In Vivo Evaluation ◽  
Peptide Conjugates ◽  
Biodistribution Studies ◽  
Target Organs ◽  
U87mg Tumor

Aims: The urokinase Plasminogen Activator Receptors (uPAR) over-expressed on tumor cells and their invasive microenvironment are clinically significant molecular targets for cancer research. uPARexpressing cancerous lesions can be suitably identified and their progression can be monitored with radiolabeled uPAR targeted imaging probes. Hence this study aimed at preparing and evaluating two 68Ga-labeled AE105 peptide conjugates, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 as uPAR PET-probes. Method: The peptide conjugates, HBED-CC-AE105-NH2 and NODAGA-AE105-NH2 were manually synthesized by standard Fmoc solid phase strategy and subsequently radiolabeled with 68Ga eluted from a commercial 68Ge/68Ga generator. In vitro cell studies for the two radiotracers were performed with uPAR positive U87MG cells. Biodistribution studies were carried out in mouse xenografts with the subcutaneously induced U87MG tumor. Results: The two radiotracers, 68Ga-NODAGA-AE105 and 68Ga-HBED-CC-AE105 that were prepared in >95% radiochemical yield and >96% radiochemical purity, exhibited excellent in vitro stability. In vivo evaluation studies revealed higher uptake of 68Ga-HBED-CC-AE105 in U87MG tumor as compared to 68Ga-NODAGAAE105; however, increased lipophilicity of 68Ga-HBED-CC-AE105 resulted in slower clearance from blood and other non-target organs. The uPAR specificity of the two radiotracers was ascertained by significant (p<0.05) reduction in the tumor uptake with a co-injected blocking dose of unlabeled AE-105 peptide. Conclusion: Amongst the two radiotracers studied, the neutral 68Ga-NODAGA-AE105 with more hydrophilic chelator exhibited faster clearance from non-target organs. The conjugation of HBED-CC chelator (less hydrophilic) resulted in negatively charged 68Ga-HBED-CC-AE105 which was observed to have high retention in blood that decreased target to non-target ratios.

Oxytocin analogues with non-coded amino acid residues in position 8: [8-Neopentylglycine]oxytocin and [8-cycloleucine]oxytocin

1987 ◽  
Vol 52 (9) ◽  
pp. 2317-2325 ◽  
Author(s):  
Jan Hlaváček ◽  
Jan Pospíšek ◽  
Jiřina Slaninová ◽  
Walter Y. Chan ◽  
Victor J. Hruby
Keyword(s):  
Amino Acid ◽  
Solid Phase Synthesis ◽  
Solid Phase ◽  
The Other ◽  
Amino Acid Residues ◽  
Phase Synthesis ◽  
Other Hand ◽  
Fragment Condensation

[8-Neopentylglycine]oxytocin (II) and [8-cycloleucine]oxytocin (III) were prepared by a combination of solid-phase synthesis and fragment condensation. Both analogues exhibited decreased uterotonic potency in vitro, each being about 15-30% that of oxytocin. Analogue II also displayed similarly decreased uterotonic potency in vivo and galactogogic potency. On the other hand, analogue III exhibited almost the same potency as oxytocin in the uterotonic assay in vivo and in the galactogogic assay.

The 1- and 2-Naphthylalanine Analogs of Oxytocin and Vasopressin

1995 ◽  
Vol 60 (12) ◽  
pp. 2170-2177 ◽  
Author(s):  
Zdenko Procházka ◽  
Jiřina Slaninová
Keyword(s):  
Amino Acids ◽  
Solid Phase ◽  
Pressor Test

Solid phase technique on p-methylbenzhydrylamine resin was used for the synthesis of four analogs of oxytocin and four analogs of vasopressin with the non-coded amino acids L- or D- and 1- or 2-naphthylalanine and D-homoarginine. [L-1-Nal2]oxytocin, [D-1-Nal2]oxytocin, [L-2-Nal2]oxytocin, [D-2-Nal2]oxytocin, [L-1-Nal2, D-Har8]vasopressin, [D-1-Nal2, D-Har8]vasopressin, [L-2-Nal2, D-Har8]vasopressin and [D-2-Nal2, D-Har8]vasopressin were synthesized. All eight analogs were found to be uterotonic inhibitors in vitro and in vivo. Analogs with 2-naphthylalanine are stronger inhibitors, particularly in the vasopressin series than the analogs with 1-naphthylalanine. Analogs with 1-naphthylalanine have no activity in the pressor test, analogs with 2-naphthylalanine are weak pressor inhibitors.

Animal Models and Alternatives in the Quality Control of Vaccines: Are In Vitro Methods or In Vivo Methods the Scientific Equivalent of the Emperor's New Clothes?

1995 ◽  
Vol 23 (1) ◽  
pp. 61-73
Author(s):  
Coenraad Hendriksen ◽  
Johan van der Gun
Keyword(s):  
Quality Control ◽  
Test Methods ◽  
In Vitro Test ◽  
Large Numbers ◽  
Alternative Approach ◽  
Inactivated Vaccines ◽  
Vitro Test

In the quality control of vaccine batches, the potency testing of inactivated vaccines is one of the areas requiring very large numbers of animals, which usually suffer significant distress as a result of the experimental procedures employed. This article deals with the potency testing of diphtheria and tetanus toxoids, two vaccines which are used extensively throughout the world. The relevance of the potency test prescribed by the European Pharmacopoeia monographs is questioned. The validity of the potency test as a model for the human response, the ability of the test to be standardised, and the relevance of the test in relation to the quality of the product are discussed. It is concluded that the potency test has only limited predictive value for the antitoxin responses to be expected in recipients of these toxoids. An alternative approach for estimating the potency of toxoid batches is discussed, in which a distinction is made between estimation of the immunogenic potency of the first few batches obtained from a seed lot and monitoring the consistency of the quality of subsequent batches. The use of animals is limited to the first few batches. Monitoring the consistency of the quality of subsequent batches is based on in vitro test methods. Factors which hamper the introduction and acceptance of the alternative approach are considered. Finally, proposals are made for replacement, reduction and/or refinement (the Three Rs) in the use of animals in the routine potency testing of toxoids.

scholarly journals 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Sinnes ◽  
Ulrike Bauder-Wüst ◽  
Martin Schäfer ◽  
Euy Sung Moon ◽  
Klaus Kopka ◽  
...  
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Solid Phase ◽  
Negative Influence ◽  
Binding Motif ◽  
Lncap Cells ◽  
Cell Binding ◽  
Binding Characteristics

Abstract Background The AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs. Results AAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay. Conclusions The AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

scholarly journals Design of PSMA ligands with modifications at the inhibitor part: an approach to reduce the salivary gland uptake of radiolabeled PSMA inhibitors?

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Veronika Barbara Felber ◽  
Manuel Amando Valentin ◽  
Hans-Jürgen Wester
Keyword(s):  
Salivary Gland ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
In Vivo Studies ◽  
Tumor Xenograft ◽  
Tumor Uptake ◽  
Lncap Cells ◽  
High Affinity

Abstract Aim To investigate whether modifications of prostate-specific membrane antigen (PSMA)-targeted radiolabeled urea-based inhibitors could reduce salivary gland uptake and thus improve tumor-to-salivary gland ratios, several analogs of a high affinity PSMA ligand were synthesized and evaluated in in vitro and in vivo studies. Methods Binding motifs were synthesized ‘on-resin’ or, when not practicable, in solution. Peptide chain elongations were performed according to optimized standard protocols via solid-phase peptide synthesis. In vitro experiments were performed using PSMA+ LNCaP cells. In vivo studies as well as μSPECT/CT scans were conducted with male LNCaP tumor xenograft-bearing CB17-SCID mice. Results PSMA ligands with A) modifications within the central Zn2+-binding unit, B) proinhibitor motifs and C) substituents & bioisosteres of the P1′-γ-carboxylic acid were synthesized and evaluated. Modifications within the central Zn2+-binding unit of PSMA-10 (Glu-urea-Glu) provided three compounds. Thereof, only natLu-carbamate I (natLu-3) exhibited high affinity (IC50 = 7.1 ± 0.7 nM), but low tumor uptake (5.31 ± 0.94% ID/g, 1 h p.i. and 1.20 ± 0.55% ID/g, 24 h p.i.). All proinhibitor motif-based ligands (three in total) exhibited low binding affinities (> 1 μM), no notable internalization and very low tumor uptake (< 0.50% ID/g). In addition, four compounds with P1′-ɣ-carboxylate substituents were developed and evaluated. Thereof, only tetrazole derivative natLu-11 revealed high affinity (IC50 = 16.4 ± 3.8 nM), but also this inhibitor showed low tumor uptake (3.40 ± 0.63% ID/g, 1 h p.i. and 0.68 ± 0.16% ID/g, 24 h p.i.). Salivary gland uptake in mice remained at an equally low level for all compounds (between 0.02 ± 0.00% ID/g and 0.09 ± 0.03% ID/g), wherefore apparent tumor-to-submandibular gland and tumor-to-parotid gland ratios for the modified peptides were distinctly lower (factor 8–45) than for [177Lu]Lu-PSMA-10 at 24 h p.i. Conclusions The investigated compounds could not compete with the in vivo characteristics of the EuE-based PSMA inhibitor [177Lu]Lu-PSMA-10. Although two derivatives (3 and 11) were found to exhibit high affinities towards LNCaP cells, tumor uptake at 24 h p.i. was considerably low, while uptake in salivary glands remained unaffected. Optimization of the established animal model should be envisaged to enable a clear identification of PSMA-targeting radioligands with improved tumor-to-salivary gland ratios in future studies.

Effect of microbial isolates on microbial yield estimation in RUSITEC system

1998 ◽  
Vol 22 ◽  
pp. 306-308
Author(s):  
M. D. Carro ◽  
E. L. Miller
Keyword(s):  
Protein Synthesis ◽  
Liquid Phase ◽  
Solid Phase ◽  
Liquid Fraction ◽  
Microbial Protein ◽  
Microbial Protein Synthesis ◽  
Continuous Culture System ◽  
The One

The estimation of rumen microbial protein synthesis is one of the main points in the nitrogen (N)-rationing systems for ruminants, as microbial protein provides proportionately 0.4 to 0.9 of amino acids entering the small intestine in ruminants receiving conventional diets (Russell et al., 1992). Methods of estimating microbial protein synthesis rely on marker techniques in which a particular microbial constituent is related to the microbial N content. Marker : N values have generally been established in mixed bacteria isolated from the liquid fraction of rumen digesta and it has been assumed that the same relationship holds in the total population leaving the rumen (Merry and McAllan, 1983). However, several studies have demonstrated differences in composition between solid-associated (SAB) and fluid-associated bacteria in vivo (Legay-Carmier and Bauchart, 1989) and in vitro (Molina Alcaide et al, 1996), as well in marker : N values (Pérez et al., 1996). This problem could be more pronounced in the in vitro semi-continuous culture system RUSITEC, in which there are three well defined components (a free liquid phase, a liquid phase associated with the solid phase and a solid phase), each one having associated microbial populations.The objective of this experiment was to investigate the effect of using different bacterial isolates (BI) on the estimation of microbial production of four different diets in RUSITEC (Czerkawski and Breckenridge, 1977), using (15NH4)2 SO4 as microbial marker, and to assess what effects any differences would have on the comparison of microbial protein synthesis between diets.This study was conducted in conjunction with an in vitro experiment described by Carro and Miller (1997). Two 14-day incubation trials were carried out with the rumen simulation technique RUSITEC (Czerkawski and Breckenridge, 1977). The general incubation procedure was the one described by Czerkawski and Breckenridge (1977) and more details about the procedures of this experiment are given elsewhere (Carro and Miller, 1997).

scholarly journals Humanin is an endogenous activator of chaperone-mediated autophagy

2017 ◽  
Vol 217 (2) ◽  
pp. 635-647 ◽  
Author(s):  
Zhenwei Gong ◽  
Inmaculada Tasset ◽  
Antonio Diaz ◽  
Jaime Anguiano ◽  
Emir Tas ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Death ◽  
Heat Shock Protein 90 ◽  
Protective Effects ◽  
Neuroprotective Effects ◽  
Selective Degradation ◽  
Cytosolic Side ◽  
Chaperone Mediated Autophagy

Chaperone-mediated autophagy (CMA) serves as quality control during stress conditions through selective degradation of cytosolic proteins in lysosomes. Humanin (HN) is a mitochondria-associated peptide that offers cytoprotective, cardioprotective, and neuroprotective effects in vivo and in vitro. In this study, we demonstrate that HN directly activates CMA by increasing substrate binding and translocation into lysosomes. The potent HN analogue HNG protects from stressor-induced cell death in fibroblasts, cardiomyoblasts, neuronal cells, and primary cardiomyocytes. The protective effects are lost in CMA-deficient cells, suggesting that they are mediated through the activation of CMA. We identified that a fraction of endogenous HN is present at the cytosolic side of the lysosomal membrane, where it interacts with heat shock protein 90 (HSP90) and stabilizes binding of this chaperone to CMA substrates as they bind to the membrane. Inhibition of HSP90 blocks the effect of HNG on substrate translocation and abolishes the cytoprotective effects. Our study provides a novel mechanism by which HN exerts its cardioprotective and neuroprotective effects.

Drug-induced liver injury classification model based on in vitro human transcriptomics and in vivo rat clinical chemistry data

2014 ◽  
Vol 2 (4) ◽  
pp. 63-70 ◽  
Author(s):  
Danyel Jennen ◽  
Jan Polman ◽  
Mark Bessem ◽  
Maarten Coonen ◽  
Joost van Delft ◽  
...  
Keyword(s):  
Liver Injury ◽  
Clinical Chemistry ◽  
Classification Model ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
Chemistry Data ◽  
Model Based ◽  
Injury Classification

Preparation of fibrils for intracerebral injection v1

2021 ◽  
Author(s):  
The Michael J Fox Foundation Pff Standardization Consortium
Keyword(s):  
Quality Control ◽  
Best Practices ◽  
External Validation ◽  
Alpha Synuclein ◽  
Intracerebral Injection ◽  
Primary Neuron ◽  
Consensus Protocol ◽  
Reference Section

This is a consensus protocol developed through discussions with Laura Volpicelli-Daley, Caryl Sortwell, Kelvin Luk, Lindsey Gottler, and Virginia Lee. This protocol is intended for research purposes only, using specially-formulated monomeric alpha-synuclein protein available for purchase at Proteos, Inc as the result of efforts by The Michael J. Fox Foundation (MJFF). Each batch of the “Alpha-Synuclein Monomer Protein for Making Pre- Formed Fibrils” has undergone internal purification and quality control at Proteos in addition to external validation to confirm successful generation of pathogenic aSyn PFFs. See Reference section for methods and results from application of alpha-synuclein pre-formed fibrils (aSyn PFFs) in primary neuron cultures in vitro or in mice in vivo. This protocol is referenced in the Polinski et al 2018 paper entitled "Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents" (doi: 10.3233/JPD-171248).

Sign in / Sign up

Export Citation Format

Share Document