Drug localization and targeting with receptor microscopic autoradiography

Stumpf, Walter E.

doi:10.1016/j.vascn.2004.09.001

Cited by 60 publications

(42 citation statements)

References 53 publications

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“…Profile of vitamin D target tissues (red) assessed in rats and mice with receptor microscopic autoradiography. This holistic view provides a basis for quantitative and functional compound characterization (Stumpf 2005) a guide for potential therapies, a change of concept for the main biological role of vitamin D beyond calcium metabolism (Stumpf 2007) • In vivo target recognition requires microscopic cellular-subcellular resolution in conjunction with integrative tissue surveys, for understanding mechanisms of action, for confirming and complementing biochemical and in vitro data, and for guiding functional and clinical follow-up. • Receptor Microscopic Autoradiography is available as a tested method that satisfies requirements for high resolution in vivo drug localization.…”

Section: Discussionmentioning

confidence: 99%

“…Instead, microscopic resolution is necessary for target recognition. Therefore, thaw-mounting of thin sections on nuclear emulsion was introduced and then developed into receptor microscopic autoradiography that has been extensively applied (Stumpf 2003(Stumpf , 2005. In microscopic autoradiography, nuclear emulsion is the detection medium for beta-particles.…”

Section: High-resolution Autoradiographymentioning

confidence: 99%

“…Receptor microscopic autoradiography provides both the histological image and the converted radiation signal into developed silver grains in the same focal plane. This allows convenient microscopic enlargement to the cellular and subcellular level and, at the same time, integrative low-resolution surveys, i.e., simultaneous recognition of molecular, cellular and tissue (organ) relationships (Stumpf 2003(Stumpf , 2005.…”

Section: High-resolution Autoradiographymentioning

confidence: 99%

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In vivo target recognition with high-resolution imaging: significance for drug development

Stumpf

2010

Eur J Drug Metab Pharmacokinet

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In vivo target identification is basic for understanding mechanisms of drug action. Target identification requires cellular resolution. Extrapolation from blood bioavailability, low-resolution scans, radioassays, or in vitro tests regularly produce false-negatives and false-positives. Common ADME procedures disrealize organ complexities. While low-specificity high-capacity sites of deposition are easily recognized, high-specificity low-capacity receptor sites remain hidden. Serious limitations of target recognition are revealed in comparative studies with three methods: high-resolution microscopic autoradiography, radioassay, and whole-body autoradiography. With radioassays and whole-body autoradiography, many targets are simply undetectable. For example, high-resolution microscopic target information for vitamin D, gained 20-30 years ago, was widely ignored. The narrow calcium focus for this multi-target and multi-function hormone was perpetuated until recently through deficient results from conventional assays together with related expert bias. Thus, follow-up has been delayed on discoveries from the use of unconventional histopharmacology methods, pointing at important actions and therapies beyond systemic calcium regulation. High-resolution 'in vivo' target identification with associated functional characterization is useful not only for understanding mechanisms of action, but also for providing leads for innovative and successful drug development and prediction.

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Section: Discussionmentioning

confidence: 99%

Section: High-resolution Autoradiographymentioning

confidence: 99%

Section: High-resolution Autoradiographymentioning

confidence: 99%

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In vivo target recognition with high-resolution imaging: significance for drug development

Stumpf

2010

Eur J Drug Metab Pharmacokinet

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“…This was the rationale for the dose and route for the application; and it was chosen so that it could be applied in well-organized clinical stroke units. Furthermore, this amount of 1,25-D 3 is known to target nuclear VDR effectively in healthy rats and mice (for review, see Stumpf 2005).…”

Section: Methodical Aspectsmentioning

confidence: 99%

1α,25-Dihydroxyvitamin D3 treatment does not alter neuronal cyclooxygenase-2 expression in the cerebral cortex after stroke

et al. 2005

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The inducible prostaglandin synthase, cyclooxygenase-2, is upregulated in response to cerebral ischemia and contributes to potentiation of oxidative injury. Cyclooxygenase-2 expression is regulated by retinoic acid receptors, which form heterodimers with vitamin D receptors and vitamin D. In addition, vitamin D has been reported to have neuroprotective qualities. The aim of this study was to examine whether the biologically active vitamin D3-metabolite 1alpha,25-dihydroxyvitamin D3 (1,25-D3), influences the expression of inducible cyclooxygenase-2 in photothrombotically lesioned brain or is part of an independent neuroprotective mechanism. We compared groups of nonlesioned control rats and infarcted animals, which were treated with either 1,25-D3 or solvent at different times postlesion. In control animals, cyclooxygenase-2 immunoreactivity was readily evident in almost all cortical neurons of layers II/III as well as in a few pyramidal cells in layer V. Following photothrombotic infarction of the right cortical hindlimb area, there was a significant, but transient, increase in cyclooxygenase-2 labeling which was restricted to neurons of the injured hemisphere in both 1,25- D3-treated and solvent-treated rats. Highest levels of cyclooxygenase-2 immunoreactivity were seen at 12 and 24 h postlesion, followed by a gradual decrease at later time points. However, no significant differences were detected between 1,25-D3-treated and solvent-treated lesioned rats, indicating that postischemic neuronal cyclooxygenase-2 upregulation is not influenced by 1,25-D3. It is concluded that the neuroprotective effect of 1,25-D3 does not depend on modulations of neuronal COX-2 expression caused by postlesional hyperexcitation.

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“…In a typical assay configuration, information about receptor distribution is obtained through antibody-mediated biotinstreptavidin interaction amplified by conjugated enzymes (eg horseradish peroxidase (HRP)). [1][2][3] Receptor autoradiography requires the use of radiolabeled ligands, with the attendant issues of frequent radiochemical synthesis and handling of radioactivity ( Figure 1b). In addition, the distribution of radioactivity is obtained on a blank background and must be superimposed on a second image, to visualize ligand binding against a background of cell structure.…”

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confidence: 99%

Enzyme-based visualization of receptor–ligand binding in tissues

Montet

Yuan

Weissleder

et al. 2006

Laboratory Investigation

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New methods of elucidating the ligand-binding activity of receptors could improve our understanding of receptor function, key events they control, and their presence in normal and pathological states. We describe a method for visualizing receptor-ligand binding in cells and tissues that substitutes fluorescein for radioactive labels, and detects receptor bound, fluoresceinated ligand with an antifluorescein/horseradish peroxidase amplification system. Receptor-bound ligand is then visualized by light microscopy against a standard hemotoxylin-stained background of cell structure. Quantitative versions of the assay provide an apparent dissociation constant and number of receptors per cell at saturation in cell or tissue specimens. Receptors examined include the folate receptor, bombesin peptide-binding receptors, the epidermal growth factor receptor, the neuropeptide Y receptor, the asialoglycoprotein receptor, and RGD peptide-binding integrins. Visualizing and quantifying the binding of ligands to receptors expressed in tissues is crucial to understanding the role receptors play and the events they control in normal and pathological tissues. Though antibody based immunohistochemistry provides valuable information about the distribution of immunoreactive structures (epitopes), it does not provide information on the presence or activity of ligand binding sites (Figure 1a). In a typical assay configuration, information about receptor distribution is obtained through antibody-mediated biotinstreptavidin interaction amplified by conjugated enzymes (eg horseradish peroxidase (HRP)).1-3 Receptor autoradiography requires the use of radiolabeled ligands, with the attendant issues of frequent radiochemical synthesis and handling of radioactivity (Figure 1b). In addition, the distribution of radioactivity is obtained on a blank background and must be superimposed on a second image, to visualize ligand binding against a background of cell structure.We hypothesized that it would be possible to substitute fluoresceinated ligands for radioactive ligands, and detect bound fluorescein using the HRP amplification system to achieve a high sensitivity. We further hypothesized that fluorescein hapten detection methodologies could be configured as a cell-based method for imaging receptors (fluorescein hapten visualization (FHV)) or as a cell-based assay for bound ligand (fluorescein hapten assay (FHA)) as shown in Figure 1c. In the visualization method (FHV), a cell monolayer or tissue specimen is incubated with the receptorbinding ligand in a slide chamber and bound ligand visualized by microscopy. In the assay method (FHA), the specimen is exposed to the fluoresceinated ligand as in FHV, but rather than proceeding with microscopy the specimen is solubilized and immunoreactive fluorescein quantified by a fluorescein immunoassay. When the number of cells is measured in the sample, the bound ligand per cell can be obtained. FHV and FHA provide information about ligand-binding receptors that can be used with different types of specimens...

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Drug localization and targeting with receptor microscopic autoradiography

Cited by 60 publications

References 53 publications

In vivo target recognition with high-resolution imaging: significance for drug development

In vivo target recognition with high-resolution imaging: significance for drug development

1α,25-Dihydroxyvitamin D3 treatment does not alter neuronal cyclooxygenase-2 expression in the cerebral cortex after stroke

Enzyme-based visualization of receptor–ligand binding in tissues

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