In Vitro System To Estimate Renal Brush Border Enzyme-Mediated Cleavage of Peptide Linkages for Designing Radiolabeled Antibody Fragments of Low Renal Radioactivity Levels

2005 ◽  
Vol 16 (6) ◽  
pp. 1610-1616 ◽  
Author(s):  
Yasushi Fujioka ◽  
Satoshi Satake ◽  
Tomoya Uehara ◽  
Takahiro Mukai ◽  
Hiromichi Akizawa ◽  
...  
Keyword(s):  
Brush Border ◽  
Antibody Fragments ◽  
In Vitro System ◽  
Brush Border Enzyme ◽  
Renal Brush Border ◽  
Radioactivity Levels

Renal Brush Border Enzyme-Cleavable Linkages for Low Renal Radioactivity Levels of Radiolabeled Antibody Fragments

2013 ◽  
Vol 24 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Hiromichi Akizawa ◽  
Mitsuo Imajima ◽  
Hirofumi Hanaoka ◽  
Tomoya Uehara ◽  
Satoshi Satake ◽  
...  
Keyword(s):  
Brush Border ◽  
Antibody Fragments ◽  
Brush Border Enzyme ◽  
Renal Brush Border ◽  
Radioactivity Levels

NAD-glycohydrolase in renal brush border membranes

1985 ◽  
Vol 249 (3) ◽  
pp. F366-F373
Author(s):  
S. A. Kempson
Keyword(s):  
Brush Border ◽  
Osmotic Shock ◽  
Phosphate Transport ◽  
Reaction Products ◽  
Brush Border Membranes ◽  
Nad Glycohydrolase ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.

Actions of NAD+ on renal brush border transport of phosphate in vivo and in vitro

1985 ◽  
Vol 249 (6) ◽  
pp. F948-F955 ◽  
Author(s):  
S. A. Kempson ◽  
S. T. Turner ◽  
A. N. Yusufi ◽  
T. P. Dousa
Keyword(s):  
Brush Border ◽  
Brush Border Membrane ◽  
Competitive Inhibition ◽  
Inhibitory Effect ◽  
Pi Transport ◽  
Renal Brush Border Membrane ◽  
Dependent Transport ◽  
Renal Brush Border

Previous studies showed that an increase in NAD+ content in renal cortex in vivo was accompanied by specific inhibition of Na+-dependent inorganic phosphate (Pi) transport across the renal brush border membrane (BBM). Further, in vitro addition of NAD+ to isolated renal BBM vesicles specifically inhibited Na+ gradient-dependent transport of Pi. The present study examined some aspects of the mechanism of this inhibition by NAD+ in vitro and in vivo. When NAD+ was increased in vivo by nicotinamide injection, the apparent Vmax was decreased, but the apparent Km was not different, indicating apparent noncompetitive inhibition. In the presence of 0.3 mM NAD+ added in vitro, the apparent Km for Na+-dependent Pi transport by BBM vesicles was increased, whereas the apparent Vmax was unchanged, indicating apparent competitive inhibition. These changes in apparent Km and apparent Vmax were identical when Pi uptake was measured either at 30-s or at 5-s (the initial rate) incubation times. Inhibition of Pi transport by BBM vesicles in vitro was due primarily to the action of intact added NAD+, although there may be some contribution by isotope dilution due to Pi released from NAD+ by enzymatic hydrolysis. Although in vitro inhibition of Pi transport by added NAD+ was reversed by washing the BBM, the inhibition due to increased NAD+ in vivo persisted after extensive washing of the isolated BBM. The specificity of the inhibitory effect of NAD+ in vivo was indicated by the finding that changes in renal cortical content of ATP or Pi, evoked by loading with glycerol or fructose, did not change BBM transport of Pi.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Evaluation of Met-Val-Lys as a Renal Brush Border Enzyme-Cleavable Linker to Reduce Kidney Uptake of 68Ga-Labeled DOTA-Conjugated Peptides and Peptidomimetics

Molecules ◽  
2020 ◽  
Vol 25 (17) ◽  
pp. 3854 ◽  
Author(s):  
Shreya Bendre ◽  
Zhengxing Zhang ◽  
Hsiou-Ting Kuo ◽  
Julie Rousseau ◽  
Chengcheng Zhang ◽  
...  
Keyword(s):  
Brush Border ◽  
Ex Vivo ◽  
Neutral Endopeptidase ◽  
Amide Bond ◽  
Detection Sensitivity ◽  
Brush Border Enzymes ◽  
Positron Emission ◽  
Brush Border Enzyme ◽  
Renal Brush Border

High kidney uptake is a common feature of peptide-based radiopharmaceuticals, leading to reduced detection sensitivity for lesions adjacent to kidneys and lower maximum tolerated therapeutic dose. In this study, we evaluated if the Met-Val-Lys (MVK) linker could be used to lower kidney uptake of 68Ga-labeled DOTA-conjugated peptides and peptidomimetics. A model compound, [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (AmBz: aminomethylbenzoyl), and its derivative, [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH, coupled with the PSMA (prostate-specific membrane antigen)-targeting motif of the previously reported HTK01166 were synthesized and evaluated to determine if they could be recognized and cleaved by the renal brush border enzymes. Additionally, positron emission tomography (PET) imaging, ex vivo biodistribution and in vivo stability studies were conducted in mice to evaluate their pharmacokinetics. [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was effectively cleaved specifically by neutral endopeptidase (NEP) of renal brush border enzymes at the Met-Val amide bond, and the radio-metabolite [68Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted via the renal pathway with minimal kidney retention. [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH retained its PSMA-targeting capability and was also cleaved by NEP, although less effectively when compared to [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH. The kidney uptake of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was 30% less compared to that of [68Ga]Ga-HTK01166. Our data demonstrated that derivatives of [68Ga]Ga-DOTA-AmBz-MVK-OH can be cleaved specifically by NEP, and therefore, MVK can be a promising cleavable linker for use to reduce kidney uptake of radiolabeled DOTA-conjugated peptides and peptidomimetics.

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