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A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

Wall JS, Williams A, Richey T, Stuckey A, Huang Y, Wooliver C, Macy S, Heidel E, Gupta N, Lee A, Rader B, Martin EB, Kennel SJ - PLoS ONE (2013)

Bottom Line: Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma.The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure.The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee, USA. jwall@utmck.edu

ABSTRACT
Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

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Peptide 125I-p5R specifically visualizes visceral AA amyloid in vivo.The biodistribution of radiolabeled peptide p5R in mouse tissues from healthy WT mice (grey) or AA amyloidotic mice (black) expressed as % injected dose per gram of tissue (%ID/g) at 1 h (A) and 4 h (B) post-injection (mean + SD, n = 3). (C) Specific localization of 125I-p5R peptide with amyloid was confirmed by micro-autoradiography where peptide was evidenced by the presence of black silver grains (original mag. 80x). (D) SPECT/CT imaging (anterior, posterior and lateral views) of mice at 2 h pi showed uptake of 125I-p5R in the liver (L), spleen (S), intestine (I), and pancreas (P).
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pone-0066181-g004: Peptide 125I-p5R specifically visualizes visceral AA amyloid in vivo.The biodistribution of radiolabeled peptide p5R in mouse tissues from healthy WT mice (grey) or AA amyloidotic mice (black) expressed as % injected dose per gram of tissue (%ID/g) at 1 h (A) and 4 h (B) post-injection (mean + SD, n = 3). (C) Specific localization of 125I-p5R peptide with amyloid was confirmed by micro-autoradiography where peptide was evidenced by the presence of black silver grains (original mag. 80x). (D) SPECT/CT imaging (anterior, posterior and lateral views) of mice at 2 h pi showed uptake of 125I-p5R in the liver (L), spleen (S), intestine (I), and pancreas (P).

Mentions: For comparison with published data for p5 peptide, the amyloid reactivity of peptide p5R was assessed in vivo using the transgenic H2/IL-6 murine model of systemic visceral AA amyloidosis. In mice with moderate to severe AA amyloidosis (3 – 4+), the biodistribution of the peptide at 1 h and 4 h post injection (pi) was indicative of the distribution of amyloid in diseased mice (Figs. 4A & B). Notably, there was significant radioactivity seen in the liver, spleen, pancreas, and intestines as compared to the WT mice at both 1 h and 4 h pi. At both time points, the major uptake of radiolabeled p5R was the pancreas (>10% ID/g) followed by the liver and spleen (5 – 10% ID/g). The specificity of the peptide binding within the tissues and organs was assessed by using micro-autoradiography (Fig. 4C). The distribution of radiolabeled peptide, evidenced by the presence of black silver grains, co-registered with the presence of amyloid deposits seen in Congo red-stained consecutive tissue sections (not shown). The 125I-p5R peptide tracer bound AA amyloid in all tissues evaluated; notably, those deposits occupying the hepatic sinusoids, the myocardium, pancreatic microvasculature, zona reticularis of the adrenal gland, renal papilla, and intestinal villi (Fig. 4C). There was no evidence of 125I-p5R associated with healthy, amyloid-free tissues with the exception of the hyaline cartilage in the tracheal rings (presumably hypersulfated chondroitin sulfate), where mild uptake was observed (not shown).


A binding-site barrier affects imaging efficiency of high affinity amyloid-reactive peptide radiotracers in vivo.

Wall JS, Williams A, Richey T, Stuckey A, Huang Y, Wooliver C, Macy S, Heidel E, Gupta N, Lee A, Rader B, Martin EB, Kennel SJ - PLoS ONE (2013)

Peptide 125I-p5R specifically visualizes visceral AA amyloid in vivo.The biodistribution of radiolabeled peptide p5R in mouse tissues from healthy WT mice (grey) or AA amyloidotic mice (black) expressed as % injected dose per gram of tissue (%ID/g) at 1 h (A) and 4 h (B) post-injection (mean + SD, n = 3). (C) Specific localization of 125I-p5R peptide with amyloid was confirmed by micro-autoradiography where peptide was evidenced by the presence of black silver grains (original mag. 80x). (D) SPECT/CT imaging (anterior, posterior and lateral views) of mice at 2 h pi showed uptake of 125I-p5R in the liver (L), spleen (S), intestine (I), and pancreas (P).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672142&req=5

pone-0066181-g004: Peptide 125I-p5R specifically visualizes visceral AA amyloid in vivo.The biodistribution of radiolabeled peptide p5R in mouse tissues from healthy WT mice (grey) or AA amyloidotic mice (black) expressed as % injected dose per gram of tissue (%ID/g) at 1 h (A) and 4 h (B) post-injection (mean + SD, n = 3). (C) Specific localization of 125I-p5R peptide with amyloid was confirmed by micro-autoradiography where peptide was evidenced by the presence of black silver grains (original mag. 80x). (D) SPECT/CT imaging (anterior, posterior and lateral views) of mice at 2 h pi showed uptake of 125I-p5R in the liver (L), spleen (S), intestine (I), and pancreas (P).
Mentions: For comparison with published data for p5 peptide, the amyloid reactivity of peptide p5R was assessed in vivo using the transgenic H2/IL-6 murine model of systemic visceral AA amyloidosis. In mice with moderate to severe AA amyloidosis (3 – 4+), the biodistribution of the peptide at 1 h and 4 h post injection (pi) was indicative of the distribution of amyloid in diseased mice (Figs. 4A & B). Notably, there was significant radioactivity seen in the liver, spleen, pancreas, and intestines as compared to the WT mice at both 1 h and 4 h pi. At both time points, the major uptake of radiolabeled p5R was the pancreas (>10% ID/g) followed by the liver and spleen (5 – 10% ID/g). The specificity of the peptide binding within the tissues and organs was assessed by using micro-autoradiography (Fig. 4C). The distribution of radiolabeled peptide, evidenced by the presence of black silver grains, co-registered with the presence of amyloid deposits seen in Congo red-stained consecutive tissue sections (not shown). The 125I-p5R peptide tracer bound AA amyloid in all tissues evaluated; notably, those deposits occupying the hepatic sinusoids, the myocardium, pancreatic microvasculature, zona reticularis of the adrenal gland, renal papilla, and intestinal villi (Fig. 4C). There was no evidence of 125I-p5R associated with healthy, amyloid-free tissues with the exception of the hyaline cartilage in the tracheal rings (presumably hypersulfated chondroitin sulfate), where mild uptake was observed (not shown).

Bottom Line: Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma.The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure.The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee, USA. jwall@utmck.edu

ABSTRACT
Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer's disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant "binding site barrier" effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.

Show MeSH
Related in: MedlinePlus