Design, synthesis and evaluation of [ 111 In]-labeled, DOTA-conjugated tetrapeptides having high affinity and selectivity for mu opioid receptors

John R. Lever, Emily A. Fergason-Cantrell, Terry L. Carmack, Lisa D. Watkinson, Fabio Gallazzi

Research output: Contribution to journalArticle

Abstract

Introduction: Peripheral mu (μ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic [ 111 In]-labeled, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated μ tetrapeptides. Methods: Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH 2 (DAPO), where Dmt = 2′,6′-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH 2 ([Dmt 1 ]DALDA). Constructs had DOTA conjugation at the Orn 4 or Lys 4 side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and [ 111 In]-radiolabeling were accomplished by standard methods. Protein binding and Log D 7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice. Results: All four indium(III)-DOTA conjugates derived from DAPO and [Dmt 1 ]DALDA showed good selectivity and subnanomolar affinity for μ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[ 111 In]DOTA)-NH 2 , showed 25% specific binding in vivo to μ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites. Conclusions: Visualizing peripheral μ opioid receptors using [ 111 In]-labeled, DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required. Advances in knowledge: This study shows in vivo labeling of peripheral μ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.

Original languageEnglish (US)
Pages (from-to)53-66
Number of pages14
JournalNuclear Medicine and Biology
Volume70
DOIs
StatePublished - Mar 1 2019
Externally publishedYes

Fingerprint

mu Opioid Receptor
Opioid Receptors
Indium
Kidney
Peptides
Protein Binding
Lysine
Mass Spectrometry
Sodium
High Pressure Liquid Chromatography
Pharmacology
Ions
Amino Acids
Pain
Neoplasms
2-(2'-(methylthio)ethyl)methionine
2',6'-dimethyltyrosyl-arginyl-phenylalanyl-lysinamide

Keywords

  • Indium-111
  • Opioid receptor
  • Peptide
  • Radiolabeling

ASJC Scopus subject areas

  • Molecular Medicine
  • Radiology Nuclear Medicine and imaging
  • Cancer Research

Cite this

Design, synthesis and evaluation of [ 111 In]-labeled, DOTA-conjugated tetrapeptides having high affinity and selectivity for mu opioid receptors . / Lever, John R.; Fergason-Cantrell, Emily A.; Carmack, Terry L.; Watkinson, Lisa D.; Gallazzi, Fabio.

In: Nuclear Medicine and Biology, Vol. 70, 01.03.2019, p. 53-66.

Research output: Contribution to journalArticle

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abstract = "Introduction: Peripheral mu (μ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic [ 111 In]-labeled, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated μ tetrapeptides. Methods: Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH 2 (DAPO), where Dmt = 2′,6′-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH 2 ([Dmt 1 ]DALDA). Constructs had DOTA conjugation at the Orn 4 or Lys 4 side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and [ 111 In]-radiolabeling were accomplished by standard methods. Protein binding and Log D 7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice. Results: All four indium(III)-DOTA conjugates derived from DAPO and [Dmt 1 ]DALDA showed good selectivity and subnanomolar affinity for μ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[ 111 In]DOTA)-NH 2 , showed 25{\%} specific binding in vivo to μ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites. Conclusions: Visualizing peripheral μ opioid receptors using [ 111 In]-labeled, DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required. Advances in knowledge: This study shows in vivo labeling of peripheral μ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.",
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T1 - Design, synthesis and evaluation of [ 111 In]-labeled, DOTA-conjugated tetrapeptides having high affinity and selectivity for mu opioid receptors

AU - Lever, John R.

AU - Fergason-Cantrell, Emily A.

AU - Carmack, Terry L.

AU - Watkinson, Lisa D.

AU - Gallazzi, Fabio

PY - 2019/3/1

Y1 - 2019/3/1

N2 - Introduction: Peripheral mu (μ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic [ 111 In]-labeled, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated μ tetrapeptides. Methods: Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH 2 (DAPO), where Dmt = 2′,6′-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH 2 ([Dmt 1 ]DALDA). Constructs had DOTA conjugation at the Orn 4 or Lys 4 side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and [ 111 In]-radiolabeling were accomplished by standard methods. Protein binding and Log D 7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice. Results: All four indium(III)-DOTA conjugates derived from DAPO and [Dmt 1 ]DALDA showed good selectivity and subnanomolar affinity for μ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[ 111 In]DOTA)-NH 2 , showed 25% specific binding in vivo to μ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites. Conclusions: Visualizing peripheral μ opioid receptors using [ 111 In]-labeled, DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required. Advances in knowledge: This study shows in vivo labeling of peripheral μ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.

AB - Introduction: Peripheral mu (μ) opioid receptors are implicated in pain, bowel dysfunction and the progression of certain cancers. In an effort to identify radioligands well suited for imaging these peripheral sites, we have prepared and evaluated four hydrophilic [ 111 In]-labeled, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugated μ tetrapeptides. Methods: Peptides were prepared by solid-phase techniques, using orthogonal strategies to achieve branching to DOTA, and then characterized by HPLC, mass spectroscopy and amino acid analysis. Scaffolds included novel peptide H-Dmt-D-Ala-Phe-Orn-NH 2 (DAPO), where Dmt = 2′,6′-dimethyltyrosine, and known peptide H-Dmt-D-Arg-Phe-Lys-NH 2 ([Dmt 1 ]DALDA). Constructs had DOTA conjugation at the Orn 4 or Lys 4 side chains, or to the C-terminal through a hexanoic acid-lysine linker. Indium(III) complexation and [ 111 In]-radiolabeling were accomplished by standard methods. Protein binding and Log D 7.4 were determined. Binding and pharmacological profiles were obtained in vitro. Biodistribution and radiometabolite studies were conducted using male CD-1 mice. Results: All four indium(III)-DOTA conjugates derived from DAPO and [Dmt 1 ]DALDA showed good selectivity and subnanomolar affinity for μ opioid receptors. One radioligand, H-Dmt-D-Ala-Phe-Orn(δ-[ 111 In]DOTA)-NH 2 , showed 25% specific binding in vivo to μ sites in mouse gut. Notably, this was the least polar of the series, and also showed low sensitivity to modulation of binding by sodium ions. All radioligands showed high kidney uptake of radiometabolites. Conclusions: Visualizing peripheral μ opioid receptors using [ 111 In]-labeled, DOTA-conjugated tetrapeptides appears feasible, but structural modifications to enhance specific binding and metabolic stability, as well as to reduce kidney uptake, will be required. Advances in knowledge: This study shows in vivo labeling of peripheral μ opioid receptors by a tetrapeptide radioligand, and provides information that should prove useful in the design of peptide radioligands having optimal properties.

KW - Indium-111

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KW - Radiolabeling

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