Synthesis and biological evaluation of a ^99mTc-labelled cyclic RGD peptide for imaging the αvβ3 expression

Summary

Aim: The αvβ3 integrin is involved in tumour induced angiogenesis and tumour metastasis. We describe the synthesis and evaluation of a ^99mTc-labelled RGD analogue for the visualisation of αvβ3 integrin expression. Methods: The linear peptides were assembled on a solid support. Cyclisation was performed under high dilution conditions. For conjugation with the chelator peptide, a water soluble carbodiimide was used. Radiolabelling was carried out due to standard procedures with high radiochemical yield and radiochemical purity. For in vivo evaluation, nude mice bearing αvβ3-positive human melanoma M21 and αv-negative human melanoma M21-L or Balb/c mice bearing αv-positive murine osteosarcoma were used. Results: Activity accumulation of ^99mTc-DKCK-RGD 240 min p. i. was 1.1% ID/g in the αvβ3-positive melanoma and 0.3% ID/g in the negative control tumour. In the osteosarcoma model 2.2% ID/g was found 240 min p. i. Planar gamma camera images allowed contrasting visualisation of αvβ3-positive tumours 240 min p. i. Blocking of the tumour using the αvβ3-selective pentapeptide cyclo(-ArgGly-Asp-D-Phe-Val-) reduces activity accumulation in the tumour to background level. However, 240 min p. i. highest activity concentration was found in kidneys resulting in low tumour/kidney ratios. Metabolite analysis 240 min p. i. showed approximately 60% intact tracer in kidneys and 80% in the tumour. Only 24% intact tracer was found in blood 30 min p. i. Conclusion: ^99mTc-DKCK-RGD allows imaging of αvβ3-positive tumours in mice. However, pharmacokinetics as well as metabolic stability of the tracer have to be improved for potential clinical application.

Zusammenfassung

Ziel: Das αvβ3-Integrin spielt eine wesentliche Rolle in der tumorinduzierten Angiogenese und der Metastasenbildung. Wir beschreiben die Synthese und Evaluierung eines ^99mTc-markierten RGD-Peptides zur Visualisierung der αvβ3-Integrin-Expression. Methoden: Die linearen Peptide wurden an der Festen Phase synthetisiert. Zyklisierung wurde in hoher Verdünnung durchgeführt. Für die Konjugation mit dem Chelatorpeptid wurde ein wasserlösliches Carbodiimid eingesetzt. Die Radio-markierung lieferte den Tracer in hohen radiochemischen Ausbeuten und radiochemischer Reinheit. Für die in vivoEvaluierung wurden Nacktmäuse mit αvβ3-positivem bzw. negativem humanem Melanom M21 bzw. M21-L oder Balb/c Mäuse, die ein αv-positives murines Osteosarkom tragen, verwendet. Ergebnisse: Die Anreiche-rung von ^99mTc-DKCK-RGD 240 min p. i. im αvβ3-positiven Melanom betrug 1,1% ID/g und im Negativkontrolltumor 0,3% ID/g. Im Osteosarkom wurden 240 min p. i. 2,2% ID/g gefunden. Planare Gamma-kameraaufnahmen 240 min p. i. ermöglichten eine kontrastreiche Darstellung des αvβ3-positiven Tumors. Die höchste Aktivitätskonzentration wurde allerdings in den Nieren gefunden, was zu ungünstigen Tumor/ Nieren-Verhältnissen führt. Die Blockade mit dem αvβ3-selektivem Peptid zyklo(-Arg-Gly-Asp-D-Phe-Val-) reduzierte die Aktivitätsanreicherung im Tumor auf Hintergrundniveau. Die Metabolitenanalyse 240 min p. i. zeigte, dass in den Nieren ca. 60% und im Tumor ca. 80% der Gesamtaktivität vom intakten Tracer stammten. Im Blut wurden 30 min p. i. nur noch 24% intakte Verbindung gefunden. Schlussfolgerung: ^99mTcDKCK-RGD erlaubt eine nicht-invasive Darstellung αvβ3-positiver Tumore im Mausmodell. Pharmakokinetik und metabolische Stabilität müssen für einen potentiellen klinischen Einsatz noch verbessert werden.

Keywords

RGD-peptides - alpha(v)beta3 - integrin - stability - angiogenesis - tumor targeting

Schlüsselwörter

RGD-Peptide - alpha(v)beta3 - Integrin - Stabilität - Angiogenese - Tumor Targeting

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