Abstract
A chelating derivative of alpha-melanocyte stimulating hormone (MSH) has been synthesised, in which two molecules of the hormone are cross-linked by diethylenetriamine pentaacetic acid (DTPA). This compound, bisMSH-DTPA, was equipotent with MSH in an in vitro tyrosinase assay with Cloudman S91 melanoma cells. When DBA/2 mice bearing the same tumour were injected with bisMSH-DTPA labelled with the gamma-emitting isotope indium-111 (111In), the radioactivity became rapidly associated with the melanoma tissue. By 24 h post-injection, radioactivity in tumour tissue was significantly higher (P less than 0.001) than in spleen, lung, brain, eye and skin. Uptake of radioactivity by the tumours was inhibited by a 200-fold molar excess of MSH, whereas uptake by liver, kidney, spleen, lung, brain, eye and skin was unaffected. We conclude that bisMSH-DTPA may offer an alternative to antibody targeting in the imaging of malignant melanoma.
Highlights
The bisMSH-diethylenetriamine pentaacetic acid (DTPA) used in most experiments was custom synthesised by Cambridge Research Biochemicals Ltd (Harston, UK)
Thin layer chromatography in both solvent systems showed that when the mixture of bisMSH-DTPA and "'In was maintained at 37°C, the "'In had become completely bound to the chelator-peptide within 15 min of mixing
Both melanocyte stimulating hormone (MSH) and bisMSH-DTPA showed maximal activity in the tyrosinase assay at a concentration of 1 x 10-7 M (Figure 2), at which point the level of enzyme had been elevated to 4-4.5 times that in the melanotropin-free controls
Summary
Fmoc amino acid reagents for peptide synthesis were from Novabiochem (UK) Ltd (Nottingham, UK) or from MilliGen/Biosearch (Watford, UK). The bisMSH-DTPA used in most experiments was custom synthesised by Cambridge Research Biochemicals Ltd (Harston, UK). Initial syntheses at the Strangeways Laboratory were of low yield and a custom synthesis was performed by Cambridge Research Biochemicals. Fmoc amino acids were coupled as their pentafluorophenyl or oxobenzotriazine esters in the presence of 1hydroxybenzotriazole (0.8 mmol of each). When assembly of the peptide was complete, the N-terminal Fmoc group was removed and the peptide-resin was treated with DTPA bisanhydride (0.3 mmol) and diisopropylethylamine (0.15 mmol). Its peptide structure was confirmed by amino acid analysis and it was shown to be the bis-peptide adduct of DTPA by fast-atom-bombardment mass spectrometry, which gave a relative molecular mass of 3,602 ± 2
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