H. P. J. Bennett

ON THE METABOLISM OF TWO ADRENOCORTICOTROPHIN ANALOGUES

The metabolism of Synacthen® (corticotrophin‐(1–24)‐tetracosapeptide) and C‐41795‐Ba ([d‐Ser1, Lys17, Lys18]‐corticotrophin‐(1–18)‐octadecapeptide amide) have been compared in the rat following intravenous injection. Using Synacthen and C‐41795‐Ba labelled with tritium it was possible to follow the tissue distribution of the two peptides. The kidney was shown to concentrate intact peptide and fragments of both peptides. Autoradiography of perfused kidney sections demonstrated the uptake of radioactivity into the lysosomes of the kidney proximal tubule cells. Comparison of the distribution of radioactive products formed from the tetracosapeptide labelled in different positions indicates that, prior to renal uptake, metabolism is taking place at other sites in the body. It is suggested that rapid cleavage occurs extracellularly at or near the N‐ and C‐termini. Then large fragments remaining in the circulation, together with any intact material, are filtered through the glomerulus and are taken up by endocytosis into the proximal tubule cells of the kidney. The synthetic N‐ and C‐terminal protection of the octadecapeptide appears to inhibit the extracellular attack and so the concentration of intact peptide in the kidneys is initially higher than the tetracosapeptide. Although concentrations of radioactivity in other tissues are low in comparison with the kidneys, the quantities are quite high when the weight of the tissues are considered. Chromatographic analysis of this radioactivity reveals that the octadecapeptide gives rise to much higher tissue levels of intact peptide and we believe that this acts as a depot and gives rise to the sustained blood concentrations and prolonged biological effects observed with this peptide.

The Catabolism of two Adrenocorticotrophin Analogues Following Intravenous Injection

We have studied the catabolic fate, following intravenous injection in the rat, of two ACTH analogues, namely corticotrophin — (1–24)—tetracosapeptide (Synacthen ®, and [D-Ser1, Lys17,1] corticotrophin — (1–18)—Octadecapeptide amide (C-41795-Ba). Using the two analogues labelled with tritium in the tyrosine of position two it was established that C-41795-Ba has a much longer plasma and tissue half-life than Synacthen, as indicated by ion exchange chromatography of tissue extracts. This confirms results previously obtained with an isolated adrenal cell bioassay.1. There is a marked concentration of radioactivity in the kidneys with both peptides which consists in each case of intact peptide and several discrete fragments. However, the maximum concentration of Synacthen and its fragments occurs after 10 min and constitutes about 10% of the injected dose whilst C-41795-Ba and its fragments reach a maximum of about 30%, of the injected dose after 30 min which has declined by 60 min. The protection from exopeptidase attack, which the D-Seryl residue at the N-terminus and amidation at the C-terminus confer on C-41795-Ba, considerably enhances its half-life. The relative importance of the kidneys as organs of clearance and degradation is also quantitatively changed by these modifications.

An Autoradiographic Study of the Renal Uptake and Metabolism of a Synthetic Adrenocorticotrophin

The cellular and subcellular events in rat kidney following intravenous injection of the radioactively labelled adrenocorticotrophic hormone analogues, [3 H-Tyr23] — Synacthen and [3 H-Tyr23]—Synacthen (see preceding abstract), have been studied using microautoradiography and quantitative electron microscope autoradiography1. The distribution of silver grains 3–7 min after injection is similar for both forms of the peptide and shows that after glomerular filtration most of the label is rapidly resorbed via endocytotic vesicles of the proximal convoluted tubule. After 22 min label from [3 H-Tyr23]—Synacthen is twice as concentrated in the lysosomes as in endocytotic vesicles whereas, at 13 and 30 min after injection of [3 H-Tyr2] — Synacthen, label is three to four times more concentrated in lysosomes than in endocytotic vesicles. After 1h label from [3H-Tyr23]—Synacthen is more randomly distributed as determined by the χ2 test showing similar lysosomal nuclear and cytoplasmic concentrations.