New potent hGH‐RH analogues with increased resistance to enzymatic degradation

Abstract

Four hGH-RH analogues containing homoarginine (Har) and/or D-Arg were obtained by solid-phase methodology using Boc-chemistry. To introduce Har residues, a Lys(Fmoc) protected Lys derivative was incorporated in the appropriate positions (11, 12, 20, 21 or 29): after assembly of the peptide chain the Fmoc group was removed and the peptide-resin was guanidinylated by treatment with N,N'-bis(tert-butoxycarbonyl)-S-methylisothiourea. The peptides were cleaved from the resin by treatment with liquid HF, and the products were purified by RP-HPLC. The peptides were subjected to digestion by trypsin, and the course of the reaction was followed by HPLC and ESI-MS. It was found that peptide bonds formed by the carboxyl group of Har are completely stable to trypsin. The course of cleavage at Lys or Arg residues depends on the position of Har in the sequence. All the analogues investigated stimulate the release of GH in rats after subcutaneous administration, and were about 50-100 times as potent as rGH-RH itself. The analogues had no effect on PRL, LH and FSH levels.

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

“…All the amino acids and reagents for peptide synthesis were obtained from Sigma Chemical Co. (St Louis, MO, USA), Fisher Scientific (Fair Lawn, NJ, USA), Bachem (Bubendorf, Switzerland), Ferak Berlin (Berlin, Germany), Aldrich (Steinheim, Germany) and IRIS Biotech (Marktredwitz, Germany). N , N ′‐Di‐ tert ‐butoxycarbonyl‐1 H ‐benzotriazole‐1‐carboxamidine (Boc 2 CABt) and N , N ′‐Di‐ tert ‐butoxycarbonyl‐1‐methyl‐2‐thiourea were synthesized as described previously10, 11. Thin layer chromatography (TLC) was performed on Merck F 254 and Silufol plates using specified solvent system.…”

Potent Trypsin‐resistant hGH‐RH Analogues

“…There is a great demand for more stable analogues which would allow a reduction in dose and frequency of administration. Numerous analogues of hGH-RH-(1-29)-NH 2 have been synthesized and tested in a search for peptides of increased metabolic stability and potency [4][5][6]. hGH-RH-(1-29)-NH 2 itself is rapidly metabolized in plasma, as a result of Ala 2 -Asp 3 peptide bond cleavage by dipeptidylpeptidase IV with the formation of an inactive 3-29 fragment [7].…”

“…Recently several biologically active analogues containing side-chain extended homoarginine (Har) instead of Arg and Lys were described. It was found that peptide bonds formed by the carboxyl group of Har are completely stable to trypsin [6].…”

“…The aim of the present work was to examine the possibility of obtaining biologically active analogues, similar to those previously described [6], by replacing all the Arg and Lys residues with non-coded amino acid residues containing a guanidino group in the side-chain: Har, 4-guanidino-2-aminobutyric acid (Gab) or 3-guanidino-2-aminopropionic acid (Gap). Orn and D-Arg residues were also incorporated.…”

Increased activity of antagonists of growth hormone-releasing hormone substituted at positions 8, 9, and 10

“…Many agonistic and antagonistic analogs of GHRH, based on the shortest bioactive sequence of hGHRH(1-29)NH 2 , have been synthesized to elucidate the structure-activity relationships and to increase their biological activity (4)(5)(6)(7)(8)(9)(10)(11)(12)(13). The first reported GHRH antagonist, termed herein as the ''standard antagonist,'' has the chemical structure of [Ac-Tyr 1 , D-Arg 2 ]hGHRH(1-29)NH 2 (7).…”