In Vitro Stability of Growth Hormone Releasing Factor (GRF) Analogs in Porcine Plasma

Summary

The stability of growth-hormone releasing factor (growth regulating factor; GRF) analogs in porcine plasma was examined. GRF analogs were incubated in porcine plasma at 37 °C, extracted and subsequently analyzed using high performance liquid chromatography (HPLC). GRF(1-29)-NH₂ was rapidly broken down in the plasma with a degradation rate of t_1/2 = 13 min. The primary degradation product was identified as GRF (3-29)-NH₂. Substitution of Gly¹⁵ by Ala¹⁵ slightly prolonged the plasma half-life (t_1/2 = 17 min) and the major degradative fragment was found to be [Ala¹⁵]GRF(3-29)-NH₂. The cleavage between the 2 and 3 position of the peptide was not inhibited by trasylol at a concentration of 1,000 KITU/ml but was dramatically reduced by the combined use of diprotin A and trasylol. Absence of the free amino group at the N-terminus and/or substitution of a D-amino acid residue at the penultimate position completely prevented cleavage between the 2 and 3 position in the structural linear GRF analogs. Side-chain to side-chain cyclization between Asp⁸ and Lys¹² amino acid residues significantly improved the stability of GRF in plasma with t_1/2 > 2 hr. An additional stability was provided by substitution of D-Ala² for Ala² in the structural cyclic analog. Cyclization between Lys²¹ and Asp²⁵ also improved the stability of the GRF peptide in the plasma. Stability was further enhanced by the presence of D-Ala² or by forming a dicyclic analog through an additional linkage between Asp⁸ and Lys¹².

Key words

Growth Hormone Releasing Factor - Dipeptidylpeptidase-IV - Plasma Stability - Cyclic Peptide