Acylation of Human Insulin With Palmitic Acid Extends the Time Action of Human Insulin in Diabetic Dogs

Abstract

To test whether the binding of insulin to an endogenous serum protein can be used to extend the time action of insulin, human insulin was acylated at the epsilon-amino group of Lys(B29) with palmitic acid to promote binding to serum albumin. Size-exclusion chromatography was used to demonstrate specific binding of the resulting analog, [N(epsilon)-palmitoyl Lys(B29)] human insulin, to serum albumin in vitro, and the time action and activity of the analog were determined in vivo using overnight-fasted, insulin-withdrawn diabetic dogs. In the diabetic animal model, the duration of action of [N(epsilon)-palmitoyl Lys(B29)] human insulin administered intravenously was nearly twice that of unmodified human insulin, and the plasma half-life was nearly sevenfold that of the unmodified protein. Administered subcutaneously, [N(epsilon)-palmitoyl Lys(B29)] human insulin had a longer duration of action; a flatter more basal plasma insulin profile; and a lower intersubject variability of response than the intermediate-acting insulin suspension Humulin L (Lilly, Indianapolis, IN). These studies support the concept that modification of insulin to promote binding to an existing serum protein can be used to extend the time action of human insulin. In addition, the time action, pattern, and decreased variability of response to [N(epsilon)-palmitoyl Lys(B29)] human insulin support the development and further testing of this soluble insulin analog as a basal insulin to increase the safety of intensive insulin therapy.

Degradation, receptor binding, insulin secreting and antihyperglycaemic actions of palmitate-derivatised native and Ala8-substituted GLP-1 analogues

“…GLP-1 that is not degraded by DPP IV is rapidly cleared from the circulation by renal filtration (Hassan et al, 1999). It has been found that insulin can be renally retained by binding to serum albumin through derivatisation with fatty acids (Kurtzhals et al, 1995;Markussen et al, 1996;Myers et al, 1997).…”

“…Spectra were recorded using a quadripole ion trap mass analyser and collected using full ion scan mode over the m/z range 150-2000. Covalent attachment of fatty acids to peptides has been explored as a potential tool to deliver soluble therapeutic agents with protracted modes of action (Hashimoto et al, 1989;Kurtzhals et al, 1995;Markussen et al, 1996;Myers et al, 1997). tration may only be as long as 3 to 4 min (Deacon et al, 1998).…”

“…Data represent the percentage of major degradation fragment, GLP-1(9-36)amide (following HPLC separation; see Figures 1 and 2), relative to the intact peptide following incubation with purified DPP IV or human plasma. Insulins acylated with fatty acids at the´-amino group of Lys B29 were found to have a prolonged action time in vivo (Markussen et al, 1996;Myers et al, 1997). Covalent attachment of fatty acids to peptides has been explored as a potential tool to deliver soluble therapeutic agents with protracted modes of action (Hashimoto et al, 1989;Kurtzhals et al, 1995;Markussen et al, 1996;Myers et al, 1997).…”

A structure–function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists

“…Because the NH 2 -terminus can adopt many different conformations with certain ones having high affinity for the PAC1-R, our approach was to systematically explore the initial 5 residues of PACAP using substitutions that resulted in conformationally-restricted analogs. We also explored the results of selective substitutions in other sites that might conformationally-restrict the PACAP-analog, which could affect degradation or could extend its half-life in the circulation, such as the attachment of long-chain fatty-acids[7,21,39]. The latter approach was included because PACAP38 has a plasma half-life of <5–10 minutes[4,4,7,31], which restricts its therapeutic potential.…”

“…Fourth, our results show that the attachment of a fatty-acid on the COOH-terminus lysine [Lys(palmitoyl) 38 ] (P12, P13 and P14) had only a minimal effect on PAC1-affinity and thus could be an important substitution for PAC1-R selective agonists in the future. One of the main problems with the potential therapeutic use of PACAP is its short half-life[4,31]; however, recent studies with other peptides (insulin, glucagon,glucagon-like peptide 1) show that e attachment of long-chain fatty acids greatly extends their half-lives[21,39,56]. …”