Development of an enzyme immunoassay for a stable amidated analog of the hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro.
The tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been shown to protect hematopoietic stem cells from the toxicity of anticancer chemotherapies. Since its pharmacological efficacy is limited by a rapid degradation by Angiotensin-I Converting Enzyme (ACE), AcSDKP analogs resistant to ACE have been synthesized. One of these compounds (AcSDKP-NH,) differs from the native AcSDKP by amidation of the C-terminus. Further evaluations of this molecule require an analytical method in order to characterize its pharmacokinetic profile. We report, here, the development of a highly specific and sensitive enzyme immunoassay (EIA) for AcSDKP-NH, thatdoes not cross-react with endogenous or exogenous AcSDKP. Using AcSDKP-NH2-acetylcholinesterase conjugate as a tracer, rabbit specific antiserum and microtiter plates coated with goat anti-rabbit immunoglobulins, this EIA allows the determination of AcSDKP-NH2 with limits of quantitation of 1 nM in mouse plasma and 100 pmol/g in tissues. Intra-day and inter-day coefficients of variations were less than 20%. The method was successfully applied to a pharmacokinetic study in order to compare plasma and tissue profiles of AcSDKP-NH2 and AcSDKP. Plasma AcSDKP-NH2 levels were found higher than those of AcSDKP, with AUCinf and Cmax values, respectively, 26- and 10-fold higher than that of AcSDKP.
Junot
C
C
CEA, CNRS, Service de Pharmacologie et d'Immunologie, Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France.
Theodoro
F
F
Thierry
J
J
Clement
G
G
Wdzieczak-Bakala
J
J
Ezan
E
E
D000577:Amides / Q000737:chemistry*; D000818:Animals; D006801:Humans; D007106:Immune Sera; D007124:Immunoenzyme Techniques / Q000379:methods*; D051379:Mice; D008808:Mice, Inbred CBA; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D011487:Protein Conformation; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000577:Amides; D007106:Immune Sera; D009842:Oligopeptides; C058504:goralatide