Preparation, purification, and characterization of a reversibly lipidized desmopressin with potentiated anti-diuretic activity

Purpose. To prepare and characterize a reversibly lipidized dipalmitoyl des mopressin (DPP), and to compare its anti-diuretic efficacy and biodistribut ion with that of unmodified desmopressin (DDAVP). Methods. Dithiothreitol (DTT) was used to reduce the intramolecular disulfi de bond in DDAVP, and the reduced DDAVP was treated with a thiopyridine-con taining disulfide lipidization reagent, Pal-CPD. The product, DPP, was puri fied by acid precipitation and, subsequently, by size-exclusion chromatogra phy. Reversed-phase HPLC was used to analyze the purity and to evaluate the hydrophobicity of the product. Mass spectrometry was employed to character ize its molecular structure. The biological activity of DPP was demonstrate d by the antidiuretic effects in vasopressin-deficient Brattleboro rats. Pr eliminary pharmacokinetic and biodistribution studies of intravenously inje cted DDAVP and DPP were carried out in CF-1 mice. Results. DDAVP was readily reduced by a 2-fold molar excess of DTT at 37 de grees C for 0.5 hr. DPP was formed by the reaction of reduced DDAVP with Pa l-CPD. Each DPP molecule contains two palmitic acid moieties, which link to the peptide via two disulfide bonds. After acid precipitation and size-exc lusion chromatography, the purity was found to be approximately 95%, and th e overall yield was 57%. When DPP was administered subcutaneously to Brattl eboro rats, the potency of the anti-diuretic activity of DDAVP was enhanced to more than 250-fold. The plasma concentration of intravenously injected DDAVP in mice decreased rapidly during the first 20 min and followed by a s low elimination rate. However, in DPP administered mice, the plasma concent ration actually increased in the first 20 min, followed by a slow eliminati on with a rate similar to that in DDAVP-injected mice. The regeneration of DDAVP was detected in the plasma of mice treated with DPP. Studies of the o rgan distribution in mice indicated that the liver retention of DPP was lon ger than that of DDAVP. On the other hand, the intestinal excretion of DPP was significantly less than that of DDAVP. Conclusions. The 250-fold increase of the anti-diuretic potency in DPP is m ost likely due to a slow elimination and prolonged tissue retention, togeth er with the regeneration of active DDAVP, in the animals. Our results indic ate that reversible lipidization is a simple and effective approach for imp roving the efficacy of many peptide drugs.