Structure-activity relationship of reversibly lipidized peptides: studies of fatty acid-desmopressin conjugates.

PURPOSE: To synthesize a series of reversible fatty acid-desmopressin (DDAVP) conjugates and to study their structure-activity relationship as anti-diuretic drugs.
METHODS: Seven fatty acid conjugates of DDAVP were prepared using various reversible lipidization reagents as described in our previous reports. All products were purified by acid precipitation and/or size-exclusion chromatography. Reversed-phase HPLC was used to evaluate their purity and lipophilicity. The anti-diuretic efficacy of these fatty acid conjugates was assessed in vasopressin-deficient Brattleboro rats. Four selected conjugates, i.e., DPA, DPH, DPD and DPP (acetic, hexanoic. decanoic, and palmitic acid conjugate, respectively), along with DDAVP itself were used in Caco-2 cell uptake studies and their degradation and the regeneration of active DDAVP were investigated using an in vitro liver slice metabolic system coupled with a HPLC assay.
RESULTS: All fatty acid-DDAVP conjugates were more lipophilic than DDAVP as examined by HPLC analyses. When cysteine was used as the linker, the capacity index (k', a measure of lipophilicity) of the conjugates was linearly correlated with the number of carbons in the fatty acid chain. The anti-diuretic activity of the conjugates was correlated with the length of the fatty acid chain, with C10 as the minimal requirement for possessing the enhanced anti-diuretic activity. Among the seven fatty acid conjugates, palmitic acid conjugate was the most potent DDAVP derivative. Removal of carboxyl group from the cysteine linker completely abolished the enhancement of the activity. The extent of cellular uptake also positively correlated with the lipophilicity of the conjugates. The metabolism of DDAVP, DPH, DPD, and DPP by liver slices all followed first order kinetics with half-life of 0.30, 0.01, 0.06 and 3.44 hr, respectively. The degradation rates of DPH and DPD in the liver slice incubation were much faster than that of DDAVP and therefore an accumulation of regenerated DDAVP in the media was observed. In contrast, DPP was metabolized much slower than DDAVP and, consequently, no significant accumulation of regenerated DDAVP could be detected.
CONCLUSION: Conjugation of DDAVP with fatty acids increased the lipophilicity and the anti-diuretic activity of this peptide drug. The anti-diuretic activity of lipidized DDAVP was dependent on the chain length of the fatty acid, as well as the structure of the linker in the conjugate. The preservation and enhancement of the in vivo antidiuretic activity of the conjugates is most likely due to a combination of an improved pharmacokinetic behavior and a concurrent regeneration of active DDAVP in tissues.