Discovery of Potent, Selective, and Short-Acting Peptidic V2 Receptor Agonists.

The vasopressin analogue desmopressin (desamino-d-arginine8 vasopressin, dDAVP, 1) is a potent vasopressin 2 (V2) receptor (V2R) agonist approved in many countries for the treatment of diabetes insipidus, primary nocturnal enuresis, nocturia, and coagulation disorders. Since 1 is primarily excreted via the kidneys, an age-related decline in kidney function leads to slower elimination, prolonged antidiuresis, and hyponatremia. In search of novel, potent, selective, and short-acting peptidic V2R agonists, we synthesized a series of C-terminally truncated analogues of [Val4]dDAVP, 2, modified in positions 2, 3, and 7 and/or at the disulfide bridge. The peptides were evaluated for in vitro potency at the human V2 receptor, selectivity versus the related receptors (human vasopressin 1a receptor, human vasopressin 1b receptor, and human oxytocin receptor), and pharmacokinetic profiles in rodents and other higher species. The truncated analogues show excellent potency at the V2R, increased systemic clearance, and shorter half-life in rats. Two compounds 19 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-Agm) and 38 (c(Bua-Cpa-Thi-Val-Asn-Cys)-Pro-d-Arg-NEt2) have been selected for clinical development for nocturia.

Targeted γ-Secretase Inhibition To Control the Notch Pathway in Renal Diseases.

Notch is a membrane inserted protein activated by the membrane-inserted γ-secretase proteolytic complex. The Notch pathway is a potential therapeutic target for the treatment of renal diseases but also controls the function of other cells, requiring cell-targeting of Notch antagonists. Toward selective targeting, we have developed the γ-secretase inhibitor-based prodrugs 13a and 15a as substrates for γ-glutamyltranspeptidase (γ-GT) and/or γ-glutamylcyclotransferase (γ-GCT) as well as aminopeptidase A (APA), which are overexpressed in renal diseases, and have evaluated them in experimental in vitro and in vivo models. In nondiseased mice, the cleavage product from Ac-γ-Glu-γ-secretase inhibitor prodrug 13a (γ-GT-targeting and γ-GCT-targeting) but not from Ac-α-Glu-γ-secretase inhibitor prodrug 15a (APA-targeting) accumulated in kidneys when compared to blood and liver. Potential nephroprotective effects of the γ-secretase inhibitor targeted prodrugs were investigated in vivo in a mouse model of acute kidney injury, demonstrating that the expression of Notch1 and cleaved Notch1 could be selectively down-regulated upon treatment with the Ac-γ-Glu-γ-secretase-inhibitor 13a.

Conformations and receptor activity of desmopressin analogues, which contain gamma-turn mimetics or a psi[CH(2)O] isostere.

Three analogues of the antidiuretic drug desmopressin ([1-desamino,8-D-arginine]vasopressin) have been prepared. In two of these, gamma-turn mimetics based on a morpholin-3-one framework have been inserted instead of residues Phe3-Asn5, whereas the third analogue has a methylene ether isostere in place of the amide bond between residues 3 and 4. The three analogues were used to probe if the structure determined for desmopressin in aqueous solution, which contains an inverse gamma-turn centered around Gln4, is important in interactions with the vasopressin V(2) receptor. Conformational studies revealed that the analogues that contain either an inverse gamma-turn mimetic or a methylene ether isostere mimicked the conformation of desmopressin fairly well and very well, respectively. Despite this, the analogues displayed only very low agonistic activities at the vasopressin V(2) receptor. Consequently, an inverse gamma-turn involving residues Phe3-Asn5 does not appear to be important when desmopressin is bound to the V(2) receptor. In addition, it was concluded that the amide bond between Phe3 and Gln4 in desmopressin is crucial for interactions with the antidiuretic V(2) receptor.

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

PURPOSE: . To prepare and characterize a reversibly lipidized dipalmitoyl desmopressin (DPP), and to compare its anti-diuretic efficacy and biodistribution with that of unmodified desmopressin (DDAVP). METHODS: Dithiothreitol (DTT) was used to reduce the intramolecular disulfide bond in DDAVP, and the reduced DDAVP was treated with a thiopyridine-containing disulfide lipidization reagent, Pal-CPD. The product, DPP, was purified by acid precipitation and, subsequently, by size-exclusion chromatography. Reversed-phase HPLC was used to analyze the purity and to evaluate the hydrophobicity of the product. Mass spectrometry was employed to characterize its molecular structure. The biological activity of DPP was demonstrated by the antidiuretic effects in vasopressin-deficient Brattleboro rats. Preliminary pharmacokinetic and biodistribution studies of intravenously injected 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 degrees C for 0.5 hr. DPP was formed by the reaction of reduced DDAVP with Pal-CPD. Each DPP molecule contains two palmitic acid moieties, which link to the peptide via two disulfide bonds. After acid precipitation and size-exclusion chromatography, the purity was found to be approximately 95%, and the overall yield was 57%. When DPP was administered subcutaneously to Brattleboro 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 slow elimination rate. However, in DPP administered mice, the plasma concentration actually increased in the first 20 min, followed by a slow elimination 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 organ distribution in mice indicated that the liver retention of DPP was longer 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 most likely due to a slow elimination and prolonged tissue retention, together with the regeneration of active DDAVP, in the animals. Our results indicate that reversible lipidization is a simple and effective approach for improving the efficacy of many peptide drugs.