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We characterized the novel, rationally designed peptide glucagon-like peptide 1 (GLP-1) receptor agonist H-HGEGTFTSDL-SKQMEEEAVRLFIEWLKNGGPSSGAPPSK KKKKK-NH 2 (ZP10A). Receptor binding studies demonstrated that the affinity of ZP10A for the human GLP-1 receptor was 4-fold greater than the affinity of GLP-1 (7-36) amide. ZP10A demonstrated dose-dependent improvement of glucose tolerance with an ED 50 value of 0.02 nmol/kg i.p. in an oral glucose tolerance test (OGTT) in diabetic db/db mice. After 42 days of treatment, ZP10A dose-dependently (0, 1, 10, or 100 nmol/kg b.i.d.; n ϭ 10/group), decreased glycosylated hemoglobin (HbA 1C ) from 8.4 Ϯ 0.4% (vehicle) to a minimum of 6.2 Ϯ 0.3% (100 nmol/kg b.i.d.; p Ͻ 0.05 versus vehicle) in db/db mice. Fasting blood glucose (FBG), glucose tolerance after an OGTT, and HbA 1C levels were significantly improved in mice treated with ZP10A for 90 days compared with vehicle-treated controls. Interestingly, these effects were preserved 40 days after drug cessation in db/db mice treated with ZP10A only during the first 50 days of the study. Real-time polymerase chain reaction measurements demonstrated that the antidiabetic effect of early therapy with ZP10A was associated with an increased pancreatic insulin mRNA expression relative to vehicle-treated mice. In conclusion, long-term treatment of diabetic db/db mice with ZP10A resulted in a dose-dependent improvement of FBG, glucose tolerance, and blood glucose control. Our data suggest that ZP10A preserves ␤-cell function. ZP10A is considered one of the most promising new drug candidates for preventive and therapeutic intervention in type 2 diabetes.

In conscious rats, intravenous (i.v.) administration of the hexapeptide Ac-RYYRWK-NH 2 , a partial agonist of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, produces a selective water diuresis without marked cardiovascular or behavioral effects. The present study examined the in vitro and in vivo pharmacodynamic profile of the novel and potentially metabolically stable NOP receptor ligand ZP120 (Ac-RYYR-WKKKKKKK-NH 2 ), which was created by conjugation of a structure-inducing probe (SIP) (i.e., K 6 sequence) to Ac-RYYRWK-NH 2 . In cells transfected with human NOP receptors, both Ac-RYYRWK-NH 2 and ZP120 displaced [ 3 H]N/OFQ (both peptides, pK i ϭ 9.6), and similar to N/OFQ inhibited forskolininduced cAMP formation (Ac-RYYRWK-NH 2 , pEC 50 ϭ 9.2; ZP120, 9.3; N/OFQ, 9.7). In the mouse vas deferens assay (MVD), Ac-RYYRWK-NH 2 and ZP120 behaved as partial agonists, inhibiting electrically induced contractions with similar pEC 50 values (9.0 and 8.6, respectively) but with submaximal efficacy compared with N/OFQ. In MVD, both peptides blocked the responses to N/OFQ, with ZP120 being approximately 50-fold more potent than Ac-RYYRWK-NH 2 . In vivo, doseresponse studies in rats showed that at doses (i.v. bolus or i.v. infusion) that produced a sodium-potassium-sparing aquaresis, ZP120 and Ac-RYYRWK-NH 2 elicited a mild vasodilatory response without reflex tachycardia. However, the renal responses to ZP120 were of greater magnitude and duration. Finally, each peptide blocked the bradycardia and hypotension to N/OFQ in conscious rats, but the effect of ZP120 was of much greater duration. Together, these findings demonstrate that ZP120 is a novel, functionally selective SIP-modified NOP receptor partial agonist with increased biological activity and sodium-potassium-sparing aquaretic activity, the actions of which may be useful in the management of hyponatremia/ hypokalemia in water-retaining states.

(RS)-5-Amino-4-(4-chlorophenyl)pentanoic acid (10) and the R-form (11) and S-form (12) of (RS)-5-amino-3-(4-chlorophenyl)pentanoic acid, which are homologues of the 4-aminobutanoic acidB (GABAB) receptor agonist (RS)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofen), were synthesized. Compound 10 was synthesized by homologation at the carboxyl end of baclofen using a seven-step reaction sequence. N-Boc-protected (4R, 5R)-4-(4-chlorophenyl)-5-hydroxy-2-piperidone (18) was deoxygenated via a modified Barton-McCombie reaction to give N-Boc-protected (R)-4-(4-chlorophenyl)-2-piperidone (20), which was ring opened and deprotected to give 11.HCl. The corresponding S-enantiomer, 12.HCl, was synthesized analogously from the 4S,5S-enantiomer of 18, compound 21. The enantiomeric purities of 11.HCl (ee = 99.8%) and 12. HCl (ee = 99.3%) were determined by chiral HPLC. Compound 10 did not show detectable affinity for GABAA or GABAB receptor sites and was inactive as an agonist or an antagonist at GABAB receptors in the guinea pig ileum. Like the enantiomers of baclofen, neither 11 nor 12 showed detectable affinity for GABAA receptor sites, and in agreement with the findings for (S)-baclofen, 12 did not interact significantly with GABAB receptor sites. Compound 11 (IC50 = 7.4 +/- 0.6 microM), a homologue of (R)-baclofen (2), was shown to be some 50 times weaker than 2 (IC50 = 0.14 +/- 0.01 microM) as an inhibitor of GABAB binding. Accordingly, 11 (EC50 = 150 +/- 23 microM) was shown to be weaker than 2 (EC50 = 11 +/- 1 microM) as an inhibitor of electrically induced contractions of the guinea pig ileum. However, whereas this effect of 2 was sensitive to the GABAB antagonist, CGP35348 (4), the inhibition by 11 was not significantly affected. Furthermore, 12 (EC50 = 310 +/- 16 microM) was shown to be one-half as potent as 11 in this test system, and this effect of 12 also was insensitive to 4. The dissimilarities of the pharmacological effects of 2 and compounds 11 and 12 were emphasized by the observation that whereas 2 only inhibits the ileum contraction by 59 +/- 5%, 11 as well as 12 were shown to inhibit this response by approximately 94%. Neither 11 nor 12 appeared to affect significantly cholinergic mechanisms in the ileum, and their mechanism(s) of action remain enigmatic.