Zyklophin, a systemically active selective kappa opioid receptor peptide antagonist with short duration of action

Abstract

The cyclic peptide zyklophin {[N-benzylTyr(1),cyclo(D-Asp(5),Dap(8))-dynorphin A-(1-11)NH(2), Patkar KA, et al. (2005) J Med Chem 48: 4500-4503} is a selective peptide kappa opioid receptor (KOR) antagonist that shows activity following systemic administration. Systemic (1-3 mg/kg s.c.) as well as central (0.3-3 nmol intracerebroventricular, i.c.v.) administration of this peptide dose-dependently antagonizes the antinociception induced by the selective KOR agonist U50,488 in C57BL/6J mice tested in the 55 degrees C warm water tail withdrawal assay. Zyklophin administration had no effect on morphine- or SNC-80-mediated antinociception, suggesting that zyklophin selectively antagonizes KOR in vivo. Additionally, the antagonism of antinociception induced by centrally (i.c.v.) administered U50,488 following peripheral administration of zyklophin strongly suggests that the peptide crosses the blood-brain barrier to antagonize KOR in the CNS. Most importantly, the antagonist activity of zyklophin (3 mg/kg s.c.) lasts less than 12 h, which contrasts sharply with the exceptionally long duration of antagonism reported for the established small-molecule selective KOR antagonists such as nor-binaltorphimine (nor-BNI) that last weeks after a single administration. Systemically administered zyklophin (3 mg/kg s.c.) also prevented stress-induced reinstatement of cocaine-seeking behavior in a conditioned place preference assay. In conclusion, the peptide zyklophin is a KOR-selective antagonist that exhibits the desired shorter duration of action, and represents a significant advance in the development of KOR-selective antagonists.

Fig. 1.

Zyklophin crosses the blood brain barrier to antagonize U50,488-induced antinociception in the mouse 55 °C warm-water tail-withdrawal test. The antinociceptive effects of U50,488 (10 mg/kg i.p.) were determined 40 min after administration in mice pretreated 1 h with vehicle or zyklophin through the (A) i.c.v. (0.3–3 nmol) or (B) s.c. route (0.3–3 mg/kg) of administration. (C) The antinociceptive effect of centrally administered U50,488 (40 nmol i.c.v.) was also antagonized in mice peripherally pretreated 60 min with zyklophin (3 mg/kg s.c.). Tail-withdrawal latencies in C were measured 20 min after injection of U50,488. Plus and minus signs under bars denote whether the listed agent was administered or not. Data are presented as the mean percent antinociception ± SEM from 6–8 mice. *, significantly different from baseline tail-withdrawal latency; ^†, significantly different from U50,488-induced antinociception after vehicle pretreatment, P < 0.05; Student's t-test.

Fig. 2.

Time course of zyklophin-mediated antagonism of U50,488-induced antinociception in the mouse 55 °C warm-water tail-withdrawal test. Antinociceptive effect of U50,488 (10 mg/kg i.p.) in mice pretreated for 1–24 h with zyklophin (3 mg/kg s.c.). Tail withdrawal latencies were determined 40 min after agonist administration. Data are presented as the mean percent antinociception ± SEM from 8–12 mice. *, significantly different from the baseline tail-withdrawal latency; ^†, significantly different from U50,488-induced antinociception without pretreatment, P < 0.05; Student's t-test.

Fig. 3.

Kappa-opioid receptor-selective antagonism by zyklophin. Antinociceptive effects of morphine (10 mg/kg i.p., left pair of white bars) or SNC-80 (12.5 mg/kg i.p., center pair of gray bars) were not reduced by a 1 h pretreatment with zyklophin (3 mg/kg s.c.), whereas the effect of U50,488 (10 mg/kg i.p., right pair of black bars) was significantly antagonized. Tail-withdrawal latencies were measured in the mouse 55 °C warm-water tail-withdrawal test 40 min after selective agonist administration. Data are presented as the mean percent antinociception ± SEM of the control animals treated only with the matching agonist (100%). n = 8–10 mice. *, Significantly different from matching agonist effect, P < 0.01; Student's t-test.

Fig. 4.

Stress-induced reinstatement of cocaine CPP prevented by zyklophin pretreatment. (A) Schematic of reinstatement and testing protocol. Vehicle (0.9% saline) or zyklophin were administered on days 28 and 29, 20 min before initial exposure to forced swim stress (diamonds) or cocaine place-conditioning (square, day 29). (B) After 4 days of cocaine (10 mg/kg s.c. daily), mice exhibited significant preference for the cocaine paired environment, with extinction occurring by 3 weeks (left bars). Mice were exposed to forced swim stress (center bars) or an additional round of cocaine place-conditioning (right bars), reinstating place preference. Zyklophin pretreatment (3, but not 1, mg/kg s.c.) prevented stress-induced reinstatement of place preference (center bars). In contrast, zyklophin pretreatment (3 mg/kg s.c.) was ineffective at preventing cocaine-induced reinstatement of CPP (rightmost bar). n = 8–17 mice; cocaine place-conditioning data on left represents combined responses of 81 mice. *, Significantly different from preconditioning place preference response (leftmost bar); ^†, significantly different from post-conditioning place preference response (second bar on left); ^‡, significantly different from stress-induced reinstatement of place preference response (striped gray bar, center), Fisher's LSD post-hoc test.