ABSTRACT
Title
Peptide and non-peptide PKR antagonists as novel analgesics
Authors
D. Maftei1, R. Lattanzi1, V. Noviello1, L.A. Giancotti1, G. Balboni2, L. Negri1
1Dept. of Physiology and Pharmacology “Vittorio Erspamer”, “Sapienza” University of Rome, Italy; 2Dept. of Toxicology, University of Cagliari, Cagliari, Italy
1Dept. of Physiology and Pharmacology “Vittorio Erspamer”, “Sapienza” University of Rome, Italy; 2Dept. of Toxicology, University of Cagliari, Cagliari, Italy
Abstract
Bv8 (an amphibian secreted protein) and prokineticins (PK1 and PK2 - mammalian homologues of Bv8) belong to a new family of chemokines, which exert their physiological functions (such as ingestive behaviours, circadian rhythms, angiogenesis, pain sensitization) through two G-protein coupled receptors (GPCRs):prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) (Negri et al., 2002). Prokineticin receptors are distributed in mammalian tissues: PKR1 is more widely distributed in the periphery and PKR2 is highly expressed throughout the brain. In rodents the activation of peripheral PKRs by Bv8 decreases the nociceptive threshold to painful stimuli and PK2 released from inflammatory granulocytes is the main mediator of inflammatory pain (Giannini et al., 2009). The aim of our research is to find PKR antagonists as novel analgesic drugs. Recently some peptide (Ala-24Bv8) (Miele et al., 2010) and non-peptide (PC1, PC7 and PC27) PKR antagonists were designed and synthesized (Balboni et al., 2008). PC1, a PKR1 preferring ligand, is our lead compound which selectively antagonizes the Bv8-induced hyperalgesia. Here we examined the pharmacological effects and the selectivity of the new ligands for the PKRs using 2 methods:
1) In PKR1-KO and PKR2-KO mice we analyzed the ability of the new compounds to antagonize Bv8-induced thermal hyperalgesia (Paw immersion test 48°C). The drugs were injected by intraplantar (i.pl.) route in a volume of 20 μl.
2) In rats we evaluated the ability of the new compounds to suppress Bv8-induced water intake: Bv8 stimulates drinking through the activation of PKR2 in subfornical organ (SFO) of the rat brain (Negri et al., 2004). The drugs were injected into the left lateral ventricle (i.c.v.) throughout a guide cannula in a volume of 5 μl.
After drugs administration the animals were observed for three hours at determinate time intervals.
Results: In KO mice i.pl. injection of Bv8 (630 fmol) decreases the nociceptive threshold to thermal stimuli. In PKR1-KO mice 150 pmol of PC1, administrated 5’ before Bv8, antagonized Bv8-induced thermal hyperalgesia, while in PKR2-KO mice a 10 folds lower dose was enough to antagonize Bv8-induced thermal hyperalgesia, confirming the selectivity of PC1 for the PKR1. In PKR2-KO mice, PC7 and PC27 antagonized Bv8-induced thermal hyperalgesia at doses 100 fold lower than that of PC1, suggesting that these molecules have still higher affinity for the PKR1.
In rats, i.c.v. injection of Bv8 (63 pmol) has a dipsogen effect mediated by PKR2. PC1 administrated i.c.v. at a dose of 15 nmol, 15’ before Bv8 administration, suppressed Bv8 induced water intake, while PC7 and PC27 suppressed Bv8-induced water intake at doses 2-3 fold higher than that of PC1.
The peptide compound Ala-24Bv8 is a partial agonist with a preferring affinity for PKR2: in mice i.pl. injection of Ala-24Bv8 at a dose 10 folds higher than Bv8, antagonized the Bv8-induced thermal hyperalgesia. However, in rats, i.c.v. administration of Ala-24Bv8 induced dipsogen effect as Bv8 did.
Balboni et al. (2008). J Med Chem. 51,7635-7639.
Giannini et al. (2009). PNAS 106 (34),14646-14651.
Miele et al. (2010). Prot Expres Purif.73, 10-14.
Negri et al. (2002). Br J Pharmacol.137, 1147-1154.
Negri et al. (2004). Br J Pharmacol. 142, 181-191.
1) In PKR1-KO and PKR2-KO mice we analyzed the ability of the new compounds to antagonize Bv8-induced thermal hyperalgesia (Paw immersion test 48°C). The drugs were injected by intraplantar (i.pl.) route in a volume of 20 μl.
2) In rats we evaluated the ability of the new compounds to suppress Bv8-induced water intake: Bv8 stimulates drinking through the activation of PKR2 in subfornical organ (SFO) of the rat brain (Negri et al., 2004). The drugs were injected into the left lateral ventricle (i.c.v.) throughout a guide cannula in a volume of 5 μl.
After drugs administration the animals were observed for three hours at determinate time intervals.
Results: In KO mice i.pl. injection of Bv8 (630 fmol) decreases the nociceptive threshold to thermal stimuli. In PKR1-KO mice 150 pmol of PC1, administrated 5’ before Bv8, antagonized Bv8-induced thermal hyperalgesia, while in PKR2-KO mice a 10 folds lower dose was enough to antagonize Bv8-induced thermal hyperalgesia, confirming the selectivity of PC1 for the PKR1. In PKR2-KO mice, PC7 and PC27 antagonized Bv8-induced thermal hyperalgesia at doses 100 fold lower than that of PC1, suggesting that these molecules have still higher affinity for the PKR1.
In rats, i.c.v. injection of Bv8 (63 pmol) has a dipsogen effect mediated by PKR2. PC1 administrated i.c.v. at a dose of 15 nmol, 15’ before Bv8 administration, suppressed Bv8 induced water intake, while PC7 and PC27 suppressed Bv8-induced water intake at doses 2-3 fold higher than that of PC1.
The peptide compound Ala-24Bv8 is a partial agonist with a preferring affinity for PKR2: in mice i.pl. injection of Ala-24Bv8 at a dose 10 folds higher than Bv8, antagonized the Bv8-induced thermal hyperalgesia. However, in rats, i.c.v. administration of Ala-24Bv8 induced dipsogen effect as Bv8 did.
Balboni et al. (2008). J Med Chem. 51,7635-7639.
Giannini et al. (2009). PNAS 106 (34),14646-14651.
Miele et al. (2010). Prot Expres Purif.73, 10-14.
Negri et al. (2002). Br J Pharmacol.137, 1147-1154.
Negri et al. (2004). Br J Pharmacol. 142, 181-191.