Regulation of Opioid Receptors by Their Endogenous Opioid Peptides.

Activation of µ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses. The three major endogenous opioid peptides, ß-endorphin, enkephalins, and dynorphins result from the processing of three main precursors: proopiomelanocortin, proenkephalin, and prodynorphin. Using a knockout approach, we sought to determine whether the absence of endogenous opioid peptides would affect the expression or activity of opioid receptors in mice lacking either proenkephalin, ß-endorphin, or both. Since gene knockout can lead to changes in the levels of peptides generated from related precursors by compensatory mechanisms, we directly measured the levels of Leu-enkephalin and dynorphin-derived peptides in the brain of animals lacking proenkephalin, ß-endorphin, or both. We find that whereas the levels of dynorphin-derived peptides were relatively unaltered, the levels of Leu-enkephalin were substantially decreased compared to wild-type mice suggesting that preproenkephalin is the major source of Leu-enkephalin. This data also suggests that the lack of ß-endorphin and/or proenkephalin does not lead to a compensatory change in prodynorphin processing. Next, we examined the effect of loss of the endogenous peptides on the regulation of opioid receptor levels and activity in specific regions of the brain. We also compared the receptor levels and activity in males and females and show that the lack of ß-endorphin and/or proenkephalin leads to differential modulation of the three opioid receptors in a region- and gender-specific manner. These results suggest that endogenous opioid peptides are important modulators of the expression and activity of opioid receptors in the brain.

Design, synthesis and pharmacological evaluation of novel polycyclic heteroarene ethers as PDE10A inhibitors: part II.

We report the design and synthesis of novel pyrrolo[3,2-b]quinoline containing heteroarene ethers as PDE10A inhibitors with good to excellent potency, selectivity and metabolic stability. Further optimization of this primary series resulted in the identification of 1-methyl-3-(4-{[3-(pyridine-4-yl)pyrazin-2-yl]oxy}phenyl)-1H-pyrrolo[3,2-b]pyridine 13a with good hPDE10A potency (IC50: 6.3 nM), excellent selectivity over other related PDEs and desirable physicochemical properties. The compound exhibited high peripheral and adequate brain levels upon oral dosing in rodents. The compound also showed excellent efficacy in multiple preclinical animal models related to psychiatric disorders, particularly schizophrenia.

Discovery of benzo[d]imidazo[5,1-b]thiazole as a new class of phosphodiesterase 10A inhibitors.

The design, synthesis and structure activity relationship studies of a series of compounds from benzo[d]imidazo[5,1-b]thiazole scaffold as phosphodiesterase 10A (PDE10A) inhibitors are discussed. Several potent analogs with heteroaromatic substitutions (9a-d) were identified. The anticipated binding mode of these analogs was confirmed by performing the in silico docking experiments. Later, the heteroaromatics were substituted with saturated heteroalkyl groups which provided a tool compound 9e with excellent PDE10A activity, PDE selectivity, CNS penetrability and with favorable pharmacokinetic profile in rats. Furthermore, the compound 9e was shown to be efficacious in the MK-801 induced psychosis model and in the CAR model of psychosis.

Isothiazole and isoxazole fused pyrimidones as PDE7 inhibitors: SAR and pharmacokinetic evaluation.

The synthesis and structure-activity relationship studies of isothiazole and isoxazole fused pyrimidones as PDE7 inhibitors are discussed. The pharmacokinetic profile of 10 and 21 with adequate CNS penetration, required for in vivo Parkinson's disease models, are disclosed.

Imidazopyridazinones as novel PDE7 inhibitors: SAR and in vivo studies in Parkinson's disease model.

The synthesis and structure-activity relationship studies of a series of compounds from imidazopyridazinone scaffold as PDE7 inhibitors are disclosed. Potent analogs such as compounds 7 (31nM), 8 (27nM), and 9 (12nM) were identified. The PDE selectivity and pharmacokinetic profile of compounds 7, 8 and 9 are also disclosed. The adequate CNS penetration of compound 7 in mice allowed it to be tested in the MPTP induced PD model and haloperidol induced catalepsy model to probe the differential pharmacology of PDE7 in the striatal pathway.

GRC-6211, a new oral specific TRPV1 antagonist, decreases bladder overactivity and noxious bladder input in cystitis animal models.

PURPOSE: We evaluated the effects of GRC-6211, an orally active TRPV1 antagonist, on the function and noxious input of naïve and inflamed bladders. MATERIALS AND METHODS: In urethane (Sigma(R)) anesthetized rats 0.5 ml GRC-6211 (0.001, 0.01, 0.1 and 1 mg/kg weight) or its vehicle (0.5% methylcellulose) were administered through a duodenal catheter and cystometry was done during infusion of saline, 100 microM capsaicin or 0.5% acetic acid (Merck, Feltham, United Kingdom). Cystometry was also performed in WT and TRPV1 knockout mice treated with 1 mg/kg GRC-6211. Cystometry was done in rats inflamed with lipopolysaccharide after receiving 0.1 mg/kg GRC-6221 or vehicle. Spinal c-fos expression induced by 0.5% acetic acid was investigated after 0.1 mg/kg GRC-6211 or vehicle administration. TRPV1 immunoreactivity was evaluated in the bladder after GRC-6211 administration. RESULTS: The reflex activity of rat and WT mice naïve bladders was unchanged by GRC-6211 up to a dose of 0.1 mg/kg. At 1 mg/kg contractions were transiently suppressed in naïve rats and WT mice but not in TRPV1 knockout mice. GRC-6211 (0.1 mg/kg) completely prevented capsaicin induced irritation, while the 0.001, 0.01 or 0.1 mg/kg dose decreased the mean +/- SD frequency of bladder contractions during acetic acid infusion from 1.5 +/- 0.3 to 1.35 +/- 0.35 (not significant), 0.9 +/- 0.2 (p <0.05) and 0.8 +/- 0.2 (p <0.05), respectively. Lipopolysaccharide inflamed rats had 1.4 +/- 0.4 and 0.8 +/- 0.1 contractions per minute after vehicle and GRC-6211, respectively (p <0.05). The c-fos expression induced by acetic acid was decreased by GRC-6211 (85.5 +/- 19.1 to 46.7 +/- 9.4, p <0.05). GRC-6211 did not change bladder TRPV1 immunoreactivity. CONCLUSIONS: GRC-6211 counteracts the bladder hyperactivity and noxious input induced by cystitis. At high doses it suppresses normal bladder activity by a TRPV1 dependent mechanism. TRPV1 antagonists might be useful for cystitis.

Design, Synthesis, and Pharmacological Evaluation of 5,6-Disubstituted Pyridin-2(1H)-one Derivatives as Phosphodiesterase 10A (PDE10A) Antagonists.

We report the design and synthesis of novel 5,6-diarylated pyridin-2(1H)-one derivatives as pharmacophoric PDE10A inhibitors. This highly potent molecular scaffold was developed from an inactive diarylpyridine-2-amine derivative 3b by extensive and systematic analogue synthesis and SAR analysis. Further optimization of the scaffold resulted in identification of pyridin-2(1H)-one 18b as a lead compound with good potency (IC50 = 1.6 nM) and selectivity (>6000-fold) over other related PDEs but with a poor pharmacokinetic profile. Careful metabolite profiling of 18b revealed that poor systemic exposure in rats (Cmax = 44 ng/mL; AUC0-t = 359 ng · h/mL) at 10 mg/kg was due to the formation of O-glucuronide conjugate by phase 2 metabolism. The structure of the glucuronide metabolite was confirmed by retention time and LC-MS/MS fragmentation matching with the synthetic glucuronide 26. The problem of low exposure of 18b was effectively addressed by its conversion to an acetate prodrug 25b, which upon oral dosing resulted in an improved pharmacokinetic profile (Cmax = 359 ng.h/mL; AUC0-t = 2436 ng.h/mL) and a desirable brain to plasma ratio of 1.2. The prodrug 25b showed good efficacy in selected rodent models of psychosis.

Role of L-type Ca(2+) channels in pertussis toxin induced antagonism of U50,488H analgesia and hypothermia.

Previous studies have shown that the kappa-opioid effects are sensitive to pertussis toxin (PTX) and affected by Ca(2+) fluxes. However, the possible involvement of Ca(2+) channels in PTX-induced inhibition of kappa-opioid effects has not been reported. The effect of intracerebroventricular (i.c.v.) treatment of pertussis toxin (1 microg/rat, PTX) or saline on the kappa-opioid agonist, U-50,488H (U5H) induced tail-flick analgesia and hypothermia in rats was determined. The effect of nimodipine (NIM), a dihydropyridine (DHP)-sensitive Ca(2+) channel blocker (CCB), on PTX-induced modulation of U5H effects was examined. The DHP ligand, [3H]PN200-110 binding was also determined in both PTX and saline treated rats to study the possible involvement of L-type Ca(2+) channels in PTX modulation of kappa-opioid agonist effects. The analgesia and change in colonic temperature were determined using tail-flick analgesiometer and telethermometer, respectively. U5H (40 mg/kg, i.p.) produced significant analgesic and hypothermic responses. PTX treatment significantly (P<0.01) antagonized the analgesic and hypothermic effects of U5H. Acute pretreatment of NIM (1 mg/kg, i.p.) 15 min prior significantly (P<0.01) reversed the PTX-induced antagonism of U5H effects. In the binding study, PTX treatment (72 h before) resulted in a significant (P<0.005) upregulation (+45% vs. saline control) of DHP binding (B(max)) with no change in affinity (K(d)). The results showed significant upregulation of DHP binding in accordance with PTX-induced antagonism of U5H effects and this blockade was reversed by NIM. Thus, present results suggest that U5H-induced analgesia and hypothermia may be mediated through PTX-sensitive transducer G-proteins (G(i/o)) coupled to L-type Ca(2+) channels.

Regulation of dihydropyridine-sensitive Ca(2+) channels during naloxone-induced opioid supersensitivity in rats.

The Ca(2+) channel blocker, nimodipine, increases the chronic naltrexone-induced supersensitivity to morphine analgesia in mice without affecting the density of up-regulated mu-opioid receptors. In the present study, the change in dihydropyridine-sensitive Ca(2+) channels associated with chronic naloxone-induced supersensitivity to morphine analgesia was studied in rat whole-brain membranes using a radiolabeled Ca(2+) channel blocker, [3H]PN200-110 [isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-methoxycarbonylpyridine-3-carboxylate] (0.02-1.0 nmol/l). Rats were chronically treated with naloxone (1 mg/kg, i.p.), nimodipine (1 mg/kg, i.p.) or their respective vehicles twice daily for 10 days. On the 11th day, 16 h after the last injection of either nimodipine or naloxone, morphine (2 mg/kg, i.p.)-induced tail-flick analgesia was determined or rats were killed for the binding study. Chronic naloxone significantly potentiated (+84%) the morphine-induced analgesia. Chronic nimodipine also potentiated (+76%) morphine analgesia. In rats treated with nimodipine and naloxone, there was an additive increase (206%) in morphine analgesia. In binding studies, chronic naloxone resulted in a significant decrease (-39%) in the density (B(max)) of [3H]PN200-110 binding with no change in K(d) value when compared to the effect of chronic vehicle. Chronic nimodipine resulted in a slight but significant (+14.5%) increase in the B(max) of [3H]PN200-110. However, when nimodipine was co-administered with naloxone, it not only reversed the down-regulation but actually up-regulated (+25%) [3H]PN200-110 binding with no change in K(d) value. Our results show significant down-regulation of [3H]PN200-110 binding in association with naloxone-induced analgesic supersensitivity to morphine. The supersensitivity was also observed following chronic blockade of up-regulated Ca(2+) channels by nimodipine. These results indicate an important role of L-type Ca(2+) channel regulation in naloxone-induced analgesic supersensitivity to morphine.

Role of L-type Ca2+ channels in attenuated morphine antinociception in streptozotocin-diabetic rats.

The role of L-type Ca2+ channels in morphine antinociception was studied in streptozotocin-induced diabetic and control rats, using [3H]PN200-110 [isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-methoxycabonylpyridine-3-caboxylate] binding (0.005-1.0 nmol/l) and rat whole brain membranes, to determine if the attenuation of morphine antinociception was related to alterations in dihydropyridine-sensitive Ca2+ channel binding characteristics. The tail-flick antinociceptive effect of morphine (4 mg/kg, i.p.) was significantly reduced in diabetic rats in comparison to that in controls. Nimodipine (0.1-3 mg/kg, i.p.) did not produce antinociception but significantly potentiated the morphine response in control rats. Nimodipine (0.3-3 mg/kg, i.p.) reversed the attenuation of morphine antinociception in a dose-dependent manner in diabetic rats. Moreover, insulin (2 U/kg, s.c.) [correction of mu/kg] reversed the attenuated morphine antinociception in streptozotocin-diabetic rats. A significant increase in the Bmax (+41%) of [3H]PN200-110 binding was observed in diabetic rat brain membranes compared to that in control rats. However, there was no change in affinity (Kd) value of [3H]PN200-110. The reduced morphine response in diabetic rats, in accordance with up-regulation of dihydropyridine sites, may be due to an increased Ca2+ influx through L-type channels. These results suggest a functional role of L-type Ca2+ channels in morphine antinociception and the diabetic state may lead to alterations in their density.

Role of Ca2+ channels on the hypothermic response produced by activation of kappa-opioid receptors.

The effect of nimodipine (NIM) and lercanidipine (LER) 1,4-dihydropyridine (DHP) calcium channel blockers (CCBs) on the hypothermic response of selective kappa-opioid receptor agonists U50,488H (U50), PD117,302 (PD) and U69,593 (U69) was determined in rats by recording colonic temperature using digital telethermometer. Intraperitoneal (i.p.) injections of U50 (7.5, 15, 22.5 and 40 mg/kg), PD (7.5, 15 and 22.5 mg/kg) and U69 (5 and 20 mg/kg) produced a dose-dependent hypothermic response. However, higher doses of U50 (60 and 80 mg/kg) produced hypothermia, which is less when compared to that produced by 22.5-mg/kg dose of U50. NIM (1 mg/kg i.p.; 15 min prior) and LER (0.3 mg/kg i.p.; 15 min prior) did not produce any change in basal colonic temperature. Treatment of NIM and LER potentiated the U50 (7.5, 15, 22.5 and 40 mg/kg)-induced hypothermic effect. NIM did not potentiate hypothermia produced by U50 (60 mg/kg). On the other hand, PD (7.5, 15 and 22.5 mg/kg)- and U69 (5 and 20 mg/kg)-induced hypothermia was unaffected by the pretreatment of either NIM or LER. This differential modulation of kappa-opioid agonist-induced hypothermia by CCBs suggest that there may be two mechanisms, Ca(2+)-sensitive and Ca(2+)-insensitive, involved in kappa-opioid agonist-induced hypothermic response.

Characterization of active and inactive states of CB1 receptor and the differential binding state modulation by cannabinoid agonists, antagonists and inverse agonists.

Cannabinoid 1 (CB1) receptors have the ability to change conformation between active (R*) and inactive (R) receptor states. Herein, we further characterize these receptor states using series of saturation radioligand binding studies and their differential displacement binding by various CB1 receptor ligands. Binding experiments were carried out in naïve rat/dog whole brain membranes using radioligands [(3)H]CP55,940 (for R* state) & [(3)H]SR141716A (both R* and R states) and various agonist, antagonist & inverse agonist ligands at CB1 receptors. In the saturation binding experiments, of the total number of CB1 receptor binding sites (R* + R) in the rat and dog whole brain membranes, only about 18.3 and 11.6% were in the active (R*) state recognized by [(3)H]CP55,940, respectively. In the competitive binding studies, all the CB1 receptor agonists investigated had significantly very high affinity for the active R* state recognized by [(3)H]CP55,940 and lower affinity for the inactive R state mainly recognized by [(3)H]SR141716A in the presence of a non-hydrolyzable analogue of GTP [Gpp(NH)p]. In contrast, various CB1 receptor antagonists/inverse agonists had similar nanomolar affinities at both [(3)H]CP55,940 and [(3)H]SR141716A recognized binding states. These results clearly characterize the significant differences between the active R* and inactive R binding states of CB1 receptors in naive rat and dog brain. In addition, these results also demonstrates that the CB1 agonists and antagonists/inverse agonists can be differentiated by their relative affinities at active (R*) and inactive (R) binding states of the CB1 receptor.

Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.