Improved efficacy, tolerance, safety, and abuse liability profile of the combination of CR4056 and morphine over morphine alone in rodent models.

BACKGROUND AND PURPOSE: Prolonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I2 receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I2 receptor ligand CR4056 could improve efficacy and safety of morphine and explored the mechanisms of the CR4056-opioid interaction. EXPERIMENTAL APPROACH: We used the complete Freund's adjuvant (CFA) model in rats to study the effects of treatments on hyperalgesia, morphine tolerance and microglia activation as measured by immunofluorescence. Opioid-induced adverse effects were assessed in rodent models of morphine-induced constipation, sedation (open field, sedation rating scale, and rotarod), physical dependence (naloxone-induced withdrawal), and abuse (conditioned place preference-associated reward). Chemiluminescence assays tested CR4056 as allosteric modulator of µ-opioid receptors. KEY RESULTS: CR4056 (ED50 = 4.88 mg·kg-1 ) and morphine (ED50 = 2.07 mg·kg-1 ) synergized in reducing CFA-induced hyperalgesia (ED50 = 0.52 mg·kg-1 ; 1:1 combination). Consistently, low doses of CR4056 (1 mg·kg-1 ) spared one third of the cumulative morphine dose administered during 4 days and prevented/reversed the development of tolerance to morphine anti-hyperalgesia. These opioid-sparing effects were associated with decreased activation of microglia, independent of CR4056 interactions on µ-opioid receptors. Importantly, the low doses of CR4056 and morphine that synergize in analgesia did not induce constipation, sedation, physical dependence, or place preference. CONCLUSION AND IMPLICATIONS: We showed selective synergism between CR4056 and morphine as analgesics. Their combination showed an improved safety and abuse liability profile over morphine alone. CR4056 could be developed as an opioid-sparing drug in multimodal analgesia.

Pharmacological characterisation of CR6086, a potent prostaglandin E2 receptor 4 antagonist, as a new potential disease-modifying anti-rheumatic drug.

BACKGROUND: Prostaglandin E2 (PGE2) acts via its EP4 receptor as a cytokine amplifier (e.g., interleukin [IL]-6) and induces the differentiation and expansion of inflammatory T-helper (Th) lymphocytes. These mechanisms play a key role in the onset and progression of rheumatoid arthritis (RA). We present the pharmacological characterisation of CR6086, a novel EP4 receptor antagonist, and provide evidence for its potential as a disease-modifying anti-rheumatic drug (DMARD). METHODS: CR6086 affinity and pharmacodynamics were studied in EP4-expressing HEK293 cells by radioligand binding and cyclic adenosine monophosphate (cAMP) production, respectively. In immune cells, IL-6 and vascular endothelial growth factor (VEGF) expression were analysed by RT-PCR, and IL-23 and IL-17 release were measured by enzyme-linked immunosorbent assay (ELISA). In collagen-induced arthritis (CIA) models, rats or mice were immunised with bovine collagen type II. Drugs were administered orally (etanercept and methotrexate intraperitoneally) starting at disease onset. Arthritis progression was evaluated by oedema, clinical score and histopathology. Anti-collagen II immunoglobulin G antibodies were measured by ELISA. RESULTS: CR6086 showed selectivity and high affinity for the human EP4 receptor (Ki = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE2-stimulated cAMP production. In models of human immune cells in culture, CR6086 reduced key cytokine players of RA (IL-6 and VEGF expression in macrophages, IL-23 release from dendritic cells, IL-17 release from Th17 cells). In the CIA model of RA in rats and mice, CR6086 significantly improved all features of arthritis: severity, histology, inflammation and pain. In rats, CR6086 was better than the selective cyclooxygenase-2 inhibitor rofecoxib and at least as effective as the Janus kinase inhibitor tofacitinib. In mice, CR6086 and the biologic DMARD etanercept were highly effective, whereas the non-steroidal anti-inflammatory drug naproxen was ineffective. Importantly, in a study of CR6086/methotrexate, combined treatment greatly improved the effect of a fully immunosuppressive dose of methotrexate. CONCLUSIONS: CR6086 is a novel, potent EP4 antagonist showing favourable immunomodulatory properties, striking DMARD effects in rodents, and anti-inflammatory activity targeted to immune-mediated inflammatory diseases and distinct from the general effects of cyclooxygenase inhibitors. These results support the clinical development of CR6086, both as a stand-alone DMARD and as a combination therapy with methotrexate. The proof-of-concept trial in patients with RA is ongoing.

Efficacy of CR4056, a first-in-class imidazoline-2 analgesic drug, in comparison with naproxen in two rat models of osteoarthritis.

PURPOSE: CR4056, (2-phenyl-6-(1H-imidazol-1yl) quinazoline), an imidazoline-2 (I2) receptor ligand, is a promising analgesic drug that has been reported to be effective in several animal models of pain. The aim of this study was to evaluate the effects of CR4056 in two well-established rat models of osteoarthritis (OA), mimicking the painful and structural components of human OA. METHODS: Knee OA was induced either by single intra-articular injection of monoiodoacetate (MIA) or by medial meniscal tear (MMT) in the right knee of male rats. In the MIA model, allodynia and hyperalgesia were measured as paw withdrawal threshold to mechanical stimulation. In the MMT model, pain behavior was analyzed as weight-bearing asymmetry (i.e. difference in hind paw weight distribution, HPWD) between the injured and the contralateral limbs. RESULTS: Acute oral administration of CR4056, 14 days after MIA injection, significantly and dose-dependently reduced allodynia and hyperalgesia 90 minutes after treatment, whereas acute naproxen administration significantly reduced allodynia but not hyperalgesia. After 7 days of repeated treatment, both CR4056 and naproxen showed significant anti-allodynic and anti-hyperalgesic effects in the MIA model. Rats undergoing MMT surgery developed a significant and progressive asymmetry in HPWD compared with sham-operated animals. Repeated treatment with CR4056 significantly reduced the progression of the pain behavior, whereas naproxen had no effects. CONCLUSION: The data presented here show that the I2 ligand CR4056 could be a new effective treatment for OA pain. The compound is currently under Phase II clinical evaluation for this indication.

Biological profile and bioavailability of imidazoline compounds on morphine tolerance modulation.

Tolerance to opioid administration represents a serious medical alert in different chronic conditions. This study compares the effects of the imidazoline compounds 1, 2, and 3 on morphine tolerance in an animal model of inflammatory pain in the rat. 1, 2, and 3 have been selected in that, although bearing a common scaffold, preferentially bind to α2-adrenoceptors, imidazoline I2 receptors, or both systems, respectively. Such compounds have been tested in vivo by measuring the paw withdrawal threshold to mechanical pressure after complete Freund's adjuvant injection. To determine the ligand levels in rat plasma, an HPLC-mass spectrometry method has been developed. All the compounds significantly reduced the induction of morphine tolerance, showing different potency and duration of action. Indeed, the selective imidazoline I2 receptor interaction (2) restored the analgesic response by maintaining the same time-dependent profile observed after a single morphine administration. Differently, the selective α2C-adrenoceptor activation (1) or the combination between α2C-adrenoceptor activation and imidazoline I2 receptor engagement (3) promoted a change in the temporal profile of morphine analgesia by maintaining a mild but long lasting analgesic effect. Interestingly, the kinetics of compounds in rat plasma supported the pharmacodynamic data. Therefore, this study highlights that both peculiar biological profile and bioavailability of such ligands complement each other to modulate the reduction of morphine tolerance. Based on these observations, 1-3 can be considered useful leads in the design of new drugs able to turn off the undesired tolerance induced by opioids.

Modulation of imidazoline I2 binding sites by CR4056 relieves postoperative hyperalgesia in male and female rats.

BACKGROUND AND PURPOSE: CR4056 is a novel imidazoline-2 (I2 ) ligand exhibiting potent analgesic activity in animal models of pain. In this study, we investigated the effects of CR4056 in a well-established model of postoperative pain where rats develop hyperalgesia in the injured hind paw. EXPERIMENTAL APPROACH: By measuring paw withdrawal threshold to mechanical pressure, we studied the pharmacology of CR4056, potential sex differences in pain perception and response to treatment, and the pharmacodynamic interaction of CR4056 with morphine. KEY RESULTS: Oral CR4056 and subcutaneous morphine dose-dependently reversed the hyperalgesic response. Analgesic effects of CR4056 were completely suppressed by the non-selective imidazoline I2 /α2 -adrenoceptor antagonist idazoxan, were partially reduced (~30%; P < 0.05) by the selective α2 -adrenoceptor antagonist yohimbine, but were not influenced by the non-selective I1 /α2 -adrenoceptor antagonist efaroxan or by the µ opioid receptor antagonist naloxone. We found no differences in responses to CR4056 or morphine between male and female rats. However, females had a lower pain threshold than males, and needed lower doses of drugs to reach a significant analgesia. When CR4056 and morphine were combined, their median effective doses were lower than expected for additive effects, both in males and in females. Isobolographic analysis confirmed a synergism between CR4056 and morphine. CONCLUSIONS AND IMPLICATIONS: CR4056 is a novel pharmacological agent under development for postoperative pain both as stand-alone treatment and in association with morphine. CR4056 has successfully completed Phase I studies for tolerability and pharmacokinetics in healthy volunteers, and is currently entering the first proof-of-concept study in patients.

CR4056, a new analgesic I2 ligand, is highly effective against bortezomib-induced painful neuropathy in rats.

Although bortezomib (BTZ) is the frontline treatment for multiple myeloma, its clinical use is limited by the occurrence of painful peripheral neuropathy, whose treatment is still an unmet clinical need. Previous studies have shown chronic BTZ administration (0.20 mg/kg intravenously three times a week for 8 weeks) to female Wistar rats induced a peripheral neuropathy similar to that observed in humans. In this animal model of BTZ-induced neurotoxicity, the present authors evaluated the efficacy of CR4056, a novel I2 ligand endowed with a remarkable efficacy in several animal pain models. CR4056 was administered in a wide range of doses (0.6-60 mg/kg by gavage every day for 2-3 weeks) in comparison with buprenorphine (Bupre) (28.8 µg/kg subcutaneously every day for 2 weeks) and gabapentin (Gaba) (100 mg/kg by gavage every day for 3 weeks). Chronic administration of BTZ reduced nerve conduction velocity and induced allodynia. CR4056, Bupre, or Gaba did not affect the impaired nerve conduction velocity. Conversely, CR4056 dose-dependently reversed BTZ-induced allodynia (minimum effective dose 0.6 mg/kg). The optimal dose found, 6 mg/kg, provided a constant pain relief throughout the treatment period and without rebound after suspension, being effective when coadministered with BTZ, starting before or after allodynia was established, or when administered alone after BTZ cessation. A certain degree of tolerance was seen after 7 days of administration, but only at the highest doses (20 and 60 mg/kg). Bupre was effective only acutely, since tolerance was evident from the fourth day onwards. Gaba showed a significant activity only at the fourth day of treatment. CR4056, over the range of concentrations of 3-30 µM, was unable to hinder BTZ cytotoxicity on several tumor cell lines, which could indicate that this substance does not directly interfere with BTZ antitumor activity. Therefore, CR4056 could represent a new treatment option for BTZ-induced neuropathic pain.

New anthranilic acid based antagonists with high affinity and selectivity for the human cholecystokinin receptor 1 (hCCK1-R).

The anthranilic acid diamides represent the most recent class of nonpeptide CCK(1) receptor (CCK(1)-R) antagonists. Herein we describe the second phase of the anthranilic acid C-terminal optimization using nonproteinogenic amino acids containing a phenyl ring in their side chain. The Homo-Phe derivative 2 (VL-0797) enhanced 12-fold the affinity for the rat CCK(1)-R affinity and 15-fold for the human CCK(1)-R relative to the reference compound 12 (VL-0395). The eutomer of 2 (6) exhibited a nanomolar range affinity toward the human CCK(1)-R and was at least 400-fold selective for the CCK(1)-R over the CCK(2)-R. Molecular docking in the modeled CCK(1)-R and its validation by site-directed mutagenesis experiments showed that the 6 binding site overlaps that occupied by the C-terminal bioactive region of the natural agonist CCK. Owing to their interesting properties, new compounds provided by this study represent a solid basis for further advances aimed at synthesis of clinically valuable CCK(1)-R antagonists.

Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain.

Two decades of investigations have failed to unequivocally clarify the functions and the molecular nature of imidazoline-2 receptors (I2R). However, there is robust pharmacological evidence for the functional modulation of monoamino oxidase (MAO) and other important enzyme activities by I2 site ligands. Some compounds of this class proved to be active experimental tools in preventing both experimental pain and opioid tolerance and dependence. Unfortunately, even though these compounds bind with high potency to central I2 sites, they fail to represent a valid clinical opportunity due to their pharmacokinetic, selectivity or side-effects profile. This paper presents the preclinical profile of a novel I2 ligand (2-phenyl-6-(1H-imidazol-1yl) quinazoline; [CR4056]) that selectively inhibits the activity of human recombinant MAO-A in a concentration-dependent manner. A sub-chronic four day oral treatment of CR4056 increased norepinephrine (NE) tissue levels both in the rat cerebral cortex (63.1% ±4.2%; P < 0.05) and lumbar spinal cord (51.3% ± 6.7%; P < 0.05). In the complete Freund's adjuvant (CFA) rat model of inflammatory pain, CR4056 was found to be orally active (ED50 = 5.8 mg/kg, by mouth [p.o.]). In the acute capsaicin model, CR4056 completely blocked mechanical hyperalgesia in the injured hind paw (ED50 = 4.1 mg/kg, p.o.; ED100 = 17.9 mg/kg, p.o.). This effect was dose-dependently antagonized by the non-selective imidazoline I2/α2 antagonist idazoxan. In rat models of neuropathic pain, oral administration of CR4056 significantly attenuated mechanical hyperalgesia and allodynia. In summary, the present study suggests a novel pharmacological opportunity for inflammatory and/or neuropathic pain treatment based on selective interaction with central imidazoline-2 receptors.

Design, synthesis, and biological evaluation of AT1 angiotensin II receptor antagonists based on the pyrazolo[3,4-b]pyridine and related heteroaromatic bicyclic systems.

Novel AT 1 receptor antagonists bearing the pyrazolo[3,4- b]pyridine bicyclic heteroaromatic system (or structurally related moieties) were designed and synthesized as the final step of a large program devoted to the development of new antihypertensive agents and to the understanding of the molecular basis of their pharmacodynamic and pharmacokinetic properties. The preliminary pharmacological characterization revealed nanomolar AT 1 receptor affinity for several compounds of the series and a potent antagonistic activity in isolated rabbit aortic strip functional assay for 7c and 8a. These results stimulated the study of the biopharmaceutical properties of some selected compounds, which were found to be characterized by a permeability from medium to high. Remarkably, the least permeable 7c showed both permeability and oral bioavailability (80%) higher than losartan, but its terminal half-life was shorter. These results suggest that the permeability is not a limiting factor in the pharmacokinetics of these AT 1 receptor antagonists.

Further studies on imidazo[4,5-b]pyridine AT1 angiotensin II receptor antagonists. Effects of the transformation of the 4-phenylquinoline backbone into 4-phenylisoquinolinone or 1-phenylindene scaffolds.

The 4-phenylquinoline fragment of novel AT(1) receptor antagonists 4 based on imidazo[4,5-b]pyridine moiety was replaced by 4-phenylisoquinolinone (compounds 5) or 1-phenylindene (compounds 6) scaffolds to investigate the structure-activity relationships. Binding studies showed that most of the synthesized compounds display high affinity for the AT(1) receptor. Because of the in vitro high potency of carboxylic acids 5b,f, they were evaluated in permeability (in Caco-2 cells) and in pharmacokinetic studies in comparison with quinoline derivatives 4b,i,j,k. The studies showed that these compounds are characterized by rapid excretion, low membrane permeability, and low oral bioavailability. The structure optimization of the indene derivatives led to compounds 6e,f possessing interesting AT(1) receptor affinities. Optimization produced polymerizing AT(1) receptor ligand 6c, which forms a thermoreversible polymer (poly-6c) and is released from the latter by a temperature-dependent kinetics. The results suggest the possibility of developing novel polymeric prodrugs based on a new release mechanism. Finally, a set of 34 AT(1) receptor antagonists was used as a new test for the evaluation of the predictive capability of the previously published qualitative and quantitative pharmacophore models.

Pharmacological profile of CR3465, a new leukotriene CysLT1 receptor antagonist with broad anti-inflammatory activity.

CR3465 (L-Tyrosine, N-[(2-quinolinyl)carbonyl]-O-(7-fluoro-2-quinolinylmethyl) sodium salt) is a potent antagonist of [3H]leukotriene D4 ([3H]LTD4) binding to guinea pig lung preparations, its Ki (4.7+/-0.7 nM) being comparable with that of montelukast (5.6+/-0.6 nM). In tracheal strips from standard or ovalbumin-sensitized guinea pigs, CR3465 caused parallel rightward shifts in the concentration-response curves obtained with either LTD4 or antigen (pA(2), 8.74 and 8.15). Intravenous (i.v.) administration of the agent both antagonized (ED50, 9.9+/-1.9 microg/kg) and reverted LTD4 -induced bronchoconstriction of anesthetized guinea pigs. CR3465 reduced inflammatory infiltrates in the bronchoalveolar lavage fluid after antigen challenge of sensitized animals, and proved also active in inhibiting phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4) activities exhibited by human platelets and neutrophils (IC50, 2.01+/-0.07 and 4.7+/-0.5 microM). In line with properties shown by phosphodiesterase inhibitors, CR3465 reduced the contractile response of guinea pig airways to histamine and decreased N-formyl-Met-Leu-Phe (fMLP)-induced degranulation of human neutrophils (IC50, 13.8 microM). Oral administration (20 mg/kg) of the compound in rats produced a significant (37%) ex vivo inhibition of tumor necrosis factor-alpha (TNF-alpha) release from lipopolysaccharide-stimulated whole blood. Pharmacokinetic data in the rat demonstrated approximately 100% bioavailability of the agent. We conclude that CR3465 represents a potent leukotriene CysLT1 receptor antagonist with enhanced effects, being also useful for counteracting spasmogenic and inflammatory stimuli other than those elicited by cysteinyl-leukotrienes (Cys-LTs).

Design, synthesis, structural studies, biological evaluation, and computational simulations of novel potent AT(1) angiotensin II receptor antagonists based on the 4-phenylquinoline structure.

Novel AT(1) receptor antagonists bearing substituted 4-phenylquinoline moieties instead of the classical biphenyl fragment were designed and synthesized as the first step of an investigation devoted to the development of new antihypertensive agents and to the understanding of the molecular basis of their pharmacodynamic and pharmacokinetic properties. The newly synthesized compounds were tested for their potential ability to displace [(125)I]Sar(1),Ile(8)-Ang II specifically bound to AT(1) receptor in rat hepatic membranes. These AT(1) receptor binding studies revealed nanomolar affinity in several of the compounds under study. The most potent ligands 4b,t were found to be equipotent with losartan and possessed either a 3-tetrazolylquinoline or a 2-amino-3-quinolinecarboxylic moiety, respectively. Moreover, some selected compounds were evaluated for antagonism of Ang II-induced contraction in rabbit aortic strips, and the most potent compounds in the binding test 4b,t were slightly more potent than losartan in inhibiting Ang II-induced contraction. Finally, the most relevant structure-affinity relationship data were rationalized by means of computational studies performed on the isolated ligands as well as by computational simulations on the ligands complexed with a theoretical AT(1) receptor model.