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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.

BACKGROUND AND PURPOSE: CR4056 is a first‐in‐class imidazoline‐2 (I(2)) receptor ligand characterized by potent analgesic activity in different experimental animal models of pain. In a recent phase II clinical trial, CR4056 effectively reduced pain in patients with knee osteoarthritis. In the present study, we investigated the effects of CR4056 on PKCε translocation in vitro and on PKCε activation in vivo in dorsal root ganglia (DRG) neurons. EXPERIMENTAL APPROACH: Effects of CR4056 on bradykinin‐induced PKCε translocation were studied in rat sensory neurons by immunocytochemistry. PKCε activation was investigated by immunohistochemistry analysis of DRG from complete Freund's adjuvant‐treated animals developing local hyperalgesia. The analgesic activity of CR4056 was tested on the same animals. KEY RESULTS: CR4056 inhibited PKCε translocation with very rapid and long‐lasting activity. CR4056 decreased hyperalgesia and phospho‐PKCε immunoreactivity in the DRG neurons innervating the inflamed paw. The effect of CR4056 on PKCε translocation was blocked by pertussis toxin, implying that the intracellular pathways involved G(i) proteins. The inhibition of PKCε translocation by CR4056 was independent of the α(2)‐adrenoeceptor and, surprisingly, was also independent of idazoxan‐sensitive I(2) binding sites. The I(2) agonist 2BFI had no effect alone but potentiated the activity of low concentrations of CR4056. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CR4056 shares the ability to inhibit PKCε translocation with other analgesics. Whether the inhibition of PKCε involves binding to specific subtype(s) of I(2) receptors should be further investigated. If so, this would be a new mode of action of a highly specific I(2) receptor ligand.

Two small series of quinoline derivatives were designed starting from previously published quinoline derivatives 7a and b in order to obtain information about their interaction with the 5-HT(4)R binding site. Initially, the structure of 7a and b was modified by replacing their basic moiety with that of partial agonist 4 (ML10302) or with that of reference ligand 6 (RS-67-333). Then, the aromatic moieties of 4-quinolinecarboxylates 7a, d–f, and h–k or 4-quinolinecarboxamides 7b, c, and g were modified into those of 2-quinolinecarboxamides 9a–e. Very interestingly, this structure–affinity relationship study led to the discovery of 7h–j as novel 5-HT(4)R ligands showing K(i) values in the subnanomolar range. The structures of all these compounds contain the N-butyl-4-piperidinylmethyl substituent, which appear to behave as an optimized basic moiety in the interaction of these 4-quinolinecarboxylates with the 5-HT(4)R binding site. However, this basic moiety was ineffective in providing 5-HT(4)R affinity in the corresponding 4-quinolinecarboxamide 7g, but it did in 2-quinolinecarboxamide ligands 9c–e. Thus, a subtle interrelationship of several structural parameters appeared to play a major role in the interaction of the ligands with the 5-HT(4)R binding site. They include the kind of basic moiety, the position of the carbonyl linking group with respect to the aromatic moiety and its orientation, which could be affected by the presence of the intramolecular H-bond as in compounds 9c–e.

Multivalency does not improve the binding and functional activities of ML10302 at 5-HT4 receptors.

5-HT4 receptors are known to form constitutive dimers in membranes. To explore whether multivalency can enhance ligand interactions and/or efficacy in 5-HT4 receptors, the structure of the partial agonist ML10302 was modified with oligo(ethylene glycol) chains, thus generating, by a gradual approach, short and long tethered bivalent or tetravalent ligands and the corresponding spanner-linked monovalent controls. Both bivalent and tetravalent ligands displayed a 10–20-fold increase in binding affinity compared to appropriate controls, but no multivalent ligand showed greater binding energy than ML10302 itself. Furthermore, the direct assessment of receptor–Gs interaction and studies of cAMP signalling indicated that multivalency does not enhance the efficacy of ML10302.

Eleonora Comi,1,2 Marco Lanza,1 Flora Ferrari,1 Valeria Mauri,1,3 Gianfranco Caselli,1 Lucio Claudio Rovati1 1Department of Pharmacology and Toxicology, Rottapharm Biotech, 2PhD Program in Neuroscience, University of Milan-Bicocca, 3Department of Surgery and Translational Medicine, University ofMilan-Bicocca, Monza,Italy 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 7days 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. Keywords: osteoarthritis, pain, CR4056, imidazoline-2 receptors, MIA, MMT

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.

Purpose: Joint pain is the cardinal symptom of OA although it poorly correlates with OA joint structure changes. Interestingly, OA pain is driven by both nociceptive and neuropathic mechanisms. The analgesic efficacy of CR4056, an I2 receptor ligand currently in phase II clinical trials, was previously reported in the OA pain model obtained by the intra-articular injection of monosodium iodoacetate (MIA) in the rat knee. Moreover, CR4056 evidenced a remarkable analgesic activity in several animal models of inflammatory, neuropathic and postoperative pain. The aim of this study was to evaluate the effect of CR4056 in a well-established model of surgically-induced OA able to mimic the structural and painful components of human OA. Methods: OA was induced in male Sprague Dawley rats (335-370 g) by transection of the medial collateral ligament and medial meniscus of the femoro-tibial right joint (MMT model). Hind paw weight bearing distribution was assessed as indirect measure of spontaneous pain by using an incapacitance tester (2Biological Instruments Snc, Besozzo, Italy), prior to surgery and 14, 28, 35 and 42 days post-surgery. Control rats were subjected to sham surgery, while treatments after MMT consisted of 6 mg/kg CR4056, or 10 mg/kg naproxen as an active control, or vehicle (9 animals/group) and they were administered orally as subacute treatment from 28 to 42 days after surgery. Statistical analysis was performed by Two-way RM ANOVA, followed by Tukey's multiple comparisons test. Results: MMT surgery resulted in a significant development of right-left hind paw weight bearing imbalance at each experimental time point, compared with the sham group (data expressed as difference of weight bearing between contralateral and ipsilateral paw). The difference in weight bearing distribution between sham and MMT rats gradually increased throughout the study, reaching its peak 42 days post-surgery in animals receiving vehicle (mean ± sem, 0.83 ± 1.87 vs. 65.92 ± 2.26, respectively). Subacute oral administration of 6 mg/kg CR4056 for 2-weeks from 28 to 42 days after surgery induced a significant reduction of right-left hind paw weight bearing imbalance, compared with the MMT control group treated with vehicle (42 days post-surgery mean ± sem, 39.84 ± 3.47 vs. 65.92 ± 2.26, respectively), as illustrated in the Figure. Conversely, 10 mg/kg naproxen was devoid of noticeable effect on weight bearing imbalance after 2-weeks subacute treatment. Conclusions: The data presented here further evidence that the imidazoline I2 receptor ligand CR4056 could represent a new highly effective analgesic treatment option for OA pain.

Cristina Meregalli,1 Cecilia Ceresa,1 Annalisa Canta,1 Valentina Alda Carozzi,1 Alessia Chiorazzi,1 Barbara Sala,1 Norberto Oggioni,1 Marco Lanza,2 Ornella Letar,i2 Flora Ferrari,2 Federica Avezza,1 Paola Marmiroli,1 GianFranco Caselli,2 Guido Cavaletti11Department of Neuroscience and Biomedical Technologies, University of Milan-Bicocca, 2Pharmacology and Toxicology Department, Rottapharm | Madaus Research Center, Monza, ItalyAbstract: 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.Keywords: imidazoline I2 receptor ligand, antinociception, allodynia, neuropathic pain, bortezomib

Summary Objective Increased levels of glutamate, the main excitatory neurotransmitter, are found in the synovial fluid of osteoarthritis (OA) patients. Our aim was to study glutamate signaling in chondrocytes, focusing on the composition, pharmacology, and functional role of N -methyl-d-aspartate (NMDA) glutamate receptors. Methods We used the human chondrocyte cell line SW1353 and, in parallel, primary rat articular chondrocytes. Glutamate release and uptake were measured by fluorimetric and radiometric methods, respectively. Gene expression was analyzed by quantitative polymerase chain reaction. NMDA receptor pharmacology was studied in binding experiments with [ 3 H]MK-801, a specific NMDA receptor antagonist. RNA interference was used to knock-down the expression of NR1, a subunit of NMDA receptors. Results Glutamate release, sodium- and calcium-dependent glutamate uptake, and the expression of a glutamate transporter were observed in chondrocytes. NR2D was the most abundant NMDA receptor subunit in these cells. Consistent with this observation, the binding affinity of [ 3 H]MK-801 was much lower in chondrocytes than in rat brain membranes (mean K d values of 700 and 2.6nM, respectively). NR1 knock-down, as well as NMDA receptor blockade with MK-801, reduced chondrocyte proliferation. Interleukin (IL)-1β significantly altered glutamate release and uptake (about 90% increase and 50% decrease, respectively, in SW1353 cells). Moreover, IL-1β induced the gene expression of cytokines and enzymes involved in cartilage degradation, and MK-801 significantly inhibited this response. Conclusions Our findings suggest that chondrocytes express a self-sufficient machinery for glutamate signaling, including a peripheral NMDA receptor with unique properties. This receptor may have a role in the inflammatory process associated with cartilage degradation, thus emerging as a potential pharmacological target in OA.

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.

Multivalency is widely used in nature in specific recognition processes. This paper describes an approach to multivalency in the pentameric 5-HT3 receptor, a ligand-gated ion channel, which constitutes an example of intrinsically multivalent biological receptors. Owing to the picomolar Ki value, TETRA-L represents an outstanding multivalent ligand for the neurotransmitter receptor. © The Partner Organisations 2014.

A small series of serotonin 5-HT4 receptor ligands has been designed from flexible 2-methoxyquinoline compounds 7a,b by applying the conformational constraint approach. Ligands 7a,b and the corresponding conformationally constrained analogues 8a-g were synthesized and their interactions with the 5-HT4 receptor were examined by measuring both binding affinity and the ability to promote or inhibit receptor-G protein coupling. Ester derivative 7a and conformationally constrained compound 8b were demonstrated to be the most interesting compounds showing a nanomolar 5-HT4R affinity similar to that shown by reference ligands cisapride (1) and RS-23,597-190 (4). The result was rationalized by docking studies in term of high similarity in the binding modalities of flexible 7a and conformationally constrained 8b. The intrinsic efficacy of some selected ligands was determined by evaluating the receptor-G protein coupling and the results obtained demonstrated that the nature and the position of substituents play a critical role in the interaction of these ligands with their receptor.

The effects of cholecystokinin sulfate octapeptide (CCK-8S) on [3H]gamma-aminobutyric acid (GABA) release have been studied in the anterior side of the rat nucleus accumbens on tissue punches exposed in superfusion to 30 mM KCl. CCK-8S in a concentration dependent manner (10-3000 nM) increased K+-evoked [3H]GABA release (EC50=192 nM). The increase caused by 1 microM CCK-8S ranged from 37% to 42%. CR 2945, (beta-[2-[[2-(8-azaspiro[4.5]dec-8-ylcarbonyl)-4,6-dimethylp henyl]-amino]-2-oxoethyl]-(R)-1-naphthalenepropanoic acid), a potent and selective nonpeptidergic CCK(B) antagonist, concentration-dependently blocked CCK-8S effect (IC50=2.16 nM). CCK-8S-induced increase in [3H]GABA overflow was completely blocked by 1 microM tetrodotoxin. Both the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) and the N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine (MK-801) antagonized the CCK-8S effect. By contrast, (+)-bicuculline, a GABA(A) receptor antagonist, was completely ineffective. Phaclofen, a selective GABA(B) antagonist, increased K+-evoked [3H]GABA release but did not affect the facilitative effect of CCK-8S. Moreover, tetrodotoxin failed to block AMPA-evoked [3H]GABA release but completely prevented the effect of NMDA (Mg2+ free conditions). The data presented suggest that CCK(B) receptors modulating [3H]GABA release from anterior accumbal punches may not be present on GABAergic terminals but could be located on glutamatergic interneurons.

Memory processes have been widely studied in recent decades. Unfortunately, although several intriguing hypotheses about the possibility of modulating cognition have been developed, no conclusive result has been obtained (69). Two different neurotransmission models are of major interest in understanding how the brain can acquire and store a single cognitive event (3,23). The main neuronal agents involved in transferring this kind of information seem to be acetylcholine and glutamic acid. These two neurotransmitters are present in all the cerebral areas involved in the memory processes and both are responsible for the stimulation currents linked to the propagation of neuronal signals (1,4). The cholinergic hypothesis of memory ( 3 ) is based on the observation that a marked degeneration of the cholinergic neuronal pathway is often associated with the development of cognitive disorders ( 12). Moreover, it has been reported that one distinctive feature of Alzheimer’s disease (AD) consists of a progressive loss of either cholinergic or adrenergic neurons’ ability to transmit stimuli received from the limbic areas to the cerebral cortex (10,12,21,75). To help preserve high levels of acetylcholine in the synaptic cleft, several antiacetylcholinesterase agents have been synthesized and tested on both biochemical and behavioral models of learning (32,46). Tacrine is certainly the most studied among these agents ( 13). Clinical trials with tacrine revealed some interesting pro-mnemic properties of this compound (42,68), but tacrine exhibits major hepatotoxicity that strongly restricts its use ( 13,73). Donepezil (E2020) is another interesting compound of this family. Two recently published clinical studies (25,63) demonstrate both the efficacy of donepezil in patients with mild-to-moderate Alzheimer’s disease and its relative liver safety. Other potent acetylcholinesterase inhibitors, such as metrifonate and SDZ ENA 7 13, are currently under clinical evaluation (28,62,65). The glutamatergic hypothesis of memory is based on a particular characteristic of the glutamatergic neuronal pathway: long-term potentiation (LTP). LTP was first defined by Bliss and Collingridge (8) as a synaptic memory model expressed as a persistent increase in the size of the synaptic component of the evoked response. The

Objective: ADAMTS5 (aggrecanase-2) has been demonstrated to be crucial in the development of osteoarthritis (OA), by use of several mouse mutants carrying either truncated, catalytically inactive enzymes or aggrecanase-resistant mutant aggrecan. We have selected recombinant monoclonal antibodies directed against ADAMTS5, by using Intracellular Antibody Capture Technology (IACT). CRB0017 revealed very high affinity for the enzyme in Biacore analyses and very good specificity in a panel of binding assays. Therefore, we tested CRB0017 in a relevant spontaneous OA model, the STR/ort mouse. Design: STR/ort male mice were recruited at 5 months of age, and treated intra-articularly in each knee with CRB0017 1.2μg, CRB0017 12μg, or vehicle. After 6 weeks, the intra-articular administration of CRB0017 was repeated with the same doses. After 3 months from recruitment, the animals were sacrificed and the femorotibial joints processed for histology and scored in a blind fashion according to both Mankin's and the OARSI methods. Results and conclusions: All histological scores were significantly decreased in the CRB0017 12μg/knee group compared to vehicle, while administration of CRB0017 1.2μg was associated with a trend to a decrease in the same parameters. Therefore, CRB0017 administered twice in 3 months could modify the course of OA in the STR/ort mouse, by delaying cartilage breakdown as assessed histologically. The procedure of blind scoring of the histological samples clearly showed that knee intra-articular administration of CRB0017, an anti-ADAMTS5 antibody, dose-dependently improved disease progression in a relevant animal model of OA. © 2013 Osteoarthritis Research Society International.

Opioid addiction is often characterized as a chronic relapsing condition due to the severe somatic and behavioral signs, associated with depressive disorders, triggered by opiate withdrawal. Since prolonged abstinence remains a major challenge, our interest has been addressed to such objective. Exploring multitarget interactions, the present investigation suggests that 3 or its (S)-enantiomer and 4, endowed with effective α2C-AR agonism/α2A-AR antagonism/5-HT1A-R agonism, or 7 and 9–11 producing efficacious α2C-AR agonism/α2A-AR antagonism/I2–IBS interaction might represent novel multifunctional tools potentially useful for reducing withdrawal syndrome and associated depression. Such agents, lacking in sedative side effects due to their α2A-AR antagonism, might afford an improvement over current therapies with clonidine-like drugs.

A new class of serotonin 5-HT1A receptor ligands related to NAN-190, buspirone and aripiprazole has been designed using our potent 5-HT3 receptor ligands as templates. The designed pyrrolidone derivatives 10a-n were prepared by means of the straightforward chemistry consisting in the reaction of the appropriate γ-haloester derivatives with the suitable arylpiperazinylalkylamines. The nanomolar 5-HT1A receptor affinity and the agonist-like profile shown by fused pyrrolidone derivatives 10k,m stimulated the rationalization of the interaction with an homology model of the 5-HT1A receptor and the evaluation of their selectivity profiles and the pharmacokinetic properties. Interestingly, the results of the profiling assays suggested for close congeners 10k,m a significantly divergent binding pattern with compound 10m showing an appreciable selectivity for 5-HT1AR.

Flora Ferrari1, Simonetta Fiorentino1, Laura Mennuni1, Paolo Garofalo1, Ornella Letari1, Stefano Mandelli2, Antonio Giordani3, Marco Lanza1, Gianfranco Caselli11Department of Pharmacology and Toxicology; 2Department of Medicinal Chemistry; 3R&D Chemistry Drug Development and OS, Rottapharm S.p.A., Monza (MB), ItalyAbstract: 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

Several clinical studies demonstrated that glucosamine sulfate (GS) is effective in controlling osteoarthritis (OA), showing a structure-modifying action. However, little is known about the molecular mechanism(s) by which GS exerts such action and about the effects of GS at a tissue level on osteoarthritic cartilage and other joint structures. Here we provide mechanistic evidence suggesting that in vitro GS attenuates NF-κB activation at concentrations in the range of those observed after GS administration to volunteers and patients, thus strengthening previous findings. Furthermore, we describe the effects of GS at a tissue level on the progression of the disease in a relevant model of spontaneous OA, the STR/ort mouse. In this model, the administration of GS at human corresponding doses was associated with a significant decrease of OA scores. Histomorphometry showed that the lesion surface was also significantly decreased, while the number of viable chondrocytes within the matrix was significantly increased. GS improved the course of OA in the STR/Ort mouse, by delaying cartilage breakdown as assessed histologically and histomorphometrically.

Konventionelles Engineering Data Management (EDM) gewährleistet zwar die Verwaltung von Informationen, unterstützt verteilte Entwicklungs- und Planungsteams aber nicht im methodischen Vorgehen durch Konstruktionsmethoden wie z.B. Axiomatic Design. Ziel einer Forschungsarbeit Der Forschungsschwerpunkt „Branchenübergreifende Informationslogistik für die Produktentwicklung in der Investitionsgüterindustrie" des Landes Baden-Württemberg dient dazu, neue Modelle, Methoden und Systeme zur Planung, Initiierung und Koordination von Informationsflüssen von der ersten Projektidee bis zur Endpräsentation zu entwickeln. war die Gestaltung eines Axiomatic Designbasierten Projektmanagements zur Unterstützung der Fertigungssystemplanung. Es wurde in ein EDM-System eingebettet.

Microdialysis in intact and denervated striatum of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats was used to investigate whether CR 3394, N -[2-(3,5-dimethyl-1-adamantyl)ethyl]acetamidine, an adamantane derivative with preferential selectivity for the NR2B subunit of the NMDA receptor, has dopamine releasing properties in vivo . We also investigated whether this NMDA antagonist can potentiate the effects of L-Dopa on extracellular dopamine in these animals. After systemic injection, there was no significant effect of CR 3394 on extracellular dopamine, at all doses studied (1, 5 and 20 mg/kg i.p.), in either intact or in denervated striatum. On the other hand, striatal perfusion with 100 µM of the compound elicited release of dopamine in intact, but not in denervated striatum. In denervated striatum of the 6-OHDA-lesioned rats, CR 3394 (5 mg/kg) significantly enhanced the dopamine release induced by L-Dopa administration (25 mg/kg i.p.) in combination with benserazide (10 mg/kg i.p.). In particular, the onset of action of L-Dopa was potentiated. However, when combined with a subthreshold dose of L-Dopa (5 mg/kg), the effects of CR 3394 were lost. We conclude that CR 3394, like other NR2B receptor antagonists, has dopamine releasing properties in vivo . It enhances the effects of suprathreshold doses of L-Dopa in the denervated striatum, but not of low doses of L-Dopa. Therefore, future studies are necessary to establish the potential of selective NR2B receptor antagonists as L-Dopa-sparing agents.

Using the patch-clamp technique, we studied the effect of two novel adamantane derivatives, N-[2-(3,5-dimethyl-1-adamantyl)ethyl] guanidine (CR 3391) and N-[2-(3,5-dimethyl-1-adamantyl) ethyl]acetamidine (CR 3394), on NMDA receptors expressed in cortical neuron cultures. Our data show that CR 3391 and CR 3394 reduce NMDA-evoked currents (IC50 = 1.7 +/- 0.6 microM and 6.7 +/- 1.5 microM, respectively). This antagonism is non-competitive and is completely reversible. The effect of CR 3394, like that of memantine, was strongly voltage dependent. HEK293 cells expressing NR1a/NR2B recombinant NMDA receptors and immature neurons (DIV 8-9) were more sensitive to CR 3394 antagonism than NR1a/NR2A expressing cells and DIV 15 neurons. CR 3394 also reduced the duration and amplitude of miniature excitatory post-synaptic currents mediated exclusively by NMDA receptors (NMDA-mEPSCs). Both memantine and CR 3394 inhibited NMDA-evoked [3H]norepinephrine release from rat hippocampal slices in a concentration-dependent manner with similar potency. CR 3394, but not memantine, increased cathecholamine resting release at low micromolar concentrations. Moreover, in an in vitro model of neurotoxicity, CR 3394 strongly reduced glutamate- and NMDA-induced neuronal death. Taken together, our data highlight pharmacological features of CR 3394 in vitro that prompt us to further evaluate it as a candidate for the treatment of neurodegenerative disorders.

Conventional Product Data Management (PDM) supports aspects of information management but don’t promote the application of target oriented design methodologies like ‘Axiomatic Design’. For Manufacturing System Design PDM has to manage the actual targets, to direct a target-oriented procedure, and to manage objects for evaluation. Targets result from customers and manufacturers. The definition of ‘Functional Requirements’ and ‘Design Parameters’ has to be organized between different core competencies at different times and places. The goal is the configuration of a PDM framework supporting distributed collaborative manufacturing system design in a ‘Axiomatic Design’ oriented way. Based on an axiomatic approach, the implementation of this methodology in a distributed PDM framework is presented. For this, the necessary procedure and controls of ‘manufacturing system design’, information architecture and data interfaces to other software tools are considered and then the paper describes necessary metamodel.

Summary -The effects of (S)-4-amino-5-[(4,4-dimethylcyclohexyl)amino]-5-oxo-pentanoic acid ((S)CR 2249), a new chemical entity selected among a series of glutamic acid derivatives, were investigated on N -methyl- d -aspartate (NMDA)-evoked release of [ 3 H]noradrenaline from rat hippocampal slices. (S)CR 2249 facilitated glycine-mediated reversion of kynurenate antagonism at strychnine-insensitive glycine receptors coupled to the NMDA receptor. The potency of glycine (EC 50 = 21.5 μM ± 4.2) was not significantly influenced by (S)CR 2249. Nevertheless, the efficacy of the glycine effect was enhanced in a concentration-dependent manner (3-10-30 μm) by (S)CR 2249. The interaction of (S)CR 2249 with NMDA receptors was also studied with binding experiments, in which we examined the effect of (S)CR 2249 on the modulation by glutamate, glycine and spermine of [ 3 H]dizocilpine (MK-801) binding. (S)CR 2249, increased [ 3 H]MK-801 binding in a concentration-dependent manner and we found positive cooperative interactions between glycine and (S)CR 2249, indicating that (S)CR 2249 probably acts at a separate allosteric site to increase NMDA receptor functionality.

The effects of S-4-amino-5-[4,4-dimethylcyclohexyl)amino]-5-oxopentanoic acid (CR 2249), a new entity selected from a new series of glutamic acid derivatives, has been investigated in different paradigms for screening nootropics. CR 2249 ameliorated the memory retention deficit produced by scopolamine in step-through-type passive avoidance in rats and by electroconvulsive shock in step-down-type passive avoidance in mice. CR 2249 was also capable of improving performance in behavioural tests of learning and memory in the absence of cholinergic hypofunction or cognitive deficit. The activity was determined using different passive and active avoidance behavioural test procedures on rats. CR 2249 was active only when given 45 min before training and did not show any effect when administered immediately after the learning training or before the retention trial. No changes in the general behaviour or motor activity of the animals were observed, indicating that CR 2249 effects cannot be attributed to sensory-motor deficit. Microdialysis experiments have shown that CR 2249 significantly increased noradrenaline release in the hippocampus of freely moving rats and reduced 3,4-dihydroxyphenylglycol efflux. These effects have led us to hypothesize that CR 2249 memory effect might be mediated by a direct or indirect action on noradrenergic transmission. These behavioural results suggest that this new agent has clinical application in memory disorders.

Software development for programmable logical controllers is usually based on low-level languages such as the instruction list or the ladder diagram. At the same time, the programmer looks at a machine or an assembly system in a bit-oriented way; he translates the operational sequences into logical and/or time based combinations of binary signals described by means of Boolean algebra. This classical method causes a lot of problems in reality so it should be improved. It is the aim of the report to show a way developing PLC-software graphically and interactively within a Virtual Reality (VR) based system (VPLC).

Objective: Bortezomib, a potent and selective proteasome inhibitor, is the first line treatment of relapsed, refractory multiple myeloma. One of the most commonly reported adverse reactions induced by this drug, is a peripheral neurotoxicity characterized by sensory and often painful neuropathy representing the major reason for a dose reduction and discontinuation of life-saving therapy. The use of currently available drugs for the treatment of neuropathic pain is largely empirical, and their mechanisms of action are not completely understood. CR4056 is a promising analgesic drug that interacts with imidazoline-2 receptors, and inhibits the activity of monoamine oxidases. Aim of this study was to evaluate if the new analgesic compound CR4056 could prevent (preventive schedule) or reverse (curative schedule) the neuropathic pain elicited by bortezomib in a rat model of chronic painful peripheral neuropathy. Methods: Neuropathy was induced in female Wistar rats by intravenous administration of Bortezomib (0.20 mg/kg, 3 times a week). CR4056 was orally administered at 6 mg/kg, once a day, in both preventive and curative schedules. In the preventive schedule animals were dosed with bortezomib and CR4056 starting from day 1 till week 8 or 10. In the curative schedule, established neuropathic animals were treated with CR4056 either from week 6 or week 8 till week 10. The sensory neuropathy was evaluated by tail nerve conduction velocity (NCV) at week 6, 8 and 10 after the first challenge with bortezomib. The development of neuropathic pain behaviour (mechanical allodynia) was followed by mechanical testing on day 1, at week 6, 8, 9 and 10. General toxicity, evaluated as body weight changes, was monitored twice a week Results: Bortezomib induced a severe reduction in NCV and a significant mechanical allodynia at all the indicated time points. Administration of CR4056, neither in preventive nor in curatives schedules, affected the NCV impairment. Conversely, CR4056 treatment significantly and completely prevented the development of mechanical allodynia in bortezomib treated-rats, in the preventive schedule, and totally reverted the established neuropathic pain in the curative schedule. The analgesic effect persisted until the last time point of observation without signs of tolerance. No remarkable effect induced by bortezomib or CR4056, alone or in co-treatment, was observed on body weight changes.Conclusions: The present study evidences a significant and long lasting analgesic effect of CR4056, which could represent a new therapeutic option for the treatment of bortezomib-induced chronic neuropathic pain Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 657. doi:10.1158/1538-7445.AM2011-657

The depolarization-evoked release of endogenous glutamate (GLU) and -aspartate (ASP) and its modulation mediated by gamma-aminobutyric acid (GABA) heteroreceptors was investigated in superfused rat cerebrocortical synaptosomes. Exposure to 12 mM K+ enhanced the release of GLU and ASP. The K(+)-evoked overflow of both amino acids was largely Ca(2+)-dependent. Exogenous GABA inhibited the K(+)-evoked overflow of GLU (EC50 2.8 microM) and ASP (EC50 2.7 microM). The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (EC50 2.0 microM for GLU and 1.3 microM for ASP release) but not by the GABAA receptor agonist muscimol, up to 100 microM. Accordingly, the GABA-induced inhibition of GLU and ASP release was not affected by the GABAA receptor antagonists, bicuculline or picrotoxin, but was antagonized by the GABAB receptor antagonist, 3-amino-propyl(diethoxymethyl)phosphinic acid (CGP 35348). The GABA effect was, however, insensitive to another GABAB receptor antagonist, phaclofen, up to 1,000 microM. It can be concluded that GABA heteroreceptors of the GABAB type regulating the depolarization-evoked release of GLU and ASP are present on cortical GLU/ASP-releasing nerve terminals. These receptors may be classified as a phaclofen-insensitive GABAB receptor subtype.

As previously reported GABA B receptors are heterogeneous. Three pharmacologically distinct receptor subtypes mediating inhibition of γ-aminobutyric acid (GABA), glutamate or somatostatin release, respectively, exist on axon terminals of rat cerebral cortex. We investigated the novel GABA B receptor antagonist, [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxy-methyl) phosphinic acid (CGP 52432), on the above receptor subtypes. The effects of (−)-baclofen on the K + -evoked release of GABA, glutamate or somatostatin from rat cortical synaptosomes were antagonized by CGP 52432. The IC 50 of the drug at GABA autoreceptors (0.085 μM) was 35- and 100-fold lower than at the receptors regulating somatostatin and glutamate overflow, respectively. At the autoreceptor the calculated pA 2 for CGP 52432 amounted to 7.70, which makes the drug about 1000-fold more potent than phaclofen at this receptor. The potency and selectivity characteristics of CGP 52432 indicate that the drug is by far the most appropriate tool to investigate the terminal GABA B autoreceptors of the rat cerebral cortex.

Bortezomib (Btz) is a highly effective and widely used antineoplastic drug for the treatment of many tumors, primarily multiple myeloma. Despite its effectiveness, the use of this proteasome inhibitor is limited by its toxicity. Among Btz side effects, peripheral neurotoxicity is a major and clinically relevant problem. In fact, since the earliest Phase I and Phase II studies, sensory peripheral neuropathy emerged as a dose-limiting side effect. Moreover, a significant proportion of Btz-treated patients developed severe neuropathic pain. The treatment of Btz-induced neuropathic pain is extremely difficult with the currently available drugs and still remains a major challenge for oncologists and neurologists. Aim of this study was to demonstrate if the new analgesic compound CR4056 could reverse the neuropathic pain induced by a chronic treatment with Btz in the rat. The anti-nociceptive and dose-dependent effect of CR4056 was assessed in a well-established experimental rat model in which Btz was administered to Wistar rats (0.20 mg/kg three times/week for 8 weeks) followed by treatment with CR4056 (6, 20 or 60 mg/kg daily p.o.) or buprenorphine administered 28,8 µg/kg/day p.o. for 2 weeks. Body weight change, hematological and histopathological parameters, nerve conduction velocity (NCV) and sensory behavioral tests were evaluated during the experiment. No remarkable effect of Btz administration or of any dose of CR4056 was observed in the hematological parameters and blood chemistry. After 8 weeks of treatment, Btz induced a severe reduction in NCV; this impairment was not reversed by CR4056 or by buprenorphine. The behavioral assessment showed a significant mechanical allodynia in rats treated with Btz for 8 weeks (delta vs. controls −6 g). Buprenorphine significantly reduced Btz-induced allodynia during the fist day by about 63%. However, this effect was lost during the following days of treatment (i.e 20% reduction on the 3rd day). The mid and high dose of CR4056 (20 and 60 mg/kg) showed a significant (i.e > 80 %) reduction of allodynia till the 3rd day of administration, afterwards a slight reduction in efficacy was recorded. On the contrary, the oral daily administration of 6 mg/kg CR4056 persistently counteracted Btz-induced allodynia, showing an even complete reversion after 2 weeks of treatment. The present study gives evidence for a relevant and persistent anti-allodynic effect of CR4056, suggesting a role for CR4056 in the management of neuropathic pain in patients treated with Btz. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3725.

What could very easily be the oldest form of Oceanographic Instrumentation, a bottle with a note in it, has now been been brought up to date with a new Loran Based Lagrangian Drifter. The needs for this sort of system are many and varied. Everything from oil spill tracking, life raft drift prediction, effluent discharge and distribution tracking, and general oceanographic current studies are all addressed by this buoy. The construction of this buoy is discussed from its two styles of drogues (surface and deep), its kevlar hull and steel instrument bay to its low power Loran receiver and high power BF or VHF transmitters. This buoy differs from most drifting buoys in that rather than acting as a simple beacon and having the receiving station have the task of triangulating the location, it takes fixes on its location and retains that information in its own memory which can later be transmitted. This buoy has a positional accuracy approaching 100 feet, and can acquire fixes as often as every 15 minutes. Both of these parameters are far superior to other techniques presently in use.

Corruption affects healthcare effectiveness and efficiency and limits equity in access to health services. Aim of the survey was to document knowledge and viewpoints on the impact of corruption on healthcare system in a sample of students attending pre-lauream healthcare professional courses at Palermo University Medical school and to evaluate any improvement in their awareness on this topic after a dedicated multidisciplinary educational intervention. An anonymous questionnaire was administered to students before and after the intervention. Absolute and relative frequency of correct and incorrect answers was computed. Chi-squared test was used to compare answers given before and after the educational intervention. Approximately less than a quarter of the respondents were able to correctly estimate impact of corruption, inappropriateness and waste in healthcare on NHS. The study documented how improving students’ knowledge of the impact of corruption in the healthcare system could represent a possible strategy to prevent corruption in healthcare.