Publications by authors named "Gianfranco Caselli"

34 Publications

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

Br J Pharmacol 2020 07 24;177(14):3291-3308. Epub 2020 Apr 24.

Rottapharm Biotech, Monza, Italy.

Background And Purpose: Prolonged use of opioids causes analgesic tolerance and adverse effects including constipation and dependence. Compounds targeting imidazoline I receptors are known to potentiate opioid analgesia in rodents. We investigated whether combination with the I 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 (ED = 4.88 mg·kg ) and morphine (ED = 2.07 mg·kg ) synergized in reducing CFA-induced hyperalgesia (ED = 0.52 mg·kg ; 1:1 combination). Consistently, low doses of CR4056 (1 mg·kg ) 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.
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http://dx.doi.org/10.1111/bph.15049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312436PMC
July 2020

CR4056, a powerful analgesic imidazoline-2 receptor ligand, inhibits the inflammation-induced PKCε phosphorylation and membrane translocation in sensory neurons.

Br J Pharmacol 2020 01 7;177(1):48-64. Epub 2019 Nov 7.

Dipartimento di Economia, Scienze e Diritto, Università degli Studi della Repubblica di San Marino, San Marino.

Background And Purpose: CR4056 is a first-in-class imidazoline-2 (I ) 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 proteins. The inhibition of PKCε translocation by CR4056 was independent of the α -adrenoeceptor and, surprisingly, was also independent of idazoxan-sensitive I binding sites. The I 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 receptors should be further investigated. If so, this would be a new mode of action of a highly specific I receptor ligand.
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http://dx.doi.org/10.1111/bph.14845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6976787PMC
January 2020

Development of subnanomolar-affinity serotonin 5-HT receptor ligands based on quinoline structures.

Medchemcomm 2018 Sep 3;9(9):1466-1471. Epub 2018 Jul 3.

Dipartimento di Biotecnologie , Chimica e Farmacia (Dipartimento di Eccellenza 2018-2022) , Università degli Studi di Siena , Via A. Moro 2 , 53100 Siena , Italy . Email: ; ; Tel: +39 0577 234320.

Two small series of quinoline derivatives were designed starting from previously published quinoline derivatives and in order to obtain information about their interaction with the 5-HTR binding site. Initially, the structure of and was modified by replacing their basic moiety with that of partial agonist (ML10302) or with that of reference ligand (RS-67-333). Then, the aromatic moieties of 4-quinolinecarboxylates , , and or 4-quinolinecarboxamides , , and were modified into those of 2-quinolinecarboxamides . Very interestingly, this structure-affinity relationship study led to the discovery of as novel 5-HTR ligands showing values in the subnanomolar range. The structures of all these compounds contain the -butyl-4-piperidinylmethyl substituent, which appear to behave as an optimized basic moiety in the interaction of these 4-quinolinecarboxylates with the 5-HTR binding site. However, this basic moiety was ineffective in providing 5-HTR affinity in the corresponding 4-quinolinecarboxamide , but it did in 2-quinolinecarboxamide ligands . Thus, a subtle interrelationship of several structural parameters appeared to play a major role in the interaction of the ligands with the 5-HTR 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 .
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http://dx.doi.org/10.1039/c8md00233aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6148519PMC
September 2018

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

Arthritis Res Ther 2018 03 1;20(1):39. Epub 2018 Mar 1.

Rottapharm Biotech, Via Valosa di Sopra 9, I-20900, Monza, MB, Italy.

Background: Prostaglandin E (PGE) 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 (K = 16.6 nM) and functioned as a pure antagonist (half-maximal inhibitory concentration, 22 nM) on PGE-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.
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http://dx.doi.org/10.1186/s13075-018-1537-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5831858PMC
March 2018

Development of Imidazole-Reactive Molecules Leading to a New Aggregation-Induced Emission Fluorophore Based on the Cinnamic Scaffold.

ACS Omega 2017 Sep 6;2(9):5453-5459. Epub 2017 Sep 6.

Dipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for Drug Discovery and Development, Università di Siena, Via A. Moro 2, 53100 Siena, Italy.

In order to obtain new fluorophores potentially useful in imidazole labeling and subsequent conjugation, a small series of Morita-Baylis-Hillman acetates () was designed, synthesized, and reacted with imidazole. The optical properties of the corresponding imidazole derivatives were analyzed both in solution and in the solid state. Although the solutions display a very weak emission, the powders show a blue emission, particularly enhanced in the case of compound possessing two methoxy groups in the cinnamic scaffold. The photophysical study confirmed the hypothesis that the molecular rigidity of the solid state enhances the emission properties of these compounds by triggering the restriction of intramolecular motions, paving the way for their applications in fluorogenic labeling.
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http://dx.doi.org/10.1021/acsomega.7b00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644839PMC
September 2017

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

J Pain Res 2017 4;10:1033-1043. Epub 2017 May 4.

Department of Pharmacology and Toxicology, Rottapharm Biotech.

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.
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http://dx.doi.org/10.2147/JPR.S132026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422496PMC
May 2017

Multivalent ligands for the serotonin 5-HT receptor.

Medchemcomm 2017 Mar 9;8(3):647-651. Epub 2017 Feb 9.

Dipartimento di Biotecnologie , Chimica e Farmacia and European Research Centre for Drug Discovery and Development , Università degli Studi di Siena , Via A. Moro 2 , 53100 Siena , Italy . Email: ; ; Tel: +39 0577 234320.

5-HT receptors are known to form constitutive dimers in membranes. To explore whether multivalency can enhance ligand interactions and/or efficacy in 5-HT 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.
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http://dx.doi.org/10.1039/c6md00458jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071950PMC
March 2017

Phenylindenone isomers as divergent modulators of p38α MAP kinase.

Bioorg Med Chem Lett 2016 11 4;26(21):5160-5163. Epub 2016 Oct 4.

Dipartimento di Scienze Chimiche e Geologiche, Università degli Studi di Modena e Reggio Emilia, Via Campi 103, 41125 Modena, Italy.

Two new fluorophenylindenone derivatives were designed as potential p38α MAPK modulators by preserving the key interactions of the vicinal pyridine/fluorophenyl pharmacophore with the enzyme protein. Interestingly, these two fluorophenylindenone isomers showed divergent activities, with compound 6 behaving as an inhibitor and 5 as a putative activator. These results were rationalized by docking studies and molecular dynamics simulations in terms of stabilization of DFG loop, by compound 5 in a conformation more accessible to phosphorylation.
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http://dx.doi.org/10.1016/j.bmcl.2016.10.001DOI Listing
November 2016

Supramolecular glycodendrimer-based hybrid drugs.

Biomacromolecules 2014 Nov 20;15(11):3985-93. Epub 2014 Oct 20.

Dipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena , Via A. Moro 2, 53100 Siena, Italy.

Specific noncovalent interactions are commonly used by nature to modulate numerous processes including cell recognition, viral adhesion, and transmembrane communications. Here we report on the design, synthesis, and preliminary characterization of new supramolecular glycodendrimer-based hybrid drugs based on adamantyl-modified glycodendrimers of third, fourth, or fifth generation (mPPI-G3-AdaB, mPPI-G4-AdaB, and mPPI-G5-AdaB) and a new heterobifunctional ligand. This component was tailored to bind through noncovalent interactions both the multimeric natural 5-HT3 receptor (through an optimized arylpiperazine pharmacophore) and the adamantyl groups located on the glycodendrimer surfaces (through a β-cyclodextrin residue) giving rise to biorelevant supramolecular constructs.
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http://dx.doi.org/10.1021/bm501057dDOI Listing
November 2014

Dendrimeric tetravalent ligands for the serotonin-gated ion channel.

Chem Commun (Camb) 2014 Aug 23;50(62):8582-5. Epub 2014 Jun 23.

Dipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy.

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.
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http://dx.doi.org/10.1039/c4cc02502dDOI Listing
August 2014

Exploring multitarget interactions to reduce opiate withdrawal syndrome and psychiatric comorbidity.

ACS Med Chem Lett 2013 Sep 22;4(9):875-9. Epub 2013 Jul 22.

School of Pharmacy, Medicinal Chemistry Unit, University of Camerino , Via S. Agostino 1, 62032 Camerino, Italy.

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.
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http://dx.doi.org/10.1021/ml400232pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027469PMC
September 2013

Synthesis and structure-activity relationship studies in serotonin 5-HT4 receptor ligands based on a benzo[de][2,6]naphthridine scaffold.

Eur J Med Chem 2014 Jul 5;82:36-46. Epub 2014 May 5.

Dipartimento di Biotecnologie, Chimica e Farmacia and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejmech.2014.05.015DOI Listing
July 2014

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

Br J Pharmacol 2014 Aug;171(15):3693-701

Department of Pharmacology & Toxicology, Rottapharm Biotech S.r.l., Monza, MB, Italy.

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.
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http://dx.doi.org/10.1111/bph.12728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128066PMC
August 2014

Silibinin hemisuccinate binding to proteins in plasma and blood cell/plasma partitioning in mouse, rat, dog and man in vitro.

Drug Metabol Drug Interact 2013 ;28(2):115-22

R&D Division, Rottapharm | Madaus, Via Valosa di Sopra 9, 20900 Monza, Italy.

Background: The determination of plasma protein binding and blood cell/plasma partitioning is important when prescribing silibinin hemisuccinate to patients concomitantly receiving other drugs and to estimate the safety margins of exposure at the no observed adverse events levels determined from toxicity studies conducted in rats and dogs.

Methods: Protein binding of [3'-14C]silibinin hemisuccinate (1, 10, 100, 1000 and 4000 μM) was evaluated in human, dog, rat and mouse plasma by ultrafiltration. Blood cell/plasma partitioning in all these species was also determined.

Results: Silibinin hemisuccinate is highly bound to plasma proteins with percentage binding ranging from 94.3% to 97.8%. Its association with blood cells was negligible (<7%) in all species. The degree of protein binding was concentration independent up to the pharmacologically effective concentration of 100 μM. The blood cell/plasma partitioning indicates that distribution into blood cells is not an important feature for the disposition of silibinin hemisuccinate.

Conclusions: No corrections for fraction unbound are needed when comparing human and preclinical pharmacokinetic and pharmacodynamic data at pharmacological doses, and it is appropriate to analyze plasma as opposed to whole blood for the determination of silibinin hemisuccinate concentrations.
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http://dx.doi.org/10.1515/dmdi-2013-0013DOI Listing
September 2013

Discovery, synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists.

Bioorg Med Chem Lett 2013 May 1;23(9):2653-8. Epub 2013 Mar 1.

Rottapharm Madaus, Medicinal Chemistry Department, via Valosa di Sopra 9, 20900 Monza, Italy.

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.
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http://dx.doi.org/10.1016/j.bmcl.2013.02.093DOI Listing
May 2013

Synthesis and structure-activity relationship studies in serotonin 5-HT(1A) receptor agonists based on fused pyrrolidone scaffolds.

Eur J Med Chem 2013 May 8;63:85-94. Epub 2013 Feb 8.

Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Università degli Studi di Siena, Via A. Moro, 53100 Siena, Italy.

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.
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http://dx.doi.org/10.1016/j.ejmech.2013.01.044DOI Listing
May 2013

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

J Pain Res 2012 20;5:151-67. Epub 2012 Jun 20.

Department of Neuroscience and Biomedical Technologies, University of Milan-Bicocca.

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.
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http://dx.doi.org/10.2147/JPR.S32122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392714PMC
October 2012

Experimental pharmacology of glucosamine sulfate.

Int J Rheumatol 2011 9;2011:939265. Epub 2011 Oct 9.

R&D, Rottapharm SpA, 20900 Monza, Italy.

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.
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http://dx.doi.org/10.1155/2011/939265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191774PMC
November 2011

Synthesis, pharmacophore modeling and in vitro activity of 10,11-dihydrodibenzo[b,f]oxepine-4-carboxamide derivatives as novel and potent antagonists of the prostaglandin EP4 receptor.

Bioorg Med Chem Lett 2011 Nov 1;21(21):6336-40. Epub 2011 Sep 1.

Rottapharm Madaus, Medicinal Chemistry Department, via Valosa di Sopra 9, Monza 20900, Italy.

The construction of a EP(4) antagonists pharmacophore model and the discovery of a highly potent oxepinic series of EP(4) antagonists is discussed. Compound 1a exhibits an excellent selectivity profile toward EP(2) receptor subtype and low cytochrome P450 inhibition potential.
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http://dx.doi.org/10.1016/j.bmcl.2011.08.102DOI Listing
November 2011

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

J Pain Res 2011 18;4:111-25. Epub 2011 Apr 18.

Department of Pharmacology and Toxicology, S.p.A., Monza (MB), Italy.

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.
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http://dx.doi.org/10.2147/JPR.S18353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3100226PMC
July 2011

Design, synthesis, and preliminary biological evaluation of pyrrolo[3,4-c]quinolin-1-one and oxoisoindoline derivatives as aggrecanase inhibitors.

ChemMedChem 2010 May;5(5):739-48

Dipartimento Farmaco Chimico Tecnologico and the European Research Centre for Drug Discovery and Development, Università di Siena, Italy.

A small set of aggrecanase inhibitors based on the pyrrolo[3,4-c]quinolin-1-one or oxoisoindoline frameworks bearing a 4-(benzyloxy)phenyl substituent and different zinc binding groups were rationally designed and evaluated in silico by docking studies using the crystal structure of the ADAMTS-5 catalytic domain (PDB code: 3B8Z). The designed compounds were synthesized via straightforward routes and tested for their potential inhibitory activity against ADAMTS-5 and ADAMTS-4. Among the compounds containing the pyrrolo[3,4-c]quinolinone tricyclic system, hydroxamate derivative 2 b inhibited both ADAMTS-5 and ADAMTS-4, with IC(50) values in the submicromolar range and an inhibitory profile very similar to that of reference carboxylate derivative 11. Conversely, the corresponding carboxylate derivative 2 a was significantly less active and unable to discriminate between ADAMTS-5 and -4. The structure-activity relationship analysis of pyrroloquinolinone derivatives 2 a-i suggests that the carboxylate or hydroxamate groups of compounds 2 a,b play a key role in the interaction of these compounds with ADAMTS-5 and -4. On the other hand, the oxoisoindoline derivatives 3 a,b lack significant ADAMTS-4 inhibitory activity and inhibit ADAMTS-5 showing IC(25) values in the micromolar range.
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http://dx.doi.org/10.1002/cmdc.200900523DOI Listing
May 2010

Antipsychotic-like effects of the N-methyl-D-aspartate receptor modulator neboglamine: an immunohistochemical and behavioural study in the rat.

Pharmacol Res 2010 May 4;61(5):430-6. Epub 2010 Jan 4.

Department of Pharmacology & Toxicology, Rottapharm S.p.A., Via Valosa di Sopra 9, 20052 Monza (MB), Italy.

Neboglamine is a functional modulator of the glycine site on the N-methyl-d-aspartate (NMDA) receptor. Dysfunction of this receptor has been associated with negative and cognitive symptoms in schizophrenia. Thus, we tested the hypothesis that neboglamine behaves as a potential antipsychotic. We compared the effects of neboglamine, D-serine, clozapine, and haloperidol on the expression of Fos-like immunoreactivity (FLI), a marker of neuronal activation, in rat forebrain. We also studied the effects of these agents on phencyclidine (PCP)-induced behaviour in rats, a model predictive of potential antipsychotic activity. Neboglamine, like haloperidol and clozapine, significantly increased the number of FLI-positive cells in the prefrontal cortex, nucleus accumbens, and lateral septal nucleus (3.2-, 4.8-, and 4.5-fold over control, respectively). Haloperidol dramatically increased FLI (390-fold over control) in the dorsolateral striatum, a brain region in which neboglamine and clozapine had no effect. The pattern of FLI induced by neboglamine closely matched that of d-serine, an endogenous agonist at the glycine site of NMDA receptors. Consistent with this finding, neboglamine restored NMDA-mediated neurotransmitter release in frontal cortex punches exposed to the NMDA antagonist PCP. In the behavioural model, all test compounds significantly inhibited PCP-induced hyperlocomotion. Unlike haloperidol and clozapine, neither neboglamine nor D-serine affected the basal levels of locomotor activity. Moreover, oral neboglamine dose-dependently inhibited both the hyperlocomotion and the frequency of rearing behaviour induced by PCP. These results, while confirming that the NMDA glycine site is a feasible target for activating the frontostriatal system, support the clinical evaluation of neboglamine as a treatment for schizophrenia.
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http://dx.doi.org/10.1016/j.phrs.2009.12.010DOI Listing
May 2010

Efficacy of CR3294, a new benzamidine derivative, in the prevention of 5-fluorouracil-induced gastrointestinal mucositis and diarrhea in mice.

Cancer Chemother Pharmacol 2010 Oct 30;66(5):819-27. Epub 2009 Dec 30.

Department of Pharmacology and Toxicology, Rottapharm, Monza, Italy.

Purpose: Gastrointestinal mucositis, commonly associated with diarrhea, is a dose-limiting toxicity of chemotherapy. The new benzamidine derivative CR3294 reduces tissue damage in animal models of intestinal inflammation. Thus, we tested whether CR3294 had the potential to prevent chemotherapy-induced mucositis.

Methods: In tests on isolated cells, reactive oxygen species (ROS) formation and cytokine release were measured by chemiluminescence and immunoassays, respectively. In studies in vivo, BDF1 mice were given oral CR3294 (2.5-20 mg/kg) for 3 days before receiving 5-fluorouracil. Intestinal crypt survival, cell apoptosis and proliferation, and diarrhea score were assessed. Additionally, nude mice bearing tumor xenografts were treated with CR3294 and/or 5-fluorouracil, and tumor growth was monitored.

Results: CR3294 significantly inhibited cytokine release from stimulated leukocytes at concentrations similar to the IC(50) (2.9 +/- 0.2 muM) for ROS production by these cells. Consistent with these molecular findings, CR3294 dose-dependently protected the intestinal mucosa against 5-fluorouracil-induced toxicity in a mouse model of mucositis. The number of surviving crypts per cross-section in mice receiving 20 mg/kg CR3294 was 2.8-fold that in vehicle-treated animals (18.1 +/- 1.9 vs. 6.5 +/- 0.9, P < 0.001). Moreover, CR3294 decreased the cumulative diarrhea score by 50%, reduced by nearly 70% the incidence of severe episodes, and increased by 3-fold the number of mice without diarrhea. CR3294 neither affected the growth of tumor xenografts nor protected tumors from the cytotoxic activity of 5-fluorouracil.

Conclusions: This study demonstrates that CR3294 acts on key molecular targets to reduce the signs of mucositis and the occurrence of diarrhea in mice exposed to the chemotherapy drug 5-fluorouracil.
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http://dx.doi.org/10.1007/s00280-009-1224-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2926937PMC
October 2010

Multivalent supramolecular dendrimer-based drugs.

Biomacromolecules 2010 Jan;11(1):182-6

Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Universitàà di Siena, Via A. Moro, 53100 Siena, Italy.

Supramolecular complexes consisting of a hydrophobic dendrimer host [DAB-dendr-(NHCONH-Ad)(64)] as well as solubilizing and bioactive guest molecules have been synthesized using a noncovalent approach. The guest-host supramolecular assembly is first preassembled in chloroform and transferred via the neat phase to aqueous solution. The bioactive guest molecules can bind to a natural (serotonin 5-HT(3)) receptor with nanomolar affinity as well as to the synthetic dendrimer receptor in aqueous solution, going toward a dynamic multivalent supramolecular construct capable of adapting itself to a multimeric receptor motif.
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http://dx.doi.org/10.1021/bm901055aDOI Listing
January 2010

Progress towards the identification of new aggrecanase inhibitors.

Curr Med Chem 2009 ;16(19):2395-415

Department of Chemistry, University of Modena and Reggio Emilia, Via G. Campi 183, Modena, Italy.

Degenerative diseases are still a challenging issue in clinical therapy; even though in several cases it is possible to treat symptoms, drugs able to block disease progression are lacking at present. Osteoarthritis (OA) and Rheumatoid Arthritis (RA) are degenerative diseases leading to serious cartilage destruction, affecting joint functions and giving rise to restricted movement, pain and chronic disability. Current clinical treatment for arthritis is confined to Non Steroidal Anti-Inflammatory Drugs (NSAIDs), which are effective in treating symptoms but fail to block the progression of the disease. Matrix Metalloproteases (MMPs) inhibitors have been clinically studied as possible drugs for cartilage degradation prevention. However, their clinical use has been limited by severe side-effects. Aggrecan, which plays a fundamental role in maintaining the structural and mechanical properties of cartilage, has recently been found to be specifically cleaved by "aggrecanases". Aggrecanases are multidomain zinc metalloproteases, different from MMPs, which cleave the aggrecan within the interglobular domain (IGD). Aggrecan breakdown at this site has been found to be crucial for cartilage degradation. These new findings re-addressed the interest of the research for new arthritis therapeutic agents focusing on aggrecanases rather than on MMPs. This review is meant to provide a critical appraisal of the ongoing developments of Zn-chelating and non chelating aggrecanase inhibitors, with a particular emphasis on the related structure-activity relationships (SARs), in the light of the protein structural information recently made available.
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http://dx.doi.org/10.2174/092986709788682092DOI Listing
November 2009

Induction of autophagic cell death by a novel molecule is increased by hypoxia.

Autophagy 2008 Nov 24;4(8):1042-53. Epub 2008 Nov 24.

Department of Cellular and Molecular Pathology, IRCCS San Raffaele Pisana, Rome, Italy.

Adaptation to hypoxia through activation of the hypoxia inducible factor-1 (HIF-1) is crucial for tumor cells survival. Here we describe the antitumoral effects of the new molecule CR 3294 on tumor cells in the presence of hypoxia. Treatment of the breast carcinoma cell line MDA-MB-231 with CR 3294 in 1% O(2) resulted in an in vivo and in vitro inhibition of tumor growth. CR 3294 induced accumulation of autophagosomes in hypoxic MDA-MB-231 cells as assessed by both transmission electron microscopy (TEM) and the autophagic marker LC3-II. TEM analysis revealed the presence of invaginations of the cytoplasm into the nucleus. Autophagosomes were present in such invaginations. Moreover, CR 3294 inhibited both the DNA binding of HIF-1alpha and VEGF mRNA synthesis. Immunoprecipitation and immunofluorescence studies showed an interaction between LC3 and HIF-1alpha. We next detailed the effect of inhibitors and activators of autophagy on both HIF-1alpha and LC3. In particular, 3 methyladenine (3MA) and wortmannin, two macroautophagic inhibitors, prevented both the decrease of HIF-1alpha protein levels and LC3 processing in cells treated with CR 3294. Bafilomycin and leupeptin, inhibitors of lysosomes, prevented HIF-1alpha decrease without affecting LC3 processing. By contrast, treating hypoxic MDA-MB-231 cells with trifluoperazine (TFP) or serum withdrawal (SW), two activators of autophagy, diminished HIF-1alpha levels and stimulated LC3 processing. These results indicate that activation of the autophagic pathway in hypoxic cells by the new molecule CR 3294, as well as by TFP or SW, can have potentially important implications for cancer treatment.
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http://dx.doi.org/10.4161/auto.7070DOI Listing
November 2008

In vivo neurochemical effects of the NR2B selective NMDA receptor antagonist CR 3394 in 6-hydroxydopamine lesioned rats.

Eur J Pharmacol 2008 Apr 19;584(2-3):297-305. Epub 2008 Feb 19.

Department of Pharmaceutical Chemistry and Drug Analysis, Research Group Experimental Pharmacology, Vrije Universiteit Brussel, Laarbeeklaan 103, B1090 Brussels, Belgium.

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 microM 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.
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http://dx.doi.org/10.1016/j.ejphar.2008.02.027DOI Listing
April 2008

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

J Med Chem 2008 Apr 5;51(7):2137-46. Epub 2008 Mar 5.

Dipartimento Farmaco Chimico Tecnologico and European Research Centre for Drug Discovery and Development, Università di Siena, Via A. Moro, Siena, Italy.

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.
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http://dx.doi.org/10.1021/jm7011563DOI Listing
April 2008

Modulation of human estrogen receptor alpha F promoter by a protein kinase C/c-Src-dependent mechanism in osteoblast-like cells.

J Mol Endocrinol 2006 Dec;37(3):489-502

Department of Experimental Medicine, University of L'Aquila, via Vetoio Coppito 2, 67100 L'Aquila, Italy.

The human estrogen receptor alpha (ERalpha) gene is driven by multiple promoters, of which the F promoter alone is found to be active in primary osteoblasts. The study was aimed at identifying new regulatory pathways affecting transcription of the receptor in this cell lineage. We generated human osteoblast-like cells, Saos-2, stably transfected with a luciferase-reporter gene downstream of the human ERalpha F promoter (Saos F-Luc), and assayed the reporter response to differentiation-related signals. Over-confluence, shown to stimulate osteoblast differentiation, caused a time-dependent increase of F-promoter activity and correlated with an inactivation of protein kinase C alpha (PKCalpha ). PKC downregulation, obtained by long-term treatment with phorbol 12-myristate 13-acetate (PMA), resulted in promoter stimulation at similar levels in sub-confluent cells. The F promoter contains a putative PMA-responsive AP-1 site, but AP-1 activation was unremarkable in over-confluent cells. Treatment with PP1, a specific inhibitor of the non-receptor tyrosine-kinase c-Src, which is a negative regulator of osteoblast differentiation, showed that the activity of this kinase inhibits the F promoter. In PP1-treated cells, F-promoter activity was not further increased by PMA. Treatment with the generic kinase inhibitor 4-dimethylaminopyridine (DMAP) resulted in a dose-dependent induction of the promoter, which matched a parallel decrease of active c-Src. The effect was c-Src dependent, as DMAP caused no further promoter induction in PP1-treated cells. Overexpression of exogenous human ERalpha resulted in modest promoter stimulation, which required the ligand-independent activator function 1 of the receptor. In murine primary osteoblasts, additional ERalpha signal was observed upon induction of F promoter. In conclusion, we demonstrated a robust PKC/c-Src-dependent and estrogen-independent mechanism modulating transcription of ERalpha in osteoblasts, probably affecting estrogen responsiveness during cell differentiation.
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http://dx.doi.org/10.1677/jme.1.02055DOI Listing
December 2006

Functional in vitro characterization of CR 3394: a novel voltage dependent N-methyl-D-aspartate (NMDA) receptor antagonist.

Neuropharmacology 2006 Mar 19;50(3):277-85. Epub 2005 Oct 19.

Department of Pharmaceutical Sciences, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy.

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.
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http://dx.doi.org/10.1016/j.neuropharm.2005.09.002DOI Listing
March 2006