33 results on '"Annalise Di Marco"'
Search Results
2. Cyclic Phosphopantothenic Acid Prodrugs for Treatment of Pantothenate Kinase-Associated Neurodegeneration
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Daniel Elbaum, Daniel O. Cicero, Paola Fezzardi, Annalise Di Marco, Steven J. Harper, Savina Malancona, Elena Bracacel, Andrea Vecchi, Ilaria Rossetti, Edith Monteagudo, Alina Ciammaichella, Giulio Auciello, Odalys Gonzalez Paz, and Maria G. Beconi
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Vitamin ,Knockout ,Pharmacology ,Inbred C57BL ,Pantothenic Acid ,Pantothenate kinase-associated neurodegeneration ,Mice ,Structure-Activity Relationship ,chemistry.chemical_compound ,Oral administration ,Drug Discovery ,medicine ,Animals ,Humans ,Coenzyme A ,Prodrugs ,Gene ,Pantothenate Kinase-Associated Neurodegeneration ,Mice, Knockout ,Animal ,Chemistry ,Regeneration (biology) ,Neurodegeneration ,Brain ,Lipid Droplets ,respiratory system ,Prodrug ,Blood-Brain Barrier ,Cyclization ,Disease Models, Animal ,Half-Life ,Hepatocytes ,Mice, Inbred C57BL ,medicine.disease ,PANK2 ,Settore CHIM/08 - Chimica Farmaceutica ,Disease Models ,Molecular Medicine - Abstract
Mutations in the human PANK2 gene are implicated in neurodegenerative diseases such as pantothenate kinase-associated neurodegeneration (PKAN) and result in low levels of coenzyme-A (CoA) in the CNS due to impaired production of phosphopantothenic acid (PPA) from vitamin B5. Restoration of central PPA levels by delivery of exogenous PPA is a recent strategy to reactivate CoA biosynthesis in PKAN patients. Fosmetpantotenate is an oral PPA prodrug. We report here the development of a new PANk2-/- knockout model that allows CoA regeneration in brain cells to be evaluated and describe two new series of cyclic phosphate prodrugs of PPA capable of regenerating excellent levels of CoA in this system. A proof-of-concept study in mouse demonstrates the potential of this new class of prodrugs to deliver PPA to the brain following oral administration and confirms incorporation of the prodrug-derived PPA into CoA.
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- 2020
3. 5,6-Dihydroxypyrimidine Scaffold to Target HIV-1 Nucleocapsid Protein
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Maria Rosaria Battista, Rajhans Sharma, Yves Mély, Marisabella Santoriello, Edith Monteagudo, Paola Fezzardi, Eleonore Real, Maurizio Zazzi, Vincenzo Summa, Mattia Mori, Maria Chiara Dasso Lang, Steven J. Harper, Savina Malancona, Antonella Cellucci, Manuel Pires, Martina Nibbio, Maurizio Botta, Roberto Speziale, Andreina Basta, Nadia Gennari, Francesco Saladini, Davide De Forni, Annalise Di Marco, Alessia Giannini, Lesia Kovalenko, Malancona, S., Mori, M., Fezzardi, P., Santoriello, M., Basta, A., Nibbio, M., Kovalenko, L., Speziale, R., Battista, M. R., Cellucci, A., Gennari, N., Monteagudo, E., Di Marco, A., Giannini, A., Sharma, R., Pires, M., Real, E., Zazzi, M., Dasso Lang, M. C., De Forni, D., Saladini, F., Mely, Y., Summa, V., Harper, S., and Botta, M.
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Nucleocapsid protein ,Scaffold ,drug resistance ,010405 organic chemistry ,antiretroviral ,Organic Chemistry ,HIV ,RNA-binding protein ,Drug resistance ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,3. Good health ,Conserved sequence ,Nordihydroguaiaretic acid ,010404 medicinal & biomolecular chemistry ,chemistry.chemical_compound ,NC inhibitor ,chemistry ,Viral replication ,Drug Discovery ,DNA ,dihydroxypyrimidine ,ADME - Abstract
[Image: see text] The HIV-1 nucleocapsid (NC) protein is a small basic DNA and RNA binding protein that is absolutely necessary for viral replication and thus represents a target of great interest to develop new anti-HIV agents. Moreover, the highly conserved sequence offers the opportunity to escape the drug resistance (DR) that emerged following the highly active antiretroviral therapy (HAART) treatment. On the basis of our previous research, nordihydroguaiaretic acid 1 acts as a NC inhibitor showing moderate antiviral activity and suboptimal drug-like properties due to the presence of the catechol moieties. A bioisosteric catechol replacement approach led us to identify the 5-dihydroxypyrimidine-6-carboxamide substructure as a privileged scaffold of a new class of HIV-1 NC inhibitors. Hit validation efforts led to the identification of optimized analogs, as represented by compound 28, showing improved NC inhibition and antiviral activity as well as good ADME and PK properties.
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- 2020
4. Efficacy of PEGylated ciliary neurotrophic factor superagonist variant in diet-induced obesity mice
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Maria Rosaria Battista, Antonella Grigoletto, Tommaso Tedeschini, Antonella Cellucci, Fabrizio Colaceci, Ralph Laufer, Gianfranco Pasut, and Annalise Di Marco
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Mice ,Multidisciplinary ,Weight Loss ,Animals ,Humans ,Mice, Obese ,Proteins ,Ciliary Neurotrophic Factor ,Obesity ,Receptor, Ciliary Neurotrophic Factor ,Diet ,Polyethylene Glycols - Abstract
Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules.
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- 2021
5. Application of an in Vitro Blood–Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington’s Disease
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Mark Rose, Odalys Gonzalez Paz, Giulio Auciello, Annalise Di Marco, Todd Herbst, Ignacio Muñoz-Sanjuán, Domenico Vignone, Edith Monteagudo, Vinod Khetarpal, Matteo Zini, Maria Rosaria Battista, Laura Orsatti, Vincenzo Summa, Celia Dominguez, Leticia M Toledo-Sherman, Ivan Fini, Antonella Cellucci, Di Marco, A., Gonzalez Paz, O., Fini, I., Vignone, D., Cellucci, A., Battista, M. R., Auciello, G., Orsatti, L., Zini, M., Monteagudo, E., Khetarpal, V., Rose, M., Dominguez, C., Herbst, T., Toledo-Sherman, L., Summa, V., and Munoz-Sanjuan, I.
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Swine ,Pharmaceutical Science ,02 engineering and technology ,Pharmacology ,efflux transporter ,030226 pharmacology & pharmacy ,brain penetration ,Rats, Sprague-Dawley ,0302 clinical medicine ,Drug Discovery ,Electric Impedance ,Coculture Technique ,Cells, Cultured ,Cerebral Cortex ,Endothelial Cell ,Tight junction ,Tight Junction ,Drug discovery ,Chemistry ,Huntington's disease ,Biological activity ,021001 nanoscience & nanotechnology ,Huntington Disease ,medicine.anatomical_structure ,Blood-Brain Barrier ,Molecular Medicine ,Central Nervous System Agent ,CNS ,Astrocyte ,0210 nano-technology ,ATP-Binding Cassette Transporter ,Central nervous system ,Blood–brain barrier ,Models, Biological ,Permeability ,Tight Junctions ,Capillary Permeability ,03 medical and health sciences ,In vivo ,Huntingtin Protein ,medicine ,Animals ,Solute Carrier Protein ,Solute Carrier Proteins ,Animal ,Endothelial Cells ,medicine.disease ,Coculture Techniques ,Rats ,Astrocytes ,transport ,Rat ,ATP-Binding Cassette Transporters ,Central Nervous System Agents - Abstract
Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting. ©
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- 2019
6. Design, Synthesis, and Biological Evaluation of New 1-(Aryl-1H-pyrrolyl)(phenyl)methyl-1H-imidazole Derivatives as Antiprotozoal Agents
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Roberto Cirilli, Daniela De Vita, Marcel Kaiser, Antonella Messore, Valentina Noemi Madia, Cristina Faggi, Claudia M. Calvet, Vincenzo Summa, Annalise Di Marco, Gareth K. Jennings, Larissa M. Podust, Alessandro De Leo, Roberta Costi, Roberto Di Santo, Luca Pescatori, Valeria Tudino, Pascal Mäser, Reto Brun, Francesco Saccoliti, Luigi Scipione, Giacomo Pepe, Maria Rosaria Battista, Saccoliti, F., Madia, V. N., Tudino, V., De Leo, A., Pescatori, L., Messore, A., De Vita, D., Scipione, L., Brun, R., Kaiser, M., Maser, P., Calvet, C. M., Jennings, G. K., Podust, L. M., Pepe, G., Cirilli, R., Faggi, C., Di Marco, A., Battista, M. R., Summa, V., Costi, R., and Di Santo, R.
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Parasitic Sensitivity Test ,Trypanosoma ,medicine.drug_class ,Stereochemistry ,Antiprotozoal Agents ,Drug Evaluation, Preclinical ,Leishmania donovani ,CYP51 ,Cytochrome P-450 Enzyme Inhibitor ,Antiprotozoal Agent ,Parasitic Sensitivity Tests ,parasitic diseases ,Drug Discovery ,medicine ,Animals ,Cytochrome P-450 Enzyme Inhibitors ,Humans ,Trypanosoma cruzi ,Imidazole ,IC50 ,biology ,Animal ,Chemistry ,Imidazoles ,Trypanosoma brucei rhodesiense ,Plasmodium falciparum ,biology.organism_classification ,antiprotozoal ,imidazole ,Mechanism of action ,Drug Design ,Antiprotozoal ,Molecular Medicine ,medicine.symptom ,Human - Abstract
We have designed and synthesized a series of new imidazole-based compounds structurally related to an antiprotozoal agent with nanomolar activity which we identified recently. The new analogues possess micromolar activities against Trypanosoma brucei rhodesiense and Leishmania donovani and nanomolar potency against Plasmodium falciparum. Most of the analogues displayed IC 50 within the low nanomolar range against Trypanosoma cruzi, with very high selectivity toward the parasite. Discussion of structure-activity relationships and in vitro biological data for the new compounds are provided against a number of different protozoa. The mechanism of action for the most potent derivatives (5i, 6a-c, and 8b) was assessed by a target-based assay using recombinant T. cruzi CYP51. Bioavailability and efficacy of selected hits were assessed in a T. cruzi mouse model, where 6a and 6b reduced parasitemia in animals >99% following intraperitoneal administration of 25 mg/kg/day dose for 4 consecutive days.
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- 2019
7. Improved Selective Class I HDAC and Novel Selective HDAC3 Inhibitors: Beyond Hydroxamic Acids and Benzamides
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Annalise Di Marco, Alberto Bresciani, Simona Ponzi, Antonella Cellucci, Jesus Maria Ontoria Ontoria, Alina Ciammaichella, Alessandra Francone, Steven J. Harper, Savina Malancona, Maria Veneziano, Vincenzo Summa, Edith Monteagudo, Federica Ferrigno, Emanuela Nizi, Ilaria Biancofiore, Ilaria Rossetti, Paola Pace, Bresciani, A., Ontoria, J. M., Biancofiore, I., Cellucci, A., Ciammaichella, A., Di Marco, A., Ferrigno, F., Francone, A., Malancona, S., Monteagudo, E., Nizi, E., Pace, P., Ponzi, S., Rossetti, I., Veneziano, M., Summa, V., and Harper, S.
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HDAC3 selective ,Hydroxamic acid ,Zinc binding ,nonbenzamide ,010405 organic chemistry ,Organic Chemistry ,HDAC3 ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,chemistry.chemical_compound ,chemistry ,Drug Discovery ,Histone deacetylase ,nonhydroxamate - Abstract
[Image: see text] The application of class I HDAC inhibitors as cancer therapies is well established, but more recently their development for nononcological indications has increased. We report here on the generation of improved class I selective human HDAC inhibitors based on an ethylketone zinc binding group (ZBG) in place of the hydroxamic acid that features the majority of HDAC inhibitors. We also describe a novel set of HDAC3 isoform selective inhibitors that show stronger potency and selectivity than the most commonly used HDAC3 selective tool compound RGFP966. These compounds are again based on an alternative ZBG with respect to the ortho-anilide that is featured in HDAC3 selective compounds reported to date.
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- 2018
8. Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro
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Annalise Di Marco, Steven J. Harper, Savina Malancona, Nadia Gennari, Simona Ponzi, Jesus Maria Ontoria Ontoria, Marcel Kaiser, Rita Graziani, Ilaria Biancofiore, Giacomo Paonessa, Alberto Bresciani, Federica Ferrigno, Vincenzo Summa, Ferrigno, F., Biancofiore, I., Malancona, S., Ponzi, S., Paonessa, G., Graziani, R., Bresciani, A., Gennari, N., Di Marco, A., Kaiser, M., Summa, V., Harper, S., and Ontoria, J. M.
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0301 basic medicine ,Antiparasitic ,medicine.drug_class ,030106 microbiology ,Clinical Biochemistry ,Plasmodium falciparum ,Trypanosoma brucei brucei ,Pharmaceutical Science ,Trypanosoma brucei ,HeLa Cell ,Biochemistry ,Piperazines ,03 medical and health sciences ,chemistry.chemical_compound ,Structure-Activity Relationship ,Sleeping sickne ,parasitic diseases ,Drug Discovery ,medicine ,Cytotoxic T cell ,Humans ,African trypanosomiasis ,Malaria, Falciparum ,Trypanosoma cruzi ,Molecular Biology ,Imidazole ,Piperazine ,biology ,2-(1H-imidazo-2-yl)piperazine ,Trypanocidal Agent ,Organic Chemistry ,Imidazoles ,Trypanosoma brucei rhodesiense ,medicine.disease ,biology.organism_classification ,Human African Trypanosomiasis (HAT) ,Virology ,Trypanocidal Agents ,030104 developmental biology ,Trypanosomiasis, African ,chemistry ,Molecular Medicine ,Growth inhibition ,Human ,HeLa Cells - Abstract
The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC50 5 nM), is not cytotoxic (HeLa CC50 > 25,000 nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC50 8120 nM, P. falciparum EC50 3624 nM).
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- 2018
9. Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide
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Philippe Neuner, Gaetano Barbato, Ralph Laufer, Karolina Zytko, Andrea M. Peier, Kunal Desai, Edith Monteagudo, Xiaobing Du, Alessandro Pocai, Donald J. Marsh, Annalise Di Marco, Ying Qian, Davide Ricci, Armin Lahm, Antonello Pessi, Fabio Talamo, Paolo Ingallinella, and Elisabetta Bianchi
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Pharmacology ,chemistry.chemical_classification ,medicine.medical_specialty ,Chemistry ,Organic Chemistry ,Endogeny ,Peptide ,General Medicine ,Human serum albumin ,Biochemistry ,Endocrinology ,Structural Biology ,In vivo ,Internal medicine ,Drug Discovery ,Anorectic ,medicine ,Molecular Medicine ,Receptor ,Molecular Biology ,Neuromedin U ,Conjugate ,medicine.drug - Abstract
Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.
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- 2013
10. PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes
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Paolo Ingallinella, Annalise Di Marco, Edith Monteagudo, Andrea M. Peier, Ying Qian, Alessandro Pocai, Donald J. Marsh, Antonella Cellucci, Xiaobing Du, Antonello Pessi, Ralph Laufer, Elisabetta Bianchi, Kunal Desai, and Karolina Zytko
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Male ,medicine.medical_specialty ,Clinical Biochemistry ,Pharmaceutical Science ,Endogeny ,Biochemistry ,Energy homeostasis ,Diabetes Mellitus, Experimental ,Polyethylene Glycols ,Mice ,Structure-Activity Relationship ,In vivo ,Internal medicine ,Drug Discovery ,medicine ,Animals ,Humans ,Glucose homeostasis ,Obesity ,Receptor ,Molecular Biology ,Mice, Knockout ,Glucose tolerance test ,Dose-Response Relationship, Drug ,medicine.diagnostic_test ,Chemistry ,Neuropeptides ,Organic Chemistry ,Glucose Tolerance Test ,Receptors, Neurotransmitter ,Mice, Inbred C57BL ,Endocrinology ,PEGylation ,Molecular Medicine ,Neuromedin U - Abstract
Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.
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- 2012
11. Discovery of a Selective Series of Inhibitors of Plasmodium falciparum HDACs
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Nadia Gennari, Emanuela Nizi, Steven J. Harper, Savina Malancona, Sergio Altamura, Edith Monteagudo, David Roberts, Ilaria Biancofiore, Paul Willis, Maria Vittoria Orsale, Ottavia Cecchetti, Antonella Cellucci, Ralph Laufer, Rita Graziani, Alberto Bresciani, Simona Ponzi, Jesus Maria Ontoria Ontoria, Maria Veneziano, Annalise Di Marco, Giacomo Paonessa, Vincenzo Summa, Federica Ferrigno, Ontoriajm, G Paonessa, G, Ponzi, S, Ferrigno, F, Nizi, E, Biancofiore, I, Malancona, S, Graziani, R, Roberts, D, Willis, P, Bresciani, A, Nadia Gennari, N, Cecchetti, O, Monteagudo, E, Orsale, Mv, Veneziano, M, Di Marco, A, Cellucci, A, Laufer, R, Sergio Altamura, S, Summa, V, and Harper, S
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0301 basic medicine ,biology ,Chemistry ,030106 microbiology ,Organic Chemistry ,Growth inhibitory ,Plasmodium falciparum ,biology.organism_classification ,Biochemistry ,03 medical and health sciences ,030104 developmental biology ,Mechanism of action ,Drug Discovery ,medicine ,medicine.symptom - Abstract
The identification of a new series of P. falciparum growth inhibitors is described. Starting from a series of known human class I HDAC inhibitors a SAR exploration based on growth inhibitory activity in parasite and human cells-based assays led to the identification of compounds with submicromolar inhibition of P. falciparum growth (EC50500 nM) and good selectivity over the activity of human HDAC in cells (up to50-fold). Inhibition of parasital HDACs as the mechanism of action of this new class of selective growth inhibitors is supported by hyperacetylation studies.
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- 2015
12. Cyclic phosphoramidates as prodrugs of 2′-C-methylcytidine
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Cristina Gardelli, Uwe Koch, Odalys Gonzalez-Paz, Barbara Pacini, Claudio Giuliano, Renzo Bazzo, Vincenzo Pucci, Joseph F. Leone, Kenneth A. Koeplinger, Michael Rowley, Frank Narjes, Sergio Altamura, Fabrizio Fiore, Malte Meppen, Ralph Laufer, and Annalise Di Marco
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viruses ,Hepatitis C virus ,Hepacivirus ,Cytidine ,medicine.disease_cause ,Antiviral Agents ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Stability ,Pegylated interferon ,Cricetinae ,Drug Discovery ,medicine ,Animals ,Humans ,Prodrugs ,Pharmacology ,biology ,Ribavirin ,Organic Chemistry ,virus diseases ,Nucleoside inhibitor ,General Medicine ,Hepatitis C ,Prodrug ,medicine.disease ,biology.organism_classification ,Virology ,digestive system diseases ,chemistry ,Hepatocytes ,Viral load ,medicine.drug - Abstract
The currently approved treatment for hepatitis C virus infections is a combination of Ribavirin and pegylated Interferon. It leads to a sustained virologic response in approximately only half of the patients treated. For this reason there is an urgent need of new therapeutic agents. 2'-C-Methylcytidine is the first nucleoside inhibitor of the HCV NS5B polymerase that was efficacious in reducing the viral load in patients infected with HCV. The application of a monophosphate prodrug approach based on unprecedented cyclic phosphoramidates is reported. Our SAR studies led to compounds that are efficiently converted to the active triphosphate in human hepatocytes.
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- 2009
13. Synthesis and evaluation of novel phosphoramidate prodrugs of 2′-methyl cytidine as inhibitors of hepatitis c virus NS5B polymerase
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Claudio Giuliano, Michael Rowley, Annalise Di Marco, Fabrizio Fiore, Monica Donghi, Cristina Gardelli, Barbara Attenni, Vincenzo Pucci, Frank Narjes, Ralph Laufer, and Joseph F. Leone
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Hepatitis C virus ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Pharmaceutical Science ,Cytidine ,Hepacivirus ,Viral Nonstructural Proteins ,medicine.disease_cause ,Biochemistry ,chemistry.chemical_compound ,Dogs ,In vivo ,Drug Discovery ,medicine ,Animals ,Humans ,Phosphoric Acids ,Prodrugs ,Nucleotide ,Molecular Biology ,Cells, Cultured ,chemistry.chemical_classification ,Organic Chemistry ,Biological activity ,Phosphoramidate ,Prodrug ,RNA-Dependent RNA Polymerase ,Amides ,In vitro ,Rats ,chemistry ,Hepatocytes ,Molecular Medicine ,Rabbits - Abstract
A variety of new prodrugs of 2'-methyl cytidine based on acyloxy ethylamino phosphoramidates have been synthesized and tested in vitro and in vivo for their biological activity. Compared with the parent drug a 10- to 20-fold increase in formation of nucleotide triphosphate in rat and human hepatocytes could be achieved.
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- 2009
14. Studies of the metabolic stability in cells of 5-(trifluoroacetyl)thiophene-2-carboxamides and identification of more stable class II histone deacetylase (HDAC) inhibitors
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Michael Rowley, Isabella Marcucci, Jesus Maria Ontoria Ontoria, Annalise Di Marco, Ester Muraglia, Federica Ferrigno, Christian Steinkühler, Philip Jones, Rita Scarpelli, Ralph Laufer, and Sergio Serafini
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medicine.drug_class ,Stereochemistry ,Clinical Biochemistry ,Cell ,Pharmaceutical Science ,Thiophenes ,Biochemistry ,Chemical synthesis ,Histone Deacetylases ,Structure-Activity Relationship ,Tubulin ,Drug Discovery ,medicine ,Humans ,Molecular Biology ,Cytochrome P-450 CYP2C9 ,chemistry.chemical_classification ,Molecular Structure ,biology ,Chemistry ,Organic Chemistry ,Histone deacetylase inhibitor ,HCT116 Cells ,Amides ,In vitro ,Histone Deacetylase Inhibitors ,medicine.anatomical_structure ,Enzyme ,Cell culture ,Enzyme inhibitor ,Drug Design ,Hepatocytes ,Microsomes, Liver ,biology.protein ,Molecular Medicine ,Aryl Hydrocarbon Hydroxylases ,Histone deacetylase ,HeLa Cells - Abstract
5-(Trifluoroacetyl)thiophene-2-carboxamides were found to be potent and selective class II HDAC inhibitors. This paper describes their further development and the investigation on the cause for the lack of cell-based activity. A rapid screening assay was set up which enabled the identification of more metabolic stable compounds as potent and selective class II HDAC inhibitors.
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- 2008
15. Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes. Kinetics and sensitivity to cytochrome P450 2D inhibitors
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Ralph Laufer, Annalise Di Marco, and Dan Yao
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Quinidine ,biology ,Chemistry ,Cytochrome P450 ,Dextromethorphan ,Pharmacology ,Biochemistry ,In vivo ,biology.protein ,medicine ,Microsome ,IC50 ,Cytochrome P-450 Enzyme Inhibitors ,medicine.drug ,Demethylation - Abstract
Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.
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- 2003
16. A New Method for Chemoselective Conjugation of Unprotected Peptides to Dauno- and Doxorubicin
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Marina Taliani, Antonello Pessi, Daniela Fattori, Annalise Di Marco, and Paolo Ingallinella
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Daunorubicin ,Stereochemistry ,Molecular Sequence Data ,Clinical Biochemistry ,Pharmaceutical Science ,Antineoplastic Agents ,Peptide ,Tripeptide ,Biochemistry ,Chemical synthesis ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Delivery Systems ,Drug Discovery ,medicine ,Moiety ,Doxorubicin ,Amino Acid Sequence ,Molecular Biology ,chemistry.chemical_classification ,Chemistry ,Organic Chemistry ,Hydrogen-Ion Concentration ,Oxime ,Molecular Medicine ,Peptides ,Drug carrier ,medicine.drug - Abstract
A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.
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- 2001
17. Enhancing B- and T-Cell Immune Response to a Hepatitis C Virus E2 DNA Vaccine by Intramuscular Electrical Gene Transfer
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Ralph Laufer, Adam J. Simon, Danilo R. Casimiro, Riccardo Cortese, Annalise Di Marco, Alfredo Nicosia, Antonella Folgori, Silvia Zucchelli, Stefania Capone, Nicola La Monica, Elena Fattori, S. Zucchelli, S. Capone, E. Fattori, A. Folgori, A. D. Marco, D. Casimiro, A. J. Simon, R. Laufer, N. L. Monica, R. Cortese, and A. Nicosia
- Subjects
Viral Hepatitis Vaccines ,Cellular immunity ,T-Lymphocytes ,Immunology ,Hepacivirus ,Biology ,Transfection ,Microbiology ,Epitope ,Virus ,DNA vaccination ,Mice ,Antigen ,Virology ,Vaccines and Antiviral Agents ,Vaccines, DNA ,Animals ,Cytotoxic T cell ,HYPERVARIABLE REGION-1, IMMUNIZATION, GLYCOPROTEIN, INTERFERON-ALPHA-2B, QUANTIFICATION, CHIMPANZEES, ENHANCEMENT, ANTIBODIES, INFECTION, RIBAVIRIN ,B-Lymphocytes ,Immunity ,Hepatitis C ,Molecular biology ,Adenovirus E2 Proteins ,Rats ,Electroporation ,Naked DNA ,Insect Science ,DNA, Viral ,Rabbits ,CD8 - Abstract
We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8+T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.
- Published
- 2000
18. Leptin receptor-mediated regulation of cholinergic neurotransmitter phenotype in cells of central nervous system origin
- Author
-
Isabelle Gloaguen, Domenico Lazzaro, Gennaro Ciliberto, Annalise Di Marco, Anna Demartis, Ralph Laufer, and Paola Delmastro
- Subjects
medicine.medical_specialty ,Leptin receptor ,biology ,Leptin ,Ciliary neurotrophic factor ,Neuropeptide Y receptor ,Biochemistry ,Choline acetyltransferase ,Endocrinology ,Internal medicine ,medicine ,biology.protein ,Cholinergic ,Cholinergic neuron ,Receptor - Abstract
Leptin is an adipocyte-secreted hormone that regulates body weight and exerts effects on hematopoiesis, reproduction, and immunity. The leptin receptor (OBR) shares sequence similarity and signaling capabilities with receptors for cytokines of the ciliary neurotrophic factor (CNTF) family. Our previous finding that CNTF and leptin exert similar anti-obesity effects and activate common neuronal signaling pathways, prompted us to investigate whether leptin may share with CNTF the ability to regulate the expression of specific neuronal genes. To this end, we established a cell line, derived from the murine septal cholinergic neuronal cell line SN-56, which stably expresses OBR. In this cell line, termed SN-56/OBR, leptin induces STAT transcription factor activation and STAT-dependent reporter gene expression in a manner similar to that of CNTF. Furthermore, in SN-56/OBR cells both CNTF and leptin produce changes in neurotransmitter and neuropeptide phenotype characteristic of cholinergic neurons, such as an increase in choline acetyltransferase and vasoactive intestinal polypeptide, and a decrease in neuropeptide Y expression. SN-56/OBR cells thus constitute an interesting new model system to investigate leptin action in cells of central nervous system origin. Possible physiological implications of OBR's intrinsic ability to regulate cholinergic phenotypic markers are discussed.
- Published
- 2000
19. Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide
- Author
-
Philippe, Neuner, Andrea M, Peier, Fabio, Talamo, Paolo, Ingallinella, Armin, Lahm, Gaetano, Barbato, Annalise, Di Marco, Kunal, Desai, Karolina, Zytko, Ying, Qian, Xiaobing, Du, Davide, Ricci, Edith, Monteagudo, Ralph, Laufer, Alessandro, Pocai, Elisabetta, Bianchi, Donald J, Marsh, and Antonello, Pessi
- Subjects
Blood Glucose ,Male ,Neuropeptides ,Drug Evaluation, Preclinical ,Serum Albumin, Human ,Cell Line ,Polyethylene Glycols ,Receptors, Neurotransmitter ,Mice, Inbred C57BL ,Mice ,Weight Loss ,Animals ,Humans ,Hypoglycemic Agents ,Anti-Obesity Agents ,Serum Albumin - Abstract
Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.
- Published
- 2013
20. ChemInform Abstract: A New Method for Chemoselective Conjugation of Unprotected Peptides to Dauno- and Doxorubicin
- Author
-
Annalise Di Marco, Antonello Pessi, Marina Taliani, Daniela Fattori, and Paolo Ingallinella
- Subjects
chemistry.chemical_classification ,chemistry.chemical_compound ,chemistry ,Stereochemistry ,Chemoselective ligation ,medicine ,Moiety ,Doxorubicin ,Peptide ,General Medicine ,Oxime ,medicine.drug - Abstract
A new approach for chemoselective ligation of peptides to dauno- and doxorubicin through an oxime bond is presented. The method does not require protecting groups on the peptide moiety.
- Published
- 2010
21. Identification of novel, selective, and stable inhibitors of class II histone deacetylases. Validation studies of the inhibition of the enzymatic activity of HDAC4 by small molecules as a novel approach for cancer therapy
- Author
-
Jesus Maria Ontoria Ontoria, Michael Rowley, Sergio Altamura, Sergio Serafini, Annalise Di Marco, Carsten Schultz-Fademrecht, Ralph Laufer, Rita Scarpelli, Ester Muraglia, Christian Steinkühler, Maria Cecilia Palumbi, Federica Ferrigno, and Philip Jones
- Subjects
Histone Deacetylase 2 ,Antineoplastic Agents ,Histone Deacetylase 1 ,Thiophenes ,Histone Deacetylase 6 ,Histone Deacetylases ,Histones ,Structure-Activity Relationship ,Tubulin ,Cell Line, Tumor ,Drug Discovery ,Humans ,IC50 ,Cell Proliferation ,biology ,Dose-Response Relationship, Drug ,Chemistry ,Acetylation ,HDAC6 ,Histone H3 deacetylation ,HDAC4 ,Isoenzymes ,Repressor Proteins ,Biochemistry ,Enzyme inhibitor ,Cancer cell ,biology.protein ,Molecular Medicine ,Histone deacetylase ,Drug Screening Assays, Antitumor - Abstract
5-Aryl-2-(trifluoroacetyl)thiophenes were identified as a new series of class II HDAC inhibitors (HDACi). Further development of this new series led to compounds such as 6h, a potent inhibitor of HDAC4 and HDAC6 (HDAC4 WT IC(50) = 310 nM, HDAC6 IC(50) = 70 nM) that displays 40-fold selectivity over HDAC1 and improved stability in HCT116 cancer cells (t(1/2) = 11 h). Compounds 6h and 2 show inhibition of alpha-tubulin deacetylation in HCT116 cells at 1 microM concentration and antiproliferation effects only at concentrations where inhibition of histone H3 deacetylation is observed.
- Published
- 2009
22. Turbulent flow chromatography TFC-tandem mass spectrometry supporting in vitro/vivo studies of NCEs in high throughput fashion
- Author
-
Annalise Di Marco, Maria Veneziano, Maria Verdirame, Fabio Bonelli, Edith Monteagudo, and Anna Alfieri
- Subjects
Bioanalysis ,Spectrometry, Mass, Electrospray Ionization ,Clinical Biochemistry ,Analytical chemistry ,Pharmaceutical Science ,Tandem mass spectrometry ,Mass spectrometry ,High-performance liquid chromatography ,Online Systems ,Analytical Chemistry ,Tandem Mass Spectrometry ,Drug Discovery ,Animals ,Humans ,Sample preparation ,Pharmacokinetics ,Solid phase extraction ,Spectroscopy ,Chromatography ,Chemistry ,Elution ,Extraction (chemistry) ,Reproducibility of Results ,Equipment Design ,High-Throughput Screening Assays ,Rats ,Pharmaceutical Preparations ,Hepatocytes - Abstract
Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. In this paper the application of the ARIA system to the analytical support of in vivo pharmacokinetics (PK) and in vitro drug metabolism studies is described, with an emphasis in high throughput optimization. For PK studies, a comparison between acetonitrile plasma protein precipitation (APPP) and TFC was carried out. Our optimized TFC methodology gave better S/N ratios and lower limit of quantification (LOQ) than conventional procedures. A robust and high throughput analytical method to support hepatocyte metabolic stability screening of new chemical entities was developed by hyphenation of TFC with mass spectrometry. An in-loop dilution injection procedure was implemented to overcome one of the main issues when using TFC, that is the early elution of hydrophilic compounds that renders low recoveries. A comparison between off-line solid phase extraction (SPE) and TFC was also carried out, and recovery, sensitivity (LOQ), matrix effect and robustness were evaluated. The use of two parallel columns in the configuration of the system provided a further increase of the throughput.
- Published
- 2009
23. High-throughput radiometric CYP2C19 inhibition assay using tritiated (S)-mephenytoin
- Author
-
Ashok Chaudhary, Ralph Laufer, Annalise Di Marco, Antonella Cellucci, and Massimiliano Fonsi
- Subjects
Tritiated water ,Pharmaceutical Science ,Tritium ,High-performance liquid chromatography ,Mixed Function Oxygenases ,chemistry.chemical_compound ,medicine ,Humans ,Mephenytoin ,Enzyme Inhibitors ,Radiometry ,Pharmacology ,Chromatography ,Chemistry ,Extraction (chemistry) ,Substrate (chemistry) ,Water ,Recombinant Proteins ,Cytochrome P-450 CYP2C19 ,NIH shift ,Microsome ,Microsomes, Liver ,Aryl Hydrocarbon Hydroxylases ,medicine.drug - Abstract
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C19 in human liver microsomes is described. The new assay, which does not require high-performance liquid chromatography (HPLC) separation or mass spectrometric detection, is based on the release of tritium as tritiated water that occurs upon CYP2C19-mediated 4′-hydroxylation of ( S )-mephenytoin labeled with tritium in the 4′ position. Because this reaction is subject to an NIH shift, tritium was also introduced into the 3′- and 5′-positions of the tracer to enhance formation of a tritiated water product. Tritiated water was separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent and sensitive to CYP2C19 inhibitors. IC 50 values for 15 diverse drugs differed less than 2.5-fold from those determined by quantification of the unlabeled 4′-hydroxy-( S )-mephenytoin product, using HPLC coupled to mass spectrometric detection. All of the steps of the new assay, namely incubation, product separation, and radioactivity counting, are performed in a 96-well format and can be automated. This assay represents a non-HPLC, high-throughput version of the classic ( S )-mephenytoin 4′-hydroxylation assay, which is the most widely used method to assess the potential for CYP2C19 inhibition of new chemical entities.
- Published
- 2007
24. Development and validation of a high-throughput radiometric CYP3A4/5 inhibition assay using tritiated testosterone
- Author
-
José Gutiérrez Pérez, Manuel Sanchez, Maria Verdirame, Annalise Di Marco, Isabella Marcucci, Fernando Pelaez, Ralph Laufer, and Ashok Chaudhary
- Subjects
Tritiated water ,Pharmaceutical Science ,Mass spectrometry ,Hydroxylation ,Tritium ,High-performance liquid chromatography ,chemistry.chemical_compound ,Automation ,Cytochrome P-450 Enzyme System ,Cytochrome P-450 CYP3A ,Cytochrome P-450 Enzyme Inhibitors ,Humans ,Testosterone ,Enzyme Inhibitors ,Radiometry ,Pharmacology ,chemistry.chemical_classification ,Chromatography ,Substrate (chemistry) ,Antibodies, Monoclonal ,Reproducibility of Results ,Water ,Assay sensitivity ,Enzyme ,chemistry ,Microsome ,Microsomes, Liver ,Hydroxytestosterones - Abstract
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 3A4/5 in human liver microsomes is described. In contrast to the conventional testosterone 6beta-hydroxylation assay, the new method does not require high-performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP3A4/5-mediated 6beta-hydroxylation of testosterone labeled with tritium in the 6beta position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. Using commercially available [1,2,6,7-(3)H]testosterone as substrate, we demonstrated that the reaction is NADPH-dependent, and sensitive to CYP3A4/5/5 inhibitors and a CYP3A4/5/5-specific inhibitory monoclonal antibody, but not to inhibitors of or antibodies against other P450 enzymes. The method was further improved by synthesis of testosterone specifically tritiated in the 6beta position, which displayed greatly improved conversion rate with an ensuing increase in assay sensitivity. Competition experiments using tritiated and unlabeled testosterone indicated that CYP3A4/5-mediated 6beta-hydroxylation exhibits positive cooperativity and a modest kinetic isotope effect. IC(50) values for more than 40 structurally diverse chemical inhibitors were not significantly different from those determined in the testosterone 6beta-hydroxylation assay, using HPLC-tandem mass spectrometry analysis. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classical testosterone 6beta-hydroxylation assay, which is the most widely used method to assess the potential for CYP3A4/5 inhibition of new chemical entities.
- Published
- 2004
25. Development and validation of a high-throughput radiometric cyp2c9 inhibition assay using tritiated diclofenac
- Author
-
Isabella Marcucci, Annalise Di Marco, Ralph Laufer, Marina Taliani, and Ashok Chaudhary
- Subjects
Diclofenac ,Tritiated water ,Pharmaceutical Science ,Hydroxylation ,Tritium ,High-performance liquid chromatography ,chemistry.chemical_compound ,Automation ,Theophylline ,medicine ,Humans ,Enzyme Inhibitors ,Radiometry ,Cytochrome P-450 CYP2C9 ,Pharmacology ,Chromatography ,Substrate (chemistry) ,Antibodies, Monoclonal ,Reproducibility of Results ,Water ,stomatognathic diseases ,chemistry ,NIH shift ,Microsome ,Microsomes, Liver ,Aryl Hydrocarbon Hydroxylases ,medicine.drug - Abstract
A rapid and sensitive radiometric assay for assessing the potential of drugs to inhibit cytochrome P450 (P450) 2C9 in human liver microsomes is described. In contrast to the conventional diclofenac 4'-hydroxylation assay, the new method does not require high performance liquid chromatography (HPLC) separation and mass spectrometry. The assay is based on the release of tritium as tritiated water that occurs upon CYP2C9-mediated 4'-hydroxylation of diclofenac labeled with tritium in the 4' position. The radiolabeled product is separated from the substrate using 96-well solid-phase extraction plates. The reaction is NADPH-dependent, and sensitive to CYP2C9 inhibitors and inhibitory monoclonal antibodies, but not to inhibitors of or antibodies against other P450 enzymes. Competition experiments using tritiated and unlabeled diclofenac indicated that CYP2C9-mediated diclofenac 4'-hydroxylation exhibits positive cooperativity and no significant kinetic isotope effect or NIH shift. IC(50) values for 18 structurally diverse chemical inhibitors were not significantly different from those determined in the diclofenac 4'-hydroxylation assay, using HPLC-tandem mass spectrometry. All the steps of the new assay, namely, incubation, product separation, and radioactivity counting, are performed in 96-well format and can be automated. This assay thus represents a high-throughput version of the classic diclofenac 4'-hydroxylation assay, which is one of the most widely used methods to assess the potential for CYP2C9 inhibition of new chemical entities.
- Published
- 2004
26. Penetrating brain injury leads to activation of ciliary neurotrophic factor receptors
- Author
-
K.L. Neitzel, A. John MacLennan, Ralph Laufer, Annalise Di Marco, and Nancy Lee
- Subjects
STAT3 Transcription Factor ,Time Factors ,Facial motor nucleus ,Poison control ,Ciliary neurotrophic factor ,Trigeminal Nuclei ,Stereotaxic Techniques ,Growth factor receptor ,medicine ,Animals ,Ciliary Neurotrophic Factor ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,Motor Neurons ,biology ,business.industry ,General Neuroscience ,Ventral Tegmental Area ,Motor neuron ,Immunohistochemistry ,Growth Inhibitors ,Rats ,DNA-Binding Proteins ,Trigeminal motor nucleus ,medicine.anatomical_structure ,Brain Injuries ,biology.protein ,Trans-Activators ,Signal transduction ,business ,Neuroscience - Abstract
Endogenous injury response mechanisms likely reduce secondary neuronal loss following CNS trauma by activating growth factor receptors. Therefore, it is important to determine which growth factor receptors are activated in vivo by CNS trauma and which signal transduction pathways are affected in which cell types. We present a model of penetrating brain injury utilizing stereotaxic insertion of a fine needle. This procedure can be used to anatomically characterize injury response mechanisms through immediate, local application of pharmacological agents. We find, through immunohistochemistry, that injury of the rat facial motor nucleus leads to activation of STAT3, a neuronal survival factor, in the dendrites, nuclei and cytoplasm of the motor neurons. A similar response was observed with the trigeminal motor nucleus. Use of the ciliary neurotrophic factor (CNTF) receptor antagonist, AADH-CNTF, indicated that the STAT3 activation resulted largely, and perhaps entirely, from injury-induced activation of CNTF receptors.
- Published
- 2004
27. SAR and pharmacokinetic studies on phenethylamide inhibitors of the hepatitis C virus NS3/NS4A serine protease
- Author
-
Ralph Laufer, Jesus Maria Ontoria Ontoria, Ian Stansfield, Odalys Gonzalez-Paz, Antonella Marchetti, Stefania Colarusso, Frank Narjes, Marina Taliani, Savina Malancona, Victor G. Matassa, Annalise Di Marco, Maria Verdirame, and Marco Poma
- Subjects
Serine Proteinase Inhibitors ,Hepacivirus ,Hepatitis C virus ,Clinical Biochemistry ,Pharmaceutical Science ,Viral Nonstructural Proteins ,medicine.disease_cause ,Biochemistry ,Virus ,Flaviviridae ,Structure-Activity Relationship ,Pharmacokinetics ,Drug Discovery ,Phenethylamines ,medicine ,Animals ,Molecular Biology ,chemistry.chemical_classification ,Serine protease ,NS3 ,biology ,Organic Chemistry ,Serine Endopeptidases ,biology.organism_classification ,Virology ,Rats ,Enzyme ,chemistry ,biology.protein ,Molecular Medicine - Abstract
SAR on the phenethylamide 1 (Ki 1.2 μM) in the P2- and the P′-position led to potent inhibitors, one of which showed good exposure and low clearance when administered intramuscularly to rat.
- Published
- 2004
28. Demethylation of radiolabelled dextromethorphan in rat microsomes and intact hepatocytes
- Author
-
Annalise, Di Marco, Dan, Yao, and Ralph, Laufer
- Subjects
Benzoflavones ,Male ,Oxidoreductases, O-Demethylating ,Quinine ,Dextromethorphan ,Methylation ,Quinidine ,Troleandomycin ,Rats ,Rats, Sprague-Dawley ,Inhibitory Concentration 50 ,Kinetics ,Cytochrome P-450 Enzyme System ,Sulfaphenazole ,Hepatocytes ,Microsomes, Liver ,Animals ,Cytochrome P-450 Enzyme Inhibitors ,Carbon Radioisotopes ,Enzyme Inhibitors ,Protein Binding - Abstract
Liver microsomal preparations are routinely used to predict drug interactions that can occur in vivo as a result of inhibition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor) at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems. Intact cells more closely reflect the environment to which CYP substrates and inhibitors are exposed in the liver, and it may therefore be desirable to assess the potential of a drug to cause CYP inhibition in isolated hepatocytes. The objective of this study was to compare the inhibitory potencies of a series of CYP2D inhibitors in rat liver microsomes and hepatocytes. For this, we developed an assay suitable for rapid analysis of CYP-mediated drug interactions in both systems, using radiolabelled dextromethorphan, a well-characterized probe substrate for enzymes of the CYP2D family. Dextromethorphan demethylation exhibited saturable kinetics in rat microsomes and hepatocytes, with apparent Km and Vmax values of 2.1 vs. 2.8 microM and 0.74 nM x min(-1) per mg microsomal protein vs. 0.11 nM x min(-1) per mg cellular protein, respectively. Quinine, quinidine, pyrilamine, propafenone, verapamil, ketoconazole and terfenadine inhibited dextromethorphan O-demethylation in rat liver microsomes and hepatocytes with IC50 values in the low micromolar range. Some of these compounds exhibited biphasic inhibition kinetics, indicative of interaction with more than one CYP2D isoform. Even though no important differences in inhibitory potencies were observed between the two systems, most inhibitors, including quinine and quinidine, displayed 2-3-fold lower IC50 in hepatocytes than in microsomes. The cell-associated concentrations of quinine and quinidine were found to be significantly higher than those in the extracellular medium, suggesting that intracellular accumulation may potentiate the effect of these compounds. Studies of CYP inhibition in intact hepatocytes may be warranted for compounds that concentrate in the liver as the result of cellular transport.
- Published
- 2003
29. Blockade of endogenous neurotrophic factors prevents the androgenic rescue of rat spinal motoneurons
- Author
-
Karen M. Gingras, Nancy G. Forger, Jun Xu, Annalise Di Marco, and Lynn Bengston
- Subjects
Testosterone propionate ,medicine.medical_specialty ,Programmed cell death ,Cell Survival ,Recombinant Fusion Proteins ,Cell Count ,Tropomyosin receptor kinase B ,Receptors, Nerve Growth Factor ,Biology ,Perineum ,Tropomyosin receptor kinase C ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Neurotrophic factors ,Internal medicine ,Reflex ,medicine ,Animals ,Receptor, trkB ,Receptor, trkC ,Testosterone ,Nerve Growth Factors ,ARTICLE ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,Motor Neurons ,Sex Characteristics ,Cell Death ,General Neuroscience ,Lumbosacral Region ,Rats ,Endocrinology ,chemistry ,nervous system ,Animals, Newborn ,Spinal Cord ,Immunoglobulin G ,biology.protein ,Androgens ,Ciliary neurotrophic factor receptor ,Female ,Neurotrophin ,Signal Transduction - Abstract
Target-derived neurotrophic factors are assumed to regulate motoneuron cell death during development but remain unspecified. Motoneuron cell death in the spinal nucleus of the bulbocavernosus (SNB) of rats extends postnatally and is controlled by androgens. We exploited these features of the SNB system to identify endogenously produced trophic factors regulating motoneuron survival. Newborn female rat pups were treated with the androgen, testosterone propionate, or the oil vehicle alone. In addition, females received trophic factor antagonists delivered either into the perineum (the site of SNB target muscles) or systemically. Fusion molecules that bind and sequester the neurotrophins (trkA-IgG, trkB-IgG, and trkC-IgG) were used to block activation of neurotrophin receptors, and AADH-CNTF was used to antagonize signaling through the ciliary neurotrophic factor receptor-α (CNTFRα). An acute blockade of trkB, trkC, or CNTFRα prevented the androgenic sparing of SNB motoneurons when antagonists were delivered to the perineum. Trophic factor antagonists did not significantly reduce SNB motoneuron number when higher doses were injected systemically. These findings demonstrate a requirement for specific, endogenously produced trophic factors in the androgenic rescue of SNB motoneurons and further suggest that trophic factor interactions at the perineum play a crucial role in masculinization of this neural system.
- Published
- 2001
30. Centrally mediated inhibition of local inflammation by ciliary neurotrophic factor
- Author
-
Maria Romano, Pia Villa, Marina Sironi, Paolo Fruscella, Annalise Di Marco, Isabelle Gloaguen, Cristina Meazza, Ralph Laufer, Jean D. Sipe, and Pietro Ghezzi
- Subjects
medicine.medical_specialty ,Hypothalamo-Hypophyseal System ,Recombinant Fusion Proteins ,Immunology ,Pituitary-Adrenal System ,Inflammation ,Endogeny ,Nerve Tissue Proteins ,Receptors, Nerve Growth Factor ,Ciliary neurotrophic factor ,Carrageenan ,chemistry.chemical_compound ,Mice ,Endocrinology ,Corticosterone ,Internal medicine ,medicine ,Animals ,Edema ,Ciliary Neurotrophic Factor ,Interleukin 6 ,Receptor, Ciliary Neurotrophic Factor ,Injections, Intraventricular ,Serum Amyloid A Protein ,biology ,Endocrine and Autonomic Systems ,business.industry ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,Air ,Anti-Inflammatory Agents, Non-Steroidal ,Antagonist ,Receptor Protein-Tyrosine Kinases ,Adrenalectomy ,Exudates and Transudates ,Neurology ,chemistry ,Gene Expression Regulation ,Injections, Intravenous ,biology.protein ,Tumor necrosis factor alpha ,medicine.symptom ,business - Abstract
Since ciliary neurotrophic factor (CNTF) inhibits the production of TNF and activates the hypothalamus-pituitary-adrenal axis (HPAA), we investigated whether CNTF can produce antiinflammatory actions and whether it may act through a central mechanism, using the murine air pouch model of inflammation. In this model, inflammation is evaluated by measuring the induction of TNF and IL-6 as well as cell recruitment in the pouch fluid 24 h after carrageenan. Intracerebroventricular injection, but not intravenous or local injection of CNTF markedly inhibited inflammation. This was associated with high serum corticosterone levels, and antiinflammatory action was not observed in adrenalectomized mice, indicating that an intact HPAA is required. A CNTF receptor antagonist increased carrageenan inflammation, suggesting that endogenous CNTF might have a centrally mediated antiinflammatory role.
- Published
- 1998
31. Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance
- Author
-
Patrizia Costa, Rita Graziani, Charles Rosenblum, Ralph Laufer, Anna Demartis, Lex H.T. Van der Ploeg, Isabelle Gloaguen, Riccardo Cortese, Domenico Lazzaro, Gennaro Ciliberto, Annalise Di Marco, Giacomo Paonessa, and Fang Chen
- Subjects
Blood Glucose ,Leptin ,Male ,medicine.medical_treatment ,Mice, Obese ,Ciliary neurotrophic factor ,Mice ,Neuroblastoma ,Hyperinsulinemia ,Insulin ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,Neurons ,Multidisciplinary ,Leptin Deficiency ,digestive, oral, and skin physiology ,Brain ,Biological Sciences ,Recombinant Proteins ,DNA-Binding Proteins ,Cytokine ,Receptors, Leptin ,hormones, hormone substitutes, and hormone antagonists ,medicine.medical_specialty ,Nerve Tissue Proteins ,Receptors, Cell Surface ,Receptors, Nerve Growth Factor ,Biology ,Hybrid Cells ,Motor Activity ,Cell Line ,Mice, Inbred AKR ,Internal medicine ,medicine ,Animals ,Humans ,Point Mutation ,Ciliary Neurotrophic Factor ,Nerve Growth Factors ,Obesity ,Leptin receptor ,Body Weight ,Arcuate Nucleus of Hypothalamus ,Proteins ,medicine.disease ,Dietary Fats ,Grooming ,Mice, Inbred C57BL ,Endocrinology ,Nerve growth factor ,Diabetes Mellitus, Type 2 ,biology.protein ,Carrier Proteins - Abstract
Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob / ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db / db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.
- Published
- 1997
32. Agonistic and antagonistic variants of ciliary neurotrophic factor (CNTF) reveal functional differences between membrane-bound and soluble CNTF α-receptor
- Author
-
Isabelle Gloaguen, Isabella Saggio, Giacomo Paonessa, Rita Graziani, Anna Demartis, Annalise Di Marco, and Ralph Laufer
- Subjects
Agonist ,Receptor complex ,medicine.medical_specialty ,Leukemia Inhibitory Factor Receptor alpha Subunit ,Receptors, OSM-LIF ,medicine.drug_class ,Protein subunit ,receptor ,Leukemia inhibitory factor receptor ,Nerve Tissue Proteins ,Receptors, Nerve Growth Factor ,Biology ,Ciliary neurotrophic factor ,Leukemia Inhibitory Factor ,Biochemistry ,Antigens, CD ,Internal medicine ,medicine ,Cytokine Receptor gp130 ,CNTF ,Humans ,Ciliary Neurotrophic Factor ,Receptors, Cytokine ,Receptor ,Receptor, Ciliary Neurotrophic Factor ,membrane ,Molecular Biology ,Lymphokines ,Membrane Glycoproteins ,Dose-Response Relationship, Drug ,Interleukin-6 ,Membrane Proteins ,Receptor Protein-Tyrosine Kinases ,Transfection ,Cell Biology ,Glycoprotein 130 ,Growth Inhibitors ,Recombinant Proteins ,Cell biology ,Endocrinology ,Models, Chemical ,Solubility ,Mutation ,biology.protein ,Biological Assay ,Protein Binding ,Signal Transduction - Abstract
Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.
- Published
- 1997
33. Subject Index Vol. 4, 1997
- Author
-
Yehuda Pollak, Sossiena Demissie, Pietro Ghezzi, Helena Haberstock-Debic, Cesare Mancuso, Werner De Potter, V.K. Singh, S. Biswas, W. Haq, Ruth M. Williams, Tetsuya Ando, Ashley B. Grossman, Pia Villa, Eleonora Wechsung, M.Y. Khan, Annalise Di Marco, Paolo Preziosi, Carolyn F. Rogers, Pierluigi Navarra, Jean D. Sipe, Raz Yirmiya, Ágota Kovács, Ron H. Stead, Valeria Rettori, Mayumi Kimura, Raymond N. Hiramoto, K. Bajpai, Isabelle Gloaguen, Hans-Rudolf Berthoud, Hrvoje Banfic, Elisabeth E. Ban, Erzsé, nos Fehér, K.B. Mathur, Nancy S. Hiramoto, bet Fehér, Ralph Laufer, Wim J. Stevens, Jianping Wang, Ronit Avitsur, Paul K.Y. Wong, Wen H. Yu, Cristina Meazza, Samuel M. McCann, Adrian J. Dunn, Maria Romano, W.S. Lynn, Katalin Gallatz, Paolo Fruscella, Luc S. De Clerck, Marina Sironi, and Vithal K. Ghanta
- Subjects
Endocrinology ,Index (economics) ,Neurology ,Endocrine and Autonomic Systems ,Immunology ,Statistics ,Subject (documents) ,Mathematics - Published
- 1997
Catalog
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