Publications by authors named "Daniela Melchiorri"

33 Publications

Combinations in the first-line treatment of patients with advanced/metastatic renal cell cancer: regulatory aspects.

ESMO Open 2020 08;5(4)

European Medicines Agency, Amsterdam, The Netherlands.

The therapeutic landscape in the treatment of advanced/metastatic renal cell cancer has evolved over the last 2 years with the advent of immune checkpoint inhibitors. In 2018 and 2019, marketing authorisations valid throughout the European Union were issued for nivolumab and ipilimumab dual checkpoint inhibition and pembrolizumab or avelumab in combination with the tyrosine kinase inhibitor axitinib. These applications presented numerous regulatory challenges.In this paper, we summarise the main regulatory considerations, originating from the assessment of the dossiers submitted from the applicants for the three combinations. The regulatory issues are grouped in four sections: clinical pharmacology, efficacy, biomarkers and safety. In each section, we describe the issues raised during the regulatory evaluation performed by the Committee for Medicinal Products for Human Use (CHMP) assessors. The CHMP assessments determine whether the medicines concerned meet the necessary quality, safety and efficacy requirements, and whether the benefit-risk balance is positive.In summary, although the overall benefit-risk was considered positive for the three combinations, the immaturity of the outcome data and the absence of long-term safety data remain issues to be addressed. Postauthorisation efficacy studies have been required to confirm the effects of the new combinations.
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http://dx.doi.org/10.1136/esmoopen-2020-000856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451283PMC
August 2020

Blinatumomab for Acute Lymphoblastic Leukemia: The First Bispecific T-Cell Engager Antibody to Be Approved by the EMA for Minimal Residual Disease.

Oncologist 2020 04 14;25(4):e709-e715. Epub 2019 Nov 14.

European Medicines Agency, Amsterdam, The Netherlands.

On November 15, 2018, the Committee for Medicinal Products for Human Use (CHMP) recommended the extension of indication for blinatumomab to include the treatment of adults with minimal residual disease (MRD) positive B-cell precursor acute lymphoblastic leukemia (ALL). Blinatumomab was authorized to treat relapsed or refractory B-precursor ALL, and the change concerned an extension of use. On March 29, 2018, the U.S. Food and Drug Administration (FDA) granted accelerated approval to blinatumomab to treat both adults and children with B-cell precursor ALL who are in remission but still have MRD. On July 26, 2018, the CHMP had originally adopted a negative opinion on the extension. The reason for the initial refusal was that although blinatumomab helped to reduce the amount of residual cancer cells in many patients, there was no strong evidence that it led to improved survival. During the re-examination, the CHMP consulted the scientific advisory group. The CHMP agreed with the expert group's conclusion that, although there was no strong evidence of patients living longer, the available data from the main study (MT103-203) indicated a good durable response to blinatumomab, with an overall complete response rate for the primary endpoint full analysis set (defined as all subjects with an Ig or T-cell receptor polymerase chain reaction MRD assay with the minimum required sensitivity of 1 × 10 at central lab established at baseline [n = 113]) as 79.6% (90/113; 95% confidence interval, 71.0-86.6), with a median time to complete MRD response of 29.0 days (range, 5-71). Therefore, the CHMP concluded that the benefits of blinatumomab outweigh its risks and recommended granting the change to the marketing authorization. The Committee for Orphan Medicinal Products, following reassessment, considered that significant benefit continued to be met and recommended maintaining the orphan designation and thus 10 years market exclusivity (the Orphan Designation is a legal procedure that allows for the designation of a medicinal substance with therapeutic potential for a rare disease, before its first administration in humans or during its clinical development). The marketing authorization holder for this medicinal product is Amgen Europe B.V. IMPLICATIONS FOR PRACTICE: Immunotherapy with blinatumomab has excellent and sustainable results, offering new hope for patients with minimal residual disease-positive acute lymphoblastic leukemia, a disease with poor prognosis. New recommendations and change of practice for treatment of this patient group are detailed.
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http://dx.doi.org/10.1634/theoncologist.2019-0559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160312PMC
April 2020

Revocation of the conditional marketing authorisation of a cancer medicine: The olaratumab experience.

Eur J Cancer 2019 12 23;123:25-27. Epub 2019 Oct 23.

European Medicines Agency, the Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.ejca.2019.09.020DOI Listing
December 2019

European Medicines Agency review of ixazomib (Ninlaro) for the treatment of adult patients with multiple myeloma who have received at least one prior therapy.

ESMO Open 2019 8;4(5):e000570. Epub 2019 Sep 8.

European Medicines Agency, Amsterdam, The Netherlands.

On 21 November 2016, the European Commission issued a marketing authorisation valid throughout the European Union for ixazomib in combination with lenalidomide and dexamethasone for the treatment of adult patients with multiple myeloma who have received at least one prior therapy. Ixazomib was evaluated in one, randomised, double-blind, phase III study comparing ixazomib plus lenalidomide and dexamethasone (n=360; ixazomib arm) versus placebo plus lenalidomide and dexamethasone (n=362; placebo arm) in adult patients with relapsed and/or refractory multiple myeloma who had received at least one prior therapy. The median progression-free survival (PFS) in the intent-to-treat population was 20.6 months in patients treated with ixazomib compared with 14.7 months for patients in the placebo arm (stratified HR=0.742, 95% CI 0.587 to 0.939, stratified p-value=0.012). The most frequently reported adverse reactions (≥20%) within the ixazomib and placebo arms were diarrhoea (42% vs 36%), constipation (34% vs 25%), thrombocytopaenia (28% vs 14%), peripheral neuropathy (28% vs 21%), nausea (26% vs 21%), peripheral oedema (25% vs 18%), vomiting (22% vs 11%) and back pain (21% vs 16%). The scientific review concluded that the gain in PFS of 5.9 months observed with ixazomib was considered clinically meaningful. Concerning the possible uncertainty about the magnitude of the effect, this uncertainty was acceptable given the favourable toxicity profile, and considering that ixazomib is the first agent to allow oral triple combination therapy in this patient population which represents a therapeutic innovation in terms of convenience for patients. Therefore, the benefit-risk for ixazomib in combination with lenalidomide and dexamethasone was considered positive, although the efficacy evidence was not as comprehensive as normally required.
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http://dx.doi.org/10.1136/esmoopen-2019-000570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735670PMC
September 2019

Optimising bench science to withstand regulatory scrutiny.

Pharmacol Res 2019 01 12;139:491-493. Epub 2018 Oct 12.

Malta Medicines Authority, Sir Temi Żammit Buildings, MaltaLife Sciences Park, San Ġwann SĠN 3000, Malta; Department of Biology, School of Pharmacy, University of Tor Vergata, Rome, Italy.

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http://dx.doi.org/10.1016/j.phrs.2018.10.014DOI Listing
January 2019

Targeting metabotropic glutamate receptors in the treatment of primary brain tumors.

Curr Opin Pharmacol 2018 02 8;38:59-64. Epub 2018 Mar 8.

Department of Physiology and Pharmacology "V. Erspamer", Sapienza University of Rome, Italy; IRCCS Neuromed, Pozzilli, Italy.

In spite of the recent advancement in the molecular characterization of malignant gliomas and medulloblastomas, the treatment of primary brain tumors remains suboptimal. The use of small molecule inhibitors of intracellular signaling pathways, inhibitors of angiogenesis, and immunotherapic agents is limited by systemic adverse effects, limited brain penetration, and, in some cases, lack of efficacy. Thus, adjuvant chemo-therapy and radiotherapy still remain the gold standard in the treatment of grade-IV astrocytoma (glioblastoma multiforme) and medulloblastoma. We review evidence that supports the development of mGlu3 receptor antagonists as add-on drugs in the treatment of malignant gliomas. These drugs appear to display pleiotropic effect on tumor cells, affecting proliferation, differentiation, and response to chemotherapy. mGlu1 and mGlu4 receptors could also be targeted by potential anticancer agents in the treatment of malignant gliomas and medulloblastoma, but extensive research is required for target validation.
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http://dx.doi.org/10.1016/j.coph.2018.02.005DOI Listing
February 2018

European Medicines Agency approval summary: Zaltrap for the treatment of patients with oxaliplatin-resistant metastatic colorectal cancer.

ESMO Open 2017 2;2(2):e000190. Epub 2017 May 2.

European Medicines Agency, London, UK.

On 1 February 2013, a marketing authorisation valid throughout the European Union was issued for aflibercept (Zaltrap) in combination with irinotecan/5-fluorouracil/folinic acid chemotherapy for the treatment of adults with metastatic colorectal cancer resistant to or progressive after an oxaliplatin-containing regimen. Aflibercept is a recombinant fusion protein which blocks the activation of vascular endothelial growth factor (VEGF) receptors and the proliferation of endothelial cells, acting as a soluble decoy receptor that binds to VEGF-A with higher affinity than its native receptors, as well as placental growth factor and VEGF-B. The use of aflibercept was studied in a randomised, double-blind, placebo-controlled phase III study, in patients with metastatic colorectal cancer (mCRC) who had previously been treated with an oxaliplatin-based treatment with or without prior bevacizumab. Aflibercept (n=612) was compared with placebo (n=614), both in combination with FOLFIRI (infusional fluorouracil, leucovorin and irinotecan). The primary endpoint of the study was overall survival (OS). The median OS in the intent-to-treat population was 13.5 months in subjects treated with aflibercept compared with 12.1 months for subjects in the control arm (stratified HR=0.817, 95% CI 0.714 to 0.935, stratified pvalue=0.0032). The frequency of adverse events was higher in the aflibercept arm compared with the placebo arm, reflecting the toxicity profile of anti-VEGF agents in combination with chemotherapy. This paper is based on the scientific review of the application leading to approval of aflibercept in the EU. The detailed scientific assessment report and product information for this product are available on the European Medicines Agency website (http://www.ema.europa.eu). NCT00561470, Results.
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http://dx.doi.org/10.1136/esmoopen-2017-000190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519808PMC
May 2017

Licensing of Generic Medicines: Are There Any Challenges Left? A Pharmaceutical Regulatory Perspective.

Sci Pharm 2014 Oct-Dec;82(4):847-56. Epub 2014 May 22.

Department of Physiology and Pharmacology, University of Rome "Sapienza", Rome, Italy.

When an innovative product (innovator) is not covered anymore by intellectual property rights, cheaper equivalent medicinal products (generic products) may be marketed and used in clinical practice. The regulation of generic products is well-established, and is primarily based on standard rules for quality, therapeutic equivalence requirements (the latter in most instances proven through a bioequivalence study), and safety data for the innovator. The extensive experience from bringing generic products to the market over the last decades allows the conclusion that they are well-accepted and provide a useful alternative option for cost-effective pharmacotherapy. While supporting this conclusion, there are a number of issues to be considered during the assessment of a generic product application. Six scenarios are described in total, from an efficacy and a safety perspective, where potential concerns with the current regulatory standards could arise in the approval of generic products. We also propose solutions to these scenarios in order to foster debate on these issues.
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http://dx.doi.org/10.3797/scipharm.1312-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475802PMC
July 2015

Implementing quality by design for biotech products: Are regulators on track?

MAbs 2015 ;7(3):451-5

a National Center for Immunobiologicals Control and Evaluation (CRIVIB); Istituto Superiore di Sanità ; Rome , Italy.

Quality by design (QbD) is an innovative approach to drug development that has started to be implemented into the regulatory framework, but currently mainly for chemical drugs. The recent marketing authorization of the first monoclonal antibody developed using extensive QbD concepts in the European Union paves the way for future further regulatory approvals of complex products employing this cutting-edge technological concept. In this paper, we report and comment on insights and lessons learnt from the non-public discussions in the European Medicines Agency's Biologicals Working Party and Committee for Medicinal Products for Human Use on the key issues during evaluation related to the implementation of an extensive QbD approach for biotechnology-derived medicinal products. Sharing these insights could prove useful for future developments in QbD for biotech products in general and monoclonal antibodies in particular.
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http://dx.doi.org/10.1080/19420862.2015.1023058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623250PMC
March 2016

Reflections on Decisions Made on the Well-Established Use of Medicinal Products by EU Regulators and the ECJ.

Sci Pharm 2014 Jul-Sep;82(3):541-54. Epub 2014 Mar 24.

Department of Physiology and Pharmacology, University of Rome "Sapienza", Rome, Italy.

Background: In the European Union (EU), a medicinal product needs a marketing authorization (MA) to be placed on the market. The EU's medicinal products' legislative framework allows for a reduced application for medicines outside their data exclusivity. One such type of application is the well-established use (WEU) medicinal product application (i.e. bibliographic applications). Recently, these MA applications have been subject to arbitration procedures at the European Medicines Agency's Committee for Medicinal Products for Human Use (CHMP) because of disagreements between member states during the authorisation process. This paper reflects on these cases and highlights their potential impact on future WEU applications.

Methods: Decisions adopted by the European Commission on WEU applications between 2009 and 2012 were identified from the EU Community Register on medicinal products for human use. Subsequently, decisions were reviewed to understand the potential serious risk to public health (PSRPH) that EU regulators raised during MA application procedures.

Results: Four decisions were adopted by the EU commission between 2009 and 2012. Three followed disagreements between member states on PSRPH grounds. One decision was the outcome of a centralised marketing authorisation application. Six key messages were identified from the four cases reviewed and presented.

Conclusion: A guideline on WEU to implement the technical specifications to fulfil Annex I of Directive 2001/83/EC for MA applications is not available. Thus, reflections on recent decisions on WEU applications provide scientific direction to the industry as well as the medicinal product regulators on the documentation required to successfully file and obtain a WEU MA.
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http://dx.doi.org/10.3797/scipharm.1402-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4318218PMC
April 2015

The European Medicines Agency Review of Pertuzumab for the treatment of adult patients with HER2-positive metastatic or locally recurrent unresectable breast cancer: summary of the scientific assessment of the committee for medicinal products for human use.

Oncologist 2014 Jul 13;19(7):766-73. Epub 2014 Jun 13.

European Medicines Agency, London, United Kingdom; Danish Health and Medicines Authority, København, Denmark; Department of Physiology and Pharmacology, Università di Roma "La Sapienza," Rome, Italy; Agenzia Italiana del Farmaco, Rome, Italy; Läkemedelsverket, Medical Products Agency, Uppsala, Sweden; Karolinska Institutet, Stockholm, Sweden; The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Pertuzumab is a recombinant humanized monoclonal antibody that specifically targets the extracellular dimerization domain (subdomain II) of HER2. Based on the positive opinion from the European Medicines Agency (EMA) on March 4, 2013, a marketing authorization valid throughout the European Union (EU) was issued for pertuzumab (Perjeta) for use in combination with trastuzumab and docetaxel for the treatment of adult patients with HER2-positive metastatic or locally recurrent unresectable breast cancer who have not received previous anti-HER2 therapy or chemotherapy for their metastatic disease. The demonstration of clinical benefit for pertuzumab was based on a single, phase III, randomized, double-blind, placebo-controlled trial comparing the efficacy and safety of pertuzumab plus trastuzumab plus docetaxel versus placebo plus trastuzumab plus docetaxel in previously untreated patients with locally advanced or metastatic HER2-positive breast cancer. In the primary analysis, median progression-free survival was 18.5 months in the pertuzumab group compared with 12.4 months in the placebo group (hazard ratio [HR]: 0.62; 95% confidence interval [CI]: 0.51-0.75; p < .0001). For the secondary endpoints, overall survival (HR: 0.66; 95% CI: 0.52-0.84; p = .0008) and objective response rate (80.2% vs. 69.3%) were also favored in the pertuzumab group. Toxicity was similar between groups except for higher incidence of diarrhea, rash, mucosal inflammation, dry skin, and neutropenia for pertuzumab compared with placebo. This paper summarizes the scientific review of the application leading to approval in the EU. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the EMA website (http://www.ema.europa.eu).
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http://dx.doi.org/10.1634/theoncologist.2013-0348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077440PMC
July 2014

Regulatory evaluation of Glybera in Europe - two committees, one mission.

Nat Rev Drug Discov 2013 Sep 19;12(9):719. Epub 2013 Aug 19.

Department of Physiology and Pharmacology, the University of Rome "Sapienza", P.le Aldo Moro 5, 00185 Rome, Italy; member of the Committee for Medicinal Products for Human Use (CHMP) at the European Medicines Agency (EMA), 7 Westferry Circus, Canary Wharf, London E14 4HB, UK.

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http://dx.doi.org/10.1038/nrd3835-c1DOI Listing
September 2013

More on influenza vaccine in young children.

N Engl J Med 2012 06;366(26):2528; author reply 2528-9

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http://dx.doi.org/10.1056/NEJMc1205643DOI Listing
June 2012

Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage.

PLoS One 2011 Jan 27;6(1):e16447. Epub 2011 Jan 27.

Department of Physiology and Pharmacology, University Sapienza, Roma, Italy.

The identification of mechanisms that mediate stress-induced hippocampal damage may shed new light into the pathophysiology of depressive disorders and provide new targets for therapeutic intervention. We focused on the secreted glycoprotein Dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway, involved in neurodegeneration. Mice exposed to mild restraint stress showed increased hippocampal levels of Dkk-1 and reduced expression of β-catenin, an intracellular protein positively regulated by the canonical Wnt signalling pathway. In adrenalectomized mice, Dkk-1 was induced by corticosterone injection, but not by exposure to stress. Corticosterone also induced Dkk-1 in mouse organotypic hippocampal cultures and primary cultures of hippocampal neurons and, at least in the latter model, the action of corticosterone was reversed by the type-2 glucocorticoid receptor antagonist mifepristone. To examine whether induction of Dkk-1 was causally related to stress-induced hippocampal damage, we used doubleridge mice, which are characterized by a defective induction of Dkk-1. As compared to control mice, doubleridge mice showed a paradoxical increase in basal hippocampal Dkk-1 levels, but no Dkk-1 induction in response to stress. In contrast, stress reduced Dkk-1 levels in doubleridge mice. In control mice, chronic stress induced a reduction in hippocampal volume associated with neuronal loss and dendritic atrophy in the CA1 region, and a reduced neurogenesis in the dentate gyrus. Doubleridge mice were resistant to the detrimental effect of chronic stress and, instead, responded to stress with increases in dendritic arborisation and neurogenesis. Thus, the outcome of chronic stress was tightly related to changes in Dkk-1 expression in the hippocampus. These data indicate that induction of Dkk-1 is causally related to stress-induced hippocampal damage and provide the first evidence that Dkk-1 expression is regulated by corticosteroids in the central nervous system. Drugs that rescue the canonical Wnt pathway may attenuate hippocampal damage in major depression and other stress-related disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0016447PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3029367PMC
January 2011

Interaction between Ephrins and mGlu5 metabotropic glutamate receptors in the induction of long-term synaptic depression in the hippocampus.

J Neurosci 2010 Feb;30(8):2835-43

Department of Physiology and Pharmacology, University Sapienza, Rome, Italy. mail:

We applied the group-I metabotropic glutamate (mGlu) receptor agonist, 3,5-dihydroxyphenylglycine (DHPG), to neonatal or adult rat hippocampal slices at concentrations (10 microM) that induced a short-term depression (STD) of excitatory synaptic transmission at the Schaffer collateral/CA1 synapses. DHPG-induced STD was entirely mediated by the activation of mGlu5 receptors because it was abrogated by the mGlu5 receptor antagonist, MPEP [2-methyl-6-(phenylethynyl)pyridine], but not by the mGlu1 receptor antagonist, CPCCOEt [7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester]. Knowing that ephrin-Bs functionally interact with group-I mGlu receptors (Calò et al., 2005), we examined whether pharmacological activation of ephrin-Bs could affect DHPG-induced STD. We activated ephrin-Bs using their cognate receptor, EphB1, under the form of a preclustered EphB1/Fc chimera. Addition of clustered EphB1/Fc alone to the slices induced a small but nondecremental depression of excitatory synaptic transmission, which differed from the depression induced by 10 microM DHPG. Surprisingly, EphB1/Fc-induced synaptic depression was abolished by MPEP (but not by CPCCOEt) suggesting that it required the endogenous activation of mGlu5 receptors. In addition, coapplication of DHPG and EphB1/Fc, resulted in a large and nondecremental long-term depression. The effect of clustered EphB1/Fc was specific because it was not mimicked by unclustered EphB1/Fc or clustered EphA1/Fc. These findings raise the intriguing possibility that changes in synaptic efficacy mediated by mGlu5 receptors are under the control of the ephrin/Eph receptor system, and that the neuronal actions of ephrins can be targeted by drugs that attenuate mGlu5 receptor signaling.
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http://dx.doi.org/10.1523/JNEUROSCI.4834-09.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633947PMC
February 2010

Differential activation of the calcium/protein kinase C and the canonical beta-catenin pathway by Wnt1 and Wnt7a produces opposite effects on cell proliferation in PC12 cells.

J Neurochem 2008 Mar 5;104(6):1588-98. Epub 2007 Nov 5.

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Rome, Italy.

We examined the effect of Wnt1 and Wnt7a on cell proliferation using undifferentiated PC12 cells, which originate from the neural crest and are widely employed as a neuronal cell model. Heterologous expression of Wnt1 enhanced [3H]thymidine incorporation and expression of cyclin D1 and cylin E in PC12 cells. Opposite effects were observed in PC12 cells expressing Wnt7a. Searching for the mechanisms underlying the opposite effects of Wnt1 and Wnt7a on PC12 cell proliferation, we examined the activation of the canonical beta-catenin/T-cell-lymphoid enhancer-binding protein transcription factor pathway and the 'calcium pathway' by co-transfecting the cells with a reporter gene controlled by either T-cell-lymphoid enhancer-binding protein transcription factor or the calcium-activated transcription factor, NFAT. Wnt1 and Wnt7a activated both pathways, but to a different extent. While Wnt1 preferentially activated the calcium pathway, Wnt7a mainly activated the canonical pathway. Pharmacological inhibition of protein kinase C, which is a component of the calcium pathway, abrogated the increase in cell proliferation induced by Wnt1 without affecting the antiproliferative action of Wnt7a. The action of Wnt7a was instead occluded by lithium ions, which mimic the activation of the canonical pathway, and was largely reduced by Dickkopf-1, which acts as an inhibitor of the canonical pathway. In addition, expression of a constitutively active mutant of beta-catenin potently activated the canonical Wnt pathway and reduced [3H]thymidine incorporation. These data challenge the view that the canonical Wnt pathway invariably supports cell growth and suggest that, at least in PC12 cells, cell proliferation is regulated by the balance between the calcium/protein kinase C pathway and the canonical pathway.
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http://dx.doi.org/10.1111/j.1471-4159.2007.05111.xDOI Listing
March 2008

Metabotropic glutamate receptors in stem/progenitor cells.

Neuropharmacology 2007 Sep 29;53(4):473-80. Epub 2007 Jun 29.

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Rome, Italy.

Functional mGlu receptor subtypes are found in stem/progenitor cells, and regulate proliferation, differentiation, and survival of these cells. Activation of mGlu5 receptors supports self-renewal of embryonic stem cells, which are pluripotent cells isolated from the blastocyst capable of generating all the body's cell lineages, including germ cells. Differentiation of embryonic stem cells into embryoid bodies is associated with the induction of mGlu4 receptors, the activation of which drives cell differentiation towards the mesoderm and endoderm lineages. Different mGlu receptor subtypes, mGlu3 and mGlu5 receptors in particular, are found in neural stem cells (stem cells resident in the CNS that give rise to neurons, astrocytes or oligodendrocytes) isolated from the developing brain or from regions of persistent neurogenesis of the adult brain (e.g. the subventricular zone lining the wall of the lateral ventricles). The evidence that activation of mGlu3 and mGlu5 receptors stimulates proliferation of these cells is particularly interesting because of the similarities between neural stem cells and putative cancer stem cells that support the growth of malignant gliomas. A link among mGlu receptors, stem cells and cancer is supported by the finding that mGlu4 receptors are expressed by cerebellar granule cell neuroprogenitors, which are the putative cells of origin of medulloblastomas. The study of mGlu receptors in stem/progenitor cells has potential applications in the optimisation of protocols of cell expansion and differentiation aimed at cell replacement strategies, and may gain new insights into the pathophysiology of neurodevelopmental disorders and brain tumours.
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http://dx.doi.org/10.1016/j.neuropharm.2007.05.031DOI Listing
September 2007

Nanomolar concentrations of anabolic-androgenic steroids amplify excitotoxic neuronal death in mixed mouse cortical cultures.

Brain Res 2007 Aug 10;1165:21-9. Epub 2007 Jul 10.

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Italy.

The use of anabolic-androgenic steroids (AASs) in the world of sport has raised a major concern for the serious, sometimes life-threatening, side effects associated with these drugs. Most of the CNS effects are of psychiatric origin, and whether or not AASs are toxic to neurons is yet unknown. We compared the effect of testosterone with that of the AASs, 19-nortestosterone (nandrolone), stanozolol, and gestrinone, on excitotoxic neuronal death induced by N-methyl-d-aspartate (NMDA) in primary cultures of mouse cortical cells. In the most relevant experiments, steroids were applied to the cultures once daily during the 4 days preceding the NMDA pulse. Under these conditions, testosterone amplified excitotoxic neuronal death only at very high concentrations (10 muM), whereas it was protective at concentrations of 10 nM and inactive at intermediate concentrations. Low concentrations of testosterone became neurotoxic in the presence of the aromatase inhibitors, i.e. anastrozole and aminoglutethimide, suggesting that the intrinsic toxicity of testosterone was counterbalanced by its aromatization into 17beta-estradiol. As opposed to testosterone, nortestosterone, stanozolol and gestrinone amplified NMDA toxicity at nanomolar concentrations; their action was insensitive to aromatase inhibitors, but was abrogated by the androgen receptor antagonist, flutamide. None of the AASs were toxic in the absence of NMDA. These data suggest that AASs increase neuronal vulnerability to an excitotoxic insult and may therefore facilitate neuronal death associated with acute or chronic CNS disorders.
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http://dx.doi.org/10.1016/j.brainres.2007.06.047DOI Listing
August 2007

Metabotropic glutamate receptors: beyond the regulation of synaptic transmission.

Psychoneuroendocrinology 2007 Aug 24;32 Suppl 1:S40-5. Epub 2007 Jul 24.

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Italy.

Metabotropic glutamate (mGlu) receptors are G-protein coupled receptors activated by glutamate, the major excitatory neurotransmitter of the CNS. A growing body of evidence suggests that the function of mGlu receptors is not restricted to the regulation of synaptic transmission. mGlu receptors are expressed in a variety of peripheral cells, including inter alia hepatocytes, pancreatic cells, osteoblasts and immune cells. Within the immunological synapses, mGlu receptors expressed by T cells might contribute to the vast array of signals generated by the antigen-presenting cells. mGlu receptors are also found in embryonic and neural stem cells. This suggests their involvement in the pathophysiology of brain tumors, which likely originates from cancer stem cells similar to neural stem cells. Ligands of mGlu3 and mGlu4 receptors are potential candidates for the experimental treatment of malignant gliomas and medulloblastomas, respectively.
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http://dx.doi.org/10.1016/j.psyneuen.2007.04.015DOI Listing
August 2007

An old threat in a new setting: High prevalence of silicosis among jewelry workers.

Am J Ind Med 2007 Aug;50(8):577-83

Section of Occupational Medicine, Respiratory Diseases and Toxicology, University of Perugia, Perugia, Italy.

Background: Silicosis is caused by inhaling free crystalline silica. Few case reports have addressed the risk of silicosis in the jewelry trade where chalk molds containing a high percentage of silica are used in casting. We conducted a cross-sectional study involving 100 goldsmiths exposed to silica.

Methods: All workers replied to a questionnaire and underwent a clinical examination, pulmonary function tests, a chest X-ray and a high-resolution CT scan.

Results: High-resolution CT visualized signs of silicosis in 23 cases, confirmed by standard chest X-rays in 10. In the 23 workers with CT evidence of silicosis Total Lung Capacity, FEV1 and the Lung Diffusing Capacity did not differ from the workers without the disease. Pulmonary function tests did not correlate with silica exposure.

Conclusion: In this study we demonstrate that use of chalk molds in casting in jewelry causes silicosis. The composition of the dust could be responsible of the high prevalence observed.
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http://dx.doi.org/10.1002/ajim.20490DOI Listing
August 2007

Anorexia in hemodialysis patients: the possible role of des-acyl ghrelin.

Am J Nephrol 2007 8;27(4):360-5. Epub 2007 Jun 8.

Department of Clinical Medicine, University La Sapienza, Rome, Italy.

Background: Anorexia is frequently found in end-stage renal disease and is a reliable predictor of morbidity and mortality in hemodialysis (HD) patients. The pathogenesis of anorexia is complex and the appetite-modulating hormone ghrelin could be involved. Two forms of circulating ghrelin have been described: acylated ghrelin (<10% of circulating ghrelin) which promotes food intake, and des-acyl ghrelin which induces a negative energy balance. The aim of this cross-sectional study is to clarify whether anorexia and body weight change in HD patients relate to plasma des-acyl ghrelin levels.

Methods: 34 HD patients and 15 healthy controls were studied. The presence of anorexia was assessed by a questionnaire. Serum des-acyl ghrelin was measured in HD patients and in 15 body mass index-, sex- and age-matched controls by ELISA. Energy intake was assessed by a 3-day dietary diary, and fat-free mass (FFM) was evaluated by body impedance analysis. Data have been statistically analyzed and are presented as mean +/- SD.

Results: 14 patients (41%) were found to be anorexic, and 20 patients (59%) non-anorexic. Energy intake (kcal/day) was significantly lower in anorexic than in non-anorexic patients (1,682 +/- 241 vs. 1,972.50 +/- 490; p < 0.05). FFM (%) was lower in anorexic than in non-anorexic patients (65.8 +/- 4.4 vs. 70.9 +/- 8.7; p = 0.05). Plasma des-acyl ghrelin levels (fmol/ml) were significantly higher in HD patients than in controls (214.88 +/- 154.24 vs. 128.93 +/- 51.07; p < 0.05), and in anorexic HD patients than in non-anorexic (301.7 +/- 162.4 vs. 159.1 +/- 115.5; p < 0.01).

Conclusion: Anorexia is highly prevalent among HD patients and des-acyl ghrelin could be involved in its pathogenesis.
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http://dx.doi.org/10.1159/000103798DOI Listing
September 2007

Metabotropic glutamate receptors: new targets for the control of tumor growth?

Trends Pharmacol Sci 2007 May 11;28(5):206-13. Epub 2007 Apr 11.

Istituto Neurologico Mediterraneo Neuromed, Località Camerelle, Pozzilli 86079, Italy.

Cancer stem cells are currently a target for the treatment of malignant tumors. Transformed neural stem-progenitor cells of the brain subventricular zone and the external granular layer of the cerebellum are the putative cells of origin of malignant gliomas and medulloblastomas, which are the most frequent malignant brain tumors in adults and children, respectively. The proliferation of neural stem-progenitor cells is regulated by metabotropic glutamate (mGlu) receptors, which are G-protein-coupled receptors that are activated by glutamate, the major excitatory neurotransmitter of the CNS. At least two receptor subtypes - mGlu(3) and mGlu(4) receptors - control the proliferation of brain tumor cells, whereas mGlu(1) receptors have been implicated in the development of melanomas. We believe that individual mGlu receptor subtypes represent new potential targets for the treatment of several malignant tumors, including brain tumors.
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http://dx.doi.org/10.1016/j.tips.2007.03.008DOI Listing
May 2007

Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells.

J Neurochem 2006 Oct;99(1):299-307

Department of Human Physiology and Pharmacology, University of Rome, Rome, Italy.

We have shown that endogenous activation of type 5 metabotropic glutamate (mGlu5) receptors supports the maintenance of a pluripotent, undifferentiated state in D3 mouse embryonic stem cells cultured in the presence of leukaemia inhibitory factor (LIF). Here, we examined the interaction between LIF and mGlu5 receptors using as a read-out the immediate early gene, c-Myc. The selective mGlu5 receptor antagonist, 2-methyl-6-(phenylenthynyl)pyridine (MPEP; 1 mum), reduced the increase in c-Myc protein levels induced by LIF by enhancing c-Myc ubiquitination. A reduction in c-Myc levels was also observed following small interfering RNA-mediated mGlu5 receptor gene silencing. MPEP reduced glycogen synthase kinase-3beta phosphorylation on Ser9, but increased phosphorylation of the phosphatidylinositol-3-kinase (PI-3-K) substrate, AKT. In our hands, activated PI-3-K reduced the stability of c-Myc, because (i) the PI-3-K inhibitor, LY294002, prevented the reduction in c-Myc levels induced by MPEP; and (ii) over-expression of AKT promoted c-Myc ubiquitination. All effects of MPEP were mimicked by protein kinase C (PKC) inhibitors and reversed by the PKC activator, tetradecanoylphorbol-13-acetate. We conclude that endogenous activation of mGlu5 receptors sustains the increase in c-Myc induced by LIF in embryonic stem cells by inhibiting both glycogen synthase kinase-3beta and PI-3-K, both effects resulting from the activation of PKC.
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http://dx.doi.org/10.1111/j.1471-4159.2006.04038.xDOI Listing
October 2006

Context-dependent regulation of embryonic stem cell differentiation by mGlu4 metabotropic glutamate receptors.

Neuropharmacology 2006 Sep 27;51(3):606-11. Epub 2006 Jun 27.

Departments of Human Physiology and Pharmacology, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.

The mGlu5 receptor is the only metabotropic glutamate receptor subtype expressed by mouse embryonic stem (ES) cells grown under non-differentiating conditions [Cappuccio, I., Spinanti, P. Porcellini, A., Desiderati, F., De Vita, T., Storto, M., Capobianco, L., Battaglia, G., Nicoletti, F., Melchiorri, D., 2005. Endogenous activation of mGlu5 metabotropic glutamate receptors supports self-renewal of cultured mouse embryonic stem cells. Neuropharmacology 1, 196-205]. We now report that ES cells differentiating into embryoid bodies (EBs) progressively lose mGlu5 receptors and begin to express mGlu4 receptors at both mRNA and proteinc level. A 4-day treatment of EBs with the mGlu4 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), increased mRNA levels of the mesoderm marker, brachyury and the endoderm marker, H19, and decreased the expression of the transcript for the primitive ectoderm marker, fibroblast-growth factor-5 (FGF-5). These effects were prevented by the mGlu4 receptor antagonists, alpha-methylserine-O-phosphate (MSOP). Plating of EBs for 4 days in vitro in ITSFn medium induced cell differentiation towards a neural lineage, as reflected by the expression of the intermediate filament protein, nestin, and the homeobox protein, Dlx-2. Pharmacological activation of mGlu4 receptors during cell incubation in ITSFn medium increased the expression of both neural markers. Similar results were obtained when neural differentiation was induced by exposure of EBs to retinoic acid. These data suggest that differentiation of cultured ES cells is associated with changes in the expression pattern of mGlu receptors and that activation of mGlu4 receptors affects cell differentiation in a context-dependent manner.
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http://dx.doi.org/10.1016/j.neuropharm.2006.05.007DOI Listing
September 2006

Interaction between ephrins/Eph receptors and excitatory amino acid receptors: possible relevance in the regulation of synaptic plasticity and in the pathophysiology of neuronal degeneration.

J Neurochem 2006 Jul;98(1):1-10

Department of Human Physiology and Pharmacology, University La Sapienza, Rome, Italy.

There is increasing evidence that Eph receptors and their transmembrane ligands, named ephrins, interact with glutamate receptors in both developing and adult neurons. EphB receptors interact with proteins that regulate the membrane trafficking of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits, and both ephrins and EphB receptors have been found to co-localize with N-methyl-d-aspartate (NMDA) receptors and to positively modulate NMDA receptor function. Moreover, pharmacologic activation of ephrin-Bs amplifies group-I metabotropic glutamate receptor signaling through mechanisms that involve NMDA receptors. The interaction with ionotropic or metabotropic glutamate receptors provides a substrate for the emerging role of ephrins and Eph receptors in the regulation of activity-dependent forms of synaptic plasticity, such as long-term potentiation and long-term depression, which are established electrophysiologic models of associative learning. In addition, these interactions explain the involvement of ephrins/Eph receptors in the regulation of pain threshold and epileptogenesis, as well as their potential implication in processes of neuronal degeneration. This may stimulate the search for new drugs that might modulate excitatory synaptic transmission by interacting with the ephrin/Eph receptor system.
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http://dx.doi.org/10.1111/j.1471-4159.2006.03844.xDOI Listing
July 2006

Comparative effects of levobupivacaine and racemic bupivacaine on excitotoxic neuronal death in culture and N-methyl-D-aspartate-induced seizures in mice.

Eur J Pharmacol 2005 Aug;518(2-3):111-5

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Ple. A. Moro 5, 00185 Rome, Italy.

We compared the neurotoxic profile of racemic bupivacaine and levobupivacaine in: (i) a mouse model of N-methyl-D-aspartate (NMDA)-induced seizures and (ii) in an in vitro model of excitotoxic cell death. When used at high doses (36 mg/kg) both bupivacaine and levobupivacaine reduced the latency to NMDA-induced seizures and increased seizure severity. However, levobupivacaine-treated animals underwent less severe seizures as compared with bupivacaine-treated animals. Lower doses of levobupivacaine and bupivacaine had opposite effects on NMDA-induced seizures. At doses of 5 mg/kg, levobupivacaine increased the latency to partial seizures and prevented the occurrence of generalized seizures, whereas bupivacaine decreased the latency to partial seizures and did not influence the development of generalized seizures. In in vitro experiments, we exposed primary cultures of mouse cortical cells, containing both neurons and astrocytes, to 100 microM NMDA for 10 min for the induction of excitotoxic neuronal death. This treatment killed 70-80% of the neuronal population, as assessed 24 h after the excitotoxic pulse. In this particular model, both levobupivacaine and bupivacaine were neuroprotective against NMDA toxicity. However, neuroprotection by levobupivacaine was seen at lower concentrations (with respect to bupivacaine) and was maintained at concentrations of 3 mM, which are much higher than the plasma security threshold for the drug in vivo. In contrast, no protection against NMDA toxicity was detected when 3 mM concentrations of bupivacaine were applied to the cultures. Our data show a better neurotoxic profile of levobupivacaine as compared to racemic bupivacaine, and are indicative of a safer profile of levobupivacaine in clinical practice.
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http://dx.doi.org/10.1016/j.ejphar.2005.06.022DOI Listing
August 2005

Endogenous activation of mGlu5 metabotropic glutamate receptors supports self-renewal of cultured mouse embryonic stem cells.

Neuropharmacology 2005 ;49 Suppl 1:196-205

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.

Cultured mouse embryonic stem (ES) cells maintained under undifferentiated conditions (i.e. grown in medium containing 15% FCS and leukemia inhibitory factor--LIF) expressed mGlu5 metabotropic glutamate receptors. Activation of these receptors with quisqualate increased [Ca2+]i but only when cultures were deprived of extracellular glutamate, indicating that the receptor was saturated by the endogenous glutamate. Pharmacological blockade of mGlu5 receptors with 2-methyl-6-(phenylethynyl)pyridine (MPEP) or antisense-induced knock-down of mGlu5 receptors decreased the expression of the two main transcription factors that sustain ES cell self-renewal, i.e. Oct-4 and Nanog, as assessed by real-time PCR and immunoblotting. Exposure of ES cell cultures to MPEP also reduced alkaline phosphatase activity, a marker of undifferentiated ES cells. These data support a critical role for mGlu receptors in early development showing that mGlu5 receptors are expressed by ES cells and their activation sustains ES cell self-renewal in culture.
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http://dx.doi.org/10.1016/j.neuropharm.2005.05.014DOI Listing
December 2005

Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is required for the development of ischemic neuronal death.

J Neurosci 2005 Mar;25(10):2647-57

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, 00185 Rome, Italy.

Expression of Dickkopf-1 (Dkk-1), a secreted protein that negatively modulates the Wnt pathway, was induced in the hippocampus of gerbils and rats subjected to transient global cerebral ischemia as well as in cultured cortical neurons challenged with an excitotoxic pulse. In ischemic animals, the temporal and regional pattern of Dkk-1 expression correlated with the profile of neuronal death, as assessed by Nissl staining and Dkk-1 immunostaining in adjacent hippocampal sections. Treatment of ischemic animals with either Dkk-1 antisense oligonucleotides or lithium ions (which rescue the Wnt pathway acting downstream of the Dkk-1 blockade) protected vulnerable hippocampal neurons against ischemic damage. The same treatments protected cultured cortical neurons against NMDA toxicity. We conclude that induction of Dkk-1 with the ensuing inhibition of the canonical Wnt signaling pathway is required for the development of ischemic and excitotoxic neuronal death.
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http://dx.doi.org/10.1523/JNEUROSCI.5230-04.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6725177PMC
March 2005

Functional characterization of WNT7A signaling in PC12 cells: interaction with A FZD5 x LRP6 receptor complex and modulation by Dickkopf proteins.

J Biol Chem 2003 Sep 11;278(39):37024-31. Epub 2003 Jul 11.

Department of Human Physiology and Pharmacology, University of Rome La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy.

WNT factors represent key mediators of many processes in animal development and homeostasis and act through a receptor complex comprised of members of the Frizzled and low density lipoprotein-related receptors (LRP). In mammals, 19 genes encoding Wingless and Int-related factor (WNTs), 10 encoding Frizzled, and 2 encoding LRP proteins have been identified, but little is known of the identities of individual Frizzled-LRP combinations mediating the effects of specific WNT factors. Additionally, several secreted modulators of WNT signaling have been identified, including at least three members of the Dickkopf family. WNT7A is a WNT family member expressed in the vertebrate central nervous system capable of modulating aspects of neuronal plasticity. Gene knock-out models in the mouse have revealed that WNT7A plays a role in cerebellar maturation, although its function in the development of distal limb structures and of the reproductive tract have been more intensely studied. To identify a receptor complex for this WNT family member, we have analyzed the response of the rat pheochromocytoma cell line PC12 to WNT7A. We find that PC12 cells are capable of responding to WNT7A as measured by increased beta-catenin stability and activation of a T-cell factor-based luciferase reporter construct and that these cells express three members of the Frizzled family (Frizzled-2, -5, and -7) and LRP6. Our functional analysis indicates that WNT7A can specifically act via a Frizzled-5.LRP6 receptor complex in PC12 cells and that this activity can be antagonized by Dickkopf-1 and Dickkopf-3.
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http://dx.doi.org/10.1074/jbc.M300191200DOI Listing
September 2003