Publications by authors named "Enrico Brunoldi"

9 Publications

  • Page 1 of 1

A Validated HPLC/MS Limit Test Method for a Potential Genotoxic Impurity in Cilostazol and its Quantification in the API and in the Commercially Available Drug Product.

Sci Pharm 2015 Apr-Jun;83(2):269-78. Epub 2015 Mar 26.

R&D Department, Dipharma Francis srl, Via Bissone 5, Baranzate (MI), Italy.

Cilostazol is a selective inhibitor of type 3 phosphodiesterase. 5-(3-Chloropropyl)-1-cyclohexyl-1H-tetrazole, used as an intermediate in the synthesis of cilostazol, has a primary alkyl chloride group, a well-known alerting function for genotoxic activity. Upon request from a regulatory agency, a limit test in accordance with ICH Q2(R1) added with the accuracy of a recovery test of 5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole in cilostazol was developed and validated. The application of the method highlighted the need to optimize the purification process to ensure levels of this potential genotoxic impurity in the final active pharmaceutical ingredient below the established limit. Also, the analytical method was suitable to determine the amount of the impurity in samples of the commercially available drug product, which showed the levels to be above the established threshold of toxicological concern (TTC).
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http://dx.doi.org/10.3797/scipharm.1502-05DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727775PMC
February 2016

Biology and chemistry of neuroprostanes. First total synthesis of 17-A4-NeuroP: validation of a convergent strategy to a number of cyclopentenone neuroprostanes.

Chem Phys Lipids 2013 Sep 27;174:64-74. Epub 2013 Jul 27.

Dipartimento di Chimica, Università di Pavia, Via Taramelli 12, 27100 Pavia, Italy.

In a process associated with ageing and neurodegeneration, radical peroxidation of docosahexaenoic acid (DHA) in neurons affords a multitude of prostaglandin-like neuroprostanes in a non-regioselective and non-stereoselective manner. In this paper, the synthesis of racemic 17-A4-NeuroP and 14-A4-NeuroP validated a general approach to several regioisomeric cyclopentenone A4- and J4-NeuroPs needed for biological tests. In preliminary experiments 17-A4-NeuroP, in analogy with 14-A4-NeuroP, readily adducted GSH free thiol, suggesting a similar mechanism of action for biological activity.
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http://dx.doi.org/10.1016/j.chemphyslip.2013.07.002DOI Listing
September 2013

The fatty acid oxidation product 15-A3t-isoprostane is a potent inhibitor of NFκB transcription and macrophage transformation.

J Neurochem 2011 Nov 23;119(3):604-16. Epub 2011 Sep 23.

Division of Clinical Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8548, USA.

Fatty acids such as eicosapentaenoic acid (EPA) have been shown to be beneficial for neurological function and human health. It is widely thought that oxidation products of EPA are responsible for biological activity, although the specific EPA peroxidation product(s) which exert these responses have not yet been identified. In this work we provide the first evidence that the synthesized representative cyclopentenone IsoP, 15-A(3t)-IsoP, serves as a potent inhibitor of lipopolysaccharide-stimulated macrophage activation. The anti-inflammatory activities of 15-A(3t)-IsoP were observed in response not only to lipopolysaccharide, but also to tumor necrosis factor alpha and IL-1b stimulation. Subsequently, this response blocked the ability of these compounds to stimulate nuclear factor kappa b (NFκB) activation and production of proinflammatory cytokines. The bioactivity of 15-A(3t)-IsoP was shown to be dependent upon an unsaturated carbonyl residue which transiently adducts to free thiols. Site directed mutagenesis of the redox sensitive C179 site of the Ikappa kinase beta subunit, blocked the biological activity of 15-A(3t)-IsoP and NFκB activation. The vasoprotective potential of 15-A(3t)-IsoP was underscored by the ability of this compound to block oxidized lipid accumulation, a critical step in foam cell transformation and atherosclerotic plaque formation. Taken together, these are the first data identifying the biological activity of a specific product of EPA peroxidation, which is formed in abundance in vivo. The clear mechanism linking 15-A(3t)-IsoP to redox control of NFκB transcription, and the compound's ability to block foam cell transformation suggest that 15-A(3t)-IsoP provides a unique and potent tool to provide vaso- and cytoprotection under conditions of oxidative stress.
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http://dx.doi.org/10.1111/j.1471-4159.2011.07422.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3640263PMC
November 2011

Isoprostane levels in urine of preterm newborns treated with ibuprofen for patent ductus arteriosus closure.

Pediatr Nephrol 2011 Jan 15;26(1):105-9. Epub 2010 Oct 15.

Department of Pediatrics, Obstetrics and Reproductive Medicine, Division of Neonatology, University of Siena, Siena, Italy.

Patent ductus arteriosus (PDA) is the most common cardiovascular abnormality of the preterm infant usually treated with ibuprofen (IBU). PDA is strictly related to oxidative stress (OS) in neonates. This study tests the hypothesis that OS occurs in neonates with PDA and that IBU treatment reduces OS. Forty-three preterm babies with gestational age (GA) <33 weeks were studied prospectively. Three urine samples were collected: at time 0 (before starting treatment), time 1 (after pharmacological PDA closure), and time 2 (7 days after the end of treatment) in all patients. OS was studied by measuring urinary isoprostane (IPs) levels. The results showed significant changes in urinary IP levels from time 0 to time 2 (Kruskal-Wallis, p=0.047). Time trend showed a significant decrease in IPs from time 0 to time 1 after IBU therapy (p=0.0067). This decrease was followed by an increase in IPs levels 7 days after treatment. IBU therapy for PDA closure reduced the risk of OS related to free-radical (FR) generation. This antioxidant effect of IBU may be beneficial in preterm babies with PDA who are at high risk for OS.
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http://dx.doi.org/10.1007/s00467-010-1651-6DOI Listing
January 2011

Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid.

J Biol Chem 2008 Jul 19;283(29):19927-35. Epub 2008 May 19.

Division of Clinical Pharmacology, and Department of Neurology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A(4)/J(4)-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A(4)/J(4)-NP, 14-A(4)-NP (A(4)-NP), potently suppresses lipopolysaccharideinduced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A(4)-NP blocks lipopolysaccharide-induced NF-kappaB activation via inhibition of Ikappa kinase-mediated phosphorylation of IkappaBalpha. Mutation on Ikappa kinase beta cysteine 179 markedly diminishes the effect of A(4)-NP, suggesting that A(4)-NP acts via thiol modification at this residue. Accordingly, the effects of A(4)-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A(4)/J(4)-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which eliminate the bioactivity of A(4)-NP, also abrogate the anti-inflammatory effects of oxidized DHA. Thus, we have demonstrated that A(4)/J(4)-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kappaB signaling and may contribute to the anti-inflammatory actions of DHA. These findings have implications for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation.
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http://dx.doi.org/10.1074/jbc.M803625200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2459280PMC
July 2008

Asymmetric synthesis of 14-A4t-neuroprostane: hunting for a suitable biomarker for neurodegenerative diseases.

J Org Chem 2007 Dec 10;72(25):9698-703. Epub 2007 Nov 10.

Department of Organic Chemistry, University of Pavia, Viale Taramelli, 10-27100 Pavia, Italy.

Oxidative stress has long been associated with aging and age-related pathologies, such as neurodegenerative diseases. One of the direct effects of oxidative stress in vivo is the formation of prostaglandin-like compounds, named isoprostanes, by the action of reactive oxygen species on membrane phospholipids. A particular subclass of isoprostanes, named neuroprostanes, is formed from docosahexaenoic acid (C22:6omega3, DHA) and is considered to be specific for neuronal oxidative stress. Since isoprostanes are considered as golden standards for oxidative stress, and due to the specificity of neuroprostanes for this condition in neurons and their relation with Alzheimer's and Parkinson's diseases, they are envisioned to be suitable biomarkers for these pathologies. Herein we describe the first total synthesis of 14-A4t-NeuroP in an enantioselective and stereoselective fashion, by means of a new and rapid approach for the installation of the omega chain based on a chemoselective Julia-Kocienski olefination. Furthermore, the construction of the 4,5-cis-disubstituted cyclopentenone moiety characteristic of class A neuroprostanes is achieved in a stereospecific fashion, and suitable reaction conditions have been tuned to avoid epimerization of the labile stereogenic centers.
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http://dx.doi.org/10.1021/jo701719fDOI Listing
December 2007

Cyclopentenone prostaglandin, 15-deoxy-Delta12,14-PGJ2, is metabolized by HepG2 cells via conjugation with glutathione.

Chem Res Toxicol 2007 Oct 14;20(10):1528-35. Epub 2007 Sep 14.

Department of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.

15-deoxy-Delta12,14-prostaglandin J2 (15-d-PGJ2) is a dehydration product of PGD2. This compound possesses a highly reactive polyunsaturated carbonyl moiety that is a substrate for Michael addition with thiol-containing biomolecules such as glutathione and cysteine residues on proteins. By reacting with glutathione and proteins, 15-d-PGJ2 is believed to exert potent biological activity. Despite the large number of publications that have ascribed bioactivity to this molecule, it is not known to what extent 15-d-PGJ2 is formed in vivo. Levels of free 15-d-PGJ2 measured in human biological fluids such as urine are low, and the biological importance of this compound has thus been questioned. Because of its reactivity, we hypothesized that 15-d-PGJ2 is present in vivo primarily as a Michael conjugate. Therefore, we undertook a detailed study of the metabolism of this compound in HepG2 cells that are known to metabolize other cyclopentenone eicosanoids. We report that HepG2 cells primarily convert 15-d-PGJ2 to a glutathione conjugate in which the carbonyl at C-11 is reduced to a hydroxyl. Subsequently, the glutathione portion of the molecule is hydrolyzed with loss of glutamic acid and glycine resulting in a cysteine conjugate. These findings confirm a general route for the metabolism of cyclopentenone eicosanoids in HepG2 cells and may pave the way for new insights regarding the formation of 15-d-PGJ2 in vivo.
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http://dx.doi.org/10.1021/tx700231aDOI Listing
October 2007

Asymmetric synthesis of a chiral building block for cyclopentanoids: a novel enantioselective synthesis of preclavulone A.

J Org Chem 2006 Oct;71(22):8459-66

Department of Organic Chemistry, University of Pavia, Viale Taramelli, 10-27100 Pavia, Italy.

A new asymmetric approach to the hydroxylactone (+)-(3aR,4R,6aS)-4-(hydroxymethyl)-3a,4-dihydro-3H-cyclopenta[b]furan-2(6aH)-one (1), a key synthetic building block for cis-1,2-disubstituted five-membered ring derivatives (i.e., isoprostanes, jasmonates, and clavulones), has been described. A remarkable control of the absolute and relative configuration of the three stereocenters was achieved. Thus, the use of the Trost's asymmetric allylic alkylation strategy secured highly enantioenriched (R)-3-(nitromethyl)cyclopent-1-ene (13), which was smoothly converted to (R)-cyclopent-2-enecarboxylic acid (15) in excellent yield and high enantiomeric purity (>98% ee). 6-exo-trig atom-transfer radical cyclizations of ((R)-cyclopent-2-enyl)methyl 2-iodoacetate (12) produced exclusively the desired cis-fused delta-lactone (4aR,7aR)-hexahydro-5-iodocyclopenta[c]pyran-3(1H)-one (11), which was subsequently elaborated to hydroxylactone 1 through a stereocontrolled Pd(II)-mediated lactonization reaction. En route to preclavulone A, a putative elusive intermediate in the biosynthesis of clavulones, the synthetic utility of compound 1 was demonstrated. The key feature in this synthesis was the installation of the lower side chain via the Knochel organozinc sp3-sp C-C coupling protocol.
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http://dx.doi.org/10.1021/jo061321hDOI Listing
October 2006