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Gerenzano, Italy

Neuner P.,I.R.B.M. P. Angeletti | Neuner P.,Novartis | Peier A.M.,Merck And Co. | Talamo F.,I.R.B.M. P. Angeletti | And 18 more authors.
Journal of Peptide Science

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. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Source

Bianchi E.,I.R.B.M. P. Angeletti | Carrington P.E.,Merck And Co. | Ingallinella P.,I.R.B.M. P. Angeletti | Ingallinella P.,DiaSorin Research Center | And 10 more authors.
Bioorganic and Medicinal Chemistry

Peptide agonists of the glucagon-like peptide 1 (GLP-1) receptor (GLP1R) are rapidly gaining favor as antidiabetic agents, since in addition to increasing glucose-dependent insulin secretion, they also cause weight loss. Oxyntomodulin (OXM), a natural peptide with sequence homology to both glucagon and GLP-1, has glucose-lowering activity in rodents and anorectic activity in rodents and humans, but its clinical utility is limited by a short circulatory half-life due to rapid renal clearance and degradation by dipeptidyl peptidase IV (DPP-IV). Here, we describe the development of a novel DPP-IV-resistant, long-acting GLP1R agonist, based on derivatization of a suitably chosen OXM analog with high molecular weight polyethylene glycol (PEG) ('PEGylation'). PEG-OXM exerts an anti-hyperglycemic effect in diet-induced obese (DIO) mice in a glucose-dependent manner, with a maximally efficacious dose of 0.1 mg/kg, and reduces food intake and body weight with a minimally efficacious dose of 1 mg/kg. If this pharmacology is recapitulated in patients with type 2 diabetes, these results indicate PEG-OXM as a potential novel once-weekly GLP-1 mimetic with both glucose-lowering activity and weight loss efficacy. © 2013 Elsevier Ltd. All rights reserved. Source

Minnucci G.,University of Milan Bicocca | Amicarelli G.,DiaSorin Research Center | Salmoiraghi S.,The Unit of Hematology | Spinelli O.,The Unit of Hematology | And 4 more authors.

Background The identification of the JAK2V617F mutation is mandatory in the diagnostic work-up of Philadelphia chromosome-negative myeloproliferative neoplasms. Several molecular techniques to detect this mutation are currently available, but each of them has some limits. Design and Methods We set up a novel molecular method for the identification of the JAK2V617F mutation based on an allele-specific loop-mediated amplification, not polymerase chain reaction analysis. This innovative technique amplifies DNA targets under isothermal conditions with high specificity, efficiency and rapidity. The method does not require either a thermal cycler or gel separation and the DNA amplification reaction is visible to the naked eye and can be monitored by turbidimetry. This method was validated on DNA from cell lines as well as from patients with myeloproliferative neoplasms. The results were compared with those obtained by conventional polymerase chain reaction methods. Results This assay detects, within 1 hour, the JAK2V617F mutation down to an allele burden of 0.1- 0.01%. All samples positive by polymerase chain reaction (n=146) proved positive when tested by allele-specific loop-mediated amplification and none of the 80 negative controls gave false positive results. In addition, six patients with essential thrombocythemia previously diagnosed as being JAK2V617F negative by polymerase chain reaction analysis were found to be positive (at a low level) by allele-specific loop-mediated amplification. Furthermore, this assay discriminated the amount of JAK2V617F tumor allele within intervals of positivity, above 50%, between 50% and 10% and below 10%. Conclusions Allele-specific loop-mediated amplification is a simple, robust and easily applicable method for the molecular diagnosis and monitoring of JAK2V617F mutation in patients with chronic myeloproliferative neoplasms. ©2012 Ferrata Storti Foundation. Source

Palmioli A.,University of Milan Bicocca | Crisma M.,University of Padua | Peggion C.,University of Padua | Brusasca P.,DiaSorin Research Center | And 4 more authors.
Tetrahedron Letters

We report the synthesis and the characterization of N-(4-aminobutyl)-N- ethyl-isoluminol (ABEI) macrocyclic lactone, an activated ester with an unusual macrocyclic structure, and its use for efficient ABEI conjugation to proteins. Compared to the equivalent reagent normally used for chemiluminescence protein labeling, the ABEI macrocyclic lactone displays improved chemical properties, including stability and reactivity. We show the simple synthesis and the use of ABEI macrocyclic lactone for efficient chemiluminescence labeling of monoclonal antibody mixtures currently used in clinical immunodiagnostic assays for the detection of the HIV p24 antigen in patients. © 2013 Elsevier Ltd. All rights reserved. Source

Ingallinella P.,I.R.B.M. P. Angeletti | Ingallinella P.,DiaSorin Research Center | Peier A.M.,Merck And Co. | Pocai A.,Merck And Co. | And 13 more authors.
Bioorganic and Medicinal Chemistry

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. © 2012 Elsevier Ltd. All rights reserved. Source

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