Ornella Curcuruto

Concurrent pharmacological MRI and in situ microdialysis of cocaine reveal a complex relationship between the central hemodynamic response and local dopamine concentration.

The mechanisms underlying the signal changes observed with pharmacological magnetic resonance imaging (phMRI) remain to be fully elucidated. In this study, we obtained microdialysis samples in situ at 5-min intervals during phMRI experiments using a blood pool contrast agent to correlate relative cerebral blood volume (rCBV) changes with changes in dopamine and cocaine concentrations following acute cocaine challenge (0.5 mg/kg iv) in the rat over a duration of 30 min. Three brain areas were investigated: the dorsal striatum (n = 8), the medial prefrontal cortex (mPFC; n = 5), and the primary motor cortex (n = 8). In the striatum and mPFC groups, cocaine and dopamine temporal profiles were tightly correlated, peaking during the first 5-min period postinjection, then rapidly decreasing. However, the local rCBV changes were uncorrelated and exhibited broader temporal profiles than those of cocaine and dopamine, attaining maximal response 5-10 min later. This demonstrates that direct vasoactivity of dopamine is not the dominant component of the hemodynamic response in these regions. In the motor cortex group, microdialysis revealed no local change in dopamine in any of the animals, despite large local cocaine increase and strong rCBV response, indicating that the central hemodynamic response following acute iv cocaine challenge is not driven directly by local dopamine changes in the motor cortex. The combination of phMRI and in situ microdialysis promises to be of great value in elucidating the relationship between the phMRI response to psychoactive drugs and underlying neurochemical changes.

Selectivity of d[Cha4]AVP and SSR149415 at human vasopressin and oxytocin receptors: evidence that SSR149415 is a mixed vasopressin V1b/oxytocin receptor antagonist.

A possible role of arginine vasopressin (AVP) V1b receptor subtype in stress‐related disorders has been recently highlighted by the discovery of the agonist [1‐deamino‐4‐cyclohexylalanine] AVP (d[Cha4]AVP) and the antagonist SSR149415. Both compounds have been proposed to target specifically V1b receptors, since the reported affinities for the related V1a, V2 and oxytocin receptors are in the micromolar or submicromolar range. In the present study, we further investigated the binding affinities of d[Cha4]AVP and SSR149415 at recombinant human vasopressin V1b (hV1b) and oxytocin (hOT) receptors expressed in Chinese hamster ovary (CHO) cells and functional properties of both compounds at hV1b, hV1a, hV2 and hOT receptors. d[Cha4]AVP bound to hV1b receptors and hOT receptors with pKi values of 9.68±0.06 and 7.68±0.09, respectively. SSR149415 showed pKi values of 9.34±0.06 at hV1b and 8.82±0.16 at hOT receptors. d[Cha4]AVP stimulated [Ca2+]i increase in hV1b‐CHO cells with a pEC50 value of 10.05±0.15. It showed pEC50 values of 6.53±0.17 and 5.92±0.02 at hV1a and hV2 receptors, respectively, and behaved as a weak antagonist at hOT receptors (pKB=6.31±0.12). SSR149415 inhibited the agonist‐induced [Ca2+]i increase with pKB values of 9.19±0.07 in hV1b‐CHO and 8.72±0.15 in hOT‐CHO cells. A functional pKi value of 7.23±0.10 was found for SSR1494151 at hV1a receptors, whereas it did not inhibit 20 nM AVP response at hV2 receptors up to 3 μM. Data obtained confirmed the high potency and selectivity of d[Cha4]AVP at hV1b receptors, but revealed that SSR149415, in addition to the high potency at hV1b receptors, displays a significant antagonism at hOT receptors.

Selective dopamine D3 receptor antagonists enhance cortical acetylcholine levels measured with high-performance liquid chromatography/tandem mass spectrometry without anti-cholinesterases

The present study compared the effects of two selective dopamine (DA) D(3) receptor antagonists, SB-277011A (3, 10 and 30 mg/kg i.p.) and SB-414796A (3, 10 and 30 mg/kg i.p.) on extracellular levels of acetylcholine (ACh) in the rat medial prefrontal cortex (mPFC) by using a LC/MS-MS analytical method that permitted the detection of ACh without the necessity of adding acetylcholinesterase inhibitors to the perfusate. Furthermore, the present LC/MS-MS method permitted the simultaneous measurement of the respective concentrations of SB-277011A and SB-414796A in the same extracellular samples from the mPFC. The systemic administration of both selective DA D(3) receptor antagonists produced a significant increase in extracellular levels of Ach compared to vehicle-treated animals, which was associated with increases in extracellular concentrations of SB-277011A and SB-414796. Overall, the present findings further strengthen the likelihood of a modulation of cortical cholinergic function through a DA D(3)-mediated mechanism and suggest that selective DA D(3) receptor antagonism may be beneficial in the treatment of psychiatric diseases, such as schizophrenia, which are characterized by cognitive dysfunction.

Electrospray Mass Spectrometry: Complexation Between 1-Anilinonaphthalene-8-sulphonate and Proteins

Complexation of the hydrophobic fluorescent dye 1-anilinonaphthalene-8-sulphonate with a number of proteins was examined by electrospray ionization mass spectrometry (ESMS). The apparent agreement between the present data and those obtained by fluorescence spectroscopy underlines the role of ESMS in the overall approach to protein folding and unfolding.

PEPTIDE NITRATION BY PEROXYNITRITE : CHARACTERISATION OF THE NITRATION SITES BY LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY

A number of recent studies have demonstrated that peroxynitrite (ONOO−) can react with a host of biomolecules, particularly those containing aromatic amino acid residues, resulting in a number of modifications which have been found in association with diverse pathological conditions. Electrospray ionisation tandem mass spectrometry with and without liquid chromatographic separation has been used to examine a series of model peptides following their treatment with peroxynitrite at physiological pH. The mass spectra of sequences containing two tyrosine residues showed the formation of both mononitrated and dinitrated species, while those with phenylalanine showed no detectable nitration. Tryptic digests of two of the investigated peptides were also examined by liquid chromatography/electrospray mass spectrometry, which yielded further information on the competition for NO2 by multiple nitration sites within a given sequence. In addition, tryptic digests of the mononitrated component of sequences containing tyrosine and tryptophan indicated that mononitration was limited to tyrosine. Furthermore, some of the presented data indicate that, as well as nitrotyrosine and nitrotryptophan formation, the reaction of ONOO− can result in oxidation as well as the formation of labile adducts with NO2. Copyright

Investigation of crudes of synthesis of neuropeptide Y by high-performance liquid chromatography-electrospray mass spectrometry

Abstract Neuropeptide Y (NPY) and its modified form, [Leu 31 , Pro 34 ]NPY, are both thirty six amino acids long and they have relative molecular masses of 4250 and 4220. Solid-phase synthesis of both peptides resulted in complex crudes of reaction, which were investigated by means of combines high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ES-MS). The combination of these two powerful analytical techniques allowed rapid and reliable identification of the target peptides and furnished comprehensive information on other reaction products, which were mainly peptidic chains containing a smaller number of amino acids compared to those present in the intact peptides. The possible origin of such side-products and the eventual purification and unambiguous identification of both peptides are discussed.

Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine β‐lactoglobulin B

Complexation of acrylamide with bovine β-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine– acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S–S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.

INVESTIGATION OF NEWLY SYNTHESIZED PEPTIDES BY CAPILLARY ZONE ELECTROPHORESIS/ELECTROSPRAY MASS SPECTROMETRY

A series of newly synthesized peptides (M(r) = 1600-2250 Da), corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor alpha subunit have been examined by capillary zone electrophoresis/electrospray-mass spectrometry (CZE/ES-MS). The separation efficiency of CZE combined with the specificity of mass spectrometry allowed rapid and reliable identification of the target peptides and a number of associated side products. In solid-phase synthesis of peptides and small proteins such side products are inevitable, therefore the use of CZE and/or high-performance liquid chromatography combined with mass spectrometry is gaining a central role in such synthesis procedures.

Pyrolysis-gas Chromatography Mass-spectrometry In the Characterization of Glycated Albumin

Abstract Pyrolysis-GC/MS has been employed to characterize the products arising from the interaction between glucose and albumin. The pyrolysis products of genuine bovine serum albumin and glycated bovine serum albumin have been compared, after an extensive study devoted to the optimization of the pyrolysis conditions. Clear differences between the pyrograms of glycated and non-glycated samples have been found, confirming the validity of such an analytical approach. The structures of some components, characteristic of glycation, have been tentatively assigned on the basis of a mass spectra library search.

Detection, identification and quantification of a new de-fluorinated impurity in casopitant mesylate drug substance during late phase development: an analytical challenge involving a multidisciplinary approach.

During late phase development of the selective NK1 receptor antagonist casopitant mesylate, a de-fluorinated impurity was discovered and quantified by an orthogonal analytical approach, using NMR and LC-MS. A dedicated (19)F NMR method was initially developed for first line identification and semi-quantification of the impurity. Subsequently, a more accurate quantification was achieved by means of a selective normal-phase LC-MS method, which was fully validated. The results obtained on the development batches of the drug substance were used by the project team to set up a suitable control strategy and ultimately to ensure patient safety and the progression of the project.