Pharmacokinetics of sumatriptan in non-respondent and in adverse drug reaction reporting migraine patients

Sumatriptan is a selectiveagonist of 5HT1 (1B/1D) receptors,which has proved to be effectiveand safe for the acute treatment ofmigraine attacks. Nevertheless, itsuse by migraine sufferers is stilllimited and some patients consideradverse reactions related to sumatriptan,especially chest symptoms,unacceptable even if not serious.Moreover, in clinical trials, almostone third and one sixth of patients,respectively, fail to experienceheadache relief either after oral orafter subcutaneous sumatriptanadministration. Our aim was to verifywhether differencies in sumatriptanpharmacokinetics couldexplain non-response and/oradverse drug reactions. Sumatriptanlevels were determined by HPLCwith electrochemical detection.Pharmacokinetic parameters werecalculated using a computer program(PK Solutions 2.0; non compartmentalPharmacokinetics DataAnalysis). After oral administration,sumatriptan is rapidly absorbed andsometimes displays multiple peaksof plasma concentration. This “multiplepeaking” gives rise to considerableinter-subject variability inthe time of reaching maximumplasma concentration.Pharmacokinetic parameters ofsumatriptan, both after oral andsubcutaneous administration, weresimilar in the three patient groups.Blood pressure and heart rate didnot show any significant differencesbetween groups. Pharmacokineticparameters and bioavailability ofsumatriptan did not seem to be correlatedeither to the lack of efficacyor the appearance of side effects.These results could depend on thelimited number of patients studied

The β-lactamase Inhibitor Boronic Acid SM23 as a new anti-Pseudomonas aeruginosa Biofilm Compound

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation can enhance bacterial resistance to antimicrobial agents due to a decreased penetration of the antibiotic and a reduced rate of bacterial cells in biofilm. Selective inhibition of biofilm formation may thus represent a novel promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa. METHODS: In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and production of virulence factors, using the P. aeruginosa bioluminescent strain P1242. RESULTS: Our results indicated that SM23: a) inhibited the development of biofilm and the production of the virulence factors pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of P. aeruginosa QS-related Autoinducers molecules 3-oxo-C12-HSL and C4-HSL by dampened lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model, closely mimicking the clinical settings. CONCLUSION: Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as potent inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections

Oxytetracycline-Protein Complex: The Dark Side of Pet Food

Background: Worldwide antibiotic abuse represents a huge burden, which can have a deep impact on pet and human health through nutrition and medicalization representing another way of antibiotic resistance transmission. Objective: We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and Oxytetracycline (OTC), an approved antibiotic for use in zootechny, which might determine a toxic effect on K562 cells. Method: Cell viability and HPLC-ESI/QqToF assays were used to assess potential toxicity of bone extract derived from OTC-treated chickens according to standard withdrawal times and from untreated chickens at 24, 48 and 72h of incubation. Results: Cell culture medium with ground bone from chickens reared in the presence of OTC (OTC-CCM) resulted significantly cytotoxic at every incubation time regardless of the bone concentration while cell culture medium with ground bone from chickens reared without OTC (BIO-CCM) resulted significantly cytotoxic only after 72h of incubation. HPLC-ESI/QqToF assay ruled out the possible presence of OTC main derivatives possibly released by bone within culture medium until 1 \u3bcg/mL. Conclusion: The presence of a protein complex with OTC is able to exert a cytotoxic effect once released in the medium after 24-48h of incubation

Identification of nuclear substrates of Akt/PKB by functional proteomics: prohibitin 2 is a target of Akt phosphorylation in human promyelocytic leukemia cells

The serine/threonine protein kinase Akt is a major signal transducer of the phosphoinositide 3-kinase (PI 3-K) pathway in all cells and tissues and plays a pivotal role in the maintenance of cellular processes including cell growth, proliferation, survival, metabolism and development of many malignancies including acute myeloid leukemia. The frequent aberrant activation of the PI 3-K/Akt pathway in human cancer has made it an attractive therapeutic target. Therefore, the study of effector proteins downstream of Akt could clarify the role of Akt in the development of myeloid leukemia. Although both localization and activity of Akt in the nuclear compartment are well documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. In this study, we applied a proteomic approach to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt (K/RXK/RXXS/T) when phosphorylated on S/T (anti-phospho-Akt substrate antibody). NB4 cells were treated with ATRA, and the putative Akt substrate proteins were isolated by immunoprecipitation with the anti-phospho-Akt substrate antibody. The proteins were separated on SDS-PAGE and analyzed by ESI-Q-TOF mass spectrometry. This analysis indicated prohibitin 2, a potential tumor suppressor protein with potent transcriptional functions in the nucleus, as a putative substrate of Akt in the nucleus of NB4 cells. The putative Akt-Prohibitin 2 interaction was validated by reverse in vivo immunoprecipitation from nuclear protein of NB4 cells. In vitro phosphorylation of endogenous prohibitin 2 by recombinant Akt further validated this result. Vandermoere F, El Yazidi-Belkoura I, Demont Y, Slomianny C, Antol J, Lemoine J, Hondermarck H. Proteomics exploration reveals that actin is a signaling target of the kinase Akt. Mol Cell Proteomics. 2007 Jan;6(1):114-24. Kasashima K, Ohta E, Kagawa Y, Endo H. Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2. J Biol Chem. 2006 Nov 24;281(47):36401-10

HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts

In this study, the composition of polyphenols (phenolic acids and flavonoids) in propolis extracts was investigated by HPLC-DAD and HPLC-ESI-MS/MS by comparing the performance of ion trap and triple quadrupole mass analyzers. The analyses were carried out on an Ascentis C18 column (250 mm×4.6 mm I.D., 5 um), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Overall, the UV spectra, the MS and MS/MS data allowed the identification of 40 compounds. In the case of flavonoids, the triple quadrupole mass analyzer provided more collision energy if compared with the ion trap, originatingproduct ions at best sensitivity.The HPLC method was validated in agreement with ICH guidelines: the correlation coefficients were >0.998; the limit of detection was in the range 1.6–4.6 ug/ml; the recovery range was 96–105%; the intra- and inter-day %RSD values for retention times and peak areas were found to be <0.3 and 1.9%, respectively.The developed technique was applied to the analysis of hydroalcoholic extracts of propolis available on the Italian market. Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.67 mg/ml and the level of total flavonoids from 2.48 to 41.10 mg/ml. The proposed method can be considered suitable for the phytochemical analysis of propolis extracts used in phytotherapy

Development, Validation and Application of an LC-MS/MS Bioanalytical Method for the Quantification of GF449, A Novel 5-HT1A Agonist, in Rat Plasma and Brain.

We have recently reported a novel class of selective 5-HT1A agonists among which GF449 emerged for its high potency and almost full agonist activity (pKi 5-HT1A = 8.8; pD2 = 9.22, %Emax = 91.6). In order to quantify GF449 in rat plasma and brain, a sensitive LC-MS/MS method was developed and validated. Solid phase extraction (SPE) or a combined protein precipitation SPE permitted an efficient analyte recovery and sample clean-up. Multiple reaction monitoring (MRM) was used to track both GF449 and its internal standard (IS), MM189. GF449 was determined and quantitated to nanomolar concentrations in both plasma and brain matrix (LOQs = 0.0025 nmol/mL). Specificity was ensured using three further MRM qualifier transitions for both analyte and IS. Linearity was found in the range of 0.0025 nmol/mL to 1.00 nmol/mL (R2 = 0.9965) and from 0.0025 nmol/mL to 50 nmol/mL (R2 = 0.9999) for plasma and brain respectively. Intraday trueness ranged from 94.0% to 117.5% for brain and from 93.7% to 108.1% for plasma, while precision values were within 3.0% - 6.7% and 2.5% - 9.2% for plasma and brain respectively. The interday trueness of plasma ranged from 89.6% to 107.7% and the precision values (CV%) ranged from 4.6% to 7.5%. Interday trueness and precision (CV%) of the brain ranged from 94.3% to 101.2% and from 1.6% to 11.5% respectively. The method was validated in accordance with the EMEA guidelines and was successfully applied to plasma and brain samples obtained from rats treated with a 10 mg/kg single oral dose of GF449, thus demonstrating its applicability to pre-clinical pharmacokinetic studies

Simultaneous determination of pregnenolone sulphate, dehydroepiandrosterone and allopregnanolone in rat brain areas by liquid chromatography-electrospray tandem mass spectrometry

Neurosteroids (NSs) are well known modulators of neuronal activity and by binding to different neuronal receptors are responsible for a broad spectrum of biological and pathophysiological conditions. Here, a sensitive liquid chromatographic-electrospray ionization-tandem mass spectrometric method (LC-ESI-MS/MS) has been developed and validated for the simultaneous determination in rat brain areas of three NSs, i.e. pregnenolone sulphate (PS), dehydroepiandrosterone (DHEA) and allopregnanolone (AP). NSs were extracted with methanol-formic acid, purified by Hybrid-SPE cartridges and subjected to LC-ESI-MS/MS without any preliminary derivatization or deconjugation procedure. Quantitation was performed by multiple reaction monitoring mode with the internal standard method, using deuterium-labelled analogues of the analyzed NSs. The proposed method provided for the first time a direct quantitative determination of PS without hydrolysis; in particular, PS concentrations were found significantly (p<0.01) higher in hippocampus, the brain area associated primarily with memory, than in cortical tissue of control rats, suggesting the important role of this NS in the process of memory formation. The developed method could be successfully applied to quantify simultaneously PS, DHEA and AP levels in brain tissue in order to study their changes during various neurodegenerative diseases and to investigate the role of PS in the brain