Z-321

Z-321, a prolyl endopeptidase inhibitor, augments the potentiation of synaptic transmission in rat hippocampal slices

The present study investigated the effects of arginine-vasopressin (AVP) and (1-[3-(2-indanylacetyl)-L-thioprolyl] pyrrolidine (Z-321), an inhibitor of prolyl endopeptidase (PEP; (EC 3.4.21.26)) which degrades AVP in vitro, on the short-lasting potentiation of the field excitatory postsynaptic potentials (EPSP) coupled with a weak tetanus. The EPSP, after the electrical stimulation of the Schaffer collateral/commissural pathway, were recorded in the CA1 region of rat hippocampal slices. AVP at 10(-8) M and Z-321 at 10(-4) M augmented the potentiation induced by the weak tetanus; the magnitude of the post-tetanic potentiation of the EPSP was enhanced and the potentiation lasted for 60 min. In contrast, the racemic D-thioprolyl compound of Z-321, which virtually lacks any inhibitory effects on PEP, failed to affect the potentiation at 10(-4) M. The facilitatory effect of Z-321 was reversed by the application of [d(CH2)5,Tyr(Me)2]AVP (10(-8) M), an antagonist of the AVP V1 receptors, indicating that the effect of Z-321 was mediated through the V1 receptors. These findings suggest that Z-321 augmented the potentiation due to its inhibitory influence on the AVP degradation by PEP.

Pharmacokinetics and safety of Z-321, a novel specific orally active prolyl endopeptidase inhibitor, in healthy male volunteers

This study investigates the pharmacokinetics and safety profile of Z-321, (4R)-3-(indan-2-ylacetyl)-4-(1-pyrrolidinyl-carbonyl)-1,3-thiazoli dine, a novel specific orally active prolyl endopeptidase (PEP) inhibitor. Following a preliminary safety evaluation wherein 2 subjects received 3.75 and 15 mg doses and 2 other subjects received 7.5 and 30 mg doses, 16 subjects were assigned to two groups of 8 subjects each. In each group, 6 subjects were to receive active treatment, and 1 or 2 subjects were to receive placebo treatment. One group received 60 mg under fasted and fed conditions. A separate group of 8 subjects received 60 mg of Z-321 or a placebo in a bid regimen for 6 days and the morning dose on day 7. The concentrations of Z-321 and its main metabolites--R- and S-sulfoxide; RR-, SS-, and RS-indanol; and indanolsulfoxides in plasma and urine--were determined by the HPLC method. In the multiple-dose study, the cholinesterase activity was gradually increased and reached above the normal range on day 8 in 3 of 6 subjects given Z-321 and gradually returned to the normal range after completion of dosing. The elevation of plasma cholinesterase activity was considered to be an action of Z-321, but this remains to be verified. In a single-dose study at a dose of 30 mg, headache and vomiting were observed in 1 of 6 subjects. In the multiple-dose study, slight skin itching and eczema in 3 and 2 of 6 subjects, respectively, and headache in 2 of 6 subjects were observed, but all symptoms were not severe. There were no other abnormal findings in objective signs and laboratory findings, including blood pressure, heart rate, electrocardiogram, body temperature, hematology, blood chemistry, and urinalysis. The Cmax of Z-321 at 30, 60, and 120 mg in the fasting state were 63.7 +/- 23.9, 102.0 +/- 43.1, and 543.3 +/- 437.0 ng/ml (mean +/- SD), respectively, at 0.9 hours after administration, and the t1/2 was about 1.8 hours. There were no dramatic changes in the pharmacokinetics of Z-321 in the presence of food. In the multiple-dose study, there was no drug accumulation trend in plasma. These results indicate that Z-321 has acceptable pharmacodynamic and pharmacokinetics profiles for clinical use without any serious adverse events, as verified in healthy young male volunteers.

Chemical and toxicological alterations of zearalenone under ozone treatment

Our work mainly studied the elimination of zearalenone (ZEN) from corn flour by ozone treatment. We found four degradation products, after separation and identification by UPLC-ESI-QTOF-MS, named as Compound 1 (m/z 335.18), Compound 2 (m/z 351.19), Compound 3 (m/z 321.19) and Compound 4 (m/z 367.18). Then, one main transformation pathway was predicted on the basis of molecular weight and the reaction mechanism of ozone. We had also detected two of the degradation products by our modified UPLC-MS/MS method in real samples-contaminated corn flour. We screened the toxicity of the parent ZEN compound as well as the ozone induced decomposition products with BALB/c mice to evaluate the safety of our method in vivo. Therefore, aqueous ozone was demonstrated to effectively degrade ZEN and chemical structure alternations of ZEN after ozone treatment were predicted in both pure ZEN and real contaminated-corn flour samples. Considering the presence of the by-products formed, differences in the toxic effects on mice exposed to ZEN before and after the ozone treatment emphasise the significance of further study on the quality and toxicity of real food samples after ozone treatment.

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESI-MS-MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid-liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS-MS in selecting m/z 321 --> 257, 321 --> 152 (35Cl2) and m/z 323 --> 257, 323 --> 152 (37Cl35Cl). Two calibration curves were constructed by plotting the area ratio of m/z 321 --> 152 versus 326 --> 157 and m/z 321 --> 257 versus 326 --> 262 against their corresponding amount ratio. Indeed, even if m/z 321 --> 152 was found to give a higher MS-MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/z 321 --> 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 microg/kg concentration levels using d5-CAP as internal standard. The decision limit (CCalpha) and detection capability (CCbeta) of CAP in milk were calculated for m/z 321 --> 152 at 0.02 microg/kg and 0.03 microg/kg, respectively, and for m/z 321 --> 257 at 0.02 microg/kg and 0.04 microg/kg, respectively. At the lowest fortification level (i.e. 0.1 microg/kg), repeatability and within-laboratory reproducibility were calculated for m/z 321 --> 257 both at 0.02 microg/kg and for m/z 321 --> 152 at 0.03 and 0.05 microg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.

Pre-electrospray ionisation manifold methylation and post-electrospray ionisation manifold cleavage/ion cluster formation observed during electrospray ionisation of chloramphenicol in solutions of methanol and acetonitrile for liquid chromatography-mass spectrometry employing a commercial quadrupole ion trap mass analyser

We have observed unusual mass spectra of chloramphenicol (CAP) in solutions of methanol or acetonitrile showing intense ions at m/z 297, m/z 311, m/z 325 and m/z 339. The observed ions were different from those which are traditionally observed in the full scan ESI mass spectra of CAP with ions of m/z 321, m/z 323 and m/z 325. We have evidence to show that this process starts with offline methylation of CAP in solutions of methanol or acetonitrile to give m/z 339. Investigations using nuclear magnetic resonance (NMR) spectroscopy showed that there is a methylene group somewhere within the CAP molecule but not attached to any of the carbon atoms when the CAP is dissolved in methanol or acetonitrile before infusion into the mass spectrometer. The possible locations of attachment were speculated to be the electronegative atoms apart from the chlorine atoms due to valence considerations. The methylene group is attached to the nitrogen atom and forms a bond as observed in the MS/MS spectra of m/z 297, m/z 311, m/z 325 and m/z 339 which give m/z 183 as the base peak in all cases. Further experiments showed that there is cleavage of the methylated CAP molecule followed by cluster ion formation involving addition of methylene groups to the CAP fragment with m/z 183 to produce ions of m/z including m/z 297, m/z 311, m/z 325 and m/z 339. This process occurs in the mass spectrometer in the region housing the tube lens and is triggered when the ions are accelerated through this region by application of a negative tube lens offset voltage. This region affords collision of the charged droplets with a collision gas in this case nitrogen to strip the droplets of their solvent molecules. Experiments to follow the intensities of m/z 183, m/z 311, m/z 321, m/z 323, m/z 325 and m/z 339 as the tube lens offset voltage was varied were done in which the intensities of m/z 311, m/z 325 and m/z 339 were observed to be at their peak when the tube lens offset voltage was set at -40 V. When the tube lens offset voltage is swung to +40 V, thus decelerating the ions through the capillary skimmer region via the tube lens, the traditionally observed spectra with m/z 321, m/z 323 and m/z 325 were observed.