Anticonvulsant Properties of 1-Diethylamino-3-phenylprop-2-en-1-one.

There is a need for novel antiepileptic agents whose modes of action differ from those of current antiepileptic drugs. The objective of this study was to determine whether 1-diethylamino-3-phenylprop-2-en-1-one (2) could prevent or at least diminish convulsions caused by different mechanisms. This amide afforded protection in the maximal electroshock and subcutaneous pentylenetetrazole screens when given intraperitoneally to both mice and rats. A number of specialized tests in mice were conducted and are explained in the text. They revealed (2) to have efficacy in the 6 Hz psychomotor seizure test, the corneal kindling model, the mouse temporal epilepsy screen and a peripheral neuronal transmission test using formalin. Three screens in rats were undertaken, which revealed that (2) blocked chloride channels, inhibited peripheral neuronal transmission (tested using sciatic ligation and von Frey fibres) and afforded protection in the lamotrigine-resistant kindled rat model. The biodata generated reveal that (2) is an important lead molecule in the quest for novel structures to combat epilepsy.

In vitro identification of the human cytochrome p450 enzymes involved in the oxidative metabolism of loxapine.

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) enzymes responsible for the oxidative metabolism of loxapine to 8-hydroxyloxapine, 7-hydroxyloxapine, N-desmethylloxapine (amoxapine) and loxapine N-oxide. These studies included use of cDNA-expressed enzymes, correlation analysis with 12 phenotyped human liver microsomal samples, and use of selective inhibitors of cytochrome P450s. The resultant data indicated that loxapine was mainly metabolized by human liver microsomes to (i) 8-hydroxyloxapine by CYP1A2, (ii) 7-hydroxyloxapine by CYP2D6, (iii) N-desmethyloxapine by CYP3A4 and (iv) loxapine N-oxide by CYP3A4. The involvement of flavin-containing monooxygenase (FMO) in the formation of loxapine N-oxide was also observed.

Characterization of glutathione conjugates of duloxetine by mass spectrometry and evaluation of in silico approaches to rationalize the site of conjugation for thiophene containing drugs.

The in vitro bioactivation of the selective serotonin and norepinephrine reuptake inhibitor duloxetine was investigated using liver microsomes and cytosol, expressed glutathione transferase, and recombinant P450 2D6 and 1A2. In the presence of glutathione, several conjugates were identified and characterized using a combination of direct infusion nanoelectrospray mass spectrometry on an LTQ/Orbitrap and liquid-chromatography mass spectrometry on a triple quadrupole. Structural characterization of these conjugates revealed that glutathione conjugation occurred on naphthalene rather than on thiophene and likely proceeded via a reactive epoxide intermediate. Experiments with recombinant P450s and the isoform specific inhibitors quinidine and furafylline suggested that both P450 2D6 and 1A2 were involved in the bioactivation of duloxetine. To explore the utility of in silico approaches to address bioactivation issues, MetaSite and two docking approaches (rigid and induced-fit docking) utilizing publicly available human P450 crystal structures or a homology model for P450 2C19 were used to predict the sites of bioactivation for duloxetine as well as the thiophene containing compounds tienilic acid, suprofen, ticlopidine, methapyrilene, and OSI-930 for which glutathione conjugates on the thiophene moiety have been reported. MetaSite and induced fit docking but not rigid docking correctly predicted that naphthalene rather than thiophene was the preferred site of bioactivation for duloxetine by P450 2D6. MetaSite predictions were also consistent with literature reports that thiophene was the site of glutathione conjugation for tienilic acid, suprofen, and OSI-930 but not for ticlopidine or methapyrilene. Of the two docking approaches investigated, induced fit docking results were consistent with thiophene as the site of bioactivation for all compounds to which it was applied. In conclusion, our investigation identified the likely bioactivation pathway for duloxetine and demonstrated the utility of in silico approaches MetaSite and induced fit docking to address potential bioactivation liabilities.

Nuclear magnetic resonance spectroscopy as a quantitative tool to determine the concentrations of biologically produced metabolites: implications in metabolites in safety testing.

Nuclear magnetic resonance (NMR) spectroscopy has traditionally been considered as an indispensable tool in elucidating structures of metabolites. With the advent of Fourier transform (FT) spectrometers, along with improvements in software and hardware (such as high-field magnets, cryoprobes, versatile pulse sequences, and solvent suppression techniques), NMR is increasingly being considered as a critical quantitative tool, despite its lower sensitivity as compared to mass spectrometry. A specific quantitative application of NMR is in determining the concentrations of biologically isolated metabolites, which could potentially be used as reference standards for further quantitative work by liquid chromatography/mass spectrometry. With the recent demands from regulatory agencies on quantitative information on metabolites, it is proposed that NMR will play a significant role in strategies aimed at addressing metabolite coverage in toxicological species. Traditionally, biologically isolated metabolites have not been considered as a way of generating "reference standards" for further quantitative work. However, because of the recent FDA guidance on safety testing of metabolites, one has to consider means of authenticating and quantitating biologically or nonbiologically generated metabolites. 1H NMR is being proposed as the method of choice, as it is able to be used as both a qualitative and a quantitative tool, hence allowing structure determination, purity check, and quantitative measurement of the isolated metabolite. In this publication, the application of NMR as a powerful and robust analytical technique in determining the concentrations of in vitro or in vivo isolated metabolites is discussed. Furthermore, to demonstrate the reliability and accuracy of metabolite concentrations determined by NMR, validation and cross-validation with gravimetric and mass spectrometric methods were conducted.

Cytotoxic and anticonvulsant aryloxyaryl Mannich bases and related compounds.

A series of 1-(4-aryloxyphenyl)-3-diethylamino-1-propanone hydrochlorides 3a-3e and related compounds 3f, 3g and 4a-4d were synthesised. In addition, a group of 4-(4-aryloxyphenyl)-3-(4-aryloxyphenylcarbonyl)-1-ethyl-4-piperidinol hydrochlorides 6a-6e were prepared which incorporated most of the structural features of 3a-3e. All of these compounds displayed cytotoxic properties towards murine L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes. A number of these compounds possessed noteworthy potencies towards seven human colon cancer cell lines. Some correlations were noted between the IC(50) values generated in the different screens and the sigma, pi and molar refractivity constants of the aryl substituents as well as with the volumes and solvent accessible surface areas of various basic groups. Molecular modelling of representative compounds revealed structural features, which may have contributed to the varying potencies noted. In general, the compounds in series 6 were well tolerated when administered to mice. Anticonvulsant properties were demonstrated by a number of compounds in the maximal electroshock (MES) screen when administered intraperitoneally to mice while 4c and 6e afforded protection in the MES test when given orally to rats.

Metabolism of prazosin in rat, dog, and human liver microsomes and cryopreserved rat and human hepatocytes and characterization of metabolites by liquid chromatography/tandem mass spectrometry.

Prazosin (2-[4-(2-furanoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline) is an antihypertensive agent that was introduced to the market in 1976. It has since established an excellent safety record. However, in vitro metabolism of prazosin has not been investigated. This study describes the in vitro biotransformation of prazosin in liver microsomes from rats, dogs, and humans, as well as rat and human cryopreserved hepatocytes and characterization of metabolites using liquid chromatography/tandem mass spectrometry. The major in vivo biotransformation pathways reported previously in rats and dogs include demethylation, amide hydrolysis, and O-glucuronidation. These metabolic pathways were also confirmed in our study. In addition, several new metabolites were characterized, including a stable carbinolamine, an iminium species, and an enamine-all formed via oxidation of the piperazine ring. Two ring-opened metabolites generated following oxidative cleavage of the furan ring were also identified. Using semicarbazide hydrochloride as a trapping agent, an intermediate arising from opening of the furan ring was captured as a pyridazine product. In the presence of glutathione, three glutathione conjugates were detected in microsomal incubations, although they were not detected in cryopreserved hepatocytes. These data support ring opening of the furan via a reactive gamma-keto-alpha,beta-unsaturated aldehyde intermediate. In the presence of UDP-glucuronic acid, prazosin underwent conjugation to form an N-glucuronide not reported previously. Our in vitro investigations have revealed additional metabolic transformations of prazosin and have shown the potential of prazosin to undergo bioactivation through metabolism of the furan ring to a reactive intermediate.

Quaternary ammonium-linked glucuronidation of 1-substituted imidazoles by liver microsomes: interspecies differences and structure-metabolism relationships.

N-Glucuronidation at an aromatic tertiary amine of 5-membered polyaza ring systems was investigated for a model series of eight 1-substituted imidazoles in liver microsomes from five species. The major objectives were to investigate substrate specificities of the series in human microsomes and interspecies variation for the prototype molecule, 1-phenylimidazole. The formed quaternary ammonium-linked metabolites were characterized by positive ion electrospray mass spectrometry. The incubation conditions for the N-glucuronidation of 1-substituted imidazoles were optimized; where for membrane disrupting agents, alamethicin was more effective than the detergents examined. The need to optimize alamethicin concentration was indicated by 4-fold interspecies variation in optimal concentration and by a change in effect from removal of glucuronidation latency to inhibition on increasing concentration. For the four species with quantifiable N-glucuronidation of 1-phenylimidazole, there were 8- and 18-fold variations in the determined apparent K(m) (range, 0.63 to 4.8 mM) and V(max) (range, 0.08 to 1.4 nmol/min/mg of protein) values, respectively. The apparent clearance values (V(max)/K(m)) were in the following order: human congruent with guinea pig congruent with rabbit > rat congruent with dog (no metabolite detected). Monophasic kinetics were observed for the N-glucuronidation of seven substrates by human liver microsomes, which suggests that one enzyme is involved in each metabolic catalysis. No N-glucuronidation was observed for the substrate containing the para-phenyl substituent with the largest electron withdrawing effect, 1-(4-nitrophenyl)imidazole. Linear correlation analyses between apparent microsomal kinetics and substrate physicochemical parameters revealed significant correlations between K(m) and lipophilicity (pi(para) or log P values) and between V(max)/K(m) and both electronic properties (sigma(para) value) and pKa.