Carbon-13 nuclear magnetic resonance investigations into the interactions of bisulfite with pyrimidine nucleosides and nucleotides

Carbon-13 NMR is utilized to demonstrate the attack of bisulfite anion on uridine, 5-fluorouridine, and uridine 5'-monophosphate. The attack produces a pair of diastereomeric adducts similar in structure to those seen in the uracil series. Intensity data from the equilibrium system give an estimate for the individual equilibrium constants. Thymidine and thymidine 5'-monophosphate show no evidence of nucleophilic attack by bisulfite. This evidence indicates that bisulfite addition to nucleosides and nucleotides models the enzymatic methylation of uridine by the enzyme thymidylate synthetase better than the uracil bisulfite system.     

Desethylaprophen: a metabolite of aprophen with antimuscarinic activities

The metabolic fate of aprophen hydrochloride (2-diethylaminoethyl 2,2-diphenylpropionate) was studied in rats after intravenous administration. Both 14C-labeled and unlabeled aprophen were used in these studies. Blood samples were collected and analyzed to determine the identities of the metabolites formed. Utilizing high-performance liquid chromatography, desethylaprophen was identified as a major metabolite in ether-extracted samples from rats, and could be detected in blood samples 1 min after intravenous administration. It was most likely formed by N-de-ethylation of aprophen by a cytochrome P-450-dependent monooxygenase. Synthetic desethylaprophen was found to possess cholinolytic activity (i.e., it functioned as a muscarinic antagonist by blocking the contraction of acetylcholine-stimulated guinea pig ileum, the release of alpha-amylase from pancreatic acinar cells stimulated by carbachol, and also by inhibiting the binding of [3H]N-methyl scopolamine to the muscarinic receptors of guinea pig ileum). It was interesting that although the biological effects of desethylaprophen were 100-fold lower than those of aprophen, it was equally able to compete for the binding sites of muscarinic receptors of the guinea pig ileum.     

Further investigations into the use of high sensitivity differential scanning calorimetry as a means of predicting drug-excipient interactions

The early prediction of drug-excipient incompatibility is vital in the pharmaceutical industry to avoid costly material wastage and time delays. We report here on the use of high sensitivity differential scanning calorimetry (HSDSC) to examine the compatibility between an experimental drug (Drug A) and common pharmaceutical excipients. Short-term HSDSC experiments (up to 25h) indicated that Drug A was stable in the presence of moisture and was compatible with both lactose monohydrate and magnesium stearate in the dry state, but showed degradation in the presence of magnesium stearate and water in combination. These results agreed with conventional stability studies, in which extensive degradation was observed in the Drug A-magnesium stearate system after storage at 40 degrees C/75% RH for 4 weeks but not under other conditions. These results indicate that HSDSC may be used to examine the compatibility of experimental drugs with conventional excipients and, in particular, illustrate the importance of incorporating humidity as an experimental variable in order to fully establish the stability profile of the material under test.     

Microcalorimetric investigations of drug-albumin interactions

Flow microcalorimetry was used to estimate primary binding constants for drug-albumin interactions. Measurements of heat of reaction at two temperatures illustrated the danger of extrapolation for pharmacokinetic purposes of measurements made at temperatures other than 37 degrees. The method could be used to predict competition between two drugs for a single binding site. Major advantages over spectroscopic techniques included direct determinations of thermodynamic parameters, and the use of physiological concentrations of albumin.     

Kinetic interactions of a neuropathy potentiator (phenylmethylsulfonyl fluoride) with the neuropathy target esterase and other membrane bound esterases

Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection, or potentiation/“promotion,” of organophosphorus delayed neuropathy (OPIDN), depending on whether it is dosed before or after an inducer of delayed neuropathy, such as mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and “ongoing inhibition” (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I ₅₀ (20 min) of 35 and 198 μM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min^?1). Four esterase components were globally identified considering also previously data with paraoxon and mipafox: EPα (4–8 %), highly sensitive to paraoxon and mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPβ (38–41 %), sensitive to paraoxon and PMSF, but practically resistant to mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39–48 %), paraoxon-resistant and sensitive to the micromolar concentration of mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed.     

Ab initio and PCILO investigations of local anaesthetic-model membrane interactions

A "double zeta" basis set ab initio method was used for investigation of the systems (trimethylamine-dimethylphosphate monoanion)H+, aniline-dimethylphosphate monoanion and formanilide-dimethylphosphate monoanion, which represent the models for associative sites of both local anaesthetics and the phospholipid part of the nerve membrane. According to the authors' calculations, complex I was found to be the most stable with a N+-H...O-hydrogen bond. Further, the PCILO method was used for investigation of the interactions of the polar groups of 1-[2-(2-methoxyphenylcarbamoyloxy)ethyl] piperidine (B) and its cation (BH) with N-methylacetamide, which represents a model of association sites of the lipoprotein part of membrane. The strongest hydrogen bond with the carbonyl group of N-methylacetamide forms a N+-H group of cationic form.     

Kinetic and thermodynamic studies on drug-DNA interactions in the ellipticine series

The temperature-jump (T-jump) method has been used to investigate the binding mechanism to calf-thymus DNA of ellipticine and some of its derivatives. The results show that the plant alkaloid, ellipticine, interacts with DNA at a unique intercalation site whereas most of its synthetic derivatives, such as ellipticinium, 9-hydroxy-ellipticinium and related alkyl-oxazolopyridocarbazoles recognize two distinct DNA sites. Parallel analysis of kinetic data and DNA lengthening abilities of these derivatives suggests that only one of these two DNA sites is an intercalation site. Owing to the determination of the genuine number of drug-DNA complexes (inferred from T-jump experiments) and with the results of thermodynamic investigations (Van't Hoff plots), further characterization of the molecular interactions involved in the binding process was proposed. Thus, the formation of the unique intercalation complex of ellipticine was found to be entropy driven whereas binding of drugs which recognize the second class of binding sites was essentially enthalpy driven. These different thermodynamic behaviors suggest that intercalation essentially results from hydrophobic solvent structure effects in contrast to the second binding mode which principally arises from hydrogen bonding interactions through DNA grooves.     

Prediction of drug metabolism and interactions on the basis of in vitro investigations

Drug metabolism profoundly affects drug action, because almost all drugs are metabolised in the body and thus their concentrations and elimination rates are dependent on metabolic activity. Drug metabolism contributes substantially to interindividual differences in drug response and is also often involved in drug interactions, resulting in either therapeutic failure or adverse effects. Knowledge about the metabolism of a new chemical entity and its affinity to drug-metabolising enzymes helps in the drug development process by providing important information for the selection of a lead compound from among a number of substances pharmacologically equally effective in their therapeutic response. In drug development protocols, metabolism characteristics should be assessed very early during the development process. This has been made possible by the advances made especially in analytical capabilities and in in vitro technologies that are employed to predict in vivo metabolite profile, pharmacokinetic parameters and drug-drug interaction potential.     

Kinetic interactions of a homologous series of bispyridinium monooximes (HGG oximes) with native and phosphonylated human acetylcholinesterase

Inhibition of acetylcholinesterase (AChE) is the main toxic mechanism of organophosphorus compounds (OP) and reactivation of OP-inhibited AChE by oximes is a mainstay of antidotal treatment. The inadequate efficacy of clinically used oximes led to the synthesis of numerous new compounds in the past decades to identify more effective reactivators. Despite of extensive in vitro reactivation studies the structural features for the development of effective oximes are not well understood. In the present study we investigated the kinetic interactions of a homologous series of bispyridinium monoximes bearing C1 to C12 alkylketone groups on the second pyridinium ring with native and cyclosarin-inhibited human AChE. We observed a correlation of the length of the alkyl side chain with an up to 20-fold increased affinity towards native AChE. The effect of the alkyl side chain on the affinity and reactivity towards phosphonylated AChE was moderate, except of a markedly reduced reactivity of C10 and C12 oximes. In comparison to the reference oxime HI-6 all HGG oximes had a lower reactivating potency and these oximes are not considered as promising compounds for the reactivation of cyclosarin-inhibited AChE.     

Biochemical investigations into the mode of action of trazodone

Trazodone is an antidepressant drug with well established clinical efficacy in the treatment of depressive disorders, in addition to a marked anxiolytic activity. In contrast to tricyclic, and certain other antidepressant drugs, trazodone has been shown to have no effects on noradrenaline (NA) reuptake mechanisms. Whereas trazodone is a weak inhibitor of the in vitro uptake of [³H]-5-hydroxy-tryptamine (5-HT) into rat occipital cortex synaptosomes, a potent effect shown by this compound is the in vivo occupation of serotonergic [³H]-spiperone binding sites in the mouse frontal cortex; an activity shared by putative 5-HT antagonists and several other antidepressant drugs. The 5-HT antagonist activity of trazodone has been behaviourally verified by the antagonism of the serotonergic head-twitch response elicited by the 5-HT agonist, MK, 212, in mice. Trazodone also interacts with alpha-noradrenergic receptors as assessed by in vitro receptor binding experiments with the radioligands [³H]-WB 4101 and [³H]-dihydroergocryptine (DHE). Ex vivo experiments indicate that, after oral administration to rats, trazodone occupies cortical alpha₂ receptor sites, but with a lower potency than either mianserin or yohimbine. Experiments with superfused rat occipital cortex mini-slices have shown that trazodone can enhance the potassium stimulated and spontaneous efflux of preloaded [³H]-NA. The combination of alpha₂ antagonism and NA releasing properties, in the absence of reuptake inhibition, may be associated with the antidepressant activity of this drug, whereas the potent 5-HT antagonist activity may explain the anxiolytic effects. Finally, trazodone has been found to be devoid of muscarinie receptor affinity as measured by [³H]-quinuclidinyl benzilate (QNB) radioreceptor assays in accord with clinical observation of the absence of anticholinergic side-effects.     

Metabonomic investigations into hydrazine toxicity in the rat

The systemic biochemical effects of oral hydrazine administration (dosed at 75, 90, and 120 mg/kg) have been investigated in male Han Wistar rats using metabonomic analysis of (1)H NMR spectra of urine and plasma, conventional clinical chemistry, and liver histopathology. Plasma samples were collected both pre- and 24 h postdose, while urine was collected predose and daily over a 7 day postdose period. (1)H NMR spectra of the biofluids were analyzed visually and via pattern recognition using principal component analysis. The latter showed that there was a dose-dependent biochemical effect of hydrazine treatment on the levels of a range of low molecular weight compounds in urine and plasma, which was correlated with the severity of the hydrazine induced liver lesions. In plasma, increases in the levels of free glycine, alanine, isoleucine, valine, lysine, arginine, tyrosine, citrulline, 3-D-hydroxybutyrate, creatine, histidine, and threonine were observed. Urinary excretion of hippurate, citrate, succinate, 2-oxoglutarate, trimethylamine-N-oxide, fumarate and creatinine were decreased following hydrazine dosing, whereas taurine, creatine, threonine, N-methylnicotinic acid, tyrosine, beta-alanine, citrulline, Nalpha-acetylcitrulline and argininosuccinate excretion was increased. Moreover, the most notable effect was the appearance in urine and plasma of 2-aminoadipate, which has previously been shown to lead to neurological effects in rats. High urinary levels of 2-aminoadipate may explain the hitherto poorly understood neurological effects of hydrazine. Metabonomic analysis of high-resolution (1)H NMR spectra of biofluids has provided a means of monitoring the progression of toxicity and recovery, while also allowing the identification of novel biomarkers of development and regression of the lesion.     

Kinetic and dynamic interactions of oral viqualine and ethanol in man

We have studied the interaction of viqualine, a 5-hydroxytryptamine (5-HT) uptake inhibitor, with ethanol in 16 healthy men aged 20 to 34 years. The subjects were randomly assigned to receive ethanol dosed to maintain blood alcohol concentrations of 17-22 mmol.l-1 (n = 8) or orange juice (n = 8) on each of two test days one week apart and preceded, in random order, by 3 days of viqualine 75 mg bd or placebo. Ethanol had no effect on steady-state viqualine concentrations or the inhibition of 5-HT uptake. Viqualine did not affect acetaldehyde concentrations or cause an aversive alcohol-sensitizing reaction. The deleterious effects of ethanol on word recall, manual tracking, body sway, and self-ratings of intoxication, sedation, and performance were not modified by the presence of viqualine. Within each beverage group performances and self-ratings on viqualine and placebo days were not different. The first dose of viqualine was associated with transient nausea. Viqualine and ethanol do not interact kinetically or dynamically on the variables examined in this study.     

Theoretical and experimental investigations into the delamination tendencies of bilayer tablets

A controlled release resinate beads of betahistine diHCl (BHCl), a short half-life freely water soluble drug, was developed to allow once-daily administration to improve patient compliance and eliminate the risk of intolerance of the drug. BHCl-resin complex was subsequently coated with Eudragit RS100. A 2(4) full factorial design was employed for optimization and to explore the effect of Eudragit RS100 concentration in the coating solution (X(1)), the coating percentage (X(2)), the speed of rotation (X(3)) and the concentration of plasticizer (PEG 400) (X(4)) on the release rate of the drug from the microcapsules. The extent of coating (Y(1)), and the percentage drug released at given times (Y(2), Y(3) and Y(4)) were selected as dependent variables. The optimization process was performed for X(1), X(2), X(3) and X(4) using target ranges of these responses determined based on target release model deduced form zero-order dissolution profile of BHCl for once-daily administration. The levels of X(1), X(2), X(3) and X(4) of optimized BHCl microcapsules are 14.42%, 50.63%, 1495rpm and 9.94%, respectively. The calculated value of f(2) for the optimized BHCl microcapsules filled into hard gelatin capsules was 67.03 indicating that the dissolution profiles of the optimized formulation is comparable to that of the target release model. It could be concluded that a promising once-daily extended-release microcapsules of the highly water soluble drug, BHCl, was successfully designed.     

Further investigations into the genotoxicity of quinoxaline-di-N-oxides and their primary metabolites

Quinoxaline-di-N-oxides (QdNOs) are potential antibacterial agents with a wide range of biological properties. Quinocetone (QCT), carbadox (CBX), olaquindox (OLA), mequindox (MEQ) and cyadox (CYA) are classical QdNOs. Though the genotoxicity of parent drugs has been evaluated, the genotoxicity of their primary N → O reduced metabolites remains unclear. In the present study, a battery of four different short-term tests, mouse lymphoma assay (MLA), Ames test, chromosomal aberration assay in vitro and bone marrow erythrocyte micronucleus assay in vivo was carried out to investigate the genotoxicity of the six primary N → O reduced metabolites. Additionally, the genotoxicity of five parent drugs was evaluated by the MLA. Strong genotoxicity of N1-MEQ, B-MEQ and B-CBX was found in three of the assays but not in the Ames assay, and the rank order was N1-MEQ>B-MEQ>B-CBX that is consistent with prototype QdNOs. Negative results for the five QdNOs were noted in the MLA. We present for the first time a comparison of the genotoxicity of primary N → O reduced metabolites, and evaluate the ability of five QdNOs to cause mutations in the MLA. The present study demonstrates that metabolites are involved in genetic toxicity mediated by QdNOs, and improve the prudent use of QdNOs for public health.