Presence Of Quinidine Research Articles

Oral bioavailability and intestinal secretion of amitriptyline: Role of P-glycoprotein?

The aim of the study was to evaluate the influence of quinidine, a P-glycoprotein inhibitor, on oral bioavailability and on intestinal secretion of amitriptyline, a tricyclic antidepressant. Amitriptyline was administrated intravenously (5 mg/kg) and orally (50 mg/kg) to rabbits, with and without quinidine. Jejunal segments of rats were mounted on diffusions chambers and the permeation of amitriptyline was measured across the tissue in luminal–serosal (LS) and serosal–luminal (SL) directions, with and without quinidine. Finally, an in situ recirculating intestinal perfusion model was performed in rabbits to study amitriptyline permeation in LS direction with and without quinidine. Absolute oral bioavailability ( F) of amitriptyline was significantly increased more than three-fold in presence of quinidine ( F = 0.6 ± 0.4% versus 1.9 ± 1.1%). The apparent permeability coefficients in SL direction were significantly higher than in LS direction ( P app (SL) = 6.01 ± 2.42 versus P app (LS) = 4.90 ± 2.73 × 10 −4 cm min −1). In presence of quinidine, the intestinal absorption was increased ( P app (LS) = 4.02 ± 2.91 versus P app (LS) = 5.99 ± 2.43 × 10 −4 cm min −1) and the intestinal secretion was decreased ( P app (SL) = 4.58 ± 0.54 versus P app (LS) = 3.63 ± 1.46 × 10 −4 cm min −1) but not significantly. In conclusion, P-glycoprotein appears to be involved in oral amitriptyline absorption but other intestinal uptake and efflux transporters maybe implicated.

Intestinal Absorptive Transport of the Hydrophilic Cation Ranitidine: A Kinetic Modeling Approach to Elucidate the Role of Uptake and Efflux Transporters and Paracellular vs. Transcellular Transport in Caco-2 Cells

The mechanism of intestinal drug transport for hydrophilic cations such as ranitidine is complex, and evidence suggests a role for carrier-mediated apical (AP) uptake and saturable paracellular mechanisms in their overall absorptive transport. The purpose of this study was to develop a model capable of describing the kinetics of cellular accumulation and transport of ranitidine in Caco-2 cells, and to assess the relative contribution of the transcellular and paracellular routes toward overall ranitidine transport. Cellular accumulation and absorptive transport of ranitidine were determined in the absence or presence of uptake and efflux inhibitors and as a function of concentration over 60 min in Caco-2 cells. A three-compartment model was developed, and parameter estimates were utilized to assess the expected relative contribution from transcellular and paracellular transport. Under all conditions, ranitidine absorptive transport consisted of significant transcellular and paracellular components. Inhibition of P-glycoprotein decreased the AP efflux rate constant (k21) and increased the relative contribution of the transcellular transport pathway. In the presence of quinidine, both the AP uptake rate constant (k12) and k21 decreased, resulting in a predominantly paracellular contribution to ranitidine transport. Increasing the ranitidine donor concentration decreased k12 and the paracellular rate constant (k13). No significant changes were observed in the relative contribution of the paracellular and transcellular routes as a function of ranitidine concentration. These results suggest the importance of uptake and efflux transporters as determinants of the relative contribution of transcellular and paracellular transport for ranitidine, and provide evidence supporting a concentration-dependent paracellular transport mechanism. The modeling approach developed here may also be useful in estimating the relative contribution of paracellular and transcellular transport for a wide array of drugs expected to utilize both pathways.

Modulation of P-Glycoprotein–Mediated Efflux by Prodrug Derivatization: An Approach Involving Peptide Transporter–Mediated Influx Across Rabbit Cornea

The aim of this study was to investigate the modulation of efflux mechanisms using transporter- targeted prodrug derivatization of a model P-gp substrate, quinidine. The L-valine, L-valine-valine esters of quinidine, val-quinidine (VQ), and val-val-quinidine (VVQ) were synthesized in our laboratory, respectively. [(14)C] erythromycin was chosen to delineate the affinity of quinidine (Q) toward P-gp. [(3)H] glycylsarcosine (GS, or glysar) was chosen as a model peptide transporter (PEPT) substrate. Uptake studies were performed on rPCEC (rabbit primary corneal epithelial culture) using 12-well plates. Transport studies were conducted with isolated rabbit corneas at 34 degrees C. Efflux of [(14)C] erythromycin was significantly increased in the presence of quinidine, whereas it was unaltered in the presence of VQ and VVQ. VVQ was more stable, both in buffers and tissue homogenate. Transport of VQ and VVQ was inhibited with GS, and their permeability values were 1.5 and 3 times higher than the permeability of quinidine, respectively. Results from this study clearly indicate that prodrug derivatization of quinidine can modulate P-gp-mediated efflux. These prodrugs have a reduced or diminished affinity toward P-gp and were further recognized by the peptide transporter- mediated process. Enhanced permeabilities of the prodrugs indicate that drug derivatization can be a viable strategy for overcoming P-gp-mediated efflux.

Quinidine and procainamide inhibit murine macrophage uptake of apoptotic and necrotic cells: A novel contributing mechanism of drug-induced-lupus

A number of mechanisms have been proposed to explain the etiology of drug-induced lupus (DIL) but the effect of apoptotic and necrotic cell handling has not been previously examined. To evaluate the effect of quinidine and procainamide at therapeutic range concentrations, on the uptake of apoptotic and necrotic thymocytes by murine peritoneal macrophages and on macrophage survival, as a novel mechanism for DIL. Thymocytes were stained and induced to undergo apoptosis by serum withdrawal. Apoptosis was evaluated using annexin V and propidum iodide (PI) and PI staining. Necrosis was induced by heating. Peritoneal macrophages were treated with quinidine or procainamide at a range of therapeutic concentrations and incubated with stained apoptotic and necrotic thymocytes. Apoptotic and necrotic cell uptake was evaluated by flow cytometry using double staining of thymocytes and macrophages and by confocal microscopy. Green fluorescent latex beads were used as controls for phagocytosis. Significantly decreased uptake of apoptotic and necrotic cells was seen in the presence of quinidine and procainamide. The documented effect was mainly on the number of apoptotic/necrotic cells per macrophage. Uptake of fluorescent latex beads offered to resident macrophages was not significantly affected by quinidine or procainamide. No pro-apoptotic effect of quinidine or procainamide on macrophages was seen. Quinidine and procainamide at therapeutic range concentrations specifically inhibit clearance of apoptotic and necrotic cells by peritoneal macrophages. Altered handling of apoptotic and necrotic cells may represent a contributing mechanism for DIL.

Effect of quinidine on the 10-hydroxylation of R-warfarin: species differences and clearance projection.

Stimulation by quinidine of warfarin metabolism in vitro was first demonstrated with liver microsomal preparations. We report herein that this drug interaction is reproducible in an animal model but that it exhibits profound species differences. Thus, using rabbit liver microsomes and a kinetic model incorporating two binding sites, the hepatic intrinsic clearance of R-warfarin via the 10-hydroxylation pathway (CL(int)(W)) was projected to be 6 +/- 1 and 128 +/- 51 microl/min/g liver, respectively, in the absence and presence of 21 microM unbound quinidine. These estimates were consistent with the results from studies in which rabbit livers (n = 5) were perfused in situ with R-warfarin or R-warfarin plus quinidine. The CL(int)(W) increased from 7 +/- 3 to 156 +/- 106 microl/min/g liver after increasing the hepatic exposure of unbound quinidine from 0 to 21 microM. In contrast, when liver microsomes or intact livers from rats were examined, R-warfarin metabolism was inhibited by quinidine, the CL(int)(W) decreasing to 26% of the control value after exposure of perfused rat livers (n = 5) to 22 microM unbound quinidine. The third example involved monkey liver microsomes, in which the rate of 10-hydroxylation of R-warfarin was little affected in the presence of quinidine (<2-fold increase). In all three species, the 10-hydroxylation of R-warfarin was catalyzed primarily by members of CYP3A, based on immuno- and chemical inhibition analyses. These findings not only highlight the variability of drug interactions among different species but also suggest that changes in hepatic clearance resulting from stimulation of cytochrome P450 activity may be projected based on estimates generated from corresponding liver microsomal preparations.

Procainamide and quinidine inhibition of the human hepatic degradation of meperidine in vitro.

Procainamide and quinidine inhibition of the degradation of meperidine in human liver was investigated by incubation of two concentrations of either drug with meperidine in homogenates of human liver over 24 and 36 h. Meperidine concentrations declined by 26% after incubation for 24 h and by 42% after incubation for 36 h. In the presence of procainamide, however, they decreased by only 15% to 18% at 24 h and by only 26% to 28% at 36 h. In the presence of quinidine, they declined by only 18% to 19% at 24 h and by only 27% to 28% at 36 h. Procainamide and quinidine may inhibit human hepatic carboxylesterase hCE-1, which is responsible for catalyzing the hydrolysis of meperidine. This inhibition may prolong the biological half-life of meperidine in patients receiving the drug together with either procainamide or quinidine.

Quinidine impairs proliferation of neurofibromatosis type 2-deficient human malignant mesothelioma cells.

Human malignant mesotheliomas (HMMs) are aggressive tumors that arise from the mesothelium. They respond poorly to conventional tumor treatment and outcome is often fatal. Inactivating mutations of the neurofibromatosis type 2 (NF2) tumor suppressor gene merlin have been described in nearly 60% of primary malignant mesothelioma and in approximately 20% of the mesothelioma cell lines. Studies regarding human NF2 schwannoma cells revealed a higher proliferation and a larger noninactivating K(+) outward current compared with controls. The enhanced proliferation of merlin-deficient NF2 schwannoma cells could be reduced in the presence of quinidine, a K(+) channel blocker, whereas the proliferation of normal Schwann cells is not affected. The current study was undertaken to evaluate the effect of quinidine on the proliferation of HMM cell lines in relation to their NF2 status. Proliferation analyses using bromodeoxyuridine incorporation was performed by immunocytochemical staining and fluorescence assisted cell sorting. The patch-clamp technique was applied for electrophysiologic characterization of the HMM cell lines. The cytochrome P450 2D6 locus, known to be mutated at high frequencies in NF2 patients and to be specifically inhibited by quinidine, was screened for mutations by cycle sequencing. Quinidine selectively reduces the proliferation of merlin-deficient HMM cell lines by causing a G(0)/G(1) arrest, whereas the proliferation rates of merlin-expressing HMM cell lines remain unchanged. The effect of quinidine on the proliferation of HMM cell lines appears to be correlated with the NF2 gene status but not with the K(+) outward current. No relation to cytochrome P450 2D6 mutations was detected. Quinidine or quinidine analogs are of potential therapeutic interest for the subset of merlin-deficient mesothelioma tumors.

Lack of interaction of digoxin and P-glycoprotein inhibitors, quinidine and verapamil in human placenta in vitro

Objective: To determine the effect of quinidine and verapamil, known antiarrhythmic agents and P-glycoprotein (Pgp) inhibitors, on digoxin transport from the maternal to the fetal compartment in the isolated perfused human placenta. Study design: Isolated placental cotyledons from normal human placentae ( n=20) were dually perfused with M199 medium enriched with albumin (0.3%) and glucose (0.1%). The maternal and the fetal circulation flow rates were 12 and 6 ml/min, respectively. Closed circulations were used to evaluate steady state transplacental gradient formation. In six placentae quinindine was added to the maternal circuit; after 45 min of perfusion, digoxin was added to the maternal circulation. The effect of verapamil on digoxin transfer from the maternal to the fetal compartments was explored in five placentae. In six additional placentae the transfer of digoxin was studied in the absence of quinidine. Transplacental passage of digoxin was calculated from repeated fetal and maternal perfusate samples. Digoxin levels were determined in perfusate samples by fluorescence polarization immunoassay. Antipyrine was added to the maternal reservoir of all placentae as reference substance. Results: The transfer of digoxin (alone) and in the presence of quinidine or verapamil was 10.93±3.71, 9.00±5.2 and 12.94±4.86%, respectively. The levels of digoxin in the fetal compartment, 0.62±0.20, 0.48±0.29 and 0.60±0.26 ng/ml, respectively, were not significantly affected by quinidine and verapamil. These Pgp modulators, also did not influence significantly the steady state levels of digoxin in the maternal compartment. Conclusion: Neither quinidine nor verapamil affected the transplacental transfer of digoxin in vitro in normal human placentae. In contrast to the other tissues, they do not inhibit Pgp activity in term human placentae.

Differences in pharmacokinetics and hepatobiliary transport of a novel anti-inflammatory agent between normal and adjuvant arthritis rats

1. The pharmacokinetics, particularly the hepatobiliary transport of T-5557 ((3-methyl-2-oxo-piperadin-3-yl)-acetic acid N '-(3-thieophen-2-yl-8-methoxy-quinazolin-1-yl)-hydrazide), a novel anti-inflammatory agent, has been examined in normal and adjuvant arthritis (AA) rats. 2. Following oral administration of T-5557, the absolute bioavailability in AA rats was increased by sixfold compared with normal rats. The extent of binding T-5557 to plasma proteins obtained from AA rats was markedly greater than in normal rats (97.0 versus 88.2%). The biliary clearance in AA rats was significantly lower than that in normal rats (1.186 versus 5.621 ml min -1 kg -1), and lower intrinsic biliary clearance was also observed in AA rats (40.33 versus 69.83 ml min -1 kg -1) . 3. Concomitant administration of T-5557 with quinidine, a potent P-glycoprotein inhibitor, to normal rats caused a significant decrease in the biliary clearance of T-5557 by 37.9%. Moreover, the transport of T-5557 for the apical-to-basal compartment in a Caco-2 cells' monolayer was fourfold lower than that for the opposite direction, and was increased in the presence of quinidine and verapamil. 4. These results suggest that P-glycoprotein is involved in the biliary excretion of T-5557 and the decrease in the transport activity as well as the increase in plasma protein binding caused the elevated plasma concentration and bioavailability of T-5557 in AA rats.

Applications of Baylis–Hillman chemistry: enantioselective synthesis of (−)-methyl 3-aryl-2-methylene-3-(prop-2-yn-1-yloxy)propanoates via chiral leaving group strategy

Asymmetric synthesis of (−)-methyl 3-aryl-2-methylene-3-(prop-2-yn-1-yloxy)propanoates in 25–40% enantiomeric purities via the reaction of methyl (2 Z)-3-aryl-2-(bromomethyl)prop-2-enoates with prop-2-yn-1-ol in the presence of quinidine is described.

The Potential for CYP2D6 Inhibition Screening Using a Novel Scintillation Proximity Assay-Based Approach

High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.

CYP2D6 is involved in O-demethylation of diltiazem

In a previous study of diltiazem (DTZ) pharmacokinetics in renal transplant patients, we speculated that a polymorphic enzyme could be involved in O-demethylation of diltiazem. The aim of this in vitro study was to investigate whether O-demethylation of DTZ is mediated by cytochrome P450-2D6 (CYP2D6). DTZ was incubated with transfected human liver epithelial (THLE) cells expressing CYP2D6 (T5-2D6 clone). Metabolism of DTZ was studied over a concentration range of 12.5-400 microM and in the presence of quinidine (a CYP2D6 inhibitor) or erythromycin (a CYP3A4 inhibitor). THLE cells lacking CYP2D6 activity (T5-neo clone) were used as control. The culture medium of the cells, in which DTZ was dissolved, was analysed for DTZ and metabolites prior to and after 8 h of incubation using high-performance liquid chromatography (HPLC, UV detection). Authentic O-demethyl-DTZ (Mx) was not available, and this metabolite was therefore not identifiable. Desacetyl-O-demethyl-DTZ (M4) was exclusively produced during incubations of DTZ with THLE cells expressing CYP2D6. The rate of M4 formation was described using Michaelis Menten kinetics in the concentration range of DTZ used. Production of M4 was inhibited by quinidine, but not erythromycin. An unidentified chromatographic peak, which was interpreted to be Mx, showed the same pattern of formation as M4 both in absence and presence of inhibitors. N-demethylated metabolites, formed by CYP3A4, were not observed in any of the cell lines. Evidence was provided in vitro that O-demethylation of DTZ is mediated by the polymorphic isoenzyme CYP2D6. Involvement of CYP2D6 in the metabolism of DTZ may have clinical implications regarding pharmacokinetic variability and interactions.

Enhancing effects of salicylate on tonic and phasic block of Na + channels by class 1 antiarrhythmic agents in the ventricular myocytes and the guinea pig papillary muscle

Objective: To study the interaction between salicylate and class 1 antiarrhythmic agents. Methods: The effects of salicylate on class 1 antiarrhythmic agent-induced tonic and phasic block of the Na + current ( I Na) of ventricular myocytes and the upstroke velocity of the action potential ( V max) of papillary muscles were examined by both the patch clamp technique and conventional microelectrode techniques. Results: Salicylate enhanced quinidine-induced tonic and phasic block of I Na at a holding potential of −100 mV but not at a holding potential of −140 mV; this enhancement was accompanied by a shift of the h ∞ curve in the presence of quinidine in a further hyperpolarized direction, although salicylate alone did not affect I Na. Salicylate enhanced the tonic and phasic block of V max induced by quinidine, aprindine and disopyramide but had little effect on that induced by procainamide or mexiletine; the enhancing effects were related to the liposolubility of the drugs. Conclusions: Salicylate enhanced tonic and phasic block of Na + channels induced by class 1 highly liposoluble antiarrhythmic agents. Based on the modulated receptor hypothesis, it is probable that this enhancement was mediated by an increase in the affinity of Na + channel blockers with high lipid solubility to the inactivated state channels.

Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line.

The mechanism of the G0/G1 arrest and inhibition of proliferation by quinidine, a potassium channel blocker, was investigated in a tissue culture cell line, MCF-7, derived from a human breast carcinoma. The earliest measurable effect of quinidine on the cell cycle was a decrease in the fraction of cells in S phase at 12 hr, followed by the accumulation of cells in G1/G0 phases at 30 hr. Arrest and release of the cell cycle established quinidine as a cell synchronization agent, with a site of arrest in early G1 preceding the lovastatin G1 arrest site by 5-6 hr. There was a close correspondence among the concentration-dependent arrest by quinidine in G1, depolarization of the membrane potential, and the inhibition of ATP-sensitive potassium currents, supporting a model in which hyperpolarization of the membrane potential and progression through G1 are functionally linked. Furthermore, the G1 arrest by quinidine was overcome by valinomycin, a potassium ionophore that hyperpolarized the membrane potential in the presence of quinidine. With sustained exposure of MCF-7 cells to quinidine, expression of the Ki67 antigen, a marker for cells in cycle, decreased, and apoptotic and necrotic cell death ensued. We conclude that MCF-7 cells that fail to progress through the quinidine-arrest site in G1 die.

Modulation of quinidine-induced arrhythmias by temperature in perfused rabbit heart.

We used low temperature to slow ion channel kinetics and studied the electrophysiological effects of quinidine at different pacing rates in isolated rabbit hearts. Fifteen epicardial electrograms together with an endocardial monophasic action potential were recorded. Epicardial activation and local recovery times were measured. Arrhythmias together with the characteristics of their mode of induction and rate were analyzed by epicardial activation sequence mapping. In the presence of quinidine, arrhythmias consistent with both triggered activity and reentry were observed. At baseline, triggered activity was not inducible, even though at 25 degrees C the recovery time was greater than that in the presence of quinidine at 36 degrees C. Also, with quinidine, the incidence of triggered activity decreased at 30 and 25 degrees C. Therefore prolongation of the recovery time per se does not cause triggered activity. Quinidine's use-dependent effects on conduction and reverse use-dependent effects on recovery time were amplified by low temperatures. These findings can be understood in terms of the known temperature sensitivities of the kinetics of the membrane ion channels responsible for activation and recovery. The results demonstrate that temperature can be used as a tool to elucidate mechanisms of drug action.

Superoxide Release is Involved in Membrane Potential Changes in Mouse Peritoneal Macrophages

Participation of reactive oxygen species (ROS) in the changes in macrophage membrane potential resulted from effects of different agonists has been studied. Treatment of macrophages with chemotactic peptide fMLP or platelet-activating factor (PAF) caused a brief depolarization followed by a long-lasting hyperpolarization. Lipopolysaccharide and interferon-γ only depolarized the plasma membrane. Chemiluminescence measurements indicated that only fMLP and PAF activated macrophages to release ROS. The hyperpolarization response of the cell was significantly decreased in the presence of superoxide dismutase (but not catalase). Moreover, the O 2 ̇ − -generating system, xanthine plus xanthine oxidase, caused a marked hyperpolarization. In all the cases, the hyperpolarization induced by fMLP, PAF and O 2 ̇ − -generating system was found to depend on the concentration of intracellular Ca 2+ and extracellular K +. Furthermore, in the presence of quinidine, a blocker of Ca 2+-dependent K + conductance fMLP and PAF caused only prolonged depolarization while the effect of O 2 ̇ − was reduced to a minimum. These data suggest that the macrophage hyperpolarization response to fMLP and PAF involves superoxide-mediated Ca 2+-dependent alteration of the relative membrane permeability to K +.

Purinergic Modulation of Rat Urinary Bladder Detrusor Smooth Muscle

1. Rat detrusor muscle was responsive to both ATP and adenosine; ATP elicited an excitatory response, whereas adenosine had an inhibitory effect. 2. ATP and adenosine had an inhibitory modulatory action on responses to acetylcholine, potassium depolarization and field stimulation. 3. Quinidine inhibited the ATP response and blocked the inhibitory effect of ATP on acetylcholine, potassium-depolarization and field-stimulation responses. The effect of adenosine remained unaltered in the presence of quinidine. 4. Caffeine and theophylline blocked the adenosine inhibition of responses to field stimulation. 5. It is concluded that excitatory P 2-type purinoreceptors mediated by ATP and inhibitory P 1-type purinoreceptors mediated by adenosine exist in rat urinary bladder detrusor smooth muscle and that both ATP and adenosine exhibit a modulatory action on detrusor muscle agonist-induced responses.

Metabolism of amitriptyline with CYP2D6 expressed in a human cell line

1. Expressed human cytochrome P450 enzyme CPY2D6 was used to metabolize amitriptyline (AMI). It was established that CYP2D6 not only catalyzed ring 10- hydroxylation of AMI, but also mediated its N -demethylation to nortriptyline (NT), as well as the formation of 10-hydroxy-NT from NT. When the metabolism of AMI by CYP2D6 was repeated in the presence of quinidine, none ofthe metabolites, 10-hydroxyAMI, NT and 10-hydroxy-NT, was formed. 2. Biochemical parameters ofNT formation from AMI were determined, yielding Km 47 48 1 32 mu M; Vmax 3 95 0 11 nmol h mg protein. The same parameters were calculated for the formation of 10-hydroxy-AMI (E Z-isomers) from AMI, yielding Km 10 70 0 20 mu M; Vmax 8 99 0 47 nmol h mg protein. 3. The formation of 10-hydroxy-NT from AMI proceeded primarily via NT and to a much lesser extent via 10-hydroxy-AMI. 4. Quantitative analyses of AMI and its metabolites were difficult to reproduce when the metabolites were analysed underivatized. Two derivatization procedures, acetylation and trifluoroacetylation, were employed to improve assay reproducibility.

Use of electrospray ionization liquid chromatography-mass spectrometry to study the role of CYP2D^ in the in vitro metabolism of 5-hydroxytryptamine receptor antagonists

An electrospray ionization liquid chromatographic-mass spectrometric (ESI-LC-MS) method has been developed to study the involvement of the cytochrome P450 isoenzyme CYP2D6 in the in vitro metabolism of the indole containing 5-hydroxytryptamine (5-HT 3) receptor antagonists tropisetron, ondansetron and dolasetron in human liver microsomes. Compounds were eluted using linear gradients of acetonitrile-20 m M ammonium acetate, solvent A, (10:90, v/v) (ph 6.0) and solvent B, (60:40, v/v) (pH 6.0) and a Nucleosil C 4 column. Microsomal incubations were analysed using selected ion monitoring of the molecular ion of parent drug and the molecular ion of hydroxylated metabolites. The involvement of CYP2D6 in drug metabolism was assessed by inhibition studies using quinidine (5 μM), a specific inhibitor of human CYP2D6, as well as by incubating compounds with microsomes prepared from celss transfected with cDNA encoding human CYP2D6. Results showed that the oxidation of all three compounds involved CYP2D6, but only that of tropisetron was inhibited by over 90% in the presence of quinidine. The present method can be applied to pre-clinical compounds, at an early stage of drug discovery, to assess the involvement of CYP2D6 in their metabolism and to screen for those compounds where CYP2D6 is the only isoenzyme implicated in the formation of major metabolites.

Cardiac transient outward potassium current: a pulse chemistry model of frequency-dependent properties.

Recent voltage-clamp studies of isolated myocytes have demonstrated widespread occurrence of a transient outward current (I(to)) carried by potassium ions. In the canine ventricle, this current is well developed in epicardial cells but not in endocardial cells. The resultant spatial dispersion of refractoriness is potentially proarrhythmic and may be amplified by channel blockade. The inactivation and recovery time constants of this channel are in excess of several hundred milliseconds, and consequently channel availability is frequency dependent at physiological stimulation rates. When the time constants associated with transitions between different channel conformations are rapid relative to drug binding kinetics, the interactions between drugs and an ion channel can be approximated by a sequence of first-order reactions, in which binding occurs in pulses in response to pulse train stimulation (pulse chemistry). When channel conformation transition time constants do not meet this constraint, analytical characterizations of the drug-channel interaction must then be modified to reflect the channel time-dependent properties. Here we report that the rate and steady-state amount of frequency-dependent inactivation of I(to) are consistent with a generalization of the channel blockade model: channel availability is reduced in a pulsatile exponential pattern as the stimulation frequency is increased, and the rate of reduction is a linear function of the pulse train depolarizing and recovery intervals. I(to) was reduced in the presence of quinidine. After accounting for the use-dependent availability of I(to) channels, we found little evidence of an additional use-dependent component of block after exposure to quinidine, suggesting that quinidine reacts with both open and closed I(to) channels as though the binding site is continuously accessible. The model provides a useful tool for assessing drug-channel interactions when the reaction cannot be continuously monitored.