In vivo assessment of black seed oil single dose on prednisolone pharmacokinetics.

OBJECTIVES: To investigate the effect of blackseed oil (BSO) single dose on prednisolone pharmacokinetics via p-gp inhibition. METHODS: Three groups of rats (n = 5) were orally administered the vehicle, verapamil (50 mg/kg) or BSO (5 ml/kg) 15 min prior to prednisolone (5 mg/kg) administration. Blood samples were collected over 24 h and quantified. Non-compartmental analysis was employed to calculate maximum plasma concentration (Cmax), area under the curve (AUC0-last), time to reach Cmax (Tmax), apparent clearance (CL/F), and half-life (t1/2). Statistical significance was considered at p<0.05. RESULTS: Prednisolone Cmax and AUC0-last decreased by 65% and 25% in the BSO group compared to the negative control (P < .0001, .0029, respectively) while they increased by 1.75-folds and 8-folds in verapamil group (P < .0001). Tmax was achieved at 0.16, 0.5, and 0.25 h in the negative control, verapamil, and BSO-treated groups, respectively. CL/F in the treatment group was 1.3-fold and 10-fold higher compared to the negative and positive control, respectively, whereas the t1/2 remained comparable. CONCLUSION: Administration of BSO decreased prednisolone Cmax and AUC0-last in rats indicating that there is a herb-drug interaction; however, p-gp inhibition cannot be concluded. Patients relying on folk medicine in chronic illnesses treatment might need to avoid combining BSO with prednisolone.

Quantitative assessment of cardiomyocyte mechanobiology through high-throughput cantilever-based functional well plate systems.

Proper regulation of the in vitro cell culture environment is essential for disease modelling and drug toxicity screening. The main limitation of well plates used for cell culture is that they cannot accurately maintain energy sources and compounds needed during cell growth. Herein, to understand the importance of perfusion in cardiomyocyte culture, changes in contractile force and heart rate during cardiomyocyte growth are systematically investigated, and the results are compared with those of a perfusion-free system. The proposed perfusion system consists of a Peltier refrigerator, a peristaltic pump, and a functional well plate. A functional well plate with 12 wells is made through injection moulding, with two tubes integrated in the cover for each well to continuously circulate the culture medium. The contractile force of cardiomyocytes growing on the cantilever surface is analysed through changes in cantilever displacement. The maturation of cardiomyocytes is evaluated through fluorescence staining and western blot; cardiomyocytes cultured in the perfusion system show greater maturity than those cultured in a manually replaced culture medium. The pH of the culture medium manually replaced at intervals of 3 days decreases to 6.8, resulting in an abnormal heartbeat, while cardiomyocytes cultured in the perfusion system maintained at pH 7.4 show improved contractility and a uniform heart rate. Two well-known ion channel blockers, verapamil and quinidine, are used to measure changes in the contractile force of cardiomyocytes from the two systems. Cardiomyocytes in the perfusion system show greater stability during drug toxicity screening, proving that the perfusion system provides a better environment for cell growth.

Verapamil hydrochloride loaded solid lipid nanoparticles: Preparation, optimization, characterisation, and assessment of cardioprotective effect in experimental model of myocardial infarcted rats.

Verapamil, a calcium channel blocker has poor bioavailability (20-30%) owing to extensive hepatic first-pass metabolism. Hence, the major objective of this research was to improve the oral bioavailability of Verapamil by Solid Lipid Nanoparticles (V-SLNs) using high shear homogenization and ultrasonication technology. A 32 factorial design was employed to statistically optimize the formulation to get minimum particle size with maximum entrapment efficiency. The average particle size was 218 nm and the entrapment efficiency was 80.32%. The V-SLN formulation exhibited biphasic behavior with a rapid release at first, then a steady release (75-80%) up to 24 h following the Korsmeyer Peppas release model. In the Isoproterenol induced myocardial necrosis model, oral administration of V-SLNs positively modulated almost all the studied hemodynamic parameters such as left ventricular end-diastolic pressure, cardiac injury markers, and tissue architecture. The cardioprotective effect was also confirmed with histopathological studies. When compared with free drugs, in-vivo pharmacokinetic studies demonstrated a rise in t1/2, AUC0-∞, and Cmax, indicating that bioavailability has improved. These encouraging results demonstrate the promising potential of developed V-SLNs for oral delivery and thereby improve the therapeutic outcome.

Simultaneous triple-parametric optical mapping of transmembrane potential, intracellular calcium and NADH for cardiac physiology assessment.

Investigation of the complex relationships and dependencies of multiple cellular processes that govern cardiac physiology and pathophysiology requires simultaneous dynamic assessment of multiple parameters. In this study, we introduce triple-parametric optical mapping to simultaneously image metabolism, electrical excitation, and calcium signaling from the same field of view and demonstrate its application in the field of drug testing and cardiovascular research. We applied this metabolism-excitation-contraction coupling (MECC) methodology to test the effects of blebbistatin, 4-aminopyridine and verapamil on cardiac physiology. While blebbistatin and 4-aminopyridine alter multiple aspects of cardiac function suggesting off-target effects, the effects of verapamil were on-target and it altered only one of ten tested parameters. Triple-parametric optical mapping was also applied during ischemia and reperfusion; and we identified that metabolic changes precede the effects of ischemia on cardiac electrophysiology.

Assessment of Drug Proarrhythmic Potential in Electrically Paced Human Induced Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes Using Multielectrode Array.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for the assessment of drug proarrhythmic potential through multielectrode array (MEA). HiPSC-CM cultures beat spontaneously with a wide range of frequencies, however, which could affect drug-induced changes in repolarization. Pacing hiPSC-CMs at a physiological heart rate more closely resembles the state of in vivo ventricular myocytes and permits the standardization of test conditions to improve consistency. In this study, we systematically investigated the time window of stable ion currents in high-purity hiPSC-derived ventricular cardiomyocytes (hiPSC-vCMs) and confirmed that these cells could be used to correctly predict the proarrhythmic risk of Comprehensive In Vitro Proarrhythmia Assay (CiPA) reference compounds. To evaluate drug proarrhythmic potentials at a physiological beating rate, we used a MEA to electrically pace hiPSC-vCMs, and we recorded regular field potential waveforms in hiPSC-vCMs treated with DMSO and 10 CiPA reference drugs. Prolongation of field potential duration was detected in cells after exposure to high- and intermediate-risk drugs; in addition, drug-induced arrhythmia-like events were observed. The results of this study provide a simple and feasible method to investigate drug proarrhythmic potentials in hiPSC-CMs at a physiological beating rate.

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (ΔΨm), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure ΔΨm across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when ΔΨm-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Telemetered common marmosets is useful for the assessment of electrocardiogram parameters changes induced by multiple cardiac ion channel inhibitors.

The objective of this study is to assess the response of telemetered common marmosets to multiple cardiac ion channel inhibitors and to clarify the usefulness of this animal model in evaluating the effects of drug candidates on electrocardiogram (ECG). Six multiple cardiac ion channel inhibitors (sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine) were orally administered to telemetered common marmosets and changes in QTc, PR interval and QRS duration were evaluated. Drugs plasma levels were determined to compare the sensitivity in common marmosets to that in humans. QTc prolongation was observed in the marmosets dosed with sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine. PR prolongation was noted after flecainide and verapamil administration, and QRS widening occurred following treatment with flecainide and quinidine. Drugs plasma levels associated with ECG changes in marmosets were similar to those in humans, except for verapamil-induced QTc prolongation. Verapamil-induced change is suggested due to body temperature decrease. These results indicate that telemetered common marmoset is a useful animal for evaluation of the ECG effects of multiple cardiac ion channel inhibitors and the influence of body temperature change should be considered in the assessment.

Assessment of the Additional Value of Verapamil to a Moxifloxacin and Linezolid Combination Regimen in a Murine Tuberculosis Model.

The favorable treatment outcome rate for multidrug-resistant tuberculosis (MDR-TB) is only 54%, and therefore new drug regimens are urgently needed. In this study, we evaluated the activity of the combination of moxifloxacin and linezolid as a possible new MDR-TB regimen in a murine TB model and the value of the addition of the efflux pump inhibitor verapamil to this backbone. BALB/c mice were infected with drug-sensitive Mycobacterium tuberculosis and were treated with human-equivalent doses of moxifloxacin (200 mg/kg of body weight) and linezolid (100 mg/kg) with or without verapamil (12.5 mg/kg) for 12 weeks. Pharmacokinetic parameters were collected during treatment at the steady state. After 12 weeks of treatment, a statistically significant decline in mycobacterial load in the lungs was observed with the moxifloxacin-linezolid regimen with and without verapamil (5.9 and 5.0 log CFU, respectively), but sterilization was not achieved yet. The spleens of all mice were culture negative after 12 weeks of treatment with both treatment modalities, and the addition of verapamil caused a significant reduction in relapse (14/14 positive spleens without versus 9/15 with verapamil, P = 0.017). In conclusion, treatment with a combination regimen of moxifloxacin and linezolid showed a strong decline in mycobacterial load in the mice. The addition of verapamil to this backbone had a modest additional effect in terms of reducing mycobacterial load in the lung as well as reducing the spleen relapse rate. These results warrant further studies on the role of efflux pump inhibition in improving the efficacy of MDR-TB backbone regimens.

Evaluation of Strategies for the Assessment of Drug-Drug Interactions Involving Cytochrome P450 Enzymes.

BACKGROUND AND OBJECTIVES: Drug-drug interactions (DDIs) can occur when one drug alters the metabolism of another drug. Drug metabolism mediated by cytochrome P450 enzymes (CYPs) is responsible for the majority of metabolism of known drugs and inhibition of CYP enzymes is a well-known cause of DDIs. In the current study, the use of various human liver microsomes (HLM)-based methods to determine occurrence of CYP-mediated metabolism-dependent inhibition (MDI) and possible follow-up studies were evaluated. METHODS: Human CYP inhibition was studied using the following methodologies: direct inhibition and (non-diluted) IC50-shift assays, a ferricyanide-based reversibility assay, a spectrophotometric metabolic intermediate complex (MIC) assay, and recording of reduced carbon monoxide (CO)-difference spectra. HLM incubations in the presence and absence of NADPH and glutathione (GSH) were performed to study the possible formation of CYP-dependent GSH adducts. HLM incubations with the radiolabeled inhibitors mifepristone and paroxetine were performed to study CYP-mediated covalent binding. RESULTS: Dihydralazine and furafylline displayed irreversible MDI of CYP1A2. Paroxetine displayed both quasi-irreversible and irreversible MDI of CYP2D6, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Mifepristone displayed irreversible MDI of CYP3A4, formation of CYP-dependent GSH adducts was observed, while CYP-mediated covalent binding occurred which was decreased in the presence of GSH. Troleandomycin and verapamil displayed quasi-irreversible MDI of CYP3A4; MIC formation was observed, while no formation of CYP-dependent GSH adducts occurred. CONCLUSIONS: This study gives a representative overview of current methodologies that can be used to study CYP inhibition. The here presented strategy can be applied as a tool during risk evaluation of CYP-mediated DDIs.

The J to T-peak interval as a biomarker in drug safety studies: A method of accuracy assessment applied to two algorithms.

OBJECTIVES: To evaluate performance of J-to-T-peak (JTP) measurements of 12-lead ECGs, in a five-arm study using drugs with various levels of electrolyte channel block. METHODS: The novel evaluation method distinguishes between different aspects of measurement. "Random noise" is the variability among repeated measurements made without changing the conditions. "Context noise" is the variability of changes in context of the measurement, e.g. T-wave morphology, autonomic nervous system state. RESULTS: The average random noise of our RR-corrected JTPc measurements in standard deviations was 3.0 ms and not dependent on the drug. The average context noise was 4.0 ms for ranolazine, verapamil, and placebo, and 8.8 ms for dofetilide and quinidine. Measurement consistency is corroborated by linear fit confidence intervals of baseline- and placebo-corrected JTPc versus drug concentration. CONCLUSIONS: Systematic differences were found in JTPc drug response between the Mortara method and published data. Residual signal component in the context noise may influence future study design.

Translational assessment of cardiac contractility by echocardiography in the telemetered rat.

INTRODUCTION: Cardiac contractility was evaluated using standard inotropic agents in rats. We compared indices of cardiac contractility, i.e. LV dP/dt max from telemetry while simultaneously collecting EF (ejection fraction) and FS (fractional shortening) measures from echocardiography. METHODS: Male Wistar rats were instrumented with telemetry devices for measurements of blood pressure and left ventricular pressure. Milrinone (PDE III inhibitor) and verapamil (L-type calcium channel blocker) at doses of 0, 3, 10, and 30 mg/kg were administered orally using a 4 × 4 Latin square crossover study design. Telemetry data were recorded at predose and continuously for 24h post-dose. Echocardiographic evaluations were conducted once at predose and at 1 and 2h after milrinone or verapamil administration, respectively. During the recording of echocardiograms, telemetry data were collected simultaneously. Blood samples were also collected to confirm plasma drug exposure. RESULTS: As expected, milrinone increased LV dP/dt max, EF and FS while verapamil decreased LV dP/dt max, EF and FS. Linear regression analysis showed a positive correlation between LV dP/dt max and EF or FS (P<0.001) with both test agents. A change in LV dP/dt max of 1000 mmHg/s was found to correspond with a change in EF and FS of 13 and 16%, respectively, in the telemetered rat. DISCUSSION: The correlation between contractility indices assessed by telemetry and echocardiographic methods in rat models has not received much attention to date. Our results with two reference compounds demonstrate that both methods are sensitive to alterations in contractility induced by inotropic agents administered to rats. The high degree of correlation between changes in LV dP/dt max and EF or FS in the rat enables a translational-element of clinical relevance following changes in contractility indices when measured with telemetry devices in preclinical studies.

Assessment of the potential drug-drug interaction between carvedilol and clopidogrel mediated through intestinal P-glycoprotein.

The most widely prescribed oral antiplatelet agent, clopidogrel, shows high interindividual variability resulting in an increased risk of cardiovascular events in the patients with reduced platelet inhibition. The purpose of this study was to investigate the role of the P-glycoprotein (P-gp) efflux pump in limiting the intestinal permeability of clopidogrel and the effect of a ß-blocker, namely, carvedilol, on its intestinal transport. Effective permeabilities (Peff) of clopidogrel and carvedilol were investigated in the proximal jejunum and distal ileum of rats using an in situ intestinal perfusion model. Peff values of clopidogrel and carvedilol were found to be concentration dependent with decreased Peff values at the low perfusate concentrations. Coperfusion with the P-gp inhibitors verapamil (100 µM) and carvedilol (10 µM) significantly increased the Peff of clopidogrel in the jejunum (8.31±0.20 x 10-5 and 6.98±0.75 x 10-5 vs. 3.60±0.51 x 10-5, respectively) and ileum (9.08±2.19 x 10-5 and 8.35±1.58 x 10-5 vs. 3.85±0.15 x 10-5, respectively). However, at the highest concentration tested (30 µM), clopidogrel exhibited 3 and 1.4 times higher Peff than those of metoprolol, an FDA high permeability reference standard, in the jejunum and ileum, respectively. Overall, this study indicates that the efflux function appears not to have a significant impact on the in vivo intestinal absorption of clopidogrel due to the saturation of P-gp, suggesting no clinically relevant interaction between carvedilol and clopidogrel mediated through P-gp at intestinal level.

Comprehensive T wave morphology assessment in a randomized clinical study of dofetilide, quinidine, ranolazine, and verapamil.

BACKGROUND: Congenital long QT syndrome type 2 (abnormal hERG potassium channel) patients can develop flat, asymmetric, and notched T waves. Similar observations have been made with a limited number of hERG-blocking drugs. However, it is not known how additional calcium or late sodium block, that can decrease torsade risk, affects T wave morphology. METHODS AND RESULTS: Twenty-two healthy subjects received a single dose of a pure hERG blocker (dofetilide) and 3 drugs that also block calcium or sodium (quinidine, ranolazine, and verapamil) as part of a 5-period, placebo-controlled cross-over trial. At pre-dose and 15 time-points post-dose, ECGs and plasma drug concentration were assessed. Patch clamp experiments were performed to assess block of hERG, calcium (L-type) and late sodium currents for each drug. Pure hERG block (dofetilide) and strong hERG block with lesser calcium and late sodium block (quinidine) caused substantial T wave morphology changes (P<0.001). Strong late sodium current and hERG block (ranolazine) still caused T wave morphology changes (P<0.01). Strong calcium and hERG block (verapamil) did not cause T wave morphology changes. At equivalent QTc prolongation, multichannel blockers (quinidine and ranolazine) caused equal or greater T wave morphology changes compared with pure hERG block (dofetilide). CONCLUSIONS: T wave morphology changes are directly related to amount of hERG block; however, with quinidine and ranolazine, multichannel block did not prevent T wave morphology changes. A combined approach of assessing multiple ion channels, along with ECG intervals and T wave morphology may provide the greatest insight into drug-ion channel interactions and torsade de pointes risk. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov/ Unique identifier: NCT01873950.

Determination of time-dependent inactivation of CYP3A4 in cryopreserved human hepatocytes and assessment of human drug-drug interactions.

Assessment of time-dependent inhibition (TDI), especially CYP3A4, is an important parameter for preclinical and clinical development. The use of human liver microsomes (HLM) is the most common in vitro matrix to assess TDI, but this often leads to an overprediction of an actual effect observed clinically. Recently, the use of human hepatocytes has been hypothesized as a more relevant and possibly predictive matrix for the assessment of CYP3A4 TDI. Our work evaluates and optimizes three different human hepatocyte assays for the assessment of CYP3A4 TDI using pooled cryopreserved human hepatocytes. Using two of the optimized methods, the time-dependent inhibition kinetic parameters (K(I) and k(inact)) for four known CYP3A4 TDI (diltiazem, erythromycin, verapamil, and troleandomycin) were determined. When comparing TDI in HLM, the K(I) values from hepatocytes were in general 4- to 13-fold higher than that in HLM, whereas the k(inact) values in human hepatocytes were similar or slightly higher or lower depending on the inhibitor. The inactivation potency (k(inact)/K(I)) for four tested CYP3A4 inactivators in human hepatocytes was generally lower than that in HLM due to either lower affinity (K(I)) or lower inactivation rate (k(inact)) or both. When drug interactions were simulated with Simcyp using either HLM or human hepatocyte data, the predictions using the kinetic parameters from human hepatocytes resulted in a much better simulated change in pharmacokinetics compared with observed clinical data.

Assessment of intestinal absorption of total flavones of Hippophae rhamnoides L. in rat using in situ absorption models.

PURPOSE: The objective of this study was to investigate the absorption behavior of total flavones of Hippophae rhamnoides L. (TFH) (the sum of isorhamnetin and quercetin as the index component) in the rat intestine using in situ circulation method. METHODS: The accumulated TFH absorption and related absorption parameters were calculated. Furthermore, the influences of Cremophor ELP and the P-glycoprotein inhibitor, verapamil, on the intestinal absorption of TFH were studied using the in situ circulation model. RESULTS AND DISCUSSION: The results showed that the absorption of TFH increased linearly with its concentration, indicating that a passive diffusion process was dominated. There were no significant differences in the absorption of TFH in three small intestine segments of duodenum, jejunum, and ileum and at different concentrations of Cremophor ELP ranging from 0.25% to 1% (P > 0.05). With the presence of P-gp inhibitor, verapamil, in the circulation fluid, the accumulated absorption of TFH did not increase significantly (P > 0.05). Further studies on the solubility and permeability enhancement of TFH should be investigated to develop new TFH products with high bioavailability.

Vasorelaxant effect in rat aortic rings through calcium channel blockage: a preliminary in vitro assessment of a 1,3,4-oxadiazole derivative.

The study was undertaken on the basis of several reports in the literature that relaxation of vascular smooth muscles is a good treatment strategy in hypertension, angina and other cardiovascular disorders. Oxadiazoles have been reported to have effect on vascular smooth muscles and calcium influx. The goals of our current in vitro study were to investigate the effect of a 1,3,4-oxadiazole derivative on vascular smooth muscles in rat aorta, and to elucidate the associated signaling pathway. NOX-1 induced a relaxation of vascular smooth muscles in both endothelium intact and denuded rat aortic rings precontracted with norepinephrine or phenylephrine or KCl. NOX-1 also significantly antagonized cumulative dose-response effect of norepinephrine, phenylephrine, KCl or calcium with reduction in submaximal contractions. Verapamil, an L-type of calcium channel blocker, effectively attenuated phenylephrine and calcium induced contractions in aortic rings. Incubation with NOX-1 and verapamil did not significantly alter the dose-response curve of phenylephrine or calcium compared to verapamil treatment alone indicating L-type Ca2+ channel blockage leads to loss of NOX-1 activity. Hence it can be concluded NOX-1 exhibited vasorelaxant action by inhibiting calcium influx from extracellular space to intracellular space through L-type of calcium channels.

[Assessment of hemorheological deformability of human red cells exposed to tert-butyl hydroperoxide, verapamil and ascorbate by ektacytometer].

BACKGROUND: Normal erythrocyte is deformable and this facilitates blood flow in the capillaries. Oxidative stress reduces the deformability of erythrocytes, and influences on blood flow in microcirculation. The objective of this study was to investigate the deformability of erythrocytes exposed to oxidative stress, the protective effects of verapamil and ascorbic acid against oxidative damages in erythrocytes, and the value of the microfluidic ektacytometer, RheoScan-D (RheoMeditech, Korea) in clinical application. METHODS: Effects of oxidative stress on erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Before exposure to tBHP, the erythrocytes were pretreated with verapamil and ascorbic acid to examine their protective effect against oxidative damages. The deformability of erythrocytes was measured by the microfluidic ektacytometer, RheoScan-D. RESULTS: When treated with tBHP, the deformability of erythrocytes was decreased (P<0.01) and methemoglobin (metHb) formation and mean corpuscular volume (MCV) of erythrocytes were increased (P<0.01, P<0.05) compared to those of the untreated control cells. Compared to the tBHP treated cells, pretreatment with verapamil increased the deformability of erythrocytes (P<0.01) and decreased metHb formation (P<0.01) and MCV (P<0.05). Likewise, pretreatment with ascorbic acid increased the deformability of erythrocytes (P<0.01) and decreased metHb formation (P<0.01). CONCLUSIONS: Oxidative stress reduces the deformability of erythrocytes and the deformability could be one of markers for oxidative damage. Verapamil and ascorbic acid have protective role against tBHP induced oxidative stress. The ektacytometer, RheoScan-D used in this study is convenient for clinical measurement and could be used in various fields of clinical medicine.

A new procedure for the quantitative assessment of p-glycoprotein efflux pump associated with human T lymphocytes.

INTRODUCTION: P-glycoprotein (P-gp), a multidrug transporter located in plasma membranes, reduces intracellular availability of some drugs. Upregulation of P-gp has been observed in some clinical situations, including chronic inflammatory disease and viral infection. However, P-gp is expressed in only a small subset of peripheral blood mononuclear cells (PBMC) and at much lower quantities than it is on P-gp-positive cell lines used in other studies. METHODS: P-gp expression was assessed by flow cytometry by using a commercially available, anti-P-gp, allophycocyanin-conjugated monoclonal antibody. Flow cytometry was also used to determine the efflux activity associated with P-gp; with this process, refluxed fluorescent P-gp substrate, rhodamine 123 (Rho123), was determined by the subsequently identified P-gp-positive PBMC subset. Use of verapamil during the dye-loading procedure maximized the amount of dye retained by the cells. RESULTS: The use of allophycocyanin-conjugated monoclonal antibody allowed for the identification of P-gp-positive PBMC subsets, even when the cells were fully loaded with Rho123. We used a logical gating strategy to identify a P-gp-positive PBMC subset, after which P-gp efflux activity of the PBMC subset could be quantitatively assessed. This new procedure enabled us to assess the P-gp efflux function of T lymphocytes in some clinical situations, which induced P-gp upregulation in vivo. CONCLUSION: This new procedure enables us to quantitatively assess the P-gp efflux activity associated with PBMC.

Assessment of intestinal absorption of vitexin-2''-o-rhamnoside in hawthorn leaves flavonoids in rat using in situ and in vitro absorption models.

The purpose of this study was to investigate the absorption mechanism of vitexin-2''-O-rhamnoside, the index component in hawthorn leaves flavonoids (HLF) in the rat intestine, using two different absorption models, the in situ single-pass intestinal perfusion and the in vitro everted gut sac model. The effective permeability coefficients (P(eff)) in the in situ single-pass intestinal perfusion experiments and the apparent permeability coefficients (P(app)) in the in vitro everted gut sac experiments were calculated. Furthermore, the influences of the P-glycoprotein inhibitors, verapamil and digoxin, on the intestinal absorption of vitexin-2''-O- rhamnoside in HLF were studied using the above-mentioned two models. Results showed that there were no significant differences in the absorption of vitexin-2''-O-rhamnoside in HLF in four segments of the rat intestine, duodenum, jejunum, ileum, and colon, and at different concentrations of HLF ranging from 0.05 mg/ml to 0.5 mg/ml (P > 0.05). The P(eff) values for vitexin-2''-O-rhamnoside in the rat jejunal perfusion at the concentration of 0.05, 0.1, 0.25, and 0.5 mg/ml were (2.48 +/- 0.33) x 10(-5); (2.23 +/- 0.67) x 10(-5); (2.18 +/- 0.48) x 10(-5); and (2.25 +/- 0.17) x 10(-5) cm/s, respectively. But there was significant difference between absence and presence of verapamil or digoxin (P < 0.05). P(eff) and P(app) values of vitexin-2''-O-rhamnoside in HLF were increased in the presence of verapamil or digoxin. In conclusion, vitexin-2''-O-rhamnoside can be classified into high permeability class drug according to the biopharmaceutical classification system. Passive diffusion dominates the absorptive transport behavior of vitexin-2''-O-rhamnoside in HLF. The absorption and secretion are mediated by the efflux transport system, P-gp. The absorption of vitexin-2''-O-rhamnoside in HLF can be enhanced administered together with P-gp inhibitors.

Comparison of guinea-pig ventricular myocytes and dog Purkinje fibres for in vitro assessment of drug-induced delayed repolarization.

INTRODUCTION: QT interval prolongation and Torsade de Pointes (TdP) arrhythmias are recognised as a potential risk with many drugs, most of which delay cardiac repolarization by inhibiting the rapidly activating K(+) current (I(Kr)). The objective of this study was to compare the effects of compounds on cardiac action potentials recorded from guinea-pig ventricular myocytes and dog Purkinje fibres. METHODS AND RESULTS: Effects of dofetilide, sotalol, cisapride, terfenadine, haloperidol and sparfloxacin, compounds known to cause QT prolongation (positive controls), and nifedipine and verapamil, not associated with QT prolongation (negative controls) were studied on intracellular action potentials recorded from guinea-pig isolated ventricular myocytes (VM) and dog isolated Purkinje fibres (PF). Prolongation of action potential duration (APD) by sotalol, dofetilide and sparfloxacin was concentration-dependent and of greater magnitude in dog PF compared to guinea-pig VM. The maximum prolongation of APD in guinea-pig VM at 0.5 and 1 Hz was approximately 25% and this was associated with complete inhibition of I(Kr) by dofetilide. Effects on APD of cisapride and haloperidol in both preparations, and terfenadine in guinea-pig VM, were biphasic, consistent with inhibition of multiple ion channels. There was no effect of terfenadine on APD in dog PF. Haloperidol increased APD by more than 25% in guinea-pig VM, consistent with effects on additional repolarizing currents. The negative controls shortened APD to a greater extent in guinea-pig VM compared to dog PF. In general, the positive control drugs increased action potential triangulation (APD(40-90)) to a greater extent than APD(90). CONCLUSION: Guinea-pig isolated VM may be more sensitive for detecting APD prolongation with compounds inhibiting multiple ion channels and action potential triangulation (APD(40-90)). Effects on repolarizing currents other than I(Kr) were also distinguished in guinea-pig VM.