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Recent studies in laboratory rodents have revealed that circadian oscillation in the physiologic functions affecting drug disposition underlies the dosing time-dependent change in pharmacokinetics. However, it is difficult to predict the circadian change in the drug pharmacokinetics in a diurnal human by using the data collected from nocturnal rodents. In this study, we used cynomolgus monkeys, diurnal active animals, to evaluate the relevance of intestinal expression of P-glycoprotein (P-gp) to the dosing time dependency of the pharmacokinetics of its substrates. The rhythmic phases of circadian gene expression in the suprachiasmatic nuclei (the mammalian circadian pacemaker) of cynomolgus monkeys were similar to those reported in nocturnal rodents. On the other hand, the expression of circadian clock genes in the intestinal epithelial cells of monkeys oscillated at opposite phases in rodents. The intestinal expression of P-gp in the small intestine of monkeys was also oscillated in a circadian time-dependent manner. Furthermore, the intestinal absorption of P-gp substrates (quinidine and etoposide) was substantially suppressed by administering the drugs at the times of day when P-gp levels were abundant. By contrast, there was no significant dosing time-dependent difference in the absorption of the non-P-gp substrate (acetaminophen). The oscillation in the intestinal expression of P-gp appears to affect the pharmacokinetics of its substrates. Identification of circadian factors affecting the drug disposition in laboratory monkeys may improve the predictive accuracy of pharmacokinetics in humans., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)

This study investigated the impact of the active efflux mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) on the predictability of the unbound brain concentration (C(u,brain)) by the concentration in the cerebrospinal fluid (CSF) (C(u,CSF)) in rats. C(u,brain) is obtained as the product of the total brain concentration and unbound fraction in the brain (f(u,brain)) determined in vitro in brain slices. Twenty-five compounds, including P-gp and/or Bcrp substrates, were given a constant intravenous infusion, and their plasma, brain, and CSF concentrations were determined. P-gp and/or Bcrp substrates, such as verapamil, loperamide, flavopiridol, genistein, quinidine, dantrolene, daidzein, cimetidine, and pefloxacin, showed a higher CSF-to-brain unbound concentration ratio (K(p,uu,CSF/brain)) compared with non-P-gp and non-Bcrp substrates. K(p,uu,CSF/brain) values of P-gp-specific (quinidine and verapamil) and Bcrp-specific (daidzein and genistein) substrates were significantly decreased in Mdr1a/1b(-/-) and Bcrp(-/-) mice, respectively. Furthermore, consistent with the contribution of P-gp and Bcrp to the net efflux at the BBB, K(p,uu,CSF/brain) values of the common substrates (flavopiridol and erlotinib) were markedly decreased in Mdr1a/1b(-/-)/Bcrp(-/-) mice, but only moderately or weakly in Mdr1a/1b(-/-) mice and negligibly in Bcrp(-/-) mice. In conclusion, predictability of C(u,brain) by C(u,CSF) decreases along with the net transport activities by P-gp and Bcrp at the BBB. C(u,CSF) of non-P-gp and non-Bcrp substrates can be a reliable surrogate of C(u,brain) for lipophilic compounds.

The purpose of this study was to examine whether in vivo drug distribution to the brain can be reconstructed by integrating P-glycoprotein (P-gp)/mdr1a expression levels, P-gp in vitro activity, and drug unbound fractions in mouse plasma and brain. For 11 P-gp substrates, in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of mouse P-gp-transfected LLC-PK1 (L-mdr1a) and parental cells. P-gp expression amounts were determined by quantitative targeted absolute proteomics. Unbound drug fractions in plasma and brain were obtained from the literature and by measuring brain slice uptake, respectively. Brain-to-plasma concentration ratios (K(p brain)) and its ratios between wild-type and mdr1a/1b(-/-) mice (K(p brain) ratio) were obtained from the literature or determined by intravenous constant infusion. Unbound brain-to-plasma concentration ratios (K(p,uu,brain)) were estimated from K(p brain) and unbound fractions. Based on pharmacokinetic theory, K(p brain) ratios were reconstructed from in vitro P-gp transport activities and P-gp expression amounts in L-mdr1a cells and mouse brain capillaries. All reconstructed K(p brain) ratios were within a 1.6-fold range of observed values. K(p brain) then was reconstructed from the reconstructed K(p brain) ratios and unbound fractions. K(p,uu,brain) was reconstructed as the reciprocal of the reconstructed K(p brain) ratios. For quinidine, loperamide, risperidone, indinavir, dexamethasone, paclitaxel, verapamil, loratadine, and diazepam, the reconstructed K(p brain) and K(p,uu,brain) agreed with observed and estimated in vivo values within a 3-fold range, respectively. Thus, brain distributions of P-gp substrates can be reconstructed from P-gp expression levels, in vitro activity, and drug unbound fractions.

A synergistic effect of P-glycoprotein (P-gp)/Abcb1a and breast cancer resistance protein (Bcrp)/Abcg2 was reported to limit the brain penetration of their common substrates. This study investigated this based on pharmacokinetics using Mdr1a/1b(-/-), Bcrp(-/-), and Mdr1a/1b(-/-)/Bcrp(-/-) mice. Comparison of the brain- and testis-to-plasma ratios (C(brain)/C(plasma) and C(testis)/C(plasma), respectively) of the reference compounds quinidine and dantrolene for P-gp and Bcrp, respectively, indicates that impairment of either P-gp and Bcrp did not cause any change in the efflux activities of Bcrp or P-gp, respectively, at both the blood-brain barrier (BBB) and blood-testis barrier (BTB). The C(brain)/C(plasma) and C(testis)/C(plasma) of the common substrates erlotinib, flavopiridol, and mitoxantrone were markedly increased in Mdr1a/1b(-/-)/Bcrp(-/-) mice even compared with Mdr1a/1b(-/-) and Bcrp(-/-) mice. Efflux activities by P-gp and Bcrp relative to passive diffusion at the BBB and BTB were separately evaluated based on the C(brain)/C(plasma) and C(testis)/C(plasma) in the knockout strains to the wild-type strain. P-gp made a larger contribution than Bcrp to the net efflux of the common substrates, but Bcrp activities were also significantly larger than passive diffusion. These parameters could reasonably account for the marked increase in C(brain)/C(plasma) and C(testis)/C(plasma) in the Mdr1a/1b(-/-)/Bcrp(-/-) mice. In conclusion, the synergistic effect of P-gp and Bcrp on C(brain)/C(plasma) and C(testis)/C(plasma) can be explained by their contribution to the net efflux at the BBB and BTB without any interaction between P-gp and Bcrp.

To investigate the role of serotonin (5-HT), an important neurotransmitter and hormone/paracrine agent in the small intestine, in the transport activity of P-glycoprotein (P-gp), the intestinal transport of quinidine, a P-gp substrate, was examined in 5-HT-depleted rats prepared by intraperitoneal administration of p-chlorophenylalanine, a specific inhibitor of tryptophan hydroxylase in 5-HT biosynthesis. In the in vitro transport study, quinidine transport across rat jejunum was significantly enhanced in both the secretory and absorptive directions under 5-HT-depleted conditions, although the secretory transport was still predominant. The electrophysiological study suggested that the quinidine transport via passive diffusion was enhanced presumably through a paracellular route. This might be due to looser tight junctions under 5-HT-depleted conditions. The voltage-clamp technique clearly indicated that the secretory transport of quinidine through the transcellular pathway was also enhanced by the depletion of 5-HT. Furthermore, 5-HT depletion increased verapamil-sensitive secretory transport of quinidine in rat jejunum. These results indicate that the secretory transport of quinidine via P-gp was significantly enhanced under 5-HT-depleted conditions. The level of ATP, an energy source for functioning P-gp, wet weight of jejunum, and total protein level in rat jejunal mucosa were not changed by 5-HT depletion, but the expression of P-gp in the brush-border membrane of rat jejunum was significantly induced, which is partly responsible for the enhancement of P-gp activity under the 5-HT-depleted condition.

The disposition of digoxin and the influence of the organic anion transporting polypeptide (Oatp)2 inhibitor rifampicin and the P-glycoprotein (P-gp) inhibitor quinidine on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner after a bolus dose of digoxin (10 microg), a dual substrate for Oatp2 and P-gp as well as CYP3A. Perfusions of digoxin were also examined in groups of rats in the presence of the inhibitors: rifampicin (100 microM) or quinidine (10 microM). In all experiments, perfusate samples were collected for 60 min. Digoxin and its primary metabolite were determined in perfusate and liver by liquid chromatography/mass spectrometry. The area under the curve (AUC) from 0 to 60 min was determined. The AUC +/- S.D. of digoxin was increased from control (3880 +/- 210 nM x min) by rifampicin (5200 +/- 240 nM x min; p < 0.01) and decreased by quinidine (3220 +/- 340 nM x min; P < 0.05). It is concluded that rifampicin limits the hepatic entrance of digoxin and reduced the hepatic exposure of digoxin to CYP3A by inhibiting the basolateral Oatp2 uptake transport, whereas quinidine increased the hepatic exposure of digoxin to CYP3A by inhibiting the canalicular P-gp transport. These data emphasize the importance of uptake and efflux transporters on hepatic drug metabolism.

The objective of the present study was to examine the time course and concentration dependence of modulation of P-glycoprotein (P-gp) activity in the blood-brain barrier (BBB) with consequent influence on substrate uptake into brain tissue. Potential P-gp inducers (rifampin and morphine) were administered subchorionically to P-gp-competent [mdr1a(+/+)] mice to induce P-gp expression in brain; the impact of rifampin pretreatment on brain penetration of verapamil also was evaluated with an in situ brain perfusion technique. In addition, the effect of single-dose rifampin on P-gp BBB transport activity was assessed with brain perfusion using verapamil and quinidine as model P-gp substrates. Chronic exposure to rifampin or morphine induced P-gp expression in mouse brain to a modest extent. However, single-dose rifampin treatment increased the brain uptake of verapamil and quinidine in mdr1a(+/+) mice in a dose- and concentration-dependent manner, consistent with P-gp inhibition. Maximum inhibition of P-gp-mediated efflux of verapamil by rifampin pretreatment in vivo (150 mg/kg) was approximately 55%, whereas there was only approximately 12% inhibition of P-gp-mediated efflux of quinidine at that rifampin dose. Coperfusion of rifampin at a concentration of 500 microM abolished P-gp-mediated efflux of verapamil at the BBB. However, only approximately 40% inhibition of P-gp-mediated efflux of quinidine was observed with coperfusion of rifampin, even at a 2-fold higher rifampin concentration (1000 microM). The present studies demonstrate that P-gp function at the BBB can be modulated by rifampin in a dose- and concentration-dependent manner. The degree to which rifampin inhibits P-gp-mediated transport is dependent on the substrate molecule.

Intracerebral microdialysis probe recovery (extraction fraction) may be influenced by several mass transport processes in the brain, including efflux and uptake exchange between brain and blood. Therefore, changes in probe recovery under various experimental conditions can be useful to characterize fundamental drug transport processes. Accordingly, the effect of inhibiting transport on probe recovery was investigated for two capillary efflux transporters with potentially different membrane localization and transport mechanisms, P-glycoprotein and an organic anion transporter. Fluorescein/probenecid and quinidine/LY-335979 were chosen as the substrate/inhibitor combinations for organic anion transport and P-glycoprotein-medicated transport, respectively. Probenecid decreased the probe recovery of fluorescein in frontal cortex, from 0.21 +/- 0.017 to 0.17 +/- 0.020 (p < 0.01). Quantitative microdialysis calculations indicated that probenecid treatment reduced the total brain elimination rate constant by 3-fold from 0.37 to 0.12 (ml/min. ml of extracellular fluid). In contrast, the microdialysis recovery of quinidine, delivered locally to the brain via the probe perfusate, was not sensitive to P-glycoprotein inhibition by systemically administered LY-335979, a potent and specific inhibitor of P-glycoprotein. Recovery of difluorofluorescein, an analog of fluorescein, was also decreased by probenecid in the frontal cortex but not in the ventricle cerebrospinal fluid. These experimental observations are in qualitative agreement with microdialysis theory incorporating mathematical models of transporter kinetics. These studies suggest that only in certain circumstances will efflux inhibition at the blood-brain barrier and blood-cerebrospinal fluid barrier influence the microdialysis probe recovery, and this may depend upon the substrate and inhibitor examined and their routes of administration, the localization and mechanism of the membrane transporter, as well as the microenvironment surrounding the probe.

We previously demonstrated that increased dietary salt markedly decreases plasma quinidine concentrations shortly after p.o. dosing, without an effect on the drug's terminal elimination half-life or concentrations after i.v. administration. These findings suggest an effect of dietary salt on intestinal metabolism or transport of the drug. Because one effect of salt loading is sympathetic inhibition, we examined the effect of beta-adrenoceptor blockade on salt-related changes in quinidine disposition. Furthermore, we examined whether the action of salt is local or systemic by determining the effect of salt loading by the i.v. route. To assess the effect of beta-blockade, quinidine disposition was studied in eight normal volunteers after a single p.o. dose of quinidine; data were obtained after 1 week on a high-salt diet (400 mEq/day) and 1 week on a low-salt diet (10 mEq/day) during chronic nadolol and compared with those previously obtained in the same subjects without the beta-blocker. beta-Blockade had no effect on oral clearance during the high-salt diet [0.28 +/- 0.1 (quinidine + nadolol) versus 0.30 +/- 0.2 liters/h/kg (quinidine alone)] but increased clearance on the low-salt diet from 0.23 +/- 0.1 to 0.29 +/- 0.1 liters/h/kg (p <. 05). For the i.v. salt study, the disposition of single p.o. and single i.v. doses of quinidine was determined on two occasions in eight subjects: once during a low-salt diet (10 mEq/day) and once during the same diet, supplemented by 400 mEq/day NaCl i.v. for 8 days. In contrast to our findings after p.o. salt loading, i.v. salt loading did not alter the pharmacokinetics of p.o. quinidine. Taken together, these data implicate a local alteration of drug-metabolizing activity and/or drug transport in the intestinal mucosa as the major effect of dietary salt on the disposition of p.o. quinidine and further suggest that beta-adrenergic activation by a low-salt diet is one component of a signaling pathway whereby intestinal drug disposition is suppressed, resulting in increased oral bioavailability.

In humans, meloxicam is metabolized mainly by cytochrome P-450 (CYP)-dependent hydroxylation of the 5'-methyl group. The predominant P-450 enzyme involved in meloxicam metabolism is CYP 2C9, with a minor contribution of CYP 3A4. Quinidine, a CYP 3A4 substrate commonly used as a selective in vitro inhibitor of CYP 2D6, was found to markedly increase the rate of meloxicam hydroxylation during in vitro experiments with human liver microsomes. A similar activation was observed with other compounds that are structurally related to quinidine. Besides quinidine, quinine and hydroquinidine were the most potent activators of meloxicam hydroxylation. Using expressed cytochrome P-450 enzymes and selective chemical inhibitors of CYP 2C9 and CYP 3A4, it was found that quinidine markedly increased the rate of CYP 3A4-mediated meloxicam hydroxylation but was virtually without effect on CYP 2C9. Kinetic analysis was performed to obtain insight into the possible mechanism of activation of CYP 3A4 and into the mutual interaction of quinidine/hydroquinidine and meloxicam. Quinidine and hydroquinidine decreased Km and increased Vmax of meloxicam hydroxylation, which was consistent with a mixed-type nonessential activation. Meloxicam, in turn, decreased both Km and Vmax of quinidine metabolism by CYP 3A4, indicating an uncompetitive inhibition mechanism. These results support the assumption that CYP 3A4 possesses at least two different substrate-binding sites. A clinically relevant effect on meloxicam drug therapy is not expected, because the most likely outcome in practice is moderately decreased meloxicam plasma concentrations.

Recent studies suggest that P-glycoprotein located on the blood-brain barrier restricts the brain uptake of its substrates. We examined the role of P-glycoprotein on the restricted entry of quinidine to the brain. Quinidine is a well known inhibitor of P-glycoprotein, although it is not yet clarified whether quinidine is the substrate for P-glycoprotein. Kinetic analysis of the uptake of quinidine into the rat brain after intravenous bolus administration revealed that the net uptake clearance is 25.5 microl/min/g brain. Intravenous administration of SDZ PSC 833, a multidrug resistance modifier, enhanced the net uptake clearance of quinidine by 15.7-fold. In contrast, no enhancement by SDZ PSC 833 was observed for the brain uptake of mannitol, a marker for the passive diffusion across the blood-brain barrier. The elimination of [3H] quinidine from the rat brain after microinjection into the cerebral cortex was inhibited by preadministered unlabeled quinidine and verapamil. In addition, the brain-to-plasma concentration ratio of quinidine at 10 min after intravenous administration was 27. 6-fold higher in mdr1a knock-out mice than in control mice. These results suggest that P-glycoprotein mediates the efflux of quinidine across the blood-brain barrier, resulting in its restricted entry to the brain.

With the single-pass, isolated, perfused rat heart preparation, we examined the effect of different perfusate albumin concentrations on the capillary permeability and pharmacodynamic effects of quinidine. Nine hearts were perfused for five consecutive 35-min phases with buffer containing quinidine and albumin concentrations of 0%, 0.1%, 1%, 6% and 0%, in that order, with a 55-min washout between each phase. Compared with 0% albumin perfusate, the equilibrium rate of quinidine perfusate output concentration (Cout) was slower with 0.1% albumin but faster with 1% and 6% albumin. Quinidine Cout, unbound fraction (fu) and perfusion flow rate (Q) for each phase of each experiment were fitted by a modified Kety-Renkin-Crone equation. Estimates of the capillary permeability-surface product (PS) for the two 0% albumin phases (14.4 +/- 5.6 and 12.3 +/- 4.4 ml/min) were significantly higher than those for the three albumin phases (5.3 +/- 1.0, 6.7 +/- 1.2 and 7.4 +/- 2.2 ml/min, respectively; P < .05). There was a direct, linear relationship between lengthening of the QT interval of the ECG and total and unbound quinidine concentrations, but the relationship for unbound concentration was independent of fu, showing that the pharmacodynamic effect was mediated by unbound concentration. In four additional experiments with antipyrine instead of quinidine, PS for the two 0% albumin phases (24.7 +/- 6.5 and 23.2 +/- 2.4 ml/min) was not significantly different than that for the three albumin phases (25.5 +/- 6.1, 22.5 +/- 2.9 and 22.9 +/- 3.6 ml/min, respectively). Albumin had no effect on the volume of distribution referenced to unbound drug for either drug.(ABSTRACT TRUNCATED AT 250 WORDS)

Mexiletine is a low clearance drug which undergoes extensive metabolism in man. In vitro studies with human liver microsomes have suggested that major oxidation pathways of mexiletine are predominantly catalyzed by the genetically determined debrisoquine 4-hydroxylase (cytochrome P450IID6) activity. In this study, we investigated the role of debrisoquine polymorphism and the effects of low dose quinidine, a selective inhibitor of cytochrome P450IID6, on the disposition of mexiletine. Fourteen healthy volunteers, 10 with the extensive metabolizer (EM) and 4 with the poor metabolizer (PM) phenotype, received a single 200-mg dose of mexiletine hydrochloride orally on two occasions (1 week apart), once alone and once under steady-state conditions for quinidine (50 mg QID). During the phase mexiletine alone, total clearance, nonrenal clearance and partial metabolic clearance of mexiletine to hydroxymethylmexiletine, to m-hydroxymexiletine and to p-hydroxymexiletine were decreased in PM compared to EM (all P less than .05). In EM, quinidine decreased mexiletine total clearance from 621 +/- 298 to 471 +/- 214 ml/min (mean +/- S.D.; P less than .05) and mexiletine nonrenal clearance from 583 +/- 292 to 404 +/- 188 ml/min (P less than .05). Moreover, quinidine increased mexiletine elimination half-life in EM from 9 +/- 1 to 11 +/- 2 h (P less than .05). In these subjects, partial metabolic clearance to hydroxymethylmexiletine, m-hydroxymexiletine and p-hydroxymexiletine were decreased by quinidine coadministration 5-, 4- and 7-fold, respectively, whereas partial metabolic clearance to N-hydroxymexiletine was unaffected. Changes induced by quinidine in EM were correlated to their debrisoquine metabolic ratio. Thus, genetically determined or pharmacologically induced modulation of cytochrome P450IID6 activity represents a major determinant of mexiletine disposition.

In cultured GH4C1 cells quinidine inhibited basal prolactin (PRL) secretion and that induced by 0.1 to 10 nM thyrotropin-releasing hormone (TRH), 30 mM medium K+ or 30% medium hyposmolarity but did not inhibit secretion induced by 100 nM 12-O-tetradecanoylphorbol 13-acetate. Inhibition of basal PRL secretion was highly correlated with the drug concentration between 30 microM to 1 mM quinidine; 50% inhibition of basal secretion occurred at 300 microM and at this concentration quinidine completely blocked PRL secretion stimulated by TRH, K+ and hyposmolarity. Significant inhibition of TRH-induced PRL secretion was produced by 15 microM quinidine, a concentration equivalent to that in plasma during standard antiarrhythmic therapy with quinidine in humans. In rats in vivo, a single injection of 2 mg i.p. of quinidine gluconate/100 g b.wt. 1 hr before TRH injection significantly inhibited induced TSH secretion by 15%. Quinidine inhibition of secretion may be caused by blocking depolarization of the cell membrane, thus depressing voltage-gated Ca++ channels and preventing a rise in intracellular Ca++ release.