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Research ArticleArticle

INTERACTION OF TRIAZOLAM AND KETOCONAZOLE IN P-GLYCOPROTEIN-DEFICIENT MICE

Lisa L. von Moltke, Brian W. Granda, Jeffrey M. Grassi, Michael D. Perloff, Daesety Vishnuvardhan and David J. Greenblatt
Drug Metabolism and Disposition August 2004, 32 (8) 800-804; DOI: https://doi.org/10.1124/dmd.32.8.800

Abstract

The role of P-glycoprotein (P-gp) on the distribution of the benzodiazepine triazolam (TRZ) and the azole antifungal agent ketoconazole (KET), and on the TRZ-KET interaction, was studied using mdr1a(-) or mdr1a/b(-/-) mice (P-gp-deficient mice) and matched controls. TRZ and KET also were studied in Caco-2 cells in Transwell culture. After single i.p. injections of TRZ or KET in separate groups of control mice, brain concentrations of TRZ exceeded those in serum [brain/serum area under the concentration curve (AUC) ratio, 5.0], whereas brain/serum AUC ratios for KET were approximately 0.5. On the basis of single time points, brain concentrations of TRZ, or brain/serum ratios, were similar in P-gp-deficient animals compared with controls, whereas P-gp-deficient animals had significantly higher KET brain concentrations and brain/serum ratios. Coadministration of KET with TRZ increased TRZ concentrations in serum, liver, and brain, both in controls and in P-gp-deficient animals, probably attributable to impairment by KET of CYP3A-mediated clearance of TRZ. However, KET did not increase brain/serum ratios of TRZ in either group. In Caco-2 cells, basal-to-apical flux of TRZ was higher than apical-to-basal flux. However, verapamil (100 μM) did not alter flux in either direction. KET inhibited basal-to-apical transport of rho-damine-123, with a 50% inhibitory concentration of 2.7 μM. Thus, TRZ does not appear to undergo measurable blood-brain barrier efflux transport by P-gp in this animal model. KET impairs clearance of TRZ but does not increase tissue uptake. However, KET itself may be a substrate for efflux transport at the blood-brain barrier.

P-Glycoprotein (P-gp; ABCB1) is a member of the ATP-binding cassette (ABC) superfamily of transport proteins and has been the focus of a number of areas of interrelated research (Kim, 2000; Ayrton and Morgan, 2001). This transporter is highly conserved across species and is found in both normal and malignantly transformed tissues. Early research involving P-gp focused on the phenomenon of multi-drug resistance in tumors, which developed resistance to a wide variety of other chemically unrelated drugs after exposure to a single agent (Wacher et al., 1995; Gottesman et al., 1996). Such resistance was presumed to be due in part to the functional capacity of P-gp to actively transport drugs out of the cell against a concentration gradient.

More recently, drug disposition has been shown to be importantly dependent on P-gp transport activity for a number of commonly used drugs. P-gp has been shown to have a role in reducing gastrointestinal absorption, enhancing elimination in both urine and bile and limiting entry of drugs that are P-gp substrates into the central nervous system (CNS) (Watkins, 1997; Schinkel, 1999; Fromm, 2000; Wacher et al., 2001). Many drugs that are transportable by P-gp are also substrates for cytochrome P450 3A (CYP3A) metabolism, and some drugs that inhibit P-gp activity have also been found to inhibit CYP3A biotransformation.

Of interest are drugs for which CNS entry appears to be greatly restricted by the presence and activity of P-gp (such as loperamide), as well agents for which CNS penetration and activity are more modestly influenced by P-gp (such as morphine) (von Moltke et al., 2000, 2001). The latter may have concentration and overall pharmacodynamic profiles that are more subtly modulated by changes in the amount or activity of the transport protein.

In this study, we evaluated whether the triazolobenzodiazepine derivative triazolam (TRZ) is a P-gp substrate in an in vitro cell culture model, and whether in vivo TRZ CNS concentrations are dependent on the activity of P-gp. The intact animal study utilized mdr1a(-) and mdr1a/b (-/-) strains of mice, neither of which express P-gp in the blood-brain barrier (Schinkel, 1998).

Materials and Methods

Cell Culture Studies. The human colon adenocarcinoma cell line (Caco-2) was kindly provided by Douglas Jefferson, Ph.D. (Tufts University School of Medicine and Tufts-New England Medical Center, Boston, MA) and used at passages 30 to 40. Caco-2 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids (Invitrogen), 100 units/ml penicillin, and 0.1 mg/ml streptomycin.

Caco-2 cells were seeded at 2 × 104/cm2 in polycarbonate membrane Transwell plates (2.5-cm diameter, 3-μm pore size) (Costar, Cambridge, MA) with 2.5 ml of media in the basolateral (B) and 1.5 ml in the apical (A) chamber. Cells were grown in a humidified chamber (37°C, 5% CO2) with media changes every 3 to 5 days. Transport experiments were conducted on days 16 to 20 after seeding. The index substrate rhodamine 123 (Rh123), presumed to be transported mainly by P-gp, was used to evaluate the effect of ketoconazole on the transport process. A methanolic solution of ketoconazole was evaporated to dryness and reconstituted in Opti-MEM serum-free media (Invitrogen), and 0.5% dimethyl sulfoxide. Rh123 was added as a concentrated methanolic solution, with a final methanol concentration of less than 0.5%.

Opti-MEM containing Rh123 (5 μM) was added to the apical (A) or basolateral (B) chamber, with ketoconazole (0 μM, and 2.5–10 μM) present in both chambers. Verapamil (100 μM), an established inhibitor of transport, was used as a positive control. Cells were incubated at 37°C in 5% CO2 for up to 180 min, and 50-μl samples were taken from the chamber, initially not containing Rh123. Samples were diluted with 500 μl of methanol, and fluorescence was determined at 500/550 nm (excitation/emission) using a Model LS50B Luminescence Spectrometer (PerkinElmer Life and Analytical Sciences, Boston, MA).

In this system, passage of Rh123 A to B is attributable to passive diffusion and occurs at a low rate. B to A passage occurs by active transport, presumed to be mediated mainly by P-gp (Perloff et al., 2001a,b, 2003; Störmer et al., 2002; von Moltke et al., 2002). The effect of ketoconazole on B to A transport of Rh123 was evaluated as a ratio of the transport rate in the presence of the ketoconazole divided by the control rate with no ketoconazole. The IC50 value for transport inhibition by ketoconazole was calculated using nonlinear regression as described previously (von Moltke et al., 2002).

The role of TRZ as a substrate for transport was evaluated by substituting TRZ (10 μM) in place of Rh123. Concentrations of TRZ in apical or basal chambers were determined by HPLC with ultraviolet detection.

Animal Studies. In preparation for interaction studies in mice, the kinetics and tissue distribution of TRZ and ketoconazole (KET) were determined after intraperitoneal injection in separate groups of CD-1 mice.

Kinetics of TRZ and KET. Preliminary studies were performed in male CD-1 mice, aged 6 to 8 weeks, weighing approximately 30 g (Charles River Laboratories, Inc., Wilmington, MA). Pure samples of TRZ and KET were kindly provided by their pharmaceutical manufacturers, or purchased from commercial sources. TRZ was dissolved in a vehicle consisting of saline/polyethylene glycol-400 (90:10). Animals received a single 0.175 mg/kg dose by intraperitoneal (i.p.) injection. Animals were sacrificed (n = 4 per group) at 20, 40, 60, 80, and 100 min after injection. Trunk blood was allowed to clot in additive-free tubes. Samples were centrifuged, and the serum was separated. Whole liver and brain were harvested, weighed, and mechanically homogenized in 2 ml of saline.

Aliquots of serum (0.1 ml) were added to extraction tubes along with a triazolobenzodiazepine internal standard (Greenblatt et al., 1981). Samples were extracted by vortex mixing with toluene/isoamyl alcohol (98.5:1.5). After centrifugation, the organic extract was transferred to a 2-ml autosampling vial and evaporated to dryness. The residue was reconstituted with toluene/isoamyl alcohol and analyzed by gas chromatography with electron capture detection as described previously (von Moltke et al., 1996). Serum TRZ concentrations were determined from a calibration curve extracted from drug-free control serum containing varying known amounts of TRZ. Analysis of TRZ in brain and liver tissue proceeded similarly, using aliquots of saline homogenates and calibration curves constructed from homogenates of drug-free control brain or liver tissue. The sensitivity limit is approximately 0.2 ng of TRZ per sample, and the within- and between-day variation of replicate samples does not exceed 8%.

KET was dissolved in polyethylene glycol-400. Animals received a single i.p. injection (50 μl total volume) of 50 mg/kg KET. Animals (n = 4 per group) were sacrificed at 20, 40, 60, 80, and 100 min after dosage, with blood, brain, and liver tissue obtained and processed as described above. For analysis of KET, dextromethorphan was added as an internal standard to study samples and to appropriate calibration standards. Samples were alkalinized with 0.1 ml of 1 N NaOH and extracted by vortex mixing with ethyl acetate/isoamyl alcohol. The organic extract was separated, evaporated to dryness, reconstituted in mobile phase, and transferred to HPLC autosampling vials with limited volume inserts. Samples were analyzed by HPLC with absorbance detection of 220 nm, using conditions described in detail previously (Greenblatt et al., 1998). The sensitivity limit is approximately 0.1 μg of KET per sample, and the within- and between-day variance of replicate samples does not exceed 10%.

Serum, brain, and liver concentrations of TRZ and KET in relation to time after dosage were analyzed using standard pharmacokinetic methods (von Moltke et al., 1996) to determine the total area under the concentration-time curve from time 0 to infinity (AUC). These were used to calculate brain/serum and liver/serum AUC ratios.

KET Distribution in mdr1a/b(-/-) Mice. Eight mdr1a/b(-/-) mice and eight genetically matched FVB control animals (Taconic Farms, Germantown, NY) received single 50 mg/kg i.p. doses of KET as described above. At 20 min after the injection, animals were sacrificed; blood, brain, and liver were obtained and processed as described above.

Interaction of TRZ and KET. Twelve mdr1a(-) mice and 12 genetically matched FVB control mice were divided into groups of three animals as indicated in Table 1. KET (50 mg/kg) or vehicle was administered i.p. at time 0. Fifteen minutes later, TRZ (0.175 mg/kg) or vehicle was given i.p. After another 15 min (30 min after KET or vehicle), animals were sacrificed; and blood, brain, and liver were obtained and processed as described above. These sampling times were based on the preliminary study, and were chosen to assure adequate systemic concentrations of KET as well as to approximately coincide with maximum levels of TRZ.

View this table:
TABLE 1

Concentrations of triazolam and ketoconazole in control mice and in mdr1a(-) mice Values are mean (±S.E.) (n = 3 per group). Administered doses were: KET, 50 mg/kg, attime zero, followed by TRZ, 0.175 mg/kg 15 min later. Animals were sacrificed 15 min after TRZ dosage (30 min after KET dosage).

Results

In Vitro Studies. In the Transwell Caco-2 cell culture system, coaddition of KET inhibited basal to apical flux of Rh123 in a concentration-dependent manner, with an IC50 value of 2.6 μM (Fig. 1). The index inhibitor, verapamil (100 μM), reduced basal to apical flux to 16% of control values.

Fig. 1.
Fig. 1.

Mean (±S.E., n = 3) basal-to-apical (B to A) flux of Rh123 in the Caco-2 Transwell cell culture system. At each ketoconazole concentration, flux was expressed as a percentage ratio versus the control value with no ketoconazole present. The 50% inhibitory concentration (IC50) was determined by nonlinear regression.

Flux of TRZ from apical to basal chambers was lower than flux from basal to apical chambers (Fig. 2). Coaddition of verapamil did not influence TRZ flux in either direction.

Fig. 2.
Fig. 2.

Mean (±S.E., n = 3) basal-to-apical (B to A) and apical-to-basal (A to B) flux of triazolam (10 μM) in the Caco-2 Transwell system. Values are shown in the control condition, and with coaddition of verapamil (100 μM).

Animal Studies.Kinetics of TRZ and KET.Figure 3 shows serum, brain, and liver concentrations of TRZ and KET after single intraperitoneal injections. Liver and brain concentrations of TRZ substantially exceeded those in serum. The brain/serum and liver/serum AUC ratios were 5.0 and 7.9, respectively. Liver concentrations of KET also exceeded those in serum (liver/serum AUC ratio, 2.7), but brain concentrations of KET were lower than serum levels (brain/serum AUC ratio, 0.23).

Fig. 3.
Fig. 3.

Mean (±S.E., n = 4 at each point) serum, brain, and liver concentrations of triazolam (left) and ketoconazole (right) after single intraperitoneal injections of triazolam or ketoconazole administered to male CD-1 mice. This preliminary study was done to establish appropriate sampling time points for subsequent studies involving P-gp-deficient animals.

KET distribution in mdr1a/b(-/-) mice. At 20 min after single i.p. doses, serum KET concentrations were not significantly different between FVB control mice and mdr1a/b(-/-) mice (Fig. 4). Liver concentrations also did not differ between groups (mean ± S.E.: 111 ± 6 μg/g versus 87 ± 10 μg/g, respectively). However, brain concentrations of KET were significantly lower in control mice compared with mdr1a/b(-/-) mice (Fig. 4), as were brain/serum concentration ratios (0.49 ± 0.08 versus 1.20 ± 0.11, respectively; p < 0.001).

Fig. 4.
Fig. 4.

Mean (±S.E., n = 8) serum and brain concentrations of ketoconazole at 30 min after a single 50 mg/kg intraperitoneal injection of ketoconazole in mdr1a/b(-/-) animals and in FVB controls. Serum ketoconazole concentrations did not differ between the two groups. However, brain concentrations were significantly higher in the P-gp-deficient animals (☆, p < 0.001).

Interaction of TRZ and KET. Among animals receiving TRZ alone, there was no difference between FVB controls and mdr1a(-) animals in serum, brain, or liver concentrations of TRZ, nor in the brain/serum or liver/serum ratios (Table 1). As in the initial kinetic study (Fig. 3), brain and liver concentrations of TRZ considerably exceeded those in serum, with brain/serum and liver/serum ratios much higher than 1.0.

Compared with animals receiving TRZ alone, coadministration of TRZ with KET caused an elevation of serum, brain, and liver concentrations of TRZ. This was true in both FVB controls and mdr1a(-) animals (Table 1; Fig. 5). Brain/serum TRZ ratios, although slightly lower in animals cotreated with KET, were not significantly different between TRZ alone and TRZ plus KET groups, either in FVB controls or in mdr1a(-) mice. Likewise, liver/serum ratios were not significantly changed by cotreatment of KET.

Fig. 5.
Fig. 5.

Mean (±S.E., n = 3) serum (left) and brain (right) concentrations of triazolam in mdr1a(-) mice and in FVB controls at 15 min following a single 0.175 mg/kg dose of triazolam, both with and without ketoconazole.

Discussion

In vitro transport studies using Caco-2 cells in Transwell culture indicate that ketoconazole is an inhibitor of Rh123 basal-to-apical flux, presumably explained by the capacity of ketoconazole to inhibit transport mediated by P-gp. This is consistent with previous studies demonstrating impairment by ketoconazole of P-gp transport activity in vitro (Takano et al., 1998; Kim et al., 1999; Raeissi et al., 1999; Yumoto et al., 1999; Wang et al., 2002), or in experimental studies of enteric or blood-brain barrier transport of P-gp substrates in intact animals (Miyama et al., 1998; Zhang et al., 1998). In one clinical study, CSF/unbound plasma concentration ratios for ritonavir, an antiretroviral drug that is a substrate for transport by P-gp, was modestly (although significantly) increased by coadministration of ketoconazole (Khaliq et al., 2000). The findings suggest that ketoconazole is a modest inhibitor of P-gp (and possibly other transporters) in addition to its well documented ability to inhibit CYP3A in humans with high potency (Venkatakrishnan et al., 2000). P-gp inhibition is also a property of the structurally similar azole antifungal agent, itraconazole (Miyama et al., 1998; Nishihara et al., 1999; Takara et al., 2000; Wang et al., 2002). However, it remains unclear whether ketoconazole itself is a substrate for transport by P-gp. Although itraconazole appears to be a P-gp substrate (Miyama et al., 1998), the work of Takano et al. (1998) showed no difference between apical-basal and basal-apical flux of ketoconazole in Caco-2 cells. Kim et al. (1999) found that basal-apical flux of ketoconazole exceeded apical-basal flux, but flux in either direction was not influenced by coaddition of a P-gp inhibitor. After single oral doses of ketoconazole, plasma and brain ketoconazole concentrations both were higher in mdr1a(-/-) mice compared with +/+ controls, but the brain/plasma ratio was not increased in mdr1a(-/-) animals (Kim et al., 1999). The present study demonstrated no difference between mdr1a/b(-/-) mice and controls in serum concentrations of ketoconazole after a single i.p. dose; however, brain concentrations and brain/serum ratios were significantly higher in mdr1a/b(-/-) animals. This finding is consistent with impaired efflux transport of ketoconazole across the blood-brain barrier in the P-gp-deficient mice. The reasons for the inconsistency among the studies is not established. The in vivo studies of Kim et al. (1999) utilized radioactive ketoconazole, whereas the present work was based on nonlabeled ketoconazole and use of an HPLC assay to assess brain/serum partitioning. In any case, further investigation will be needed to clarify the possible role of ketoconazole as a substrate for transport by P-gp.

Ketoconazole coadministration increased triazolam concentrations in serum, brain, and liver, both in the control animals and in the mdr1a(-) mice. Triazolam biotransformation in mice is mediated mainly by CYP3A isoforms and is strongly inhibited by ketoconazole (Fahey et al., 1998; Perloff et al., 1999, 2000). Therefore, the triazolam-ketoconazole interaction in the present study is most likely attributable to impairment of triazolam clearance by ketoconazole. We also observed that triazolam concentrations without coadministration of ketoconazole were similar between control and mdr1a(-) animals, consistent with in vitro studies showing that CYP3A expression and function is not impaired in P-gp knockout mice compared with controls (Perloff et al., 1999; Schuetz et al., 2000). Brain concentrations of triazolam, as well as the brain/serum concentration ratio, were not different between control and mdr1a(-) animals, and brain/serum ratios were not increased by ketoconazole. This indicates that triazolam is unlikely to be a substrate for transport by P-gp. Our in vitro studies demonstrated that flux of triazolam in the basal-to-apical direction exceeded flux in the apical-to-basal direction. However, flux in either direction was not affected by coaddition of verapamil. This suggests that differential flux, possibly attributable to transport, may be operative in the Caco-2 Transwell system, but the insensitivity to verapamil inhibition indicates that any operative transport is likely to be mediated by transporters other than P-gp (Taipalensuu et al., 2001; Nakamura et al., 2002). One previous in vitro study of midazolam transport yielded similar results (Takano et al., 1998). Other in vitro studies of midazolam (Kim et al., 1999), diazepam (Yamazaki et al., 2001), and flunitrazepam (Schinkel et al., 1996) indicated little or no differential transport. Possible discrepancies between in vitro and intact animal studies may be explained by inherent limitations in the Transwell cell culture model, such that substrate flux in both directions may be strongly influenced by lipophilicity of the substrate (Lentz et al., 2000; Doan et al., 2002). Triazolam could also be a substrate for transport by other transporters expressed in Caco-2 cells (Taipalensuu et al., 2001; Nakamura et al., 2002) that are not expressed in blood-brain barrier cells in vivo.

The present study, along with other available data, indicates that ketoconazole inhibits triazolam clearance and elevates triazolam serum and tissue concentration in vivo, both in control mice and P-gp-deficient animals. Ketoconazole inhibits P-gp-mediated transport in vitro but does not alter triazolam brain uptake in vivo. Furthermore, triazolam tissue distribution, including brain uptake, is not different between control and P-gp-deficient mice. Triazolam brain/serum ratios are inherently very high (Arendt et al., 1987) and are not importantly influenced by active efflux transport across the blood-brain barrier.

Footnotes

  • This work was supported in part by Grants MH-58435, DA-05258, DA-13209, DK/AI-58496, DA-13834, AG-17880, AT-01381, and RR-00054 from the Department of Health and Human Services.

  • ABBREVIATIONS: P-gp, P-glycoprotein; CNS, central nervous system; TRZ, triazolam; B, basolateral; A, apical; KET, ketoconazole; Rh123, rhodamine 123; HPLC, high-performance liquid chromatography; IC50, 50% inhibitory concentration; AUC, area under the concentration curve.

    • Received January 30, 2004.
    • Accepted April 15, 2004.

References

  1. ↵
    Arendt RM, Greenblatt DJ, Liebisch DC, Luu MD, and Paul SM (1987) Determinants of benzodiazepine brain uptake: lipophilicity versus binding affinity. Psychopharmacology 93: 72-76.
    OpenUrlCrossRefPubMed
  2. ↵
    Ayrton A and Morgan P (2001) Role of transport proteins in drug absorption, distribution and excretion. Xenobiotica 31: 469-497.
    OpenUrlCrossRefPubMed
  3. ↵
    Fahey JM, Pritchard GA, von Moltke LL, Pratt JS, Grassi JM, Shader RI, and Greenblatt DJ (1998) The effects of ketoconazole on triazolam pharmacokinetics, pharmacodynamics and benzodiazepine receptor binding in mice. J Pharmacol Exp Ther 285: 271-276.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Fromm MF (2000) P-glycoprotein: a defense mechanism limiting oral bioavailability and CNS accumulation of drugs. Int J Clin Pharmacol Ther 38: 69-74.
    OpenUrlPubMed
  5. ↵
    Gottesman MM, Pastan I, and Ambudkar SV (1996) P-glycoprotein and multidrug resistance. Curr Opin Genet Dev 6: 610-617.
    OpenUrlCrossRefPubMed
  6. ↵
    Greenblatt DJ, Divoll M, Moschitto LJ, and Shader RI (1981) Electron-capture gas chromatographic analysis of the triazolobenzodiazepines alprazolam and triazolam. J Chromatogr 225: 202-207.
    OpenUrlPubMed
  7. ↵
    Greenblatt DJ, von Moltke LL, Harmatz JS, Mertzanis P, Graf JA, Durol ALB, Counihan M, Roth-Schechter B, and Shader RI (1998) Kinetic and dynamic interaction study of zolpidem with ketoconazole, itraconazole and fluconazole. Clin Pharmacol Ther 64: 661-671.
    OpenUrlCrossRefPubMed
  8. Hunter J and Hirst BH (1997) Intestinal secretion of drugs. The role of P-glycoprotein and related drug efflux systems in limiting oral drug absorption. Adv Drug Delivery Rev 25: 129-157.
    OpenUrlCrossRef
  9. ↵
    Khaliq Y, Gallicano K, Venance S, Kravcik S, and Cameron DW (2000) Effect of ketoconazole on ritonavir and saquinavir concentrations in plasma and cerebrospinal fluid from patients infected with human immunodeficiency virus. Clin Pharmacol Ther 68: 637-646.
    OpenUrlCrossRefPubMed
  10. ↵
    Kim RB (2000) Transporters and drug disposition. Curr Opin Drug Discovery Dev 3: 94-101.
    OpenUrlPubMed
  11. ↵
    Kim RB, Wandel C, Leake B, Cvetkovic M, Fromm MF, Dempsey PJ, Roden MM, Belas F, Chaudhary AK, Roden DM, et al. (1999) Interrelationship between substrates and inhibitors of human CYP3A and P-glycoprotein. Pharm Res (NY) 16: 408-414.
    OpenUrl
  12. Kohji T, Yusuke T, Fusao K, Kohshi N, Toshiyuki S, and Katsuhiko O (2000) Nicardipine and itraconazole inhibited transcellular transport of digoxin. Pharm Pharmacol Commun 6: 167-171.
    OpenUrl
  13. ↵
    Lentz KA, Polli JW, Wring SA, Humphreys JE, and Polli JE (2000) Influence of passive permeability on apparent P-glycoprotein kinetics. Pharm Res (NY) 17: 1456-1460.
    OpenUrlCrossRef
  14. Mahar Doan KM, Humphreys JE, Webster LO, Wring SA, Shampine LJ, Serabjit-Singh CJ, Adkison KK, and Polli JW (2002) Passive permeability and P-glycoprotein-mediated efflux differentiate central nervous system (CNS) and non-CNS marketed drugs. J Pharmacol Exp Ther 303: 1029-1037.
    OpenUrlAbstract/FREE Full Text
  15. Matheny CJ, Lamb MW, Brouwer KL, and Pollack GM (2001) Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy 21: 778-796.
    OpenUrlCrossRefPubMed
  16. ↵
    Miyama T, Takanaga H, Matsuo H, Yamano K, Yamamoto K, Iga T, Naito M, Tsuruo T, Ishizuka H, Kawahara Y, and Sawada Y (1998) P-glycoprotein-mediated transport of itraconazole across the blood-brain barrier. Antimicrob Agents Chemother 42: 1738-1744.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Nakamura T, Sakaeda T, Ohmoto N, Tamura T, Aoyama N, Shirakawa T, Kamigaki T, Kim KI, Kim SR, Kuroda Y, et al. (2002) Real-time quantitative polymerase chain reaction for MDR1, MRP1, MRP2, and CYP3A-mRNA levels in Caco-2 cell lines, human duodenal enterocytes, normal colorectal tissues and colorectal adenocarcinomas. Drug Metab Dispos 30: 4-6.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    Nishihara K, Hibino J, Kotaki H, Sawada Y, and Iga T (1999) Effect of itraconazole on the pharmacokinetics of digoxin in guinea pigs. Biopharm Drug Dispos 20: 145-149.
    OpenUrlCrossRefPubMed
  19. ↵
    Perloff MD, Störmer E, von Moltke LL, and Greenblatt DJ (2003) Rapid assessment of P-glycoprotein inhibition and induction in vitro. Pharm Res (NY) 20: 1177-1183.
    OpenUrl
  20. ↵
    Perloff MD, von Moltke LL, Cotreau MM, and Greenblatt DJ (1999) Unchanged CYP 3A expression and metabolism of midazolam, triazolam and dexamethasone in mdr(-/-) mouse liver microsomes. Biochem Pharmacol 57: 1227-1232.
    OpenUrlCrossRefPubMed
  21. ↵
    Perloff MD, von Moltke LL, Court MH, Kotegawa T, Shader RI, and Greenblatt DJ (2000) Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms. J Pharmacol Exp Ther 292: 618-628.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Perloff MD, von Moltke LL, Marchand JE, and Greenblatt DJ (2001a) Ritonavir induces P-glycoprotein expression, multidrug resistance-associated protein (MRP1) expression and drug transporter-mediated activity in a human intestinal cell line. J Pharm Sci 90: 1829-1837.
    OpenUrlCrossRefPubMed
  23. ↵
    Perloff MD, von Moltke LL, Störmer E, Shader RI, and Greenblatt DJ (2001b) Saint John's wort: an in vitro analysis of P-glycoprotein induction due to extended exposure. Br J Pharmacol 134: 1601-1608.
    OpenUrlCrossRefPubMed
  24. ↵
    Raeissi SD, Hidalgo IJ, Segura-Aguilar J, and Artursson P (1999) Interplay between CYP3A-mediated metabolism and polarized efflux of terfenadine and its metabolites in intestinal epithelial Caco-2 (TC7) cell monolayers. Pharm Res (NY) 16: 625-632.
    OpenUrl
  25. ↵
    Schinkel AH (1998) Pharmacological insights from P-glycoprotein knockout mice. Int J Clin Pharmacol Ther 36: 9-13.
    OpenUrlPubMed
  26. ↵
    Schinkel AH (1999) P-glycoprotein, a gatekeeper in the blood-brain barrier. Adv Drug Delivery Rev 36: 179-194.
    OpenUrlCrossRefPubMed
  27. ↵
    Schinkel AH, Wagenaar E, Mol CA, and van Deemter L (1996) P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs. J Clin Investig 97: 2517-2524.
    OpenUrlCrossRefPubMed
  28. ↵
    Schuetz EG, Umbenhauer DR, Yasuda K, Brimer C, Nguyen L, Relling MV, Schuetz JD, and Schinkel AH (2000) Altered expression of hepatic cytochromes P-450 in mice deficient in one or more mdr1 genes. Mol Pharmacol 57: 188-197.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Störmer E, von Moltke LL, Perloff MD, and Greenblatt DJ (2002) Differential modulation of P-glycoprotein expression and activity by non-nucleoside HIV-1 reverse transcriptase inhibitors in cell culture. Pharm Res (NY) 19: 1038-1045.
    OpenUrl
  30. ↵
    Taipalensuu J, Törnblom H, Lindberg G, Einarsson C, Sjöqvist F, Melhus H, Garberg P, Sjöström B, Lundgren B, and Artursson P (2001) Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial caco-2 cell monolayers. J Pharmacol Exp Ther 299: 164-170.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Takano M, Hasegawa R, Fukuda T, Yumoto R, Nagai J, and Murakami T (1998) Interaction with P-glycoprotein and transport of erythromycin, midazolam and ketoconazole in Caco-2 cells. Eur J Pharmacol 358: 289-294.
    OpenUrlCrossRefPubMed
  32. ↵
    Venkatakrishnan K, von Moltke LL, and Greenblatt DJ (2000) Effects of the antifungal agents on oxidative drug metabolism in humans: clinical relevance. Clin Pharmacokinet 38: 111-180.
    OpenUrlCrossRefPubMed
  33. von Moltke LL and Greenblatt DJ (2000) Drug transporters in psychopharmacology—are they important? J Clin Phychopharmacol 20: 291-294.
    OpenUrlCrossRefPubMed
  34. von Moltke LL and Greenblatt DJ (2001) Drug transporters revisited. J Clin Phychopharmacol 21: 1-3.
    OpenUrlCrossRefPubMed
  35. ↵
    von Moltke LL, Greenblatt DJ, Harmatz JS, Duan SX, Harrel LM, Cotreau-Bibbo MM, Pritchard GA, Wright CE, and Shader RI (1996) Triazolam biotransformation by human liver microsomes in vitro: effects of metabolic inhibitors and clinical confirmation of a predicted interaction with ketoconazole. J Pharmacol Exp Ther 276: 370-379.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    von Moltke LL, Weemhoff JL, Perloff MD, Hesse LM, Harmatz JS, Roth-Schechter BF, and Greenblatt DJ (2002) Effect of zolpidem on human cytochrome P450 activity and on transport mediated by P-glycoprotein. Biopharm Drug Dispos 23: 361-367.
    OpenUrlCrossRefPubMed
  37. ↵
    Wacher VJ, Wu C-Y, and Benet LZ (1995) Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery and activity in cancer chemotherapy. Mol Carcinog 13: 129-134.
    OpenUrlCrossRefPubMed
  38. ↵
    Wang EJ, Lew K, Casciano CN, Clement RP, and Johnson WW (2002) Interaction of common azole antifungals with P glycoprotein. Antimicrob Agents Chemother 46: 160-165.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Watkins PB (1997) The barrier function of CYP3A4 and P-glycoprotein in the small bowel. Adv Drug Delivery Rev 27: 161-170.
    OpenUrlCrossRefPubMed
  40. ↵
    Yamazaki M, Neway WE, Ohe T, Chen I, Rowe JF, Hochman JH, Chiba M, and Lin JH (2001) In vitro substrate identification studies for p-glycoprotein-mediated transport: species difference and predictability of in vivo results. J Pharmacol Exp Ther 296: 723-735.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Yumoto R, Murakami T, Nakamoto Y, Hasegawa R, Nagai J, and Takano M (1999) Transport of rhodamine 123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers in the presence of cytochrome P-450 3A-related compounds. J Pharmacol Exp Ther 289: 149-155.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Zhang Y, Hsieh Y, Izumi T, Lin ET, and Benet LZ (1998) Effects of ketoconazole on the intestinal metabolism, transport and oral bioavailability of K02, a novel vinylsulfone peptidomimetic cysteine protease inhibitor and a P450 3A, P-glycoprotein dual substrate, in male Sprague-Dawley rats. J Pharmacol Exp Ther 287: 246-252.
    OpenUrlAbstract/FREE Full Text
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INTERACTION OF TRIAZOLAM AND KETOCONAZOLE IN P-GLYCOPROTEIN-DEFICIENT MICE

Lisa L. von Moltke, Brian W. Granda, Jeffrey M. Grassi, Michael D. Perloff, Daesety Vishnuvardhan and David J. Greenblatt
Drug Metabolism and Disposition August 1, 2004, 32 (8) 800-804; DOI: https://doi.org/10.1124/dmd.32.8.800
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