Evaluation of Limiting Brain Penetration Related to P-glycoprotein and Breast Cancer Resistance Protein Using [11C]GF120918 by PET in Mice

Purpose

GF120918 has a high inhibitory effect on P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). We developed [¹¹C]GF120918 as a positron emission tomography (PET) probe to assess if dual modulation of P-gp and BCRP is useful to evaluate brain penetration.

Procedures

PET studies using [¹¹C]GF120918 were conducted on P-gp and/or Bcrp knockout mice as well as wild-type mice.

Results

In PET studies, the AUC_{brain [0?C60 min]} and K ₁ value in P-gp/Bcrp knockout mice were nine- and 26-fold higher than that in wild-type mice, respectively. These results suggest that brain penetration of [¹¹C]GF120918 is related to modulation of P-gp and BCRP and is limited by two transporters working together.

Conclusions

PET using [¹¹C]GF120918 may be useful for evaluating the function of P-gp and BCRP. PET using P-gp/Bcrp knockout mice may be an effective method to understand the overall contributions the functions of P-gp and BCRP.     

The role of the breast cancer resistance protein (ABCG2) in the distribution of sorafenib to the brain

ATP-binding cassette transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) have been shown to work in concert to restrict brain penetration of several tyrosine kinase inhibitors. It has been reported that P-gp is dominant in limiting transport of many dual P-gp/BCRP substrates across the blood-brain barrier (BBB). This study investigated the influence of P-gp and BCRP on the central nervous system (CNS) penetration of sorafenib, a multitargeted tyrosine kinase inhibitor currently being evaluated in clinical trials for glioma. In vitro studies showed that BCRP has a high affinity for sorafenib. Sorafenib inhibited P-gp, but did not seem to be a P-gp substrate in vitro. CNS distribution studies showed that transport of sorafenib to the brain was restricted because of active efflux at the BBB. The brain-to-plasma equilibrium-distribution coefficient (area under the concentration-time profiles for plasma/area under the concentration-time profiles for brain) was 0.06 in wild-type mice. Steady-state brain-to-plasma concentration ratio of sorafenib was approximately 0.36 ± 0.056 in the Bcrp1(-/-) mice, 0.11 ± 0.021 in the Mdr1a/b(-/-) mice, and 0.91 ± 0.29 in the Mdr1a/b(-/-)Bcrp1(-/-) mice compared with 0.094 ± 0.007 in the wild-type mice. Sorafenib brain-to-plasma ratios increased on coadministration of the dual P-gp/BCRP inhibitor elacridar such that the ratio in wild-type mice (0.76 ± 0.24), Bcrp1(-/-) mice (1.03 ± 0.33), Mdr1a/b(-/-) mice (1.3 ± 0.29), and Mdr1a/b(-/-)Bcrp1(-/-) mice (0.73 ± 0.35) were not significantly different. This study shows that BCRP and P-gp together restrict the brain distribution of sorafenib with BCRP playing a dominant role in the efflux of sorafenib at the BBB. These findings are clinically relevant to chemotherapy in glioma if restricted drug delivery to the invasive tumor cells results in decreased efficacy.     

P-glycoprotein and mutlidrug resistance-associated proteins limit the brain uptake of saquinavir in mice

Efflux transporters such as P-glycoprotein (P-gp) and multidrug resistance-associated proteins (Mrps) and their contributions to saquinavir (SQV) brain uptake were characterized. Cerebral flow rate was estimated from diazepam uptake and brain vascular volume was assessed using inulin. Mice brains were perfused with buffer containing SQV alone or coperfused with different concentrations of GF120918, a P-gp inhibitor or MK571, a specific Mrp family inhibitor. Inulin, a nonabsorbable marker, was also coperfused in all studies to assess whether the inhibitors altered the physical integrity of the blood-brain barrier (BBB). The estimated cerebral flow rate using diazepam was 250 ml.100g(-1.)min(-1). The brain vascular volume, estimated using inulin, was almost constant (0.94 +/- 0.03 ml.100 g(-1), n = 12) during the perfusion study. SQV uptake kinetics was linear during the sampling period. Inclusion of 10 muM GF120918 in the perfusate resulted in a more than 7-fold increase in the brain distributional volume (i.e., uptake) of SQV. Inclusion of 100 muM MK571 in the perfusate increased SQV apparent brain uptake by more than 4.4-fold, suggesting, for the first time, that Mrp transporters may play an important role in the brain uptake and retention of SQV. Neither GF120918 nor MK571 altered the integrity of the BBB during the time course of the study. Although the current results reaffirm that SQV is a P-gp substrate, this is the first report implicating the Mrp transporter family in the limited brain uptake and retention of SQV in vivo in mice.     

Effect of the ATP-binding cassette drug transporters ABCB1, ABCG2, and ABCC2 on erlotinib hydrochloride (Tarceva) disposition in in vitro and in vivo pharmacokinetic studies employing Bcrp1-/-/Mdr1a/1b-/- (triple-knockout) and wild-type mice

We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATP-binding cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2 (MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral pharmacokinetics of erlotinib hydrochloride in vivo. In vitro cell survival, drug transport, accumulation, and efflux of erlotinib were done using Madin-Darby canine kidney II [MDCKII; wild-type (WT), MDR1, Bcrp1, and MRP2] and LLCPK (WT and MDR1) cells and monolayers as well as the IGROV1 and the derived human BCRP-overexpressing T8 cell lines. In vivo, the pharmacokinetics of erlotinib after p.o. and i.p. administration was studied in Bcrp1/Mdr1a/1b(-/-) (triple-knockout) and WT mice. In vitro, erlotinib was actively transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was observed. In vivo, systemic exposure (P = 0.01) as well as bioavailability of erlotinib after oral administration (5 mg/kg) were statistically significantly increased in Bcrp1/Mdr1a/1b(-/-) knockout mice (60.4%) compared with WT mice (40.0%; P = 0.02). CONCLUSION: Erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib need to be addressed.     

Roles of P-glycoprotein, Bcrp, and Mrp2 in biliary excretion of spiramycin in mice

The multidrug resistance proteins P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein 2 (Mrp2) are the three major canalicular transport proteins responsible for the biliary excretion of most drugs and metabolites. Previous in vitro studies demonstrated that P-gp transported macrolide antibiotics, including spiramycin, which is eliminated primarily by biliary excretion. Bcrp was proposed to be the primary pathway for spiramycin secretion into breast milk. In the present study, the contributions of P-gp, Bcrp, and Mrp2 to the biliary excretion of spiramycin were examined in single-pass perfused livers of male C57BL/6 wild-type, Bcrp-knockout, and Mrp2-knockout mice in the presence or absence of GF120918 (GW918), a P-gp and Bcrp inhibitor. Spiramycin was infused to achieve steady-state conditions, followed by a washout period, and parameters governing spiramycin hepatobiliary disposition were recovered by using pharmacokinetic modeling. In the absence of GW918, the rate constant governing spiramycin biliary excretion was decreased in Mrp2(-) knockout mice (0.0013 +/- 0.0009 min(-1)) relative to wild-type mice (0.0124 +/- 0.0096 min(-1)). These data are consistent with the approximately 8-fold decrease in the recovery of spiramycin in the bile of Mrp2-knockout mice and suggest that Mrp2 is the major canalicular transport protein responsible for spiramycin biliary excretion. Interestingly, biliary recovery of spiramycin in Bcrp-knockout mice was increased in both the absence and presence of GW918 compared to wild-type mice. GW918 significantly decreased the rate constant for spiramycin biliary excretion and the rate constant for basolateral efflux of spiramycin. In conclusion, the biliary excretion of spiramycin in mice is mediated primarily by Mrp2 with a modest P-gp component.     

Impact of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on the brain distribution of a novel BRAF inhibitor: vemurafenib (PLX4032)

Vemurafenib [N-(3-{[5-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]carbonyl}-2,4-difluorophenyl)propane-1-sulfonamide(PLX4032)] is a novel small-molecule BRAF inhibitor, recently approved by the Food and Drug Administration for the treatment of patients with metastatic melanoma with a BRAF(V600E) mutation. The objective of this study was to investigate the role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in the distribution of vemurafenib to the central nervous system. In vitro studies conducted in transfected Madin-Darby canine kidney II cells show that the intracellular accumulation of vemurafenib is significantly restricted because of active efflux by P-gp and BCRP. Bidirectional flux studies indicated greater transport in the basolateral-to-apical direction than the apical-to-basolateral direction because of active efflux by P-gp and BCRP. The selective P-gp and BCRP inhibitors zosuquidar and (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino(1',2':1,6)pyrido(3,4-b)indole-3-propanoic acid-1,1-dimethylethyl ester (Ko143) were able to restore the intracellular accumulation and bidirectional net flux of vemurafenib. The in vivo studies revealed that the brain distribution coefficient (area under the concentration time profile of brain/area under the concentration time profile of plasma) of vemurafenib was 0.004 in wild-type mice. The steady-state brain-to-plasma ratio of vemurafenib was 0.035 ± 0.009 in Mdr1a/b(-/-) mice, 0.009 ± 0.006 in Bcrp1(-/-) mice, and 1.00 ± 0.19 in Mdr1a/b(-/-)Bcrp1(-/-) mice compared with 0.012 ± 0.004 in wild-type mice. These data indicate that the brain distribution of vemurafenib is severely restricted at the blood-brain barrier because of active efflux by both P-gp and BCRP. This finding has important clinical significance given the ongoing trials examining the efficacy of vemurafenib in brain metastases of melanoma.     

In vivo evaluation of P-glycoprotein function at the blood-brain barrier in nonhuman primates using [11C]verapamil

P-glycoprotein (P-gp) is a major efflux transporter contributing to the efflux of a range of xenobiotic compounds at the blood-brain barrier (BBB). In the present study, we evaluated the P-gp function at the BBB using positron emission tomography (PET) in nonhuman primates. Serial brain PET scans were obtained in three rhesus monkeys after intravenous administration of [(11)C]verapamil under control and P-gp inhibition conditions ([PSC833 ([3'-keto-Me-Bmt(1)]-[Val(2)]-cyclosporin) 20 mg/kg/2 h]). The parent [(11)C]verapamil and its metabolites in plasma were determined by HPLC with a positron detector. The initial brain uptake clearance calculated from the integration plot was used for the quantitative analysis. After intravenous administration, [(11)C]verapamil was taken up rapidly into the brain (time to reach the peak, 0.58 min). The blood level of [(11)C]verapamil decreased rapidly, and it underwent metabolism with time. The inhibition of P-gp by PSC833 increased the brain uptake of [(11)C]verapamil 4.61-fold (0.141 versus 0.651 ml/g brain/min, p < 0.05). These results suggest that PET measurement with [(11)C]verapamil can be used for the evaluation of P-gp function at the BBB in the living brain.     

Differential involvement of Mrp2 (Abcc2) and Bcrp (Abcg2) in biliary excretion of 4-methylumbelliferyl glucuronide and sulfate in the rat

The hepatic excretion of hydrophilic conjugates, end products of phase II metabolism, is not completely understood. In the present studies, transport mechanism(s) responsible for the biliary excretion of 4-methylumbelliferyl glucuronide (4MUG) and 4-methylumbelliferyl sulfate (4MUS) were studied. Isolated perfused livers (IPLs) from Mrp2-deficient (TR(-)) Wistar rats were used to examine the role of Mrp2 in the biliary excretion of 4MUG and 4MUS. After a 30-micromol dose of 4-methylumbelliferone, cumulative biliary excretion of 4MUG was extensive in wild-type rat IPLs (25 +/- 3 micromol) but was negligible in TR(-) livers (0.4 +/- 0.1 micromol); coadministration of the Bcrp and P-glycoprotein inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] had no effect on 4MUG biliary excretion in wild-type rat IPLs. In contrast, biliary excretion of 4MUS was partially maintained in Mrp2-deficient rat IPLs. Recovery of 4MUS in bile was approximately 50 to 60% lower in both control TR(-) (149 +/- 8 nmol) and wild-type IPLs with GF120918 coadministration (176 +/- 30 nmol) relative to wild-type control livers (378 +/- 37 nmol) and was nearly abolished in TR(-) IPLs in the presence of GF120918 (13 +/- 8 nmol). These changes were the result of decreased rate constants governing 4MUG and 4MUS biliary excretion. In vitro assays and perfused livers from Bcrp and P-glycoprotein gene-knockout mice indicated that 4MUS did not interact with P-glycoprotein but was transported by Bcrp in a GF120918-sensitive manner. In the rat liver, Mrp2 mediates the biliary excretion of 4MUG, whereas both Mrp2 and Bcrp contribute almost equally to the transport of 4MUS into bile.     

Limited distribution of new quinolone antibacterial agents into brain caused by multiple efflux transporters at the blood-brain barrier

Transport of new quinolone antibacterial agents (quinolones) at the blood-brain barrier (BBB) was studied in vitro by using immortalized rat brain capillary endothelial cells RBEC1, and in vivo by using the brain perfusion method in rats and multidrug-resistant mdr1a/1b gene-deficient mice. The permeability coefficient of grepafloxacin measured by brain perfusion was increased by an excess of unlabeled grepafloxacin, suggesting a participation of a saturable BBB efflux system. Uptake coefficients of [(14)C]grepafloxacin, [(14)C]sparfloxacin, and [(14)C]levofloxacin by RBEC1 cells at the steady state were increased in the presence of the unlabeled quinolones. The steady-state uptake of [(14)C]grepafloxacin was increased in the presence of various quinolones. Brain distributions of [(14)C]grepafloxacin and [(14)C]sparfloxacin evaluated in terms of the brain-to-plasma free concentration ratio in mdr1a/1b gene-deficient mice were significantly higher than those in wild-type mice, demonstrating an involvement of P-glycoprotein as the efflux transporter. Anionic compounds, including 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and genistein, increased the steady-state uptake of [(14)C]grepafloxacin by RBEC1 cells. Because [(14)C]grepafloxacin was transported by multidrug resistance-associated protein (MRP), in MRP1-overexpressing cells and because RBEC1 and primary cultured brain capillary endothelial cells expressed MRP1, this protein may be an additional efflux transporter for quinolones. Furthermore, the permeability coefficient of [(14)C]grepafloxacin across the BBB was increased by DIDS or in the absence of bicarbonate ions in the brain perfusion method. DIDS or bicarbonate ion did not affect MRP1 function. Accordingly, the brain distribution of quinolones is restricted by the action of multiple efflux transporters, including P-glycoprotein, MRP1, and an unknown anion exchange transporter.     

Ixabepilone, a novel microtubule-targeting agent for breast cancer, is a substrate for P-glycoprotein (P-gp/MDR1/ABCB1) but not breast cancer resistance protein (BCRP/ABCG2)

Ixabepilone is the first epothilone to be approved for clinical use. Current data suggest the epothilones have a role in treating taxane-resistant cancers and ixabepilone is unaffected by at least some of the mechanisms underlying chemoresistance. Here, we report a series of cytotoxicity and transport studies to assess the potential role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in ixabepilone resistance. A significant decrease in ixabepilone-mediated cytotoxicity was observed in Madin-Darby canine kidney cells transfected with human multidrug resistance 1 (MDR1) comparative with the parental cells (IC(50) > 2000 nM versus 90 nM). Overexpression of P-gp also resulted in significantly decreased cell susceptibility to docetaxel, paclitaxel, and vinblastine. Bidirectional transport of ixabepilone across monolayers of porcine kidney-derived cells expressing human MDR1 showed a significantly increased efflux ratio relative to the parental cells. A BCRP-overexpressing cell line was developed by transfecting human embryonic kidney (HEK)-293 cells with BCRP cDNA and confirmed by immunoblotting and bodipy prazosin and mitoxantrone uptake. Neither P-gp nor multidrug resistance protein 2 was detected in the cells by corresponding polyclonal antibodies. This HEK-BCRP cell line demonstrated resistance to docetaxel, paclitaxel, vinblastine, and mitoxantrone, in comparison with the parental cell line (7.3, 4.3, 2.9, and 11.9 resistance factor, respectively). Transport inhibition by BCRP inhibitor fumitremorgin C and broad efflux inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) restored drug sensitivity. In contrast, ixabepilone was far less susceptible to BCRP-mediated resistance, resulting in a resistance factor of only 1.2-fold. In summary, these results suggest that P-gp could cause resistance to ixabepilone in tumors and affect the disposition of the drug, but it is unlikely that BCRP mediates any drug resistance to ixabepilone.     

Brain distribution of cediranib is limited by active efflux at the blood-brain barrier

Cediranib is an orally active tyrosine kinase inhibitor that targets the vascular endothelial growth factor receptor family. Because of its potent antiangiogenic and antitumor activities, cediranib has been evaluated for therapy in glioma, a primary brain tumor. This study investigated the influence of two important efflux transporters at the blood-brain barrier, P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp), on the delivery of cediranib to the central nervous system. In vitro studies indicated that cediranib is a dual substrate for both P-gp and Bcrp. It is noteworthy that in spite of the in vitro data the in vivo mouse disposition studies conclusively showed that P-gp was the dominant transporter restricting the brain distribution of cediranib. The brain-to-plasma partitioning (AUC(brain)/AUC(plasma), where AUC is area under the curve) and the steady-state brain-to-plasma concentration ratio of cediranib were approximately 20-fold higher in Mdr1a/b?/? and Mdr1a/b?/?Bcrp1?/? mice compared with wild-type and Bcrp1?/? mice. Moreover, there was no significant difference in brain distribution of cediranib between wild-type and Bcrp1?/? mice and between Mdr1a/b?/? and Mdr1a/b?/?Bcrp1?/? mice. These results show that, unlike other tyrosine kinase inhibitors that are dual substrates for P-gp and Bcrp, Bcrp does not restrict the distribution of cediranib across the blood-brain barrier. We also show that inhibition of P-gp using specific or nonspecific inhibitors resulted in significantly enhanced delivery of cediranib to the brain. Concurrent administration of cediranib with chemical modulators of efflux transporters can be used as a strategy to enhance delivery and thus efficacy of cediranib in the brain. These findings are clinically relevant to the efficacy of cediranib chemotherapy in glioma.     

Expression of ATP-binding cassette membrane transporters in rodent and human sertoli cells: relevance to the permeability of antiretroviral therapy at the blood-testis barrier

The blood-testis barrier (BTB), composed primarily of Sertoli cells, is responsible for protecting developing germ cells from xenobiotic exposure. ATP-binding cassette (ABC) membrane-associated drug efflux transporters, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the multidrug resistance-associated proteins (Mrps), have been shown to restrict antiretroviral drug permeability at blood-tissue barriers such as the blood-brain barrier. However, it remains unclear whether these transporters are functional at the level of Sertoli cells and can regulate anti-HIV drug permeability at the BTB. This study investigated the functional expression of ABC transporters in a mouse Sertoli cell line system (TM4) and in primary cultures of human Sertoli cells (HSECs). Expression of multidrug resistance Mdr1a/1b/MDR1/P-gp, Mrp1/MRP1, and Mrp4/MRP4 is confirmed by quantitative polymerase chain reaction and immunoblotting analysis in TM4 cells and HSECs. Immunofluorescence studies revealed plasma membrane localization of P-gp, Mrp1/MRP1, and Mrp4/MRP4 in both cell systems. However, Bcrp expression and localization was only detected in rodent cells. Accumulation of 1) rhodamine-6G (R-6G), a fluorescent P-gp substrate, 2) [3H]atazanavir, a HIV protease inhibitor and known P-gp substrate, 3) 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein (BCECF), a fluorescent Mrp substrate, and 4) [3H]mitoxantrone, a BCRP substrate, by TM4 monolayer cells in the presence of established inhibitors demonstrates that these transporters are functional. In addition, several anti-HIV drugs significantly enhance the accumulation of R-6G, [3H]atazanavir, BCECF, and [3H]mitoxantrone by TM4 cells. This study provides the first evidence of ABC transporter expression and activity in Sertoli cells and suggests that these transporters could play an important role in restricting antiretroviral drug permeability at the BTB.     

Quantitative evaluation of the impact of active efflux by p-glycoprotein and breast cancer resistance protein at the blood-brain barrier on the predictability of the unbound concentrations of drugs in the brain using cerebrospinal fluid concentration as a surrogate

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.     

Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems

[D-Pen2,D-Pen5]-Enkephalin (DPDPE) is excreted extensively into the bile. Although DPDPE is transported by P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (Mrp2) has been identified as an important mechanism for DPDPE transport across the canalicular membrane of the hepatocyte. The present studies determined the relative impact of Mrp2 and P-gp on the hepatobiliary disposition of [3H]DPDPE in isolated perfused rat livers (IPLs). Perfusate clearance of [3H]DPDPE was not different between livers from control and Mrp2-deficient (TR-) rats. Biliary excretion of [3H]DPDPE in IPLs from Wistar control rats was rapid and extensive. However, when [3H]DPDPE was administered to livers from TR- rats, the rate and extent of excretion decreased significantly. Surprisingly, in the presence of the P-gp inhibitor GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], biliary excretion of [3H]DPDPE was not inhibited in control livers. In contrast, administration of GF120918 to TR- livers further reduced the maximal excretion rate and decreased net biliary excretion of [3H]DPDPE by 87%. GF120918 administration caused an unexpected increase in perfusate clearance in both control and TR- rat livers. At distribution equilibrium, [3H]DPDPE liver/perfusate partitioning was higher in GF120918-treated livers. Results of pharmacokinetic modeling were consistent with the hypothesis that GF120918 inhibited a [3H]DPDPE basolateral excretion mechanism. Mrp2 is the primary mechanism for [3H]DPDPE biliary excretion, and P-gp facilitates excretion of [3H]DPDPE only in the absence of functional Mrp2. [3H]DPDPE is a substrate for a basolateral efflux mechanism that is sensitive to inhibition by GF120918. These data emphasize the importance of using appropriate model systems and comprehensive pharmacokinetic modeling in elucidating the complex interplay between multiple transport systems.     

Impact of p-glycoprotein inhibition and lipopolysaccharide administration on blood-brain barrier transport of colistin in mice

The aim of this study was to investigate the factors limiting the blood-brain barrier (BBB) transport of colistin in healthy mice and to assess the impact of systemic inflammation on the transport of this antibiotic across the BBB. Colistin sulfate (40 mg/kg) was administered subcutaneously to Swiss outbred mice as single and multiple doses to determine any relationship between brain uptake and plasma concentrations of colistin. To assess the effect of P-glycoprotein (P-gp) on BBB transport, colistin sulfate (5 mg/kg) was concomitantly administered intravenously with PSC833 or GF120918 (10 mg/kg). Systemic inflammation was induced by three intraperitoneal injections of lipopolysaccharide (LPS; 3 mg/kg), and BBB transport of colistin was subsequently measured following subcutaneous administration and by an in situ brain perfusion. The brain uptake of colistin was low following single and multiple subcutaneous doses, with brain-to-plasma concentration ratios ranging between 0.021 and 0.037, and this was not significantly enhanced by coadministration of GF120918 or PSC833 (P > 0.05). LPS significantly increased the brain uptake of subcutaneously administered colistin with area under the brain concentration time curve (AUC(brain)) values of 11.7 ± 2.7 μg·h/g and 4.0 ± 0.3 μg·h/g for LPS- and saline-treated mice, respectively (mean ± standard deviation). Similarly, in situ perfusion of colistin led to higher antibiotic brain concentrations in LPS-treated animals than in saline-treated animals, with colistin brain-to-perfusate concentration ratios of 0.019 ± 0.001 and 0.014 ± 0.001, respectively. This study demonstrates that the BBB transport of colistin is negligible in healthy mice; however, brain concentrations of colistin can be significantly enhanced during systemic inflammation, as might be observed in infected patients.     

A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology.     

Expression and transport activity of breast cancer resistance protein (Bcrp/Abcg2) in dually perfused rat placenta and HRP-1 cell line

Breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette transporter family that recognizes a variety of chemically unrelated compounds. Its expression has been revealed in many mammal tissues, including placenta. The purpose of this study was to describe its role in transplacental pharmacokinetics using rat placental HRP-1 cell line and dually perfused rat placenta. In HRP-1 cells, expression of Bcrp, but not P-glycoprotein, was revealed at mRNA and protein levels. Cell accumulation studies confirmed Bcrp-dependent uptake of BODIPY FL prazosin. In the placental perfusion studies, a pharmacokinetic model was applied to distinguish between passive and Bcrp-mediated transplacental passage of cimetidine as a model substrate. Bcrp was shown to act in a concentration-dependent manner and to hinder maternal-to-fetal transport of the drug. Fetal-to-maternal clearance of cimetidine was found to be 25 times higher than that in the opposite direction; this asymmetry was partly eliminated by BCRP inhibitors fumitremorgin C (2 microM) or N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918; 2 microM) and abolished at high cimetidine concentrations (1000 microM). When fetal perfusate was recirculated, Bcrp was found to actively remove cimetidine from the fetal compartment to the maternal compartment even against a concentration gradient and to establish a 2-fold maternal-to-fetal concentration ratio. Based on our results, we propose a two-level defensive role of Bcrp in the rat placenta in which the transporter 1) reduces passage of its substrates from mother to fetus but also 2) removes the drug already present in the fetal circulation.     

Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C

Inhibitors of the breast cancer resistance protein (BCRP/ABCG2) multidrug transporter are of interest as chemosensitizers for clinical drug resistance, for improving the pharmacokinetics of substrate chemotherapeutic drugs, and in functional assays of BCRP activity for tailoring chemotherapy. The fungal toxin fumitremorgin C (FTC) is a potent and specific inhibitor of BCRP, but its neurotoxic effects preclude use in vivo. We have therefore evaluated a new tetracyclic analogue of FTC, Ko143, as a practical inhibitor of BCRP, comparing it with two other analogues in the same class and with GF120918. All three FTC analogues are effective inhibitors of both mouse Bcrp1 and human BCRP, proving highly active for increasing the intracellular drug accumulation and reversing Bcrp1/BCRP-mediated multidrug resistance. Indeed, Ko143 appears to be the most potent BCRP inhibitor known thus far. In contrast, the compounds have only low activity against P-glycoprotein, the multidrug resistance-associated protein (MRP1), or other known drug transporters. They are nontoxic in vitro at useful concentrations and evinced no signs of toxicity in mice at high oral or i.p. doses. Administered p.o. to inhibit intestinal Bcrp1, Ko143 markedly increased the oral availability of topotecan in mice. It is thus the first highly potent and specific BCRP inhibitor applicable in vivo. As such, Ko143 and other FTC analogues of this type represent valuable reagents for analysis of drug resistance mechanisms and may be candidates for development as clinical BCRP inhibitors.     

Assessing safety and efficacy of directed P-glycoprotein inhibition to improve the pharmacokinetic properties of saquinavir coadministered with ritonavir

Using a mouse model, we tested the effects of in vivo P-glycoprotein inhibition to enhance the oral uptake and penetration into pharmacological sanctuary sites of the human immunodeficiency virus protease inhibitor (HPI) saquinavir. The HPI ritonavir is frequently coadministered with saquinavir to improve saquinavir plasma levels since it strongly reduces the cytochrome P450 3A4-mediated metabolism of saquinavir. Previously, we demonstrated that ritonavir is not an efficient P-glycoprotein inhibitor in vivo, evidenced by the limited oral uptake of saquinavir and its penetration into brain and fetus. Increasing drug concentrations in these sites using more effective P-gp inhibitors might improve therapy but could also lead to toxicity. We orally coadministered ritonavir and saquinavir to mice, with or without the potent P-glycoprotein inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Upon GF120918 coadministration, two of seven P-glycoprotein-deficient animals died. Using a decreased ritonavir dose, GF120918 coadministration led to a 4.4-fold increase in the saquinavir plasma area under the curve in wild-type mice, whereas no such effect was observed in P-glycoprotein-deficient mice. Despite the decreased ritonavir dose, all mice did suffer from impaired gastric emptying. Including GF120918 in a multiple (twice daily) dosing regimen, we found continued accumulation of saquinavir in brain over several days, resulting in 10-fold higher levels compared with vehicle-treated mice. Transient ritonavir-related neurotoxicity, however, was observed after the fourth and final drug dosing. Clinical attempts to efficiently inhibit P-glycoprotein function for improved HPI disposition may therefore be feasible, but they should be performed without ritonavir and monitored carefully for unexpected toxicities.     

HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2)

Breast cancer resistance protein (BCRP) is a recently discovered ATP-binding cassette drug transporter. Hence, the full spectrum of therapeutic agents that interact with BCRP remains to be elucidated. Because human immunodeficiency virus protease inhibitors (HPIs) are well known P-glycoprotein (P-gp) substrates, and there is an overlap in substrate specificity between P-gp and BCRP, this study was performed to investigate whether HPIs are substrates and/or inhibitors of BCRP. First, the effect of HPIs on BCRP efflux activity in human embryonic kidney (HEK) cells stably expressing wild-type BCRP (482R) and its two mutants (482T and 482G) was studied by measuring intracellular mitoxantrone fluorescence using flow cytometry. We found that ritonavir, saquinavir, and nelfinavir were effective inhibitors of wild-type BCRP (482R) with IC50 values of 19.5 +/- 0.8 microM, 19.5 +/- 7.6 microM, and 12.5 +/- 4.1 microM, respectively. Ritonavir, saquinavir, and nelfinavir inhibited 482T and 482G with IC50 values that were approximately 2 times greater than that for 482R. Indinavir and amprenavir had no significant inhibition on BCRP activity. Direct efflux of radiolabeled HPIs in HEK cells was measured to determine whether the HPIs are substrates of BCRP. None of the HPIs were found to be transported by BCRP. Together, ritonavir, saquinavir, nelfinavir, indinavir, and amprenavir are not substrates for BCRP. However, ritonavir, saquinavir, and nelfinavir are effective inhibitors of the transporter. These results suggest that BCRP may play an important role in drug-drug interactions involving coadministration of the HPIs with drugs that are substrates of the transporter.