ABC transporters Mdr1a/1b, Bcrp1, Mrp2 and Mrp3 determine the sensitivity to PhIP/DSS-induced colon carcinogenesis and inflammation.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is an abundant dietary carcinogen, formed during high-temperature cooking of meat. In this study, we investigated whether clinically relevant ATP-binding cassette (ABC) efflux transporters can modulate PhIP-induced colorectal carcinogenesis in vivo using wild-type (WT), Bcrp1-/-; Mrp2-/-; Mrp3-/- and Bcrp1-/-; Mdr1a/b-/-; Mrp2-/- mice. We used a physiological mouse model of colorectal cancer; a combination of a single high-dose oral PhIP administration (200 mg/kg), followed by administering a colonic inflammatory agent, dextran sodium sulfate (DSS), in drinking water for 7 days. Pilot experiments showed that both knockout strains were more sensitive to DSS-induced colitis compared to WT mice. Lack of these transporters in mice also led to clearly altered disposition of activated PhIP metabolites after a high-dose oral PhIP administration. The results suggest that Mdr1a/1b, Bcrp1 and Mrp2 contributed to biliary excretion and Mrp3 to sinusoidal secretion of the pre-carcinogenic metabolite N2-OH-PhIP. The levels of a genotoxicity marker, PhIP-5-sulphate, were at least 4- and 17-fold reduced in the intestinal tissue and intestinal content of both knockout strains compared to WT mice. In line with these findings, the level of colon carcinogenesis was reduced by two- to four-fold in both knockout strains compared to WT mice when PhIP and DSS treatments were combined. Thus, perhaps counterintuitively, reduced activity of these ABC transporters may in part protect from PhIP-induced colon carcinogenesis. Collectively, these data suggest that ABC transporters are important in protecting the body from inflammatory agents such as DSS, in the disposition of carcinogenic metabolites, and in determining the sensitivity to dietary PhIP-induced carcinogenesis.

Brain organic cation transporter 2 controls response and vulnerability to stress and GSK3ß signaling.

Interactions between genetic and environmental factors, like exposure to stress, have an important role in the pathogenesis of mood-related psychiatric disorders, such as major depressive disorder. The polyspecific organic cation transporters (OCTs) were shown previously to be sensitive to the stress hormone corticosterone in vitro, suggesting that these transporters might have a physiologic role in the response to stress. Here, we report that OCT2 is expressed in several stress-related circuits in the brain and along the hypothalamic-pituitary-adrenocortical (HPA) axis. Genetic deletion of OCT2 in mice enhanced hormonal response to acute stress and impaired HPA function without altering adrenal sensitivity to adrenocorticotropic hormone (ACTH). As a consequence, OCT2(-/-) mice were potently more sensitive to the action of unpredictable chronic mild stress (UCMS) on depression-related behaviors involving self-care, spatial memory, social interaction and stress-sensitive spontaneous behavior. The functional state of the glycogen synthase kinase-3ß (GSK3ß) signaling pathway, highly responsive to acute stress, was altered in the hippocampus of OCT2(-/-) mice. In vivo pharmacology and western blot experiments argue for increased serotonin tonus as a main mechanism for impaired GSK3ß signaling in OCT2(-/-) mice brain during acute response to stress. Our findings identify OCT2 as an important determinant of the response to stress in the brain, suggesting that in humans OCT2 mutations or blockade by certain therapeutic drugs could interfere with HPA axis function and enhance vulnerability to repeated adverse events leading to stress-related disorders.

Oral co-administration of elacridar and ritonavir enhances plasma levels of oral paclitaxel and docetaxel without affecting relative brain accumulation.

BACKGROUND: The intestinal uptake of the taxanes paclitaxel and docetaxel is seriously hampered by drug efflux through P-glycoprotein (P-gp) and drug metabolism via cytochrome P450 (CYP) 3A. The resulting low oral bioavailability can be boosted by co-administration of P-gp or CYP3A4 inhibitors. METHODS: Paclitaxel or docetaxel (10 mg/kg) was administered to CYP3A4-humanised mice after administration of the P-gp inhibitor elacridar (25 mg kg(-1)) and the CYP3A inhibitor ritonavir (12.5 mg kg(-1)). Plasma and brain concentrations of the taxanes were measured. RESULTS: Oral co-administration of the taxanes with elacridar increased plasma concentrations of paclitaxel (10.7-fold, P<0.001) and docetaxel (four-fold, P<0.001). Co-administration with ritonavir resulted in 2.5-fold (paclitaxel, P<0.001) and 7.3-fold (docetaxel, P<0.001) increases in plasma concentrations. Co-administration with both inhibitors simultaneously resulted in further increased plasma concentrations of paclitaxel (31.9-fold, P<0.001) and docetaxel (37.4-fold, P<0.001). Although boosting of orally applied taxanes with elacridar and ritonavir potentially increases brain accumulation of taxanes, we found that only brain concentrations, but not brain-to-plasma ratios, were increased after co-administration with both inhibitors. CONCLUSIONS: The oral availability of taxanes can be enhanced by co-administration with oral elacridar and ritonavir, without increasing the brain penetration of the taxanes.

P-glycoprotein (MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) restrict brain accumulation of the JAK1/2 inhibitor, CYT387.

CYT387 is an orally bioavailable, small molecule inhibitor of Janus family of tyrosine kinases (JAK) 1 and 2. It is currently undergoing Phase I/II clinical trials for the treatment of myelofibrosis and myeloproliferative neoplasms. We aimed to establish whether the multidrug efflux transporters P-glycoprotein (P-gp; MDR1; ABCB1) and breast cancer resistance protein (BCRP;ABCG2) restrict oral availability and brain penetration of CYT387. In vitro, CYT387 was efficiently transported by both human MDR1 and BCRP, and very efficiently by mouse Bcrp1 and its transport could be inhibited by specific MDR1 inhibitor, zosuquidar and/or specific BCRP inhibitor, Ko143. CYT387 (10 mg/kg) was orally administered to wild-type (WT), Bcrp1(-/-), Mdr1a/1b(-/-) and Bcrp1;Mdr1a/1b(-/-) mice and plasma and brain concentrations were analyzed. Over 8h, systemic exposure of CYT387 was similar between all the strains, indicating that these transporters do not substantially limit oral availability of CYT387. Despite the similar systemic exposure, brain accumulation of CYT387 was increased 10.5- and 56-fold in the Bcrp1;Mdr1a/1b(-/-) mice compared to the WT strain at 2 and 8h after CYT387 administration, respectively. In single Bcrp1(-/-) mice, brain accumulation of CYT387 was more substantially increased than in Mdr1a/1b(-/-) mice, suggesting that CYT387 is a slightly better substrate of Bcrp1 than of Mdr1a at the blood-brain barrier. These results indicate a marked and additive role of Bcrp1 and Mdr1a/1b in restricting brain penetration of CYT387, potentially limiting efficacy of this compound against brain (micro) metastases positioned behind a functional blood-brain barrier.

Quantification of docetaxel and its metabolites in human plasma by liquid chromatography/tandem mass spectrometry.

RATIONALE: During drug development accurate quantification of metabolites in biological samples using mass spectrometry is often hampered by the lack of metabolites of chemically pure quality. However, quantification of metabolites can be useful for assessment and interpretation of (pre)clinical data. We now describe an approach to quantify docetaxel metabolites in human plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using docetaxel calibration standards. METHODS: Metabolites (M1/M3, M2 and M4) were generated using microsomal incubations. Retention times of docetaxel and its metabolites were assessed using an LC/UV assay and peak identification was performed by LC/MS(n). Samples containing isolated metabolites from human faeces were quantified by LC/UV and used as references for spiking human plasma samples. LC/MS/MS was applied to sensitively quantify docetaxel and its metabolites in human plasma using docetaxel calibration standards in a range of 0.25-500 ng/mL. RESULTS: Because ionisation of docetaxel and its metabolites differed, correction factors were established to quantify the metabolites using docetaxel calibration samples. During method validation, accuracy and precision of the metabolites were within ±7.7% and ≤17.6%, respectively, and within ±14.3% and ≤10.1%, respectively, for docetaxel. Metabolites were found to be unstable in human plasma at ambient temperature. After storage up to 1 year at -20 °C, recovered metabolite concentrations were within ±25%. CONCLUSIONS: Development and validation of an LC/MS/MS assay for the quantification of docetaxel and its metabolites M1/M3, M2 and M4 using docetaxel calibration standards is described. The same approach may be used for quantification of metabolites of other drugs by LC/MS/MS when chemically pure reference substances are unavailable.

Development and validation of a UPLC-MS/MS method with a broad linear dynamic range for the quantification of morphine, morphine-3-glucuronide and morphine-6-glucuronide in mouse plasma and tissue homogenates.

The aim of this study was to develop and to validate a UPLC-MS/MS method for the quantification of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in mouse plasma and tissue homogenates to support preclinical pharmacokinetic studies. The sample preparation consisted of protein precipitation with cold (2-8 °C) methanol:acetonitrile (1:1, v/v), evaporation of the supernatant to dryness, and reconstitution of the dry-extracts in 4 mM ammonium formate pH 3.5. Separation was achieved on a Waters UPLC HSS T3 column (150 × 2.1 mm, 1.8 µm) maintained at 50 °C and using gradient elution with a total runtime of 6.7 min. Mobile phase A consisted of 4 mM ammonium formate pH 3.5 and mobile phase B of 0.1% formic acid in methanol:acetonitrile (1:1, v/v). Detection was carried out by tandem mass spectrometry with electrospray ionization in the positive ion mode. The method was validated within a linear range of 1-2,000 ng/mL, 10-20,000 ng/mL, and 0.5-200 ng/mL for morphine, morphine-3-glucuronide, and morphine-6-glucuronide, respectively. In human plasma, the intra- and inter-run precision of all analytes, including the lower limit of quantification levels, were ≤ 15.8%, and the accuracies were between 88.1 and 111.9%. It has been shown that calibration standards prepared in control human plasma can be used for the quantification of the analytes in mouse plasma and tissue homogenates. The applicability of the method was successfully demonstrated in a preclinical pharmacokinetic study in mice.

Development and validation of an LC-MS/MS method with a broad linear dynamic range for the quantification of tivozanib in human and mouse plasma, mouse tissue homogenates, and culture medium.

The first bioanalytical assay for tivozanib in human and mouse plasma, mouse tissue homogenates and culture medium was developed and validated over a linear dynamic range from 0.5 to 5000 ng/mL. The extended concentration range will cover the quantification of tivozanib in the majority of study samples, reducing the need for reanalysis which is often not possible due to limited amount of sample in preclinical studies. A simple and fast pretreatment method was used consisting of protein precipitation with acetonitrile followed by dilution of the supernatant. The final extract was injected onto an Ultra-Performance Liquid Chromatography (UPLC) BEH C18 column with gradient elution of formic acid in water and formic acid in acetonitrile mobile phase. Chromatographic separation was followed by detection with a triple-quadrupole mass spectrometer operating in the positive ion-mode. By simultaneously monitoring the sensitive conventional [M + H]+ isotopologue- product transition for quantification of low concentrations and a less abundant [M + H]++1 isotopologue- product transition to reduce the sensitivity for quantification of high concentrations, we were able to extend the overall linear dynamic range up to 0.5-5000 ng/mL. A full validation was performed in human plasma and a partial validation was executed for the other matrices. All results were within the acceptance criteria of the European Medicines Agency (EMA) guidelines and the US Food and Drug Administration (FDA) guidance, except for the carry-over. This was solved by the analysis of extra matrix blanks and by grouping study samples containing a high tivozanib concentration in the sample sequence. In this way carry-over did not impact the data integrity. We demonstrated that by measuring two multiple reaction monitoring (MRM) transitions for tivozanib, the linear dynamic range could be extended from two to four decades. The assay was successfully applied in pharmacokinetic studies in mice and a transport assay.

Liquid chromatography-tandem mass spectrometric assay for the quantification of galunisertib in human plasma and the application in a pre-clinical study.

Galunisertib is an anti-cancer drug currently evaluated in phase I and II clinical trials. This study describes the development and validation of a bioanalytical assay to quantify galunisertib in human plasma using HPLC-MS/MS. Stable isotope labelled galunisertib was added as internal standard and the analyte and internal standard were extracted from the matrix by protein precipitation using acetonitrile-methanol (50:50, v/v). Final extracts were injected onto a C18 column, gradient elution was applied for chromatographic separation and detection was performed using a triple quadrupole mass spectrometer operating in the positive ion mode. The assay was linear over the range 0.05-10 ng/mL, with acceptable accuracy (bias ranging from -6.1 to 3.1%) and precision (below 5.7% C.V.) values. The applicability of the assay was demonstrated in a pharmacokinetic experiment in mice.

Mitochondrial RNA polymerase: dual role in transcription and replication.

Mitochondrial RNA polymerases from humans, Xenopus laevis and Saccharomyces cerevisiae are very similar in protein composition and function. They consist of a nonspecific core RNA polymerase and a protein factor that confers promoter selectivity on the core component, and they participate in transcription as well as in DNA replication. Amino acid sequence comparisons indicate that the yeast mitochondrial core component is related to bacteriophage T3 and T7 RNA polymerases; mitochondrial and phage polymerases may therefore belong to a family of related polymerases.

Genetic dissection of the function of mammalian P-glycoproteins.

Mammalian P-glycoproteins are plasma membrane proteins belonging to the superfamily of ATP-binding cassette transporters. They were discovered as drug pumps in multidrug-resistant cancer cells, but are also present in many normal tissues. Genetic approaches have helped to dissect the physiological functions and mode of action of P-glycoproteins. Disruption of both genes for the drug-transporting P-glycoproteins in mice has no effect on the normal sheltered life of these mice, but renders them hypersensitive to many drugs. P-glycoprotein appears to be especially important in protecting the brain and in limiting uptake of hydrophobic drugs from the gut. Recent experiments with polarized cells support the idea that drug-transporting P-glycoproteins act by flipping drugs from the inner to the outer leaflet of the plasma membrane.

Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A.

We have previously shown that absence of the mouse mdr1a (also called mdr3) P-glycoprotein in mdr1a (-/-) "knockout" mice has a profound effect on the tissue distribution and elimination of vinblastine and ivermectin, and hence on the toxicity of these compounds. We show here that the mouse mdr1a and the human MDR1 P-glycoprotein actively transport ivermectin, dexamethasone, digoxin, and cyclosporin A and, to a lesser extent, morphine across a polarized kidney epithelial cell layer in vitro. Injection of these radio-labeled drugs in mdr1a (-/-) and wild-type mice resulted in markedly (20- to 50-fold) higher levels of radioactivity in mdr1a (-/-) brain for digoxin and cyclosporin A, with more moderate effects for dexamethasone (2- to 3-fold) and morphine (1.7-fold). Digoxin and cyclosporin A were also more slowly eliminated from mdr1a (-/-) mice. Our findings show that P-glycoprotein can be a major determinant for the pharmacology of several medically important drugs other than anti-cancer agents, especially in the blood-brain barrier. These results may explain a range of pharmacological interactions observed between various drugs in patients.

P-glycoprotein in the blood-brain barrier of mice influences the brain penetration and pharmacological activity of many drugs.

The mouse mdr1a (also called mdr3) P-GP is abundant in the blood-brain barrier, and its absence in mdr1a (-/-) mice leads to highly increased levels of the drugs ivermectin, vinblastine, digoxin, and cyclosporin A in the brain. We show here that the drugs loperamide, domperidone, and ondansetron are transported substrates for the mouse mdr1a P-GP and its human homologue MDR1. Phenytoin is a relatively weaker substrate for each, and the drugs haloperidol, clozapine, and flunitrazepam are transported hardly or not at all. Tissue distribution studies demonstrated that the relative brain penetration of radiolabeled ondansetron and loperamide (and their metabolites) is increased four- and sevenfold, respectively, in mdr1a (-/-) mice. A pilot toxicity study with oral loperamide showed that this peripherally acting antidiarrheal agent gains potent opiatelike activity in the central nervous system of mdr1a (-/-) mice. mdr1a (-/-) mice also showed increased sensitivity to neurolepticlike side effects of oral domperidone. These results point to the possible role that the drug-transporting P-GP(s) may play in the clinical use of many drugs, especially those with potential targets in the central nervous system.

Regulation of biliary lipid secretion by mdr2 P-glycoprotein in the mouse.

Disruption of the mdr2 gene in mice leads to a complete absence of phospholipid from bile (Smit, J. J. M., et al. 1993. Cell. 75:451-462). We have investigated the control of both mdr2 P-glycoprotein (Pgp) expression and bile salt secretion on biliary lipid secretion in the mouse. Lipid secretion was monitored at various bile salt output rates in wild-type mice (+/+), heterozygotes (+/-), and homozygotes (-/-) for mdr2 gene disruption. In (-/-) mice, phospholipid secretion was negligible at all bile salt output rates. In (+/-) mice, a curvilinear relation between bile salt and phospholipid secretion was observed similar to that in (+/+) mice; however, at all bile salt secretion rates phospholipid secretion was reduced compared to (+/+) mice, indicating that mdr2 Pgp exerts a strong control over secretion. Infusion of increasing amounts of taurocholate up to maximal secretory rate led to a decline in the phospholipid and cholesterol secretion in both (+/+) and (+/-) mice in accordance to what has been observed in other species. In contrast, in (-/-) mice cholesterol secretion increased under these conditions while phospholipid output remained extremely low. The increased cholesterol secretion may represent extraction of cholesterol from the canalicular plasma membrane by taurocholate micelles as opposed to the concomitant secretion of both phospholipid and cholesterol in the presence of a functional mdr2 Pgp. Increased bile flow in (-/-) mice could be attributed completely to an increase in the bile salt-independent fraction and may therefore be caused by the bile duct proliferation in these mice.

Full blockade of intestinal P-glycoprotein and extensive inhibition of blood-brain barrier P-glycoprotein by oral treatment of mice with PSC833.

Mice lacking mdr1-type P-glycoproteins (mdr1a/1b [-/-] mice) display large changes in the pharmacokinetics of digoxin and other drugs. Using the kinetics of digoxin in mdr1a/1b (-/-) mice as a model representing a complete block of P-glycoprotein activity, we investigated the activity and specificity of the reversal agent SDZ PSC833 in inhibiting mdr1-type P-glycoproteins in vivo. Oral PSC833 was coadministered with intravenous [3H]digoxin to wild-type and mdr1a/1b (-/-) mice. The direct excretion of [3H]digoxin mediated by P-glycoprotein in the intestinal mucosa of wild-type mice was abolished by administration of PSC833. Hepatobiliary excretion of [3H]digoxin was markedly decreased in both wild-type and mdr1a/1b (-/-) mice by PSC833, the latter effect indicating that in vivo, PSC833 inhibits not only mdr1-type P-glycoproteins, but also other drug transporters. Upon coadministration of PSC833, brain levels of [3H]digoxin in wild-type mice showed a large increase, approaching (but not equaling) the levels found in brains of PSC833-treated mdr1a/1b (-/-) mice. Thus, orally administered PSC833 can inhibit blood-brain barrier P-glycoprotein extensively, and intestinal P-glycoprotein completely. These profound pharmacokinetic effects of PSC833 treatment imply potential risks, but also promising pharmacological applications of the use of effective reversal agents.

Absence or pharmacological blocking of placental P-glycoprotein profoundly increases fetal drug exposure.

It was recently shown that naturally occurring Mdr1a mutant fetuses of the CF-1 outbred mouse stock have no placental Mdr1a P-glycoprotein (P-gp) and that this absence is associated with increased sensitivity to avermectin, a teratogenic pesticide. To further define the role of placental drug-transporting P-gp in toxicological protection of the fetus, we used mice with a targeted disruption of the Mdr1a and Mdr1b genes. Mdr1a(+/-)/1b(+/-) females were mated with Mdr1a(+/-)/1b(+/-) males to obtain fetuses of 3 genotypes (Mdr1a(+/+)/1b(+/+), Mdr1a(+/-)/1b(+/-), and Mdr 1a(-/-)/1b(-/-)) in a single mother. Intravenous administration of the P-gp substrate drugs [(3)H]digoxin, [(14)C]saquinavir, or paclitaxel to pregnant dams revealed that 2.4-, 7-, or 16-fold more drug, respectively, entered the Mdr1a(-/-)/1b(-/-) fetuses than entered wild-type fetuses. Furthermore, placental P-gp activity could be completely inhibited by oral administration of the P-gp blockers PSC833 or GG918 to heterozygous mothers. Our findings imply that the placental drug-transporting P-gp is of great importance in limiting the fetal penetration of various potentially harmful or therapeutic compounds and demonstrate that this P-gp function can be abolished by pharmacological means. The latter principle could be applied clinically to improve pharmacotherapy of the unborn child.

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.

The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.

Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier.

Multidrug resistance protein 1 (MRP1) is a transporter protein that helps to protect normal cells and tumor cells against the influx of certain xenobiotics. We previously showed that Mrp1 protects against cytotoxic drugs at the testis-blood barrier, the oral epithelium, and the kidney urinary collecting duct tubules. Here, we generated Mrp1/Mdr1a/Mdr1b triple-knockout (TKO) mice, and used them together with Mdr1a/Mdr1b double-knockout (DKO) mice to study the contribution of Mrp1 to the tissue distribution and pharmacokinetics of etoposide. We observed increased toxicity in the TKO mice, which accumulated etoposide in brown adipose tissue, colon, salivary gland, heart, and the female urogenital system. Immunohistochemical staining revealed the presence of Mrp1 in the oviduct, uterus, salivary gland, and choroid plexus (CP) epithelium. To explore the transport function of Mrp1 in the CP epithelium, we used TKO and DKO mice cannulated for cerebrospinal fluid (CSF). We show here that the lack of Mrp1 protein causes etoposide levels to increase about 10-fold in the CSF after intravenous administration of the drug. Our results indicate that Mrp1 helps to limit tissue distribution of certain drugs and contributes to the blood-CSF drug-permeability barrier.

Reduced hepatic uptake and intestinal excretion of organic cations in mice with a targeted disruption of the organic cation transporter 1 (Oct1 [Slc22a1]) gene.

The polyspecific organic cation transporter 1 (OCT1 [SLC22A1]) mediates facilitated transport of small (hydrophilic) organic cations. OCT1 is localized at the basolateral membrane of epithelial cells in the liver, kidney, and intestine and could therefore be involved in the elimination of endogenous amines and xenobiotics via these organs. To investigate the pharmacologic and physiologic role of this transport protein, we generated Oct1 knockout (Oct1(-/-)) mice. Oct1(-/-) mice appeared to be viable, healthy, and fertile and displayed no obvious phenotypic abnormalities. The role of Oct1 in the pharmacology of substrate drugs was studied by comparing the distribution and excretion of the model substrate tetraethylammonium (TEA) after intravenous administration to wild-type and Oct1(-/-) mice. In Oct1(-/-) mice, accumulation of TEA in liver was four to sixfold lower than in wild-type mice, whereas direct intestinal excretion of TEA was reduced about twofold. Excretion of TEA into urine over 1 h was 53% of the dose in wild-type mice, compared to 80% in knockout mice, probably because in Oct1(-/-) mice less TEA accumulates in the liver and thus more is available for rapid excretion by the kidney. In addition, we found that absence of Oct1 leads to decreased liver accumulation of the anticancer drug metaiodobenzylguanidine and the neurotoxin 1-methyl-4-phenylpyridium. In conclusion, our data show that Oct1 plays an important role in the uptake of organic cations into the liver and in their direct excretion into the lumen of the small intestine.

Availability of PSC833, a substrate and inhibitor of P-glycoproteins, in various concentrations of serum.

BACKGROUND: P-glycoproteins are membrane-associated transporters that can render cells resistant to a variety of chemotherapeutic drugs. Reversal agents are (preferably nontoxic) drugs that can inhibit these P-glycoproteins and thereby overcome multidrug resistance. PSC833, a cyclosporin A analog, is a reversal agent that has shown potential in in vitro experiments and in clinical trials. We tested PSC833 to determine whether it is a transported substrate of human and murine P-glycoproteins associated with multidrug resistance (encoded by the human MDR1 gene and its murine homolog, mdr1a) and whether it can completely inhibit these P-glycoproteins under simulated in vivo conditions. METHODS: Monolayers of polarized LLC-PK1 pig kidney cells transfected with complementary DNA containing either MDR1 or mdr1a sequences were used to measure the directional transport of P-glycoprotein substrates under various serum conditions. RESULTS: In contrast to two previous studies, we found that PSC833 is transported by both the MDR1 and the mdr1a P-glycoproteins, albeit at a low rate. PSC833 has a very high affinity for the MDR1 P-glycoprotein, and its Michaelis constant (Km) for transport is 50 nM, fourfold lower than for cyclosporin A. Inhibition of drug transport by PSC833 is approximately eightfold less effective in 100% fetal bovine serum than in tissue culture medium containing 10% serum. The concentration of PSC833 necessary to fully inhibit transport of digoxin and paclitaxel (Taxol) under complete (i.e., 100%) serum conditions is higher than the plasma concentrations achieved in clinical trials. CONCLUSIONS: Although PSC833 binds efficiently to the MDR1 P-glycoprotein and is released only sluggishly, the high concentrations of PSC833 necessary to inhibit this P-glycoprotein under complete serum conditions in our in vitro system suggest that it may be difficult for PSC833 alone to produce total inhibition of P-glycoprotein activity in patients.