Els Wagenaar

Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs

We have generated mice homozygous for a disruption of the mdr1a (also called mdr3) gene, encoding a drug-transporting P-glycoprotein. The mice were viable and fertile and appeared phenotypically normal, but they displayed an increased sensitivity to the centrally neurotoxic pesticide ivermectin (100-fold) and to the carcinostatic drug vinblastine (3-fold). By comparison of mdr1a (+/+) and (-/-) mice, we found that the mdr1a P-glycoprotein is the major P-glycoprotein in the blood-brain barrier and that its absence results in elevated drug levels in many tissues (especially in brain) and in decreased drug elimination. Our findings explain some of the side effects in patients treated with a combination of carcinostatics and P-glycoprotein inhibitors and indicate that these inhibitors might be useful in selectively enhancing the access of a range of drugs to the brain.

Homozygous disruption of the murine MDR2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease

Two types of P-glycoprotein have been found in mammals: the drug-transporting P-glycoproteins and a second type, unable to transport hydrophobic anticancer drugs. The latter is encoded by the human MDR3 (also called MDR2) and the mouse mdr2 genes, and its tissue distribution (bile canalicular membrane of hepatocytes, B cells, heart, and muscle) suggests a specialized metabolic function. We have generated mice homozygous for a disruption of the mdr2 gene. These mice develop a liver disease that appears to be caused by the complete inability of the liver to secrete phospholipid into the bile. Mice heterozygous for the disrupted allele had no detectable liver pathology, but half the level of phospholipid in bile. We conclude that the mdr2 P-glycoprotein has an essential role in the secretion of phosphatidylcholine into bile and hypothesize that it may be a phospholipid transport protein or phospholipid flippase.

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.

The Drosophila homology of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless

We have isolated the Drosophila melanogaster homolog (Dint-1) of int-1, a conserved cellular oncogene implicated in viral mammary tumorigenesis in mice. The deduced Dint-1 protein sequence contains 468 amino acids and starts with a hydrophobic leader; it is 54% identical to the int-1 sequence, and all 23 cysteine residues are conserved. The putative Drosophila protein has an extra sequence of 85 amino acids, encoded on an additional exon. Dint-1 is expressed throughout development, but transcripts are barely detectable in adult flies. Hybridization in situ to embryos reveals a segmented pattern of expression. We show that Dint-1 and the segment polarity gene wingless are identical and map to the same location. The sequence of the gene suggests that the Dint-1/wingless protein functions in morphogenesis as a signal in cell-cell communication.

The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria

The breast cancer resistance protein (BCRP/ABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1/Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1−/− mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1−/− mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents.

Substantial excretion of digoxin via the intestinal mucosa and prevention of long-term digoxin accumulation in the brain by the mdrla P-glycoprotein

1 We have used mice with a disrupted mdrla P‐glycoprotein gene (mdrla (—/—)mice) to study the role of P‐glycoprotein in the pharmacokinetics of digoxin, a model P‐glycoprotein substrate. 2 [3H]‐digoxin at a dose of 0.2 mg kg−1 was administered as a single i.v. or oral bolus injection. We focussed on intestinal mucosa and brain endothelial cells, two major pharmacological barriers, as the mdrla P‐glycoprotein is the only P‐glycoprotein normally present in these tissues. 3 Predominant faecal excretion of [3H]‐digoxin in wild‐type mice shifted towards predominantly urinary excretion in mdrla (—/—) mice. 4 After interruption of the biliary excretion into the intestine, we found a substantial excretion of [3H]‐digoxin via the gut mucosa in wild‐type mice (16% of administered dose over 90 min). This was only 2% in mdrla (—/—) mice. Biliary excretion of [3H]‐digoxin was not dramatically decreased (24% in wild‐type mice versus 16% in mdrla (—/—) mice). 5 After a single bolus injection, brain levels of [3H]‐digoxin in wild‐type mice remained very low, whereas in mdrla (—/—) mice these levels continuously increased over a period of 3 days, resulting in a ∼200 fold higher concentration than in wild‐type mice. 6 These data demonstrate the in vivo contribution of intestinal P‐glycoprotein to direct elimination of [3H]‐digoxin from the systemic circulation and to the pattern of [3H]‐digoxin disposition, and they underline the importance of P‐glycoprotein for the blood‐brain barrier.

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.

Reduced Hepatic Uptake and Intestinal Excretion of Organic Cations in Mice with a Targeted Disruption of the Organic Cation Transporter 1 (Oct1 [Slc22a1]) Gene

ABSTRACT 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. InOct1 −/− 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 inOct1 −/− 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.

Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver

Bilirubin, a breakdown product of heme, is normally glucuronidated and excreted by the liver into bile. Failure of this system can lead to a buildup of conjugated bilirubin in the blood, resulting in jaundice. The mechanistic basis of bilirubin excretion and hyperbilirubinemia syndromes is largely understood, but that of Rotor syndrome, an autosomal recessive disorder characterized by conjugated hyperbilirubinemia, coproporphyrinuria, and near-absent hepatic uptake of anionic diagnostics, has remained enigmatic. Here, we analyzed 8 Rotor-syndrome families and found that Rotor syndrome was linked to mutations predicted to cause complete and simultaneous deficiencies of the organic anion transporting polypeptides OATP1B1 and OATP1B3. These important detoxification-limiting proteins mediate uptake and clearance of countless drugs and drug conjugates across the sinusoidal hepatocyte membrane. OATP1B1 polymorphisms have previously been linked to drug hypersensitivities. Using mice deficient in Oatp1a/1b and in the multispecific sinusoidal export pump Abcc3, we found that Abcc3 secretes bilirubin conjugates into the blood, while Oatp1a/1b transporters mediate their hepatic reuptake. Transgenic expression of human OATP1B1 or OATP1B3 restored the function of this detoxification-enhancing liver-blood shuttle in Oatp1a/1b-deficient mice. Within liver lobules, this shuttle may allow flexible transfer of bilirubin conjugates (and probably also drug conjugates) formed in upstream hepatocytes to downstream hepatocytes, thereby preventing local saturation of further detoxification processes and hepatocyte toxic injury. Thus, disruption of hepatic reuptake of bilirubin glucuronide due to coexisting OATP1B1 and OATP1B3 deficiencies explains Rotor-type hyperbilirubinemia. Moreover, OATP1B1 and OATP1B3 null mutations may confer substantial drug toxicity risks.

Deficiency in the Organic Cation Transporters 1 and 2 (Oct1/Oct2 (Slc22a1/Slc22a2)) in Mice Abolishes Renal Secretion of Organic Cations

ABSTRACT The polyspecific organic cation transporters 1 and 2 (Oct1 and -2) transport a broad range of substrates, including drugs, toxins, and endogenous compounds. Their strategic localization in the basolateral membrane of epithelial cells in the liver, intestine (Oct1), and kidney (Oct1 and Oct2) suggests that they play an essential role in removing noxious compounds from the body. We previously showed that in Oct1−/− mice, the hepatic uptake and intestinal excretion of organic cations are greatly reduced. Since Oct1 and Oct2 have extensively overlapping substrate specificities, they might be functionally redundant. To investigate the pharmacologic and physiologic roles of these proteins, we generated Oct2 single-knockout and Oct1/2 double-knockout mice. Oct2 −/− and Oct1/2 −/− mice are viable and fertile and display no obvious phenotypic abnormalities. Absence of Oct2 in itself had little effect on the pharmacokinetics of tetraethylammonium (TEA), but in Oct1/2 −/− mice, renal secretion of this compound was completely abolished, leaving only glomerular filtration as a TEA clearance mechanism. As a consequence, levels of TEA were substantially increased in the plasma of Oct1/2 −/− mice. This study shows that Oct1 and Oct2 together are essential for renal secretion of (small) organic cations. A deficiency in these proteins may thus result in increased drug sensitivity and toxicity.