Johan W. Smit

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.

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.

Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other drugs

Background: Various drug transporters of the ATP-binding cassette (ABC) family restrict the oral bioavailability and cellular, brain, testis, cerebrospinal fluid and fetal penetration of substrate drugs. MDRI P-glycoprotein (P-gp) has been demonstrated to transport most HIV protease inhibitors (HPI) and to reduce their oral bioavailability and lymphocyte, brain, testis and fetal penetration, possibly resulting in major limiting effects on the therapeutic efficacy of these drugs. Objectives: To investigate whether the ABC transporters MRP1, MRP2, MRP3, MRP5 and breast cancer resistance protein 1 (Bcrp1) are efficient transporters of the HPI saquinavir, ritonavir and indinavir. Methods: Polarized epithelial non-human (canine) cell lines transduced with human or murine complementary DNA (cDNA) for each of the transporters were used to study transepithelial transport of the HPI. Results: MRP2 efficiently transported saquinavir, ritonavir and indinavir and this transport could be enhanced by probenecid. Sulfinpyrazone was also able to enhance MRP2-mediated saquinavir transport. In contrast, MRP1, MRP3, MRP5, or Bcrp1 did not efficiently transport the HPI tested. Conclusions: Human MRP2 actively transports several HPI and could, based on its known and assumed tissue distribution, therefore reduce HPI oral bioavailability. It may also limit brain and fetal penetration of these drugs and increase their hepatobiliary, intestinal and renal clearance. MRP2 function and enhancement of its activity could adversely affect the therapeutic efficacy, including the pharmacological sanctuary penetration, of HPI. In vivo inhibition of MRP2 function might, therefore, improve HIV/AIDS therapy.

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.

Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.

Ectopic fat and insulin resistance: pathophysiology and effect of diet and lifestyle interventions.

The storage of triglyceride (TG) droplets in nonadipose tissues is called ectopic fat storage. Ectopic fat is associated with insulin resistance and type 2 diabetes mellitus (T2DM). Not the triglycerides per se but the accumulation of intermediates of lipid metabolism in organs, such as the liver, skeletal muscle, and heart seem to disrupt metabolic processes and impair organ function. We describe the mechanisms of ectopic fat depositions in the liver, skeletal muscle, and in and around the heart and the consequences for each organs function. In addition, we systematically reviewed the literature for the effects of diet-induced weight loss and exercise on ectopic fat depositions.

Hepatobiliary elimination of cationic drugs: the role of P-glycoproteins and other ATP-dependent transporters

Abstract The liver plays a central role in the elimination of basic drugs. To fulfil this role, the liver is equipped with a number of uptake and excretion mechanisms for organic cations. A number of these transporters have been identified functionally and at the molecular level. At the level of uptake, at least three transporters have been identified, of which two have been cloned recently and seem to be involved in the uptake of cationic compounds; Octl and Oatp. Both proteins are discussed with regard to their contribution to cation uptake, along with other mechanisms that have been detected. At the level of the bile canaliculi, members of the ATP binding cassette (ABC) superfamily as well as a cation:proton antiporter seem to be involved in excretion of cationic drugs into bile. With regard to this aspect, current evidence for the involvement of various isoforms of P-glycoprotein and the multidrug resistance associated protein (MRP) in the biliary excretion process of cationic drugs is presented. The physiological and pharmacological role of P-glycoprotein in liver, including substrate specificity, regulation and induction of the transport protein are discussed. P-glycoprotein gene disruption studies in our laboratory demonstrate the essential function of this ATP-dependent transport system in hepatobiliary and intestinal transport of cationic drugs.

Interactions between P‐glycoprotein substrates and other cationic drugs at the hepatic excretory level

In the present study it was tested whether known P‐glycoprotein (P‐gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tri‐n‐butylmethylammonium (TBuMA) (a relatively small type 1 organic cation), rocuronium (Roc) (a bulky type 2 organic cation) and the classical P‐gp substrate doxorubicin (Dox). Inhibitors were given in a 4 fold molar excess to the substrate studied. To minimize an interaction of the substrates at the hepatic uptake level, the competing compounds were added when over 55% to 85% of the administered dose of the model compounds had been removed from the perfusate and taken up by the liver. We found a mutual interaction between TBuMA and procainamidethobromide (PAEB), both type 1 cationic compounds during biliary excretion. Interestingly, type 2 compounds, such as rocuronium, clearly inhibited type 1 cationic drugs as well as Dox secretion into bile, whereas type 1 compounds did not significantly inhibit type 2 drug excretion into bile. The type 1 cations PAEB and TBuMA only moderately inhibited Dox biliary excretion. Dox did not inhibit the biliary excretion of the type 2 agent rocuronium whereas rocuronium reduced Dox biliary excretion by 50% compared to controls. MDR substrates/reversal agents like verapamil, quinine, quinidine and vinblastine strongly reduced both type 1 and type 2 organic cation excretion into bile. Dox secretion into bile was also profoundly reduced by these drugs, vinblastine being the most potent inhibitor in general. The lack of mutual inhibition observed in some combinations of substrates may indicate that major differences in affinity of the substrates for a single excretory system exist. Alternatively, multiple organic cation transport systems with separate substrate specificities may be involved in the biliary excretion of amphiphilic drugs. Furthermore, the present study revealed a clear positive correlation between the lipophilicity of the potential inhibitors studied and their respective inhibitory activity on the biliary excretion of the model drugs investigated. Our data are compatible with a potential involvement of P‐glycoprotein in the hepatobiliary excretion of doxorubicin as well as of some type 1 and type 2 organic cations. Furthermore we postulate that the hydrophobic properties of the amphiphilic cationic drugs studied play a crucial role in the accommodation of these agents by P‐glycoprotein and/or other potential cationic drug carrier proteins in the canalicular membrane.

Distinct effects of pioglitazone and metformin on circulating sclerostin and biochemical markers of bone turnover in men with type 2 diabetes mellitus

OBJECTIVE Patients with type 2 diabetes mellitus (T2DM) have an increased risk of fractures and thiazolidinediones (TZDs) increase this risk. TZDs stimulate the expression of sclerostin, a negative regulator of bone formation, in vitro. Abnormal sclerostin production may, therefore, be involved in the pathogenesis of increased bone fragility in patients with T2DM treated with TZDs. METHODS We measured serum sclerostin, procollagen type 1 amino-terminal propeptide (P1NP), and carboxy-terminal cross-linking telopeptide of type I collagen (CTX) in 71 men with T2DM treated with either pioglitazone (PIO) (30 mg once daily) or metformin (MET) (1000 mg twice daily). Baseline values of sclerostin and P1NP were compared with those of 30 healthy male controls. RESULTS Compared with healthy controls, patients with T2DM had significantly higher serum sclerostin levels (59.9 vs 45.2 pg/ml, P<0.001) but similar serum P1NP levels (33.6 vs 36.0 ng /ml, P=0.39). After 24 weeks of treatment, serum sclerostin levels increased by 11% in PIO-treated patients and decreased by 1.8% in MET-treated patients (P=0.018). Changes in serum sclerostin were significantly correlated with changes in serum CTX in all patients (r=0.36, P=0.002) and in PIO-treated patients (r=0.39, P=0.020), but not in MET-treated patients (r=0.17, P=0.31). CONCLUSIONS Men with T2DM have higher serum sclerostin levels than healthy controls, and these levels further increase after treatment with PIO, which is also associated with increased serum CTX. These findings suggest that increased sclerostin production may be involved in the pathogenesis of increased skeletal fragility in patients with T2DM in general and may specifically contribute to the detrimental effect of TZDs on bone.

MR Imaging Evaluation of Cardiovascular Risk in Metabolic Syndrome

Metabolic syndrome has become an important public health problem and has reached epidemic proportions globally. Metabolic syndrome is characterized by a cluster of metabolic abnormalities in an individual, with insulin resistance as the main characteristic. The major adverse consequence of metabolic syndrome is cardiovascular disease, which is often already present without clinical signs or symptoms. In this early stage of disease, interventions (eg, lifestyle intervention, medication) can be used to prevent further cardiovascular deterioration or even to reverse cardiovascular disease. Therefore, risk stratification on an individual basis and early detection of cardiovascular disease are essential. Magnetic resonance (MR) imaging is a powerful tool for demonstrating cardiovascular risk factors in metabolic syndrome, such as increased fat depots and arterial stiffening. Furthermore, MR imaging is an established modality for the assessment of myocardial function. This review provides a summary of the current MR applications in metabolic syndrome and discusses how these MR techniques can be used to identify subclinical cardiovascular damage.