The Xenobiotic Transporter Mdr1 Enforces T Cell Homeostasis in the Presence of Intestinal Bile Acids.

CD4+ T cells are tightly regulated by microbiota in the intestine, but whether intestinal T cells interface with host-derived metabolites is less clear. Here, we show that CD4+ T effector (Teff) cells upregulated the xenobiotic transporter, Mdr1, in the ileum to maintain homeostasis in the presence of bile acids. Whereas wild-type Teff cells upregulated Mdr1 in the ileum, those lacking Mdr1 displayed mucosal dysfunction and induced Crohn's disease-like ileitis following transfer into Rag1-/- hosts. Mdr1 mitigated oxidative stress and enforced homeostasis in Teff cells exposed to conjugated bile acids (CBAs), a class of liver-derived emulsifying agents that actively circulate through the ileal mucosa. Blocking ileal CBA reabsorption in transferred Rag1-/- mice restored Mdr1-deficient Teff cell homeostasis and attenuated ileitis. Further, a subset of ileal Crohn's disease patients displayed MDR1 loss of function. Together, these results suggest that coordinated interaction between mucosal Teff cells and CBAs in the ileum regulate intestinal immune homeostasis.

Characterization and Validation of Canine P-Glycoprotein-Deficient MDCK II Cell Lines for Efflux Substrate Screening.

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.

Pharmacokinetic Modeling of the Impact of P-glycoprotein on Ondansetron Disposition in the Central Nervous System.

PURPOSE: Modulation of 5-HT3 receptor in the central nervous system (CNS) is a promising approach for treatment of neuropathic pain. The goal was to evaluate the role of P-glycoprotein (Pgp) in limiting exposure of different parts of the CNS to ondansetron (5-HT3 receptor antagonist) using wild-type and genetic knockout rat model. METHODS: Plasma pharmacokinetics and CNS (brain, spinal cord, and cerebrospinal fluid) disposition was studied after single 10 mg/kg intravenous dose. RESULTS: Pgp knockout resulted in significantly higher concentrations of ondansetron in all tested regions of the CNS at most of the time points. The mean ratio of the concentrations between KO and WT animals was 2.39-5.48, depending on the region of the CNS. Male and female animals demonstrated some difference in ondansetron plasma pharmacokinetics and CNS disposition. Mechanistic pharmacokinetic model that included two systemic disposition and three CNS compartments (with intercompartmental exchange) was developed. Pgp transport was incorporated as an efflux from the brain and spinal cord to the central compartment. The model provided good simultaneous description of all data sets, and all parameters were estimated with sufficient precision. CONCLUSIONS: The study provides important quantitative information on the role of Pgp in limiting ondansetron exposure in various regions of the CNS using data from wild-type and Pgp knockout rats. CSF drug concentrations, as a surrogate to CNS exposure, are likely to underestimate the effect of Pgp on drug penetration to the brain and the spinal cord.

ABCB5 is a limbal stem cell gene required for corneal development and repair.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

ABCB5 promotes melanoma metastasis through enhancing NF-κB p65 protein stability.

Melanoma is the most aggressive type of skin cancer. Melanoma has an extremely poor prognosis because of its high potential for vascular invasion, metastasis and recurrence. The mechanism of melanoma metastasis is not well understood. ATP-binding cassette sub-family B member 5 (ABCB5) plays a key role in melanoma growth. However, it is uncertain what function ABCB5 may exert in melanoma metastasis. In this report, we for the first time demonstrate ABCB5 as a crucial factor that promotes melanoma metastasis. ABCB5 positive (ABCB5+) malignant melanoma initiating cells (MMICs) display a higher metastatic potential compared with ABCB5 negative (ABCB5-) melanoma subpopulation. Knockdown of ABCB5 expression reduces melanoma cell migration and invasion in vitro and melanoma pulmonary metastasis in tumor xenograft mice. ABCB5 and NF-κB p65 expression levels are positively correlated in both melanoma tissues and cell lines. Consequently, ABCB5 activates the NF-κB pathway by inhibiting p65 ubiquitination to enhance p65 protein stability. Our finding highlights ABCB5 as a novel pro-metastasis factor and provides a potential therapeutic target for melanoma.

Transintestinal cholesterol excretion is an active metabolic process modulated by PCSK9 and statin involving ABCB1.

OBJECTIVE: Transintestinal cholesterol excretion (TICE) is an alternate pathway to hepatobiliary secretion. Our study aimed at identifying molecular mechanisms of TICE. APPROACH AND RESULTS: We studied TICE ex vivo in mouse and human intestinal explants, and in vivo after bile diversion and intestinal cannulation in mice. We provide the first evidence that both low-density lipoprotein (LDL) and high-density lipoprotein deliver cholesterol for TICE in human and mouse jejunal explants at the basolateral side. Proprotein convertase subtilisin kexin type 9 (PCSK9)(-/-) mice and intestinal explants show increased LDL-TICE, and acute injection of PCSK9 decreases TICE in vivo, suggesting that PCSK9 is a repressor of TICE. The acute repression was dependent on the LDL receptor (LDLR). Further, TICE was increased when mice were treated with lovastatin. These data point to an important role for LDLR in TICE. However, LDLR(-/-) mice showed increased intestinal LDL uptake, contrary to what is observed in the liver, and tended to have higher TICE. We interpret these data to suggest that there might be at least 2 mechanisms contributing to TICE; 1 involving LDL receptors and other unidentified mechanisms. Acute modulation of LDLR affects TICE, but chronic deficiency is compensated for most likely by the upregulation of the unknown mechanisms. Using mice deficient for apical multidrug active transporter ATP-binding cassette transporter B1 a and b, and its inhibitor, we show that these apical transporters contribute significantly to TICE. CONCLUSIONS: TICE is operative in human jejunal explants. It is a metabolically active process that can be acutely regulated, inversely related to cholesterolemia, and pharmacologically activated by statins.

[(11)C-carbonyl]CEP-32496: radiosynthesis, biodistribution and PET study of brain uptake in P-gp/BCRP knockout mice.

CEP-32496 is a novel, orally active serine/threonine-protein kinase B-raf (BRAF) (V600E) kinase inhibitor that is being investigated in clinical trials for the treatment of some cancers in patients. In this study, we developed [(11)C-carbonyl]CEP-32496 as a novel positron emission tomography (PET) probe to study its biodistribution in the whole bodies of mice. [(11)C]CEP-32496 was synthesized by the reaction of 5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-amine hydrochloride (1·HCl) with [(11)C]phosgene, followed by treatment with 3-(6,7-dimethoxyquinozolin-4-yloxy)aniline (2). Small-animal PET studies with [(11)C]CEP-32496 indicated that radioactivity levels (AUC0-90 min, SUV×min) accumulated in the brains of P-gp/BCRP knockout mice at a 8-fold higher rate than in the brains of wild-type mice.

Impact of genetic deficiencies of P-glycoprotein and breast cancer resistance protein on pharmacokinetics of aripiprazole and dehydroaripiprazole.

1. We investigated how deficiencies in P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) affect the pharmacokinetics of atypical antipsychotics aripiprazole and its active metabolite (dehydroaripiprazole) using normal Friend leukemia virus strain B (FVB) mice, BCRP knockout (Bcrp[-/-]) mice, and P-gp and BCRP triple knockout (Mdr1a/1b[-/-]Bcrp[-/-]) mice. 2. While plasma concentrations of aripiprazole and dehydroaripiprazole after oral administration were slightly higher in both Bcrp(-/-) and Mdr1a/1b(-/-)/Bcrp(-/-) mice than in normal FVB mice, the difference was not marked. The increase in absolute bioavailability (F) compared with normal mice (approximately 1.3-fold increase) was comparable between Bcrp(-/-) and Mdr1a/1b(-/-)/Bcrp(-/-) mice. This finding suggests that BCRP may be involved in the intestinal absorption of aripiprazole in mice, albeit with minimal contribution to absorption at best. 3. In contrast, the brain-to-plasma concentration ratio (Kp,brain) for aripiprazole and dehydroaripiprazole after oral administration was significantly higher in Mdr1a/1b(-/-)/Bcrp(-/-) mice than in normal mice, whereas Bcrp(-/-) mice exhibited Kp,brain values similar to those in normal mice. In addition, the Kp,brain values in Mdr1a/1b(-/-)/Bcrp(-/-) mice were not drastically different from those previously reported in Mdr1a/1b(-/-) mice, suggesting that brain penetration of aripiprazole and dehydroaripiprazole can be affected by P-gp, but with little synergistic effect of BCRP.

Imaging of activity of multidrug resistance-associated protein 1 in the lungs.

Multidrug resistance-associated protein 1 (MRP1) transports various xenobiotics and metabolites across cell membranes, and the alteration of MRP1 expression is associated with certain lung diseases. This study sought to examine the feasibility of imaging pulmonary MRP1 activity using 6-bromo-7-[(11)C]methylpurine ([(11)C]1). A positron emission tomography study with [(11)C]1 was performed in wild-type, Mrp1 knockout (KO), and P-glycoprotein/breast cancer resistance protein (Pgp/Bcrp) KO mice. Lung radioactivity in wild-type and Mrp1 KO mice reached a maximum level immediately after the administration of [(11)C]1. Thereafter, radioactivity rapidly decreased in the lungs of wild-type mice, whereas it was mostly retained in the lungs of Mrp1 KO mice. The kinetics in the lungs of Pgp/Bcrp KO mice was quite similar to that of wild-type mice. Analysis of the chemical form confirmed that radioactive compounds in the lungs of Mrp1 KO mice were nearly completely composed of a glutathione conjugate, a MRP1 substrate, 5 minutes after the intravenous administration of [(11)C]1. The effect of an MRP1 inhibitor, MK571, on the kinetics of [(11)C]1 was also examined. Treatment with MK571 delayed the elimination of radioactivity from the lungs, compared with control mice. These results suggest that [(11)C]1 diffuses into the lung tissue after administration and undergoes conversion into the hydrophilic conjugate, which is then specifically expelled by MRP1. In conclusion, [(11)C]1 allows for the imaging of in vivo MRP1 activity in lungs.

P-glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel.

Paclitaxel is avidly transported by P-glycoprotein (P-gp/MDR1/ABCB1). This results in low oral bioavailability, which can be boosted by coadministration of P-gp inhibitors. Unlike paclitaxel, docetaxel is extensively metabolized by CYP3A4 and its oral bioavailability can be enhanced in mice and humans by coadministration of the potent CYP3A inhibitor ritonavir. Unexpectedly, ritonavir also enhances the oral bioavailability of paclitaxel in humans. We aimed to resolve the mechanism underlying this enhancement. Using mice lacking Cyp3a and/or P-gp, we investigated the combined and separate restricting roles of Cyp3a and P-gp in the oral bioavailability of paclitaxel, and the boosting effect of ritonavir. CYP3A4-humanized mice were used for translation to the human situation. P-gp had a dominant effect (11.6-fold, p < 0.001) over Cyp3a (<1.5-fold, n.s.) in limiting plasma concentrations of oral paclitaxel. However, in the absence of P-gp, Cyp3a decreased paclitaxel plasma concentrations twofold (p < 0.001). Coadministered ritonavir inhibited Cyp3a-mediated metabolism, but not P-gp-mediated transport of paclitaxel. Owing to the dominant effect of P-gp, ritonavir enhanced only paclitaxel plasma concentrations in P-gp-deficient mice. Mouse liver microsomes metabolized paclitaxel far less efficiently than human or CYP3A4-transgenic liver microsomes, revealing much lower efficiency of paclitaxel metabolism by mouse than by human CYP3As. Accordingly, ritonavir could enhance the oral bioavailability of paclitaxel in CYP3A4-humanized mice, despite the fact that these mice are P-gp-proficient. Our results show that CYP3A4 inhibition most likely underlies the boosting effect of ritonavir on oral paclitaxel bioavailability in humans. Furthermore, CYP3A4-humanized mice allow improved understanding of CYP3A4-mediated paclitaxel metabolism in humans.

Mechanisms limiting distribution of the threonine-protein kinase B-RaF(V600E) inhibitor dabrafenib to the brain: implications for the treatment of melanoma brain metastases.

Brain metastases are a common cause of death in stage IV metastatic melanoma. Dabrafenib is a BRAF (gene encoding serine/threonine-protein kinase B-Raf) inhibitor that has been developed to selectively target the valine 600 to glutamic acid substitution (BRAF(V600E)), which is commonly found in metastatic melanoma. Clinical trials with dabrafenib have shown encouraging results; however, the central nervous system distribution of dabrafenib remains unknown. Thus, the objective of the current study was to evaluate the brain distribution of dabrafenib in mice, and to see whether active efflux by P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) restricts its delivery across the blood-brain barrier (BBB). In vitro accumulation studies conducted in Madin-Darby canine kidney II cells indicate that dabrafenib is an avid substrate for both P-gp and BCRP. Directional flux studies revealed greater transport in the basolateral to apical direction with corrected efflux ratios greater than 2 for both P-gp and Bcrp1 transfected cell lines. In vivo, the ratio of area under the concentration-time curve (AUC)(brain) to AUC(plasma) (K(p)) of dabrafenib after an i.v. dose (2.5 mg/kg) was 0.023, which increased by 18-fold in Mdr1 a/b(-/-)Bcrp1(-/-) mice to 0.42. Dabrafenib plasma exposure was ∼2-fold greater in Mdr1 a/b(-/-)Bcrp1(-/-) mice as compared with wild-type with an oral dose (25 mg/kg); however, the brain distribution was increased by ~10-fold with a resulting K(p) of 0.25. Further, compared with vemurafenib, another BRAF(V600E) inhibitor, dabrafenib showed greater brain penetration with a similar dose. In conclusion, the dabrafenib brain distribution is limited in an intact BBB model, and the data presented herein may have clinical implications in the prevention and treatment of melanoma brain metastases.

Pharmacodynamic effects of aripiprazole and ziprasidone with respect to p-glycoprotein substrate properties.

INTRODUCTION: Aripiprazole, an atypical antipsychotic drug with mixed antagonism and agonism on dopamine D2 and serotonin receptors, is a substrate of the efflux transporter P-glycoprotein (P-gp). Here we tested the pharmacodynamic consequences of these properties in a P-gp deficient mouse model by studying the effects of aripiprazole and of ziprasidone on motor coordination. METHODS: The motor behaviour of wild-type (WT) and P-gp deficient [abcb1ab(-/-)] mice was investigated on a RotaRod. Mice received acute injections of either aripirazole or ziprasidone. For comparison, the dopamine receptor antagonist haloperidol and serotonin receptor ligands buspirone and ketanserin were also applied. RESULTS: Pharmacokinetic analyses revealed P-gp activity for aripiprazole and ziprasidone. This was indicated by 3.1- and 1.9-fold higher ratios of brain to plasma concentrations of drugs in knock-out to WT animals. Acute doses of ariprazole or ziprasidone impaired motor behaviour on the RotaRod. Effects were similar after injection of haloperidol, whereas the serotonin receptor ligands buspirone and ketanserin enhanced RotaRod performance. Genotype dependent differences of motor performance were found for aripiprazole but not for ziprasidone. DISCUSSION: Evidence was given that P-gp substrate properties have pharmacodynamic consequences for aripiprazole but not for ziprasidone and thus affect dopamine receptor related motor behaviour.

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict brain accumulation of the active sunitinib metabolite N-desethyl sunitinib.

N-desethyl sunitinib is a major and pharmacologically active metabolite of the tyrosine kinase inhibitor and anticancer drug sunitinib. Because the combination of N-desethyl sunitinib and sunitinib represents total active drug exposure, we investigated the impact of several multidrug efflux transporters on plasma pharmacokinetics and brain accumulation of N-desethyl sunitinib after sunitinib administration to wild-type and transporter knockout mice. In vitro, N-desethyl sunitinib was a good transport substrate of human ABCB1 and ABCG2 and murine Abcg2, but not ABCC2 or Abcc2. At 5 µM, ABCB1 and ABCG2 contributed almost equally to N-desethyl sunitinib transport. In vivo, the systemic exposure of N-desethyl sunitinib after oral dosing of sunitinib malate (10 mg/kg) was unchanged when Abcb1 and/or Abcg2 were absent. However, brain accumulation of N-desethyl sunitinib was markedly increased (13.7-fold) in Abcb1a/1b(-/-)/Abcg2(-/-) mice, but not in Abcb1a/1b(-/-) or Abcg2(-/-) mice. In the absence of the ABCB1 and ABCG2 inhibitor elacridar, brain concentrations of N-desethyl sunitinib were detectable only in Abcb1a/1b(-/-)/Abcg2(-/-) mice after sunitinib administration. Combined elacridar plus N-desethyl sunitinib treatment increased N-desethyl sunitinib plasma and brain exposures, but not brain-to-plasma ratios in wild-type mice. In conclusion, brain accumulation of N-desethyl sunitinib is effectively restricted by both Abcb1 and Abcg2. The effect of elacridar treatment in improving brain accumulation of N-desethyl sunitinib in wild-type mice was limited compared with its effect on sunitinib brain accumulation.

Quantitative proteomics of transporter expression in brain capillary endothelial cells isolated from P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice.

The objective of this study was to quantitatively examine the protein expression of relevant transporters and other proteins in the brain capillary endothelial cells isolated from wild-type mice and P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and P-gp/Bcrp knockout mice. After the isolation of brain capillary endothelial cells, a highly sensitive liquid chromatography-tandem mass spectrometry method with multiple reaction monitoring was used to determine the quantitative expression of membrane transporters at the blood-brain barrier (BBB) of the various mouse genotypes. Quantitative expression of 29 protein molecules, including 12 ATP-binding cassette transporters, 10 solute carrier transporters, five receptors, and two housekeeping proteins, was examined by quantitative proteomics in the four mouse genotypes. There was no significant difference in the expression of P-gp between the wild-type and Bcrp1(-/-) mice. Likewise, Bcrp expression was not significantly different between the wild-type and Mdr1a/b(-/-) mice. There was no significant difference in the expression of any of the measured proteins in the brain capillary endothelial cells across the genotypes, except for the lack of expression of the corresponding protein in the mice that had a genetic deletion of P-gp or Bcrp. In conclusion, using a quantitative proteomic approach, we have shown that there are no changes in the expression of several relevant transporters in brain capillary endothelial cells isolated from single and combination knockout mice. These data suggest that the mechanism behind the functional compensation between P-gp and Bcrp at the BBB is not related to compensatory changes in transporter expression.

Acitretin, an enhancer of alpha-secretase expression, crosses the blood-brain barrier and is not eliminated by P-glycoprotein.

BACKGROUND: ADAM10 (a disintegrin and metalloproteinase 10) has been demonstrated to act as the main physiological α-secretase. Enzymatic activity of the α-secretase on the one hand prevents the formation of toxic Aß peptides and on the other hand promotes the secretion of a neurotrophic and neuroprotective amyloid precursor protein fragment (APPs-α) by cleaving the amyloid precursor protein within its Aß sequence. Enhancement of ADAM10's gene expression may therefore present a valuable therapeutic approach for the treatment of Alzheimer's disease (AD), where Aß peptides are severely involved in the pathogenesis. OBJECTIVE: In cell culture and in a transgenic mouse model of AD, retinoids led to increased ADAM10 expression and activity. We therefore endeavor to develop a clinical application of synthetic retinoids such as acitretin in AD. METHODS: The effect of synthetic retinoids on ADAM10 gene expression was analyzed by reporter gene assays in human neuroblastoma cell line SH-SY5Y. Penetrance of acitretin into the murine brain was analyzed by high-performance liquid chromatography. P-glycoprotein (P-gp) double-knockout mice with a deficiency in both isoforms, mdr1a and 1b, were used to analyze a possible role of P-gp-dependent efflux on acitretin distribution. RESULTS: Acitretin and tamibarotene are both potent activators of ADAM10 promoter activity. Acitretin crosses the murine blood-brain barrier and its level in the mouse brain is not reduced by P-gp. CONCLUSION: Synthetic retinoids and especially acitretin seem to be ideal candidates to establish an ADAM10-based AD treatment, and therefore have already entered first clinical trials.

Inhibition of leukotriene C4 action reduces oxidative stress and apoptosis in cardiomyocytes and impedes remodeling after myocardial injury.

UNLABELLED: Tissue damage leads to release of pro-inflammatory mediators. Among these, leukotriene C(4) (LTC(4)) is a powerful, intracellularly induced mediator of inflammation, which requires inside-out transport of LTC(4). We investigated whether release of LTC(4)via the multidrug resistance related protein 1 (MRP1) induces apoptosis in cardiomyocytes in vitro and in vivo. METHODS AND RESULTS: Incubation of cultured embryonic cardiomyocytes (eCM) with recombined LTC(4) caused enhanced rates of reactive oxygen species (ROS) release measured via L012-luminescence method and apoptosis. Pharmacologic LTC(4) receptor blockade antagonized this effect in vitro. To evaluate the relevance of MRP1 mediated LTC(4) release after myocardial injury in vivo, MRP1(-/-) mice and FVB wildtype mice (WT) received cryoinjury of the left ventricle. Fourteen days after injury, left-ventricular ejection fraction (EF), end-diastolic volume (EDV), and akinetic myocardial mass (AMM) were quantified via echocardiography. MRP1(-/-) mice demonstrated increased EF (MRP1(-/-): 39 ± 3%, WT: 29 ± 4%) and reduced AMM (MRP1(-/-): 13 ± 2% WT: 16 ± 4%), indicating reduced post-infarction remodeling. Mechanistically, LTC(4) serum concentrations and levels of cellular apoptosis were increased in myocardial cryosections of FVB WT mice as compared to MRP1(-/-) mice. To identify key targets for pharmacological inhibition of LTC(4) actions, WT mice were treated with the specific Cys-LT1-receptor blocker Montelukast or the MRP1-Inhibitor MK571. Treatment of WT mice resulted in significant increase of EF (WT(Montelukast): 40 ± 5%, WT(MK571): 39 ± 3%, WT(vehicle): 33 ± 3% and decrease of AMM (WT(Montelukast): 12 ± 1%, WT(MK571): 10 ± 3%, WT(vehicle): 15 ± 5%) compared to untreated WT mice. CONCLUSION: Inhibition of leukotriene C(4) reduces levels of oxidative stress and apoptosis and demonstrates beneficial effects on myocardial remodeling after left ventricular injury.

Paclitaxel in self-micro emulsifying formulations: oral bioavailability study in mice.

The anticancer drug paclitaxel is formulated for i.v. administration in a mixture of Cremophor EL and ethanol.Its oral bioavailability is very low due to the action of P-glycoproteinin the gut wall and CYP450 in gut wall and liver.However, proof-of-concept studies using the i.v. formulation diluted in drinking water have demonstrated the feasibility of the oral route as an alternative when given in combination with inhibitors of P-glycoprotein and CYP450. Because of the unacceptable pharmaceutical properties of the drinking solution, a better formulation for oral application is needed.We have evaluated the suitability of various self-micro emulsifying oily formulations (SMEOF's) of paclitaxel for oral application using wild-type and P-glycoprotein knockout mice and cyclosporin A (CsA) as P-glycoprotein and CYP450 inhibitor. The oral bioavailability of paclitaxel in all SMEOF's without concomitant CsA was low in wild-type mice, showing that this vehicle does not enhance intestinal uptake by itself.Paclitaxel (10 mg/kg) in SMEOF#3 given with CsA resulted in plasma levels that were comparable to the Cremophor ELethanol containing drinking solution plus CsA. Whereas the AUC increased linearly with the oral paclitaxel dose in P-glycoprotein knockout mice, it increased less than proportional in wild-type mice given with CsA. In both strains more unchanged paclitaxel was recovered in the feces at higher doses. This observation most likely reflects more profound precipitation of paclitaxel within the gastro-intestinal tract at higher doses. The resulting absolute reduction in absorption of paclitaxel from the gut was possibly concealed by partial saturation of first-pass metabolism when P-glycoprotein was absent. In conclusion, SMEOF's maybe a useful vehicle for oral delivery of paclitaxel in combination with CsA, although the physical stability within the gastro-intestinal tract remains a critical issue, especially when applied at higher dose levels.

[(76) Br]BMK-152, a nonpeptide analogue, with high affinity and low nonspecific binding for the corticotropin-releasing factor type 1 receptor.

Corticotropin-releasing factor (CRF), a neuropeptide, regulates endocrine and autonomic responses to stress through G-protein coupled receptors, CRF(1) or CRF(2) . A PET ligand able to monitor changes in CRF(1) receptor occupancy in vivo would aid in understanding the pathophysiology of stress-related diseases as well as in the clinical development of nonpeptide antagonists with therapeutic value. We have radiolabeled the CRF(1) receptor ligand, [8-(4-bromo-2,6-dimethoxyphenyl)-2,7-dimethylpyrazolo[1,5-α][1,3,5]triazin-4-yl]-N,N-bis-(2-methoxyethyl)amine (BMK-152) (ClogP = 2.6), at both the 3 and 4 position with [(76) Br]. Using in vitro autoradiography saturation studies the 4-[(76) Br]BMK-152 exhibited high affinity binding to both rat (K(d) = 0.23 ± 0.07 nM; n = 3) and monkey frontal cortex (K(d) = 0.31 ± 0.08 nM; n = 3) consistent with CRF(1) receptor regional distribution whereas with the 3-[(76) Br]BMK-152, the K(d) s could not be determined due to high nonspecific binding. In vitro autoradiography competition studies using [(125) I]Tyr(0) -o-CRF confirmed that 3-Br-BMK-152 (K(i) = 24.4 ± 4.9 nM; n = 3) had lower affinity (70-fold) than 4-Br-BMK-152 (K(i) = 0.35 ± 0.07 nM; n = 3) in monkey frontal cortex and similiar studies using [(125) I]Sauvagine confirmed CRF(1) receptor selectivity. In vivo studies with P-glycoprotein (PGP) knockout mice (KO) and their wild-type littermates (WT) showed that the brain uptake of 3-[(76) Br]BMK/4-[(76) Br]BMK was increased less than twofold in KO versus WT indicating that 3-[(76) Br]BMK-152/4-[(76) Br]BMK was not a Pgp substrate. Rat brain uptakes of 4-[(76) Br] BMK-152 from ex vivo autoradiography studies showed regional localization consistent with known published CRF(1) receptor distribution and potential as a PET ligand for in vivo imaging of CRF(1) receptors.

Strategies to lower the Pgp efflux liability in a series of potent indole azetidine MCHR1 antagonists.

A series of potent indolyl azetidine rMCHR1 antagonists were found to show poor CNS penetration due to Pgp efflux. We envisioned a strategy which included: lowering basicity; changing the conformational flexibility motif; and removal of a hydrogen bond donor, in an attempt to optimize this property while maintaining target receptor efficacy. This work resulted in mitigation of Pgp efflux, and led us to identify 1-dihydroindolyl azetidine derivatives with CNS penetration and excellent rMCHR1 binding affinity.

[11C]sorafenib: radiosynthesis and preliminary PET study of brain uptake in P-gp/Bcrp knockout mice.

Sorafenib (Nexavar, BAY43-9006, 1) is a second-generation, orally active multikinase inhibitor that is approved for the treatment of some cancers in patients. In this Letter, we developed [(11)C]1 as a novel positron emission tomography (PET) probe, and evaluated the influence of ABC transporters-mediated efflux on brain uptake using PET with [(11)C]1 in P-glycoprotein (P-gp)/breast cancer resistance protein (Bcrp) knockout mice versus wild-type mice. [(11)C]1 was synthesized by the reaction of hydrochloride of aniline 2 with [(11)C]phosgene ([(11)C]COCl(2)) to give isocyanate [(11)C]6, followed by reaction with another aniline 3. Small-animal PET study with [(11)C]1 indicated that the radioactivity level (AUC(0-60 min), SUV×min) in the brains of P-gp/Bcrp knockout mice was about three times higher than in wild-type mice.