Thalidomide does not interact with P-glycoprotein

Background: There is growing clinical interest in thalidomide for the treatment of various disorders due to its anti-inflammatory, immunomodulatory, and anti-angiogenic properties. In numerous clinical trials thalidomide is used as an adjunct to standard therapy. Therefore, clinicians should be aware of all possible drug-drug interactions that might occur with this drug. P-glycoprotein (P-gp), a drug efflux transporter that is expressed in many tissues, is the cause of several drug-drug interactions. P-gp induction or inhibition can lead to ineffective therapy or side-effects. In this study, we investigated thalidomide's potential to cause drug-drug interactions on the level of P-gp. Methods: LS180 cells were incubated with thalidomide for 72h in order to determine P-gp induction using real-time RT-PCR. A human leukaemia cell line over-expressing MDR1 (CCRF-CEM/MDR1) was used to measure uptake of rhodamine 123, a P-gp substrate, in the presence of thalidomide. Dose-dependent and bi-directional transport of thalidomide through Caco-2 cell monolayers was performed to assess site-directed permeability. Transport rates were determined using HPLC including chiral separation of the thalidomide enantiomers. Results: Thalidomide did not induce P-gp expression in LS180 cells. The uptake of rhodamine 123 in CCRF cells over-expressing MDR1 was not influenced by co-incubation with thalidomide. The transport through Caco-2 monolayers was linear and the permeability was similar for both directions. No differences between the thalidomide enantiomers were observed. Conclusions: Our study indicates that thalidomide is neither a substrate, nor an inhibitor or an inducer of P-gp. Therefore, P-gp-related drug-drug interactions with thalidomide are not likel

Modulation of transendothelial permeability and expression of ATP-binding cassette transporters in cultured brain capillary endothelial cells by astrocytic factors and cell-culture conditions

Confluent cell monolayers of brain capillary endothelial cells (BCEC) are used widely as an in vitro cell culture model of the blood-brain barrier. The present study describes the influence of cell-culture conditions on tight junctions, filamentous-actin cytoskeleton, and expression of ATP-binding cassette (ABC) transporters in primary cell cultures of porcine BCEC. Astrocyte as well as C6 glioma-conditioned cell culture medium was used in combination with retinoic acid, dexamethasone, cyclic adenosine monophosphate (cAMP) analogs, or 1,25-dihydroxyvitamin D3. It was shown that C6-conditioned medium led to a reorganization of filamentous actin and to an improved staining of zonula occludens-associated protein-1 (ZO-1). Further optimization of these culture conditions was achieved with cAMP analogs and dexamethasone. Retinoic acid, as well as 1,25-dihydroxyvitamin D3, did not improve cellular tight junctions as judged by filamentous actin, ZO-1 rearrangement, and transcellular electrical resistance (TER) measurements. However, these morphological changes did not influence the paracellular permeability of the extracellular marker sucrose. Expression of ABC transporters such as P-glycoprotein, multidrug resistance-associated protein-1( MRP1), and MRP2 were compared by measuring messenger RNA (mRNA) levels in whole-brain tissue, isolated brain capillaries, and cultured cells. In freshly isolated BCEC, mRNA levels of MRP2 and P-glycoprotein dropped by two- to sevenfold, respectively, whereas MRP1 mRNA levels were slightly increased. During cell culture, mRNA levels of MRP1 and MRP2 decreased by up to fivefold, while P-glycoprotein levels remained constant. These results were unaltered by different cell-culture conditions. In conclusion, the present study suggests that paracellular permeability, as well as mRNA expression of the studied ABC transporters in primary cultures, of porcine BCEC are insensitive toward changes in cell-culture condition

TRANSPORT OF THE ␤-LACTAM ANTIBIOTIC BENZYLPENICILLIN AND THE DIPEPTIDE GLYCYLSARCOSINE BY BRAIN CAPILLARY ENDOTHELIAL CELLS IN VITRO

This paper is available online at http://www.dmd.org ABSTRACT: Peripherally administered ␤-lactam antibiotics, which are structural analogs of tripeptides, may cause neurotoxic reactions or induce seizures. Previous in vivo studies provided evidence for brain uptake of these antibiotics. In the present work, we studied the extent and mechanism of the uptake of benzylpenicillin and glycylsarcosine by brain microvessel endothelial cells in vitro, using freshly isolated and cultured porcine brain capillary endothelial cells. Characterization of the cell culture model demonstrated the functional expression of the system transporting the neutral amino acids leucine and phenylalanine. The initial rate of uptake of benzylpenicillin was >3-fold greater than the rate of uptake of the extracellular marker sucrose (ratio, 3.29 ؎ 0.37), whereas uptake of glycylsarcosine did not differ from that of sucrose. The differences in cellular uptake correlated with the octanol/buffer partition coefficients for glycylsarcosine and benzylpenicillin (1.16 ؋ 10 ؊3 for glycylsarcosine and 6.83 ؋ 10 ؊2 for benzylpenicillin). The concentration-dependent uptake of benzylpenicillin (1-2000 M) was not saturable and was not sensitive to shifts in pH or temperature. The permeability-surface area product for the uptake of benzylpenicillin at pH 7.4 was determined from these experiments and was found to be 8.1 ؋ 10 ؊5 ml/sec/g of brain. This value was very close to the value determined in in vivo studies. Uptake of benzylpenicillin and glycylsarcosine was not reduced in the presence of 1 mM ceftibuten or 100 M probenecid. The findings with cultured cell monolayers were confirmed using freshly isolated endothelial cells. These in vitro data are compatible with benzylpenicillin, but not glycylsarcosine, being able to penetrate endothelial cells. Uptake of benzylpenicillin by brain capillary endothelial cells occurs by a slow nonsaturable process, with no evidence for carriermediated transport

Decreased expression of breast cancer resistance protein in the duodenum in patients with obstructive cholestasis

Background/Aims: The expression of transporters involved in bile acid homeostasis is differentially regulated during obstructive cholestasis. Since the drug efflux transporter breast cancer resistance protein (BCRP) is known to transport bile acids, we investigated whether duodenal BCRP expression could be altered during cholestasis. Methods: Using real-time RT-PCR analysis we determined mRNA expression levels in duodenal tissue of 19 cholestatic patients. Expression levels were compared to 14 healthy subjects. BCRP protein staining was determined in biopsies of 6 cholestatic and 6 healthy subjects by immunohistochemistry. Results: We found that in patients with obstructive cholestasis mean duodenal BCRP mRNA levels were significantly reduced to 53% and mean protein staining was reduced to 57%. Conclusions: BCRP, a transporter for bile acids and numerous drugs, appears to be down-regulated in the human duodenum during cholestasis. The clinical impact of these results has to be investigated in further studies. Copyright (c) 2006 S. Karger AG, Basel

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

A New Intestinal Cell Culture Model To Discriminate the Relative Contribution of P-gp and BCRP on Transport of Substrates Such as Imatinib

P-glycoprotein (P-gp/MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) play an important role in transport of a wide variety of endogenous compounds, drugs and toxins. Transport of some drugs, for example the tyrosine kinase inhibitor imatinib, is influenced by both P-gp and BCRP. Establishing an intestinal Caco-2 cell culture model with specific knock-downs of P-gp and BCRP and double knock-down of both proteins, we aimed to elucidate the impact of each transporter on transport of imatinib. Stable single and double knock-downs of P-gp and BCRP were obtained by RNA interference (RNAi). Transporter expression was measured on RNA and protein level using real-time RT-PCR and Western blot, respectively. Functional activity was quantified by transport of specific substrates across Caco-2 cells. MDR1 and BCRP mRNA expression was reduced to 75% and 90% compared to wild-type control in single MDR1- and BCRP-knock-down clones, respectively. In double knock-down clones, MDR1 expression decreased to 95% and BCRP expression to 80%. Functional activity of P-gp and BCRP was diminished as transport of the P-gp-specific substrate (3)H-digoxin and the BCRP-specific substrate (14)C-PhIP was augmented in the opposite direction, when the respective transporter was knocked down. Similar effects were observed by chemical inhibition of the respective transporter. Bidirectional transport studies with (14)C-imatinib revealed an abrogation of asymmetric transport when P-gp was knocked down, either in single or double knock-down clones compared to wild-type cells. This was not observed in single BCRP-knock-down clones. In conclusion, this newly established cell system with single and concomitant knock-down of P-gp and BCRP can be used to quantify the specific partial impact of the transporters on transport of substrates that are transported by both proteins. For imatinib transport, the contribution of P-gp seems to be more important compared to BCRP in this Caco-2 cell sys

Interaction of the antiviral drug telaprevir with renal and hepatic drug transporters

Telaprevir is a new, direct-acting antiviral drug that has been approved for the treatment of chronic hepatitis C viral infection. First data on drug-drug interactions with co-medications such as cyclosporine, tacrolimus and atorvastatin have been reported recently. Drug transporting proteins have been shown to play an important role in clinically observed drug-drug interactions. The aim of this study was therefore to systematically investigate the potential of telaprevir to inhibit drug transporting proteins. The effect of telaprevir on substrate uptake mediated by drug transporters located in human kidney and liver was investigated on a functional level in HEK293 cell lines that over-express single transporter. Telaprevir was shown to exhibit significant inhibition of the human renal drug transporters OCT2 and MATE1 with IC50 values of 6.4 µM and 23.0 µM, respectively, whereas no inhibitory effect on OAT1 and OAT3 mediated transport by telaprevir was demonstrated. Liver drug transporters were inhibited with IC50 of 2.2 µM for OATP1B1, 6.8 µM for OATP1B3 and 20.7 µM for OCT1. Our data show that telaprevir exhibited significant potential to inhibit human drug transporters. In view of the inhibitory potential of telaprevir, clinical co-administration of telaprevir together with drugs that are substrates of renal or hepatic transporters should be carefully monitored