Evaluation of transport of common antiepileptic drugs by human multidrug resistance-associated proteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistant epilepsy

Resistance to antiepileptic drugs (AEDs) is one of the most serious problems in the treatment of epilepsy. Accumulating experimental evidence suggests that increased expression of the drug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier may be involved in the mechanisms leading to AED resistance. In addition to Pgp, increased expression of several multidrug resistance-associated proteins (MRPs) has been determined in epileptogenic brain regions of patients with pharmacoresistant epilepsy. However, it is not known whether AEDs are substrates for MRPs. In the present experiments, we evaluated whether common AEDs are transported by human MRPs (MRP1, 2 and 5) that are overexpressed in AED resistant epilepsy. For this purpose, we used a highly sensitive assay (concentration equilibrium transport assay; CETA) in polarized kidney cell lines (LLC, MDCKII) transfected with human MRPs. The assay was validated by known MRP substrates, including calcein-AM (MRP1), vinblastine (MRP2) and chloromethylfluorescein diacetate (CMFDA; MRP5). The directional transport determined with these drugs in MRP-transfected cell lines could be blocked with the MRP inhibitor MK571. However, in contrast to transport of known MRP substrates, none of the common AEDs (carbamazepine, valproate, levetiracetam, phenytoin, lamotrigine and phenobarbital) used in this study was transported by MRP1, MRP2 or MRP5. A basolateral-to-apical transport of valproate, which could be inhibited by MK571 and probenecid, was determined in LLC cells (both wildtype and transfected), but the specific transporter involved was not identified. The data indicate that common AEDs are not substrates for human MRP1, MRP2 or MRP5, at least in the in vitro models used in this study.     

Do ATP-binding cassette transporters cause pharmacoresistance in epilepsy? Problems and approaches in determining which antiepileptic drugs are affected

Resistance to multiple antiepileptic drugs (AEDs) is a common problem in epilepsy, affecting at least 30% of patients. One prominent hypothesis to explain this resistance suggests an inadequate penetration or excess efflux of AEDs across the blood - brain barrier (BBB) as a result of overexpressed efflux transporters such as P-glycoprotein (Pgp), the encoded product of the multidrug resistance- 1 (MDR1, ABCB1) gene. Pgp and MDR1 are markedly increased in epileptogenic brain tissue of patients with AED-resistant partial epilepsy and following seizures in rodent models of partial epilepsy. In rodent models, AED-resistant rats exhibit higher Pgp levels than responsive animals; increased Pgp expression is associated with lower brain levels of AEDs; and, most importantly, co-administration of Pgp inhibitors reverses AED resistance. Thus, it is reasonable to conclude that Pgp plays a significant role in mediating resistance to AEDs in rodent models of epilepsy - however, whether this phenomenon extends to at least some human refractory epilepsy remains unclear, particularly because it is still a matter of debate which AEDs, if any, are transported by human Pgp. The difficulty in determining which AEDs are substrates of human Pgp is mainly a consequence of the fact that AEDs are highly permeable compounds, which are not easily identified as Pgp substrates in in vitro models of the BBB, such as monolayer (Transwell(?)) efflux assays. By using a modified assay (concentration equilibrium transport assay; CETA), which minimizes the influence of high transcellular permeability, two groups have recently demonstrated that several major AEDs are transported by human Pgp. Importantly, it was demonstrated in these studies that Pgp-mediated transport highly depends on the AED concentration and may not be identified if concentrations below or above the therapeutic range are used. In addition to the efflux transporters, seizure-induced alterations in BBB integrity and activity of drug metabolizing enzymes (CYPs) affect the brain uptake of AEDs. For translating these findings to the clinical arena, in vivo imaging studies using positron emission tomography (PET) with (11)C-labelled AEDs in epileptic patients are under way.     

Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein

In view of the important role of P-glycoprotein (Pgp) and other drug efflux transporters for drug distribution and resistance, the identification of compounds as substrates of Pgp-mediated transport is one of the key issues in drug discovery and development, particularly for compounds acting on the central nervous system. In vitro transport assays with Pgp-transfected kidney cell lines are widely used to evaluate the potential of compounds to act as Pgp substrates or inhibitors. Furthermore, such cell lines are also frequently utilized as a substitute for more labor-intensive in vitro or in vivo models of the blood-brain barrier (BBB). Overexpression of Pgp or members of the multidrug resistance protein (MRP) family at the BBB has been implicated in the mechanisms underlying resistance to antiepileptic drugs (AEDs) in patients with epilepsy. Therefore, it is important to know which AEDs are substrates for Pgp or MRPs. In the present study, we used monolayers of polarized MDCKII dog kidney or LLC-PK1 pig kidney cells transfected with cDNA containing either human MDR1, MRP2 or mouse mdr1a and mdr1b sequences to measure the directional transport of AEDs. Cyclosporin A (CsA) and vinblastine were used as reference standards for Pgp and MRP2, respectively. The AEDs phenytoin and levetiracetam were directionally transported by mouse but not human Pgp, whereas CsA was transported by both types of Pgp. Carbamazepine was not transported by any type of Pgp and did not inhibit the transport of CsA. In contrast to vinblastine, none of the AEDs was transported by MRP2 in transfected kidney cells. The data indicate that substrate recognition or transport efficacy by Pgp differs between human and mouse for certain AEDs. Such species differences, which are certainly not restricted to human and mouse, may explain, at least in part, the controversial data which have been previously reported for AED transport by Pgp in preparations from different species. However, because transport efficacy of efflux transporters such as Pgp or MRP2 may not only differ between species but also between tissues, the present data do not exclude that the AEDs examined are weak substrates of Pgp or MRP2 at the human BBB.     

The Antiepileptic Drug Topiramate is a Substrate for Human P-glycoprotein but Not Multidrug Resistance Proteins

Purpose  

Resistance to antiepileptic drugs (AEDs) is the major problem in the treatment of epilepsy. One of the candidate mechanisms of pharmacoresistance is the limitation of AED access to the seizure focus by overexpression of efflux transporters, including P-glycoprotein (Pgp) and multidrug resistance proteins (MRPs). In this respect, it is important to know which AEDs are substrates for such drug transporters in humans.     

Marked Differences in the Effect of Antiepileptic and Cytostatic Drugs on the Functionality of P-Glycoprotein in Human and Rat Brain Capillary Endothelial Cell Lines

Purpose

The expression of P-glycoprotein (Pgp) is increased in brain capillary endothelial cells (BCECs) of patients with pharmacoresistant epilepsy. This may restrict the penetration of antiepileptic drugs (AEDs) into the brain. However, the mechanisms underlying increased Pgp expression in epilepsy patients are not known. One possibility is that AEDs induce the expression and functionality of Pgp in BCECs. Several older AEDs that induce human cytochrome P450 enzymes also induce Pgp in hepatocytes and enterocytes, but whether this extends to Pgp at the human BBB and to newer AEDs is not known.

Methods

This prompted us to study the effects of various old and new AEDs on Pgp functionality in the human BCEC line, hCMEC/D3, using the rhodamine 123 (Rho123) efflux assay. For comparison, experiments were performed in two rat BCEC lines, RBE4 and GPNT, and primary cultures of rat and pig BCECs. Furthermore, known Pgp inducers, such as dexamethasone and several cytostatic drugs, were included in our experiments.

Results

Under control conditions, GPNT cells exhibited the highest and RBE4 the lowest Pgp expression and Rho123 efflux, while intermediate values were determined in hCMEC/D3. Known Pgp inducers increased Rho123 efflux in all cell lines, but marked inter-cell line differences in effect size were observed. Of the various AEDs examined, only carbamazepine (100 μM) moderately increased Pgp functionality in hCMEC/D3, while valproate (300 μM) inhibited Pgp.

Conclusions

These data do not indicate that treatment with AEDs causes a clinically relevant induction in Pgp functionality in BCECs that form the BBB.     

Functional evaluation of polymorphisms in the human ABCB1 gene and the impact on clinical responses of antiepileptic drugs

OBJECTIVE: The ABCB1 haplotype combinations have been demonstrated to be associated with epilepsy treatment outcomes. The aim of this study is to investigate whether ABCB1 haplotype combinations would affect P-glycoprotein (Pgp) function and impact the clinical responses of antiepileptic drugs (AEDs). METHODS AND RESULTS: Transport of substrate rhodamine 123 and calcein-AM by human Pgp carrying 12 haplotype combinations of 1236C>T, 2677G>T/A and 3435C>T were assayed in the absence and presence of known inhibitors and AEDs. The inhibitory potency of the tested drugs from the dose-response relationships was cyclosporin A>verapamil> phenytoin> carbamazepine> lamotrigine>phenobarbital>valproic acid, levetiracetam, gabapentin. The silent polymorphisms combination (1236T-3435T) and triple haplotypes (1236T-2677A/T-3435T) resulted in profoundly less effective inhibition against substrates with significantly lower intracellular substrate concentration. These results confirmed that ABCB1 polymorphisms were associated with clinical responses of AEDs. CONCLUSION: Our findings demonstrated that human ABCB1 polymorphisms may alter the interactions between Pgp and substrates, and provided functional evidence for ABCB1 haplotypes-associated epilepsy treatment responses.     

Cerebral expression of drug transporters in epilepsy

Over-expression of drug efflux transporters at the level of the blood-brain barrier (BBB) has been proposed as a mechanism responsible for multidrug resistance. Drug transporters in epileptogenic tissue are not only expressed in endothelial cells at the BBB, but also in other brain parenchymal cells, such as astrocytes, microglia and neurons, suggesting a complex cell type-specific regulation under pathological conditions associated with epilepsy. This review focuses on the cerebral expression patterns of several classes of well-known membrane drug transporters such as P-glycoprotein (Pgp), and multidrug resistance-associated proteins (MRPs) in the epileptogenic brain. Both experimental and clinical evidence of epilepsy-associated cerebral drug transporter regulation and the possible mechanisms underlying drug transporter regulation are discussed. Knowledge of the cerebral expression patterns of drug transporters in normal and epileptogenic brain will provide relevant information to guide strategies attempting to overcome drug resistance by targeting specific transporters.     

The power of the pump: Mechanisms of action of P-glycoprotein (ABCB1)

Members of the superfamily of ATP-binding cassette (ABC) transporters mediate the movement of a variety of substrates including simple ions, complex lipids and xenobiotics. At least 18 ABC transport proteins are associated with disease conditions. P-glycoprotein (Pgp, ABCB1) is the archetypical mammalian ABC transport protein and its mechanism of action has received considerable attention. There is strong biochemical evidence that Pgp moves molecular cargo against a concentration gradient using the energy of ATP hydrolysis. However, the molecular details of how the energy of ATP hydrolysis is coupled to transport remain in dispute and it has not been possible to reconcile the data from various laboratories into a single model. The functional unit of Pgp consists of two nucleotide binding domains (NBDs) and two trans-membrane domains which are involved in the transport of drug substrates. Considerable progress has been made in recent years in characterizing these functionally and spatially distinct domains of Pgp. In addition, our understanding of the domains has been augmented by the resolution of structures of several non-mammalian ABC proteins. This review considers: (i) the role of specific conserved amino acids in ATP hydrolysis mediated by Pgp; (ii) emerging insights into the dimensions of the drug binding pocket and the interactions between Pgp and the transport substrates and (iii) our current understanding of the mechanisms of coupling between energy derived from ATP binding and/or hydrolysis and efflux of drug substrates.     

Impact of P-glycoprotein-mediated intestinal efflux kinetics on oral bioavailability of P-glycoprotein substrates

Studies of many P-glycoprotein (Pgp) substrates have demonstrated a significant effect of Pgp-mediated efflux on intestinal drug transport. However, most of these studies were designed to detect whether a particular drug is a Pgp substrate and thus were conducted at very low concentrations. We performed two simulations to evaluate the effect of Pgp-mediated efflux on oral drug absorption at various concentrations. In the first simulation, a steady-state model allowed us to predict whether the contribution of Pgp to oral drug absorption would be significant at clinically relevant concentrations. Our second simulation investigated the role of Pgp-mediated efflux in oral absorption with a dynamic compartmental absorption and transit model linked to a pharmacokinetic model. For high-solubility drugs, Pgp-mediated efflux altered the bioavailability only at drug concentrations corresponding to doses much lower than the usual clinical dose. The ratio of transporter-mediated transport to passive transport determined whether intestinal Pgp transporters would reduce the bioavailability of high-solubility drugs.     

Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport

Background and purpose:

P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport.

Experimental approach:

Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine.

Key results:

In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells.

Conclusions and implications:

Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport.     

Evaluation of the role of P-glycoprotein in the uptake of paroxetine, clozapine, phenytoin and carbamazapine by bovine retinal endothelial cells

Expression of the drug transport proteins, including P-glycoprotein (Pgp), in the brain vascular endothelium represents a challenge for the effective delivery of drugs for the treatment of several central nervous system (CNS) disorders including depression, schizophrenia and epilepsy. It has been hypothesized that Pgp plays a major role in drug efflux at the blood-brain barrier, and may be an underlying factor in the variable responses of patients to CNS drugs. However, the role of Pgp in the transport of many CNS drugs has not been directly demonstrated. To explore the role of Pgp in drug transport across an endothelial cell barrier derived from the central nervous system, the expression and activity of Pgp in bovine retinal endothelial cells (BRECs) and the effects of representative CNS drugs on Pgp activity were examined. Significant Pgp expression in BRECs was demonstrated by western analyses, and expression was increased by treatment of the cells with hydrocortisone. Intracellular accumulation of the well-characterized Pgp-substrate Taxol was markedly increased by the non-selective transporter inhibitor verapamil and the Pgp-selective antagonist PGP-4008, demonstrating that Pgp is active in these endothelial cells. In contrast, neither verapamil nor PGP-4008 affected the intracellular accumulation of [3H]paroxetine, [14C]phenytoin, [3H]clozapine or [14C]carbamazapine, indicating that these drugs are not substrates for Pgp. Paroxetine, clozapine and phenytoin were shown to be Pgp inhibitors, while carbamazapine did not inhibit Pgp at any concentration tested. These results indicate that Pgp is not likely to modulate patient responses to these drugs.     

PET and SPECT radiotracers to assess function and expression of ABC transporters in vivo

Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer's and Parkinson's disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications.     

Caco-2 permeability,P-glycoprotein transport ratios and brain penetration of heterocyclic drugs

In this study the gastrointestinal absorption and P-glycoprotein (Pgp) efflux transport of heterocyclic drugs was investigated with the Caco-2 cell model. Based on the calculation of the physico-chemical properties a good oral absorption was predicted for all the drugs tested in this study which corresponded well with the measured Caco-2 permeabilities (Papp). Generally a high permeability of the tested heterocyclic drugs was measured being in agreement with earlier published human in vivo absorption data. Based on the transport data of domperidone and verapamil it was found that the Pgp efflux transporter was expressed in the Caco-2 cells. Many of the drugs tested were indicated to be potential Pgp efflux substrates. Since Pgp is expressed at the Blood Brain Barrier (BBB) as well, it was expected that CNS penetration will be impaired if a drug is a Pgp substrate. However, no correlation could be found between brain penetration in rats and the Pgp efflux ratio as measured with the Caco-2 cells. From the data it is concluded that Pgp efflux ratio's as determined in in vitro High Throughput Screening (HTS) tests, where the transport conditions are fixed (pH gradient, concentration, etc.), cannot routinely be used to predict a possible limited brain penetration.     

In vitro absorption and secretory quotients: practical criteria derived from a study of 331 compounds to assess for the impact of P-glycoprotein-mediated efflux on drug candidates

The absorptive (AQ) and secretory (SQ) quotients have been proposed as a novel experimental approach to quantify the modulation of intestinal absorption and secretion by P-glycoprotein (Pgp). Because these unidirectional assays inherently assess for the impact of Pgp, conclusions as to whether a compound is a Pgp substrate will be made from the data. Therefore, the objective of this study was to establish the relationship between AQ/SQ and the bidirectional efflux assay and to derive criteria to classify a compound as a Pgp substrate. AQ and SQ parameters were calculated for 331 compounds that had previously been evaluated in the bidirectional assay and the concordance of Pgp substrate classification between these methods assessed by establishing AQ/SQ criteria of increasing magnitude. The AQ and SQ values correctly identified 80 and 85% of the compounds as Pgp substrates/nonsubstrates relative to the bidirectional efflux assay. This study demonstrates that the optimal AQ and SQ value to classify compounds as Pgp substrates was 0.3 and provides a basis to deploy unidirectional efflux assays in the early stages of drug discovery, which would benefit from the twofold increase in throughput over current bidirectional transport assays.     

Pharmacologic management of epilepsy in the elderly

OBJECTIVE: To review the epidemiology and pharmacologic management of epilepsy in elderly patients. DATA SOURCES: Controlled trials, case studies, and review articles identified via MEDLINE using the search terms epilepsy, seizures, elderly, phenobarbital, primidone, phenytoin, carbamazepine, valproic acid, felbamate, gabapentin, lamotrigine, topiramate, tiagabine, levetiracetam, oxcarbazepine, and zonisamide. Recently published standard textbooks on epilepsy were also consulted. DATA SYNTHESIS: Epilepsy is a common neurologic disorder in the elderly. Cerebrovascular and neurodegenerative diseases are the most common causes of new-onset seizures in these patients. Alterations in protein binding, distribution, elimination, and increased sensitivity to the pharmacodynamic effects of antiepileptic drugs (AEDs) are relatively frequent, and these factors should be assessed at the initiation, and during adjustment, of treatment. Drug-drug interactions are also an important issue in elderly patients, because multiple drug use is common and AEDs are susceptible to many interactions. In addition to understanding age-related changes in the pharmacokinetics and pharmacodynamics of AEDs, clinicians should know the common seizure types in the elderly and the spectrum of AED activity for these seizure types. AEDs with activity against both partial-onset and generalized seizures include felbamate, lamotrigine, levetiracetam, topiramate, valproic acid, and zonisamide. Other AEDs discussed in this review (carbamazepine, gabapentin, phenobarbital, phenytoin, primidone, and tiagabine) are most useful for partial-onset seizures. CONCLUSION: The provision of safe and effective drug therapy to elderly patients requires an understanding of the unique age-related changes' in the pharmacokinetics and pharmacodynamics of AEDs as well as an appreciation of common seizure types and the drugs that are effective for the specific types seen in the elderly.     

Delineating the contribution of secretory transporters in the efflux of etoposide using Madin-Darby canine kidney (MDCK) cells overexpressing P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and canalicular multispecific organic anion transporter (cMOAT).

Multidrug resistance conferred to cancer cells is often mediated by the expression of efflux transporter "pumps". It is also believed that many of the same transporters are involved in drug efflux from numerous normal endothelial and epithelial cell types in the intestine, brain, kidney, and liver. Etoposide transport kinetics were characterized in Caco-2 cells and in well established Madin-Darby canine kidney (MDCKII) cell lines that were stably-transfected with a human cDNA encoding P-glycoprotein (Pgp), human multidrug resistance protein (MRP1), or the canalicular multispecific organic anion (cMOAT) transporters to determine the roles of these transporters in etoposide efflux. Etoposide transport kinetics were concentration-dependent in the MDCKII-MDR1 and MDCKII-cMOAT cells. The apparent secretory Michaelis constant (Km) and carrier-mediated permeability (Pc) values for Pgp and cMOAT were 254.96 +/- 94.39 microM and 5.96 +/- 0.41 x 10(-6) cm/s and 616.54 +/- 163.15 microM and 1.87 +/- 0.10 x 10(-5) cm/s, respectively. The secretory permeability of etoposide decreased significantly in the basal to apical (B to A) (i.e., efflux) direction, whereas the permeability increased 2.3-fold in the apical to basal (A to B) direction in MDCKII-MDR1 cells in the presence of elacridar (GF120918). Moderate inhibition of etoposide efflux by leukotriene C4 (LTC4) was observed in MDCKII-cMOAT cells. Furthermore, etoposide inhibited LTC4 efflux, confirming the involvement of cMOAT. The flux of etoposide in MDCKII-MRP1 cells was similar to that in MDCKII/wt control cells. The current results demonstrate that the secretory transport mechanism of etoposide involves multiple transporters, including Pgp and cMOAT but not MRP1. These results demonstrate that Pgp and cMOAT are involved in the intestinal secretory transport of etoposide. Since the intestinal secretion of etoposide was previously reported in the literature, it also suggests that they may be involved in the in vivo intestinal secretion of etoposide; however, mechanistic in vivo studies are required to confirm this.     

ABC transporters: unvalidated therapeutic targets in cancer and the CNS

The discovery of the multidrug transporter P-glycoprotein (Pgp) over 35 years ago in drug resistant cells prompted several decades of work attempting to overcome drug resistance by inhibition of drug efflux. Despite convincing laboratory data showing that drug transport can be inhibited in vitro, efforts to translate this discovery to the clinic have not succeeded. Since overexpression of Pgp and related transporters including ABCG2 and members of the ABCC family have been linked with poor outcome, it remains a reasonable hypothesis that this poor outcome is linked to reduction of drug exposure by efflux, and thus to drug resistance. In this review, we will discuss the question of whether ABC transporters mediate drug resistance in cancer through a reduction in drug accumulation in tumors, and whether the "Pgp inhibition hypothesis" might be wrong. The hypothesis, which holds that increased chemotherapy effectiveness can be achieved by inhibiting Pgp-mediated drug efflux has only been validated in model systems. Possible explanations for the failure to validate this clinically include the existence of other modulators of drug accumulation and uptake in tumors. Despite these difficulties, a potential role has emerged for drug transporters as therapeutic targets in the central nervous system (CNS). Both lines of investigation point to the need for imaging agents to facilitate the study of drug accumulation in human cancer. This is a critical need for targeted therapies where an important dose-response relationship is likely to exist, and where drug resistance renders many of the novel targeted agents ineffective in a subset of patients.     

Utility of unbound plasma drug levels and P-glycoprotein transport data in prediction of central nervous system exposure

1. Drug concentrations in cerebrospinal fluid have been assumed to be a natural surrogate for total drug exposures in the central nervous system. The present communication reports a data set from a study of 30 compounds in mice. An attempt was made to correlate cerebrospinal fluid and unbound plasma drug concentrations via incorporation of in vitro P-glycoprotein (Pgp)-mediated transport data.

2. Pgp-deficient (Pgp –/–) and wild-type mice were dosed with compounds of interest by oral gavage (orally) at 5?mg kg^?1. Plasma and cerebrospinal fluid samples were collected at 1?h post-dosing, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for drug concentrations. Mouse and human Pgp-mediated transport were evaluated in vitro by a bi-directional (B to A and A to B) transport assay using LLC-PK1 cells expressing mouse (mdr1a) and human (MDR1) forms of Pgp, respectively.

3. Compounds with B to A/A to B transport ratios in vitro via equilibrium dialysis. Of the 30 compounds, 13 were identified to be mouse Pgp substrates, all of which were also human Pgp substrates, demonstrating a good agreement between mouse and human data.

4. In Pgp wild-type mice, the unbound plasma and cerebrospinal fluid concentrations of the non-Pgp substrates correlated well, with a regression slope of approximately 1.0. A similar relationship existed for Pgp substrates in Pgp –/– mice. On the other hand, an improved correlation of cerebrospinal fluid and systemic exposures of the Pgp substrates in Pgp wild-type mice was observed when the unbound plasma concentrations were normalized to the corresponding B to A/A to B transport ratios.

5. These results reinforce the premise that a combined use of unbound plasma drug concentrations and in vitro Pgp transport data may be of value for the estimation of central nervous system exposures.

     

Detection and functional characterization of Pgp1 (ABCB1) and MRP3 (ABCC3) efflux transporters in the PLHC-1 fish hepatoma cell line

The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in the metabolism of xenobiotics. However, the presence of the ABC transport proteins possibly involved in the MultiXenobiotic Resistance (MXR) mechanism as phase III of cellular detoxification has never been described in the PLHC-1 cells. The main goal of this study was the detection and functional characterization of toxicologically relevant xenobiotic efflux transporters from ABCB and ABCC subfamily in the PLHC-1 cells. Using specific primer pairs two PCR products 1769 and 1023bp in length were successfully cloned and sequenced. Subsequent multiple alignment and phylogenetic analysis showed that these sequences share a high degree of homology with the P-glycoprotein (Pgp1; ABCB1) and the MRP3 (ABCC3). Functional experiments with fluorescent model substrates and specific inhibitors were used to verify that transport activities of Pgp- and MRP-related proteins are indeed present in PLHC-1 cells. Accumulation or efflux/retention rates of rhodamine 123, calcein-AM or monochlorbimane were time- and concentration-dependent. Cyclosporine A, MK571, verapamil, reversine 205, indomethacine and probenecid were used as specific inhibitors of Pgp1 and/or MRPs transport activities, resulting in a dose dependent inhibition of related transport activities in PLHC-1 cells. Similar to mammalian systems, the obtained IC(50) values were in the lower micromolar range. Taken together these data demonstrate that: (1) the PLHC-1 cells do express a functional MXR mechanism mediated by toxicologically relevant ABC efflux transporters; and (2) the presence of all three critical phases of cellular detoxification additionally affirms the PLHC-1 cells as a reliable in vitro model in aquatic toxicology.