In vitro transport assays of rufinamide, pregabalin, and zonisamide by human P-glycoprotein
Introduction
Despite the development of over 20 antiepileptic drugs (AEDs), epilepsy does not respond to AED treatment in approximately one third of patients, due to mechanisms that remain unclear (Kwan and Brodie, 2000). The ABCB1 or MDR1 gene encodes P-glycoprotein (Pgp), which transports xenobiotics across the blood–brain barrier (BBB) from the basolateral to the apical side. Pgp is expressed on the luminal membrane of brain capillary endothelium and is overexpressed at epileptic foci (Kwan and Brodie, 2005, Loscher and Potschka, 2005). In drug-resistant patients, Pgp is expressed at greater levels than in drug-responsive patients (Kwan et al., 2010, Aronica et al., 2012, Feldmann et al., 2013). Reports demonstrate several AEDs to be substrates or inhibitors of Pgp (Weiss et al., 2003, Baltes et al., 2007a, Baltes et al., 2007b, Luna-Tortos et al., 2008, Zhang et al., 2010, Zhang et al., 2011), including phenytoin, phenobarbital, topiramate, lamotrigine, levetiracetam, oxcarbazepine, eslicarbazepine acetate, and the drug metabolites carbamazepine-10,11-epoxide, and S-licarbazepine (Luna-Tortos et al., 2008, Luna-Tortos et al., 2009, Zhang et al., 2010, Zhang et al., 2011). Thus, Pgp might play a role in epilepsy drug-resistance (Kwan et al., 2011).
Efforts to develop new AEDs have resulted in the approval in recent years of drugs including zonisamide (ZNS), pregabalin (PGB), and rufinamide (RFM). The Pgp substrate status of these drugs is largely unknown. There has been only one published study employing OS2.4/Doxo cells (canine osteosarcoma cells induced by exposure to doxorubicin to highly express Pgp) which showed that ZNS did not affect the uptake of Rho123 (West and Mealey, 2007), suggesting that ZNS is not a Pgp substrate. No evidence has been reported to indicate the substrate status of PGB or RFM in animal models or epilepsy patients. Therefore, using the concentration equilibrium transport assay, we studied whether they are substrates of Pgp, which may help in understanding the molecular basis of pharmacoresistance.
Section snippets
Chemicals
ZNS, PGB, and RFM were supplied by 3B Pharmachem International Co., Ltd (Wuhan, China). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) was dissolved in water, and other drugs were dissolved in DMSO (dimethyl sulfoxide, <0.1% in final solution). ZNS, PGB, and RFM were tested at concentrations covering the ranges of therapeutic plasma concentrations. Acetonitrile (Labscan Asia, Thailand), ethanol (TEDIA Company, Inc., USA), and methanol (TEDIA Company,
Results
The cytotoxicities of PGB, RFM and ZNS were tested by the MTT assay. The three drugs were not toxic to the four cell lines for at least 4 h at tested concentrations. Integrity of monolayers of the cell lines was verified by testing that the apparent permeability values (Papp) of propranolol and atenolol were within the range of 15 × 10−6 to 40 × 10−6 cm/s and 0.5 × 10−6 to 1.5 × 10−6 cm/s respectively, which were comparable to those previously published (Crespi et al., 2000, Wang et al., 2008, Thiel-Demby
Discussion
Resistance to AEDs is a serious problem in epilepsy treatment. The efflux transport of AEDs from the brain by Pgp might play a role (Kwan et al., 2011). Publicly-accessible studies on the Pgp substrate status of AEDs performed for drug agencies such as the US Food and Drug Administration were not found, however there is in vitro and in vivo evidence from academic studies that a number of established and newer AEDs are transported by Pgp (Zhang et al., 2012).
However, AEDs were designed to have a
Acknowledgements
Cell lines were kindly provided by Prof. P. Borst, The Netherlands Cancer Institute. Financial support was provided by CUHK Direct Grant 2008.1.078.
References (27)
- et al.
Cerebral expression of drug transporters in epilepsy
Adv. Drug. Deliv. Rev.
(2012) - et al.
Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein
Neuropharmacology
(2007) - et al.
Analysis of drug transport and metabolism in cell monolayer systems that have been modified by cytochrome P4503A4 cDNA-expression
Eur. J. Pharm. Sci.
(2000) - et al.
P-glycoprotein expression and function in patients with temporal lobe epilepsy: a case–control study
Lancet Neurol.
(2013) - et al.
Association between temporal lobe P-glycoprotein expression and seizure recurrence after surgery for pharmacoresistant temporal lobe epilepsy
Neurobiol. Dis.
(2010) - et al.
Several major antiepileptic drugs are substrates for human P-glycoprotein
Neuropharmacology
(2008) - et al.
Impact of P-glycoprotein and breast cancer resistance protein on the brain distribution of antiepileptic drugs in knockout mouse models
Eur. J. Pharmacol.
(2013) - et al.
Application and limitation of inhibitors in drug-transporter interactions studies
Int. J. Pharm.
(2008) - et al.
The transport of antiepileptic drugs by P-glycoprotein
Adv. Drug. Deliv. Rev.
(2012) - et al.
In vitro concentration dependent transport of phenytoin and phenobarbital, but not ethosuximide, by human P-glycoprotein
Life Sci.
(2010)
Mechanistic study on the intestinal absorption and disposition of baicalein
Eur. J. Pharm. Sci.
Valproic acid is not a substrate for P-glycoprotein or multidrug resistance proteins 1 and 2 in a number of in vitro and in vivo transport assays
J. Pharmacol. Exp. Ther.
A silent polymorphism in the MDR1 gene changes substrate specificity
Science
Cited by (12)
Cell culture and pharmacokinetic evaluation of a solid dosage formulation containing a water-insoluble orphan drug manufactured by FDM-3DP technology
2022, International Journal of PharmaceuticsCitation Excerpt :The obtained results are presented in Fig. 5. A review of literature revealed no MTT test results for Caco-2 or Hep G2 cell lines for the active substance, rufinamide, although in a study of MDCK (Madin-Darby Canine Kidney cells) at concentrations of 20, 10 and 5 µg/mL, it was stated that the active substance was not cytotoxic (Chan et al., 2014). It is thought that the reason why the improved 3DP tablet formula is less cytotoxic than the pure active substance may be related to solubility, the solubility of the formulation being considerably higher than that of the pure active substance.
Long-term safety and seizure outcome in Japanese patients with Lennox-Gastaut syndrome receiving adjunctive rufinamide therapy: An open-label study following a randomized clinical trial
2016, Epilepsy ResearchCitation Excerpt :It was recently suggested that the mechanism of drug resistance in some types of epilepsy (including LGS) involves over-expression of P-glycoprotein in the endothelium of the blood–brain barrier (Zhang et al., 2012; Kumar et al., 2014). It is thought that penetration of the blood–brain barrier by rufinamide is not affected because it is not a substrate of human P-glycoprotein (Chan et al., 2014), and this pharmacological characteristic may be related to its long-term effectiveness for LGS. During the present long-term study, the safety profile of rufinamide was similar to that revealed by the preceding placebo-controlled study (Ohtsuka et al., 2014) and we did not find any new late-onset AEs.
The antiepileptic drug lamotrigine is a substrate of mouse and human breast cancer resistance protein (ABCG2)
2015, NeuropharmacologyCitation Excerpt :Second, Cerveny et al. (2006) used a conventional bidirectional transport assay, which is less sensitive than the CETA to identify highly lipophilic transporter substrates (Löscher et al., 2011). After we first described the CETA in 2008 (Luna-Tortós et al., 2008), this modified transport assay is now used by several other groups to study drug transport by efflux transporters (Zhang et al., 2012; Dickens et al., 2013; Chan et al., 2014). In contrast to lamotrigine, none of the other AEDs tested in our study was transported by mouse Bcrp, confirming the experiments of Cerveny et al. (2006) with human BCRP.
Dose-dependent pharmacokinetics and brain penetration of rufinamide following intravenous and oral administration to rats
2015, European Journal of Pharmaceutical SciencesCitation Excerpt :Sometimes high protein binding in plasma associated with low protein binding in brain tissue can also produce low Kp, although this hypothesis could be refuted by the low plasma binding percentage of 26.2–34.8% of the compound (Perucca et al., 2008). Efflux transports can also be involved, although recent findings indicate that rufinamide is not a substrate of P-glycoprotein (Chan et al., 2014), but other transport proteins – multidrug resistance proteins (MRPs), breast cancer related proteins (BCRPs), organic anion transporting-polypeptide family proteins (OATPs), major vault protein (MVP) – remain to be verified (Löscher and Potschka, 2005). Regardless, Kp of rufinamide was independent of dose, route of administration, or post-dosing time, so a linear correlation between brain concentrations and plasma concentrations was established.
Increased amisulpride serum concentration in a patient treated with concomitant pregabalin and trazodone: a case report
2022, Therapeutic Advances in PsychopharmacologyPregabalin for chemotherapy-induced neuropathy: background and rationale for further study
2022, Supportive Care in Cancer
- 1
Current address: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, The University of Georgia, Athens, GA 30606, USA.