Drug discovery and resistanceInhibitory potential of tuberculosis drugs on ATP-binding cassette drug transporters
Introduction
Tuberculosis (TB) remains a prevalent global health problem. In 2013, an estimated 9.0 million people developed active TB and 1.5 million died from the disease [1]. Treatment of TB requires long and intensive combination drug therapy. The first-line regimen for drug-sensitive TB contains four drugs, namely rifampicin, isoniazid, pyrazinamide and ethambutol [2] (Table 1). For multiple drug resistant TB (MDR TB), a range of second-line TB drugs are in use (Table 1), which are administered for longer periods, generally have less potency and are associated with more toxicity [3]. In recent years, novel TB drugs have been developed (bedaquiline and delamanid) and available TB drugs are being repurposed or optimized as first-line TB drugs (e.g. high dose rifamycins, moxifloxacin and clofazimine). In addition, various new compounds are still being evaluated for treatment of both drug-sensitive and resistant TB [3], [4]. Overall, in TB drug development the focus is on development of regimens rather than just single new TB drugs, and in this context, drug-drug interactions (DDIs) between available and (potential) new TB drugs need to be assessed.
Because TB is increasingly associated with co-morbidities, drug therapy is becoming even more complex. An estimated 1.1 million (13%) of the people who developed TB in 2013 were HIV-positive [1]. In addition, the relative burden of type 2 diabetes mellitus (DM) to the TB epidemic is increasing, as the incidence of DM is rapidly rising in countries where TB is endemic and DM is a prominent risk factor for active TB [5]. The concurrent use of antimycobacterial, antiretroviral, and antidiabetic drugs further increases the likelihood of DDIs to occur in this patient population [6].
Inhibition of membrane transporter activity is one of the mechanisms known to result in relevant DDIs. In general, drugs need to cross multiple cellular barriers in order to reach their sites of action. Uptake and efflux via membrane transporters play a vital role in the passage across these barriers and, therefore, in the absorption, distribution and excretion of drugs. Efflux transporters also protect sanctuary sites such as brain and placenta against xenobiotics and increase their elimination via liver and kidney. Many transporters of the ATP binding cassette (ABC) transporter protein family are involved in the cellular efflux of drugs [7]. ABC proteins are primary active transporters that mediate the translocation of substrates across the membrane at the expense of ATP hydrolysis. Interaction of a drug with such a transporter could alter the systemic or local disposition of other compounds that are substrates of the same transporter [8]. Consequently, drug concentrations of the other substrate drug can be increased or decreased and the safety and efficacy of this drug may be at risk [8], [9]. As an example for an interaction affecting systemic exposure, the antimycobacterial drug clarithromycin (classified as a group V anti-TB drug) has demonstrated to increase the oral bioavailability and reduce the nonglomerular renal clearance of digoxin, a P-gp substrate, probably by inhibiting intestinal and renal P-glycoprotein [10]. This inhibitory effect has also been demonstrated in an in vitro model [11] and may form a reasonable explanation for the reports of clarithromycin induced digoxin intoxication [12].
Considering the likelihood of DDIs to occur in TB treatment, we aimed to clarify the in vitro interaction potential of available TB drugs with the major ABC transporters relevant to drug transport, namely P-gp, BCRP, BSEP and MRP1-5 [13]. For this purpose, we used membrane vesicles isolated from transporter-overexpressing HEK293 cells. In drug development, inside-out membrane vesicles are recognized as an appropriate in vitro model to assess whether a drug is an in vivo transporter substrate or inhibitor [13]. This model has as a major advantage that drugs can be directly applied to the cytoplasmic compartment and influx, rather than efflux, is measured. In the field of TB treatment, vesicular transport data are especially valuable, because of the limited knowledge on interactions of TB drugs with ABC transporters and the current emphasis on development of TB drug regimens. Next to first and second-line TB drugs, we assessed the inhibitory potential of three so-called mycobacterial efflux pump inhibitors (EPIs) [14], i.e. thioridazine, timcodar and SQ109 (Table 1). EPIs are proposed to work as co-adjuvant drugs to increase intramycobacterial concentrations of TB drugs, which may even reverse Mycobacterium tuberculosis efflux pump induced resistance [14], [15], [16], [17], [18], [19], [20].
Section snippets
Chemicals
The following TB drugs were purchased from Sigma–Aldrich (Zwijndrecht, the Netherlands): rifampicin, isoniazid, pyrazinamide, ethambutol, amikacin, moxifloxacin hydrochloride, cycloserin, ethionamide, 4-aminosalicylic acid (PAS), amoxicillin, clofazimine, linezolid and thioridazine hydrochloride. SQ109 was kindly provided by Sequella Inc. (Rockville, MD, USA) and timcodar by Vertex Pharmaceuticals Inc. (Boston, MA, USA), respectively. P-gp substrate [3H]-N-methyl quinidine ([3H]-NMQ) and
Screening for ABC transporter activity inhibition
Membrane vesicles isolated from transporter-overexpressing HEK293 cells were used to study the inhibitory action of TB drugs and EPIs on the transport of model radio-labeled substrates [3H]-NMQ (P-gp); [3H]-E1S (BCRP); [3H]-TCA (BSEP); [3H]-E217βG; (MRP1, 3 and 4) and [3H]-MTX (MRP2 and 5). Figure 1 shows the results of screening for the inhibitory potency of TB drugs and EPIs at a concentration of 200 μM against ATP-dependent uptake of the model substrates. Of all transporters, P-gp-mediated [3
Discussion
We assessed the in vitro inhibitory potential of a range of currently used TB drugs and novel EPIs on various ABC transporters that are important in drug transport, namely P-gp, BCRP, BSEP and MRP1-5. Inhibition of these membrane transporters may be an important mechanism underlying clinically relevant drug-drug interactions (DDIs). Overall, we demonstrated a strong inhibition (IC50 < 15 μM) of P-gp, BCRP and MRP1 by clofazimine; P-gp and BCRP by SQ109; BCRP by thioridazine and inhibition of
Acknowledgments
Sequella Inc, USA is acknowledged for providing SQ109 for this study and Vertex Pharmaceuticals Incorporated, USA for providing timcodar. Reinout van Crevel, Radboudumc, The Netherlands, is thanked for reviewing the manuscript.
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