TRAM-34, a Putatively Selective Blocker of Intermediate-Conductance, Calcium-Activated Potassium Channels, Inhibits Cytochrome P450 Activity

TRAM-34, a clotrimazole analog characterized as a potent and selective inhibitor of intermediate-conductance, calcium-activated K+ (IKCa) channels, has been used extensively in vitro and in vivo to study the biological roles of these channels. The major advantage of TRAM-34 over clotrimazole is the reported lack of inhibition of the former drug on cytochrome P450 (CYP) activity. CYPs, a large family of heme-containing oxidases, play essential roles in endogenous signaling and metabolic pathways, as well as in xenobiotic metabolism. However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform. To test the hypothesis that TRAM-34 may inhibit some CYP isoforms, the effects of this compound were presently studied on the activities of four rat and five human CYP isoforms. TRAM-34 inhibited recombinant rat CYP2B1, CYP2C6 and CYP2C11 and human CYP2B6, CYP2C19 and CYP3A4 with IC50 values ranging from 0.9 µM to 12.6 µM, but had no inhibitory effects (up to 80 µM) on recombinant rat CYP1A2, human CYP1A2, or human CYP19A1. TRAM-34 also had both stimulatory and inhibitory effects on human CYP3A4 activity, depending on the substrate used. These results show that low micromolar concentrations of TRAM-34 can inhibit several rat and human CYP isoforms, and suggest caution in the use of high concentrations of this drug as a selective IKCa channel blocker. In addition, in vivo use of TRAM-34 could lead to CYP-related drug-drug interactions.


Introduction
Clotrimazole and related azole antimycotic agents are well known inhibitors of cytochrome P450 (CYP) enzymes [1]. CYPs, which are members of a large family of heme-containing oxidases, are key elements of endogenous biosynthetic and signaling pathways involving steroids, prostaglandins, and fatty acid derivatives, and also play essential roles in xenobiotic metabolism [2]. Each CYP has a specific profile of catalytic activities across a number of substrates. These profiles are important for understanding potential drug-drug interactions due to CYP inhibition, as well as induction [3].
Clotrimazole is also a highly potent blocker of intermediate conductance Ca 2+ -activated K + channels (IK Ca ) [4]. These channels (also known as IK1, SK4, IK Ca 3.1 or KCNN4) are expressed in various non-excitable cell types throughout the body. IK Ca channels play a vital role in the loss of cellular water [5] as well as the migration of microglia [6] and mast cells [7]. Because of clotrimazole's potent IK Ca channel blocking activity, this drug has been used clinically for treating several disorders related to abnormal ion channel activity, such as sickle cell disease [8]. However, clotrimazole's potent anti-CYP activities account for numerous side effects and systemic toxicity [9].
Because of the toxicity of clotrimazole, efforts have been made to develop more selective IK Ca blockers devoid of CYP-related side effects. Wulff et al. [10] characterized TRAM-34 (1-[(2chlorophenyl) diphenylmethyl]-1H-pyrazole), a triarylmethane pyrazole analog of clotrimazole, as a selective and potent blocker of the IK Ca channel (K d = 20 nM). The finding that TRAM-34 did not inhibit CYP activity was the basis for the claimed increase in selectivity [10]. These results suggest that TRAM-34 could have a pharmacological profile similar to that of clotrimazole, without its anti-CYP side effects. Consequently, TRAM-34 is a marketed research tool which is widely used as a highly selective IK Ca blocker in vitro and in vivo (see Discussion). The drug is currently not in human use. Since published studies of TRAM-34's activity on CYPs are limited to one isoform (human CYP3A4), and recent studies have reported effects of TRAM-34 which are independent of IK Ca [11], we further explored the effects of this compound on several human and rat CYP isoforms.

Fluorescence-based CYP Assays
These assays were performed by methods similar to those described by VanAlstine and Hough [12], Stresser et al. [13], Wulff et al. [10], Henderson et al. [14], and Crespi et al. [15], with minor modifications. All incubations were performed in a final volume of 200 mL of 50 mM potassium phosphate buffer at pH 7.4 containing 1% acetonitrile. CYP inhibitor studies were performed according to the parameters of Table 1. Substrate concentrations were chosen to be near known K m values. As described in the references cited, two different NADPH regenerating systems were used depending on the substrates used (Table 1). Inhibitors were added to either ''low'' NADPH-regenerating system (final concentration of 8.1 mM NADP + , 0.4 mM glucose-6phosphate, 0.4 mM magnesium chloride, 0.2 U/mL glucose-6phosphate dehydrogenase) or to ''high'' NADPH-regenerating system (final concentration of 1.3 mM NADP + , 3.3 mM glucose-6-phosphate, 3.3 mM magnesium chloride, 0.4 U/mL glucose-6phosphate dehydrogenase) in a total of 0.1 mL buffer. Samples were pre-incubated at 37uC for the designated times, and the reactions were initiated with the addition of a substrate and enzyme mixtures (Table 1). For assays utilizing ''low'' regenerating systems, product formation was monitored continuously by a Victor3 1420 Multilabel Plate Counter at the designated wavelengths (Table 1). For the assays utilizing the ''high'' regenerating system, enzyme reactions were terminated at the designated time by the addition of 75 mL of 2N NaOH, followed by a 2 h post-incubation at 37uC. Product formation was then measured at the designated wavelengths (Table 1). For the ''low'' regenerating system assays, blanks and standards were prepared in 0.1 mL buffer without the regenerating system. For the ''high'' regenerating system assays, blanks and standards containing 0.1 mL of buffer received 75 mL of 2N NaOH following preincubation. Enzyme activity was linear with incubation time in all assays. TRAM-34 (30 and 100 mM) did not significantly alter the fluorescent signal from any of the CYP products listed in Table 1.

CYP3A4 Assay with LVS as Substrate
CYP3A4-containing Supersomes (2 pmol) were incubated with LVS (10 mM) in a 200 ml reaction mixture containing 0.1 M potassium phosphate buffer, pH 7.4, 1.0 mM NADPH, and 3 mM MgCl 2 for 20 min at 37uC. The reaction was initiated by the addition of NADPH, and terminated by the addition of 400 mL of acetonitrile to the reaction mixture. Samples were extracted and analyzed for the two major LVS metabolites 69bhydroxy LVS and 69-exomethylene LVS by LC-MS-MS exactly as described [16].

Effects of TRAM-34 on Rat CYP Activity
TRAM-34 was tested on CYP activities from 4 rat and 5 human isoforms. Surprisingly, concentration-dependent inhibition by TRAM-34 was seen with 3 rat CYP isoforms. TRAM-34 potently inhibited CYP2C6 and CYP2B1 (IC 50 = 2.9 mM and 3.0 mM respectively, Figs. 1A and 1D). The drug showed weaker inhibition on CYP2C11 with an IC 50 value of 12.6 mM (Fig. 1B). Clotrimazole, used as a positive control, was a potent inhibitor of CYP2C6, CYP2B1, and CYP2C11, as expected. TRAM-34 showed no inhibition at concentrations up to 80 mM on CYP1A2r (Fig. 1C). Clotrimazole only partly inhibited CYP1A2r activity (Fig. 1C). Other inhibitors (fluvoxamine and miconazole) did not inhibit CYP1A2r activity up to 80 mM (not shown).

Effects of TRAM-34 on Human CYP Activity
TRAM-34 also showed concentration-dependent inhibition of some human CYP isoforms. The drug potently inhibited CYP2C19 and CYP2B6 (IC 50 = 1.8 mM and 0.9 mM respectively, Figs. 2A and 2D). Clotrimazole, used as a positive control for CYP2C19 and CYP2B6, was a potent inhibitor of enzyme activities, as expected. TRAM-34 (up to 80 mM) showed little to no inhibition on CYP19A1h or CYP1A2h ( Fig. 2B and 2C). Ketoconazole was a potent inhibitor of CYP19A1h activity, as expected. Also, unlike the results with CYP1A2r, fluvoxamine was a potent inhibitor of CYP1A2h (Fig. 2C).

Effects of TRAM-34 on CYP3A4 Activity
TRAM-34 was tested on CYP3A4 with three different substrates. TRAM-34 showed potent and concentration-dependent inhibition of CYP3A4 with DBF (IC 50 = 3.6 mM, Fig. 3A). Ketoconazole, used as a positive control, was a potent inhibitor of this CYP3A4 activity. Because of the inhibition seen with TRAM-34 in Fig. 3A, TRAM-34 was also tested on CYP3A4 with another substrate, BFC (used previously with CYP3A4 [10]). Surprisingly, TRAM-34 exerted concentration-dependent stimulation of CYP3A4 with BFC. The magnitude of the stimulation was up to ,200% (Fig. 3B). Ketoconazole, used as a positive control, potently inhibited CYP3A4 activity with BFC (Fig. 3B).
CYP3A4 activity was further monitored with the clinicallyrelevant substrate LVS, an anti-hypercholesterolemia drug (Fig. 4). TRAM-34 demonstrated concentration-dependent inhibition of the formation of two major lovastatin metabolites, 69b-hydroxy LVS and 69-exomethylene LVS, with IC 50 values approximately 1 mM.

Discussion
TRAM-34 has been used extensively in research as a selective blocker of IK Ca channels [10,17]. Initial studies reported this drug to have a high selectivity for IK Ca and to lack inhibitory activity against CYP3A4 [10]. Subsequently, TRAM-34 has been used in vitro to study IK Ca channels in many biological roles, including fibroblast proliferation [17], microglia-mediated toxicity [18], tumor growth [19] and epithelial cell membrane function [20]. In vivo studies with this drug suggested the importance of IK Ca channels in experimental autoimmune encephalomyelitis [21], kidney allograft rejection [22], atherosclerosis [23], and restenosis [24]. Because of the importance of TRAM-34 as a research tool, we presently investigated the effects of this drug on several rat and human CYP isoforms.
The   human and rat CYP2C isoforms by somewhat higher concentrations of drug (IC 50 values = 1.8 to 12.6 mM, Figs. 1-2) also has consequences for the selectivity of TRAM-34 for IK Ca . While conducting the present study, we became aware of an abstract reporting TRAM-34 induced inhibition of CYP2B1, CYP2C6 and CYP3A2r [25]. Reported potencies were in the low micromolar ranges, similar to the present results. The effects of TRAM-34 on CYP3A4 are interesting because of the importance of this enzyme in drug metabolism [2]. The present results show clear, potent, actions of TRAM-34 on CYP3A4. Wulff et al. [10] reported that TRAM-34 (10 mM) did not inhibit CYP3A4 when the enzyme was assayed with BFC as substrate. Surprisingly, the current findings showed that TRAM-34 produced concentration-dependent activation of this enzyme when BFC was used as substrate (Fig. 3B). While the present results are technically in agreement with Wulff et al. [10] (i.e. no CYP inhibition), they clearly demonstrate modulation of CYP3A4 activity by TRAM-34. Wulff et al [10] did not report CYP3A4 activation by TRAM-34, but their data were not shown. Inhibition of CYP3A4 by TRAM-34 was confirmed when either DBF (Fig. 3A) or LVS (Fig. 4) were used as substrates. These results, showing that the same drug can exert opposing actions on CYP3A4 depending on the substrate used (Fig. 3A, 3B and 4), are reminiscent of earlier studies on this enzyme [13]. Such results have been explained by the property of substrate-specific positive cooperativity known to occur with CYP3A4 [26].
Imidazole-containing drugs are well known inhibitors of many CYPs [1]. TRAM-34 was developed by modification of the potent IK Ca blocker and CYP inhibitor clotrimazole [1]. Replacing the imidazole in clotrimazole with a pyrazole led to TRAM-34, which retained the ability to inhibit IK Ca but was reported to not inhibit CYP activity. Although pyrazoles like TRAM-34 have less inhibitory activity on CYPs as compared to clotrimazole, this pyrazole-containing drug remains a CYP inhibitor. Previous studies have also shown some pyrazoles to be even more potent inhibitors of various CYP isoforms than their imidazole congeners [27].
Wulff et al. [10] reported that TRAM-34 is up to 200-fold less potent on other potassium channels (such as the Kv1.2 channel) vs. the IK Ca channel (K d = 20 nM). Our results, showing TRAM-34 modulation of CYP activity in the low micromolar range, suggest a selectivity less than 200-fold for this drug on the IK Ca channel. Current results suggest that in vitro concentrations of 0.2-0.8 mM of TRAM-34 would not inhibit the presently studied CYPs, implying 10-to 40-fold selectivity. It should be noted that many additional CYP isoforms exist [28], and should be studied for further evaluation of TRAM-34 selectivity.
The present findings, showing TRAM-34 modulation of CYP activity in the low micromolar range, suggest that some conclusions made by earlier studies using this drug as a selective IK Ca channel blocker may need to be reevaluated. For example, numerous previous in vitro studies have used TRAM-34 at concentrations $10 mM [20,[29][30][31][32][33][34][35][36][37][38][39]. At these concentrations, some CYP isoforms are clear targets of TRAM-34. Previous in vivo studies have also used TRAM-34 to inhibit the effects of the IK Ca channel. Although, these studies found plasma concentra- tions of TRAM-34 to be in the nanomolar range [21,22,24], drug concentrations in the liver and subcutaneous-fat 48 h later can be quite substantially higher [24].
The current findings also add to the already developing literature of novel targets for TRAM-34. The drug has been found to inhibit non-selective cation channels [40] and may directly interact with the estrogen receptor in mammary adenocarcinoma cell lines [41]. In addition, Abdullaev et al. [11] found that that the effects of micromolar levels of TRAM-34 on proliferation of malignant cells are completely independent of its effects on IK Ca channels and therefore involve unknown, off-target actions. Our results and these other studies show that TRAM-34 needs to be used with caution even at low micromolar concentrations.
Currently, TRAM-34 is used as an important tool to probe the physiological roles of the IK Ca channel, but the present results suggest that interactions of TRAM-34 with CYPs also need to be considered in both in vitro and in vivo studies. TRAM-34 is not currently used clinically, since a similar triarylmethane drug (ICA-17304, Senicopoc) is in clinical trials for treating sickle cell disease. However, the activities of TRAM-34 on drug-metabolizing enzymes show that in vivo use of this drug for research in animals may produce unexpected drug-drug interactions.