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TRAM-34, a putatively selective blocker of intermediate-conductance, calcium-activated potassium channels, inhibits cytochrome P450 activity.

Agarwal JJ, Zhu Y, Zhang QY, Mongin AA, Hough LB - PLoS ONE (2013)

Bottom Line: However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York, United States of America.

ABSTRACT
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.

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Concentration-dependent inhibition and activation of CYP3A4 by TRAM-34 with two substrates.Recombinant enzyme CYP3A4, substrates DBF (A) or BFC (B) and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Pmol of product (ordinate) is plotted versus the log of inhibitor concentration (abscissa) for the incubation times specified in parenthesis. All TRAM-34 data points represent the mean ±SEM of 3 experiments performed in triplicate. Data from ketoconazole represent the mean ±SEM of triplicates from a single experiment. The TRAM-34 IC50 value was determined by non-linear regression and are shown in brackets (A). Control data points (i.e. no inhibitor, C on abscissa) represent the mean ±SEM from 3 experiments.
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pone-0063028-g003: Concentration-dependent inhibition and activation of CYP3A4 by TRAM-34 with two substrates.Recombinant enzyme CYP3A4, substrates DBF (A) or BFC (B) and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Pmol of product (ordinate) is plotted versus the log of inhibitor concentration (abscissa) for the incubation times specified in parenthesis. All TRAM-34 data points represent the mean ±SEM of 3 experiments performed in triplicate. Data from ketoconazole represent the mean ±SEM of triplicates from a single experiment. The TRAM-34 IC50 value was determined by non-linear regression and are shown in brackets (A). Control data points (i.e. no inhibitor, C on abscissa) represent the mean ±SEM from 3 experiments.

Mentions: TRAM-34 was tested on CYP3A4 with three different substrates. TRAM-34 showed potent and concentration-dependent inhibition of CYP3A4 with DBF (IC50 = 3.6 µM, 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).


TRAM-34, a putatively selective blocker of intermediate-conductance, calcium-activated potassium channels, inhibits cytochrome P450 activity.

Agarwal JJ, Zhu Y, Zhang QY, Mongin AA, Hough LB - PLoS ONE (2013)

Concentration-dependent inhibition and activation of CYP3A4 by TRAM-34 with two substrates.Recombinant enzyme CYP3A4, substrates DBF (A) or BFC (B) and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Pmol of product (ordinate) is plotted versus the log of inhibitor concentration (abscissa) for the incubation times specified in parenthesis. All TRAM-34 data points represent the mean ±SEM of 3 experiments performed in triplicate. Data from ketoconazole represent the mean ±SEM of triplicates from a single experiment. The TRAM-34 IC50 value was determined by non-linear regression and are shown in brackets (A). Control data points (i.e. no inhibitor, C on abscissa) represent the mean ±SEM from 3 experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646888&req=5

pone-0063028-g003: Concentration-dependent inhibition and activation of CYP3A4 by TRAM-34 with two substrates.Recombinant enzyme CYP3A4, substrates DBF (A) or BFC (B) and varying concentrations of TRAM-34 were incubated in the presence of 50 mM potassium phosphate buffer and regenerating system at 37°C according to the methods described. Pmol of product (ordinate) is plotted versus the log of inhibitor concentration (abscissa) for the incubation times specified in parenthesis. All TRAM-34 data points represent the mean ±SEM of 3 experiments performed in triplicate. Data from ketoconazole represent the mean ±SEM of triplicates from a single experiment. The TRAM-34 IC50 value was determined by non-linear regression and are shown in brackets (A). Control data points (i.e. no inhibitor, C on abscissa) represent the mean ±SEM from 3 experiments.
Mentions: TRAM-34 was tested on CYP3A4 with three different substrates. TRAM-34 showed potent and concentration-dependent inhibition of CYP3A4 with DBF (IC50 = 3.6 µM, 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).

Bottom Line: However, previously published work has only characterized the effects of TRAM-34 on a single CYP isoform.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus