Contribution of Monocarboxylate Transporter 6 to the Pharmacokinetics and Pharmacodynamics of Bumetanide in Mice

Abstract

Bumetanide, a sulfamyl loop diuretic, is used for the treatment of edema in association with congestive heart failure. Being a polar, anionic compound at physiologic pH, bumetanide uptake and efflux into different tissues is largely transporter-mediated. Of note, organic anion transporters (SLC22A) have been extensively studied in terms of their importance in transporting bumetanide to its primary site of action in the kidney. The contribution of one of the less-studied bumetanide transporters, monocarboxylate transporter 6 (MCT6; SLC16A5), to bumetanide pharmacokinetics (PK) and pharmacodynamics (PD) has yet to be characterized. The affinity of bumetanide for murine Mct6 was evaluated using Mct6-transfected Xenopus laevis oocytes. Furthermore, bumetanide was intravenously and orally administered to wild-type mice (Mct6^+/+) and homozygous Mct6 knockout mice (Mct6^-/-) to elucidate the contribution of Mct6 to bumetanide PK/PD in vivo. We demonstrated that murine Mct6 transports bumetanide at a similar affinity compared with human MCT6 (78 and 84 μM, respectively, at pH 7.4). After bumetanide administration, there were no significant differences in plasma PK. Additionally, diuresis was significantly decreased by ∼55% after intravenous bumetanide administration in Mct6^-/- mice. Kidney cortex concentrations of bumetanide were decreased, suggesting decreased Mct6-mediated bumetanide transport to its site of action in the kidney. Overall, these results suggest that Mct6 does not play a major role in the plasma PK of bumetanide in mice; however, it significantly contributes to bumetanide's pharmacodynamics due to changes in kidney concentrations. SIGNIFICANCE STATEMENT: Previous evidence suggested that MCT6 transports bumetanide in vitro; however, no studies to date have evaluated the in vivo contribution of this transporter. In vitro studies indicated that mouse and human MCT6 transport bumetanide with similar affinities. Using Mct6 knockout mice, we demonstrated that murine Mct6 does not play a major role in the plasma pharmacokinetics of bumetanide; however, the pharmacodynamic effect of diuresis was attenuated in the knockout mice, likely because of the decreased bumetanide concentrations in the kidney.

Graphical abstract

Fig. 1.

Chemical structure of bumetanide.

Fig. 2.

Time (A) and concentration-dependent (B) uptake of bumetanide in murine Mct6-transfected X. laevis oocytes (N = 4 to 5 oocytes per data point). Experiment was performed at least three separate times with at least two different ovaries. Data are presented as mean ± S.D. Closed circles, murine Mct6 cRNA-injected; open circles, water-injected; open squares, Mct6-mediated uptake (Mct6 cRNA-injected minus water-injected). Dashed line represents model fitting using eq. 1 to Mct6-mediated uptake data.

Fig. 3.

Concentration-time profiles of bumetanide after intravenous (A) and oral (B) administration in Mct6^+/+ wild-type mice. N = 3 to 4 mice per group. Data are plotted as mean ± S.D.

Fig. 4.

Concentration-time profiles of bumetanide after 10 mg/kg i.v. (A) and 25 mg/kg oral (B) administration in Mct6^+/+ (closed symbols) and Mct6^−/− (open symbols) mice. N = 3 to 4 mice per group. Data are plotted as mean ± S.D.

Fig. 5.

Amount of bumetanide eliminated unchanged in the urine (A_e,24-hour) after different dose administrations [intravenous (A and B); oral (C and D)]. Urine was collected after a 24-hour urine collection. N = 4 mice per group. Data are plotted as mean ± S.D. An unpaired Student’s t test was performed to test for statistical significance.

Fig. 6.

Urinary output after administration of bumetanide in Mct^+/+ and Mct6^−/− mice after doses of 25 mg/kg orally (A), 2.5 mg/kg orally (B), 10 mg/kg i.v. (C) and 1 mg/kg i.v. (D) . N = 4 mice per group. Data are plotted as mean ± S.D. An unpaired Student’s t test was performed to test for statistical significance * p < 0.05; ** p < 0.01.

Fig. 7.

Bumetanide tissue concentrations in (A) liver at 60 minutes after oral dose administration of 25 mg/kg and (B) kidney cortex at 30 minutes after intravenous dose administration of 10 mg/kg. N = 4 mice per group. Data are plotted as mean ± S.D. An unpaired Student’s t test was performed to test for statistical significance * p<0.05.