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

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.

Bumepamine, a brain-permeant benzylamine derivative of bumetanide, does not inhibit NKCC1 but is more potent to enhance phenobarbital's anti-seizure efficacy.

Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.

Detection of bumetanide in an over-the-counter dietary supplement.

Bumetanide is a loop diuretic used clinically to treat heart failure, acute renal failure, high blood pressure, and edema. However, diuretics may also be used by athletes as masking agents and to decrease weight. Taken as masking agents, diuretics increase urine production and decrease urinary concentrations of banned performance-enhancing agents, such as anabolic steroids. StarCaps is an over-the-counter dietary supplement marketed as a diet aid. The manufacturer claims that the product contains only natural cleansing agents and emphasizes that it is free from traditional appetite suppressants such as sympathomimetic amines. However, no such disclaimer is made concerning diuretic agents. A single StarCaps capsule was administered to two male and two female volunteers, and their urine specimens were collected at discrete intervals (2, 4, 8, and 12 h) post administration. The specimens were analyzed by a high-performance liquid chromatography-mass spectrometry quadrupole (HPLC-MS) method, and bumetanide was detected in all specimens (4.6 to 351.3 ng/mL). Adjusting the bumetanide concentrations for creatinine content did little to normalize the excretion profiles. Bumetanide was also detected in the StarCaps capsules at concentrations approaching therapeutic doses. HPLC-quadrupole-time-of-flight mass spectrometry was used to confirm the presence of bumetanide in the urine samples and StarCaps capsules. The results showed that unregulated dietary supplements may put consumers at risk for unwitting consumption of prescription medications, and that it is possible for athletes to inadvertently test positive for bumetanide and face disciplinary actions.