Multiple blood-brain barrier transport mechanisms limit bumetanide accumulation, and therapeutic potential, in the mammalian brain

There is accumulating evidence that bumetanide, which has been used over decades as a potent loop diuretic, also exerts effects on brain disorders, including autism, neonatal seizures, and epilepsy, which are not related to its effects on the kidney but rather mediated by inhibition of the neuronal Na-K-C1 cotransporter isoform NKCC1. However, following systemic administration, brain levels of bumetanide are typically below those needed to inhibit NKCC1, which critically limits its clinical use for treating brain disorders. Recently, active efflux transport at the blood-brain barrier (BBB) has been suggested as a process involved in the low brain:plasma ratio of bumetanide, but it is presently not clear which transporters are involved. Understanding the processes explaining the poor brain penetration of bumetanide is needed for developing strategies to improve the brain delivery of this drug. In the present study, we administered probenecid and more selective inhibitors of active transport carriers at the BBB directly into the brain of mice to minimize the contribution of peripheral effects on the brain penetration of bumetanide. Furthermore, in vitro experiments with mouse organic anion transporter 3 (Oat3)-overexpressing Chinese hamster ovary cells were performed to study the interaction of bumetanide, bumetanide derivatives, and several known inhibitors of Oats on Oat3-mediated transport. The in vivo experiments demonstrated that the uptake and efflux of bumetanide at the BBB is much more complex than previously thought. It seems that both restricted passive diffusion and active efflux transport, mediated by Oat3 but also organic anion-transporting polypeptide (Oatp) Oatpla4 and multidrug resistance protein 4 explain the extremely low brain concentrations that are achieved after systemic administration of bumetanide, limiting the use of this drug for targeting abnormal expression of neuronal NKCC1 in brain diseases

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

Correction Volume: 143 Pages: 349-350 DOI: 10.1016/j.neuropharm.2018.10.012Based 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.Peer reviewe

Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality

A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelial cells as blood–brain barrier models for drug transport studies

Background!#!Predictive in vitro models of the human blood-brain barrier (BBB) are essential in early drug discovery and development. Among available immortalized human brain capillary endothelial cell lines (BCECs), the hCMEC/D3 cell line has become the most widely used in vitro BBB model. However, monolayers of hCMEC/D3 cells form only moderately restrictive barriers, most likely because the major tight junction protein, claudin-5, is markedly downregulated. Thus, hCMEC/D3 monolayers cannot be used for vectorial drug transport experiments, which is a major disadvantage of this model.!##!Methods!#!Here we transduced hCMEC/D3 cells with a claudin-5 plasmid and compared the characteristics of these cells with those of hCMEC/D3 wildtype cells and primary cultured porcine BCECs.!##!Results!#!The claudin-5 transduced hCMEC/D3 exhibited expression levels (and junctional localization) of claudin-5 similar to those of primary cultured porcine BCECs. The transduced cells exhibited increased TEER values (211 Ω cm!##!Conclusions!#!The claudin-5 transduced hCMEC/D3 cells provide a tool to studying the contribution of claudin-5 to barrier tightness and how this can be further enhanced by additional transfections or other manipulations of this widely used in vitro model of the BBB

Structure-activity relationships of bumetanide derivatives:correlation between diuretic activity in dogs and inhibition of the human NKCC2A transporter

BACKGROUND AND PURPOSE: The N-K-Cl cotransporters (NKCCs) mediate the coupled, electroneutral movement of Na(+), K(+) and Cl(−) ions across cell membranes. There are two isoforms of this cation co-transporter, NKCC1 and NKCC2. NKCC2 is expressed primarily in the kidney and is the target of diuretics such as bumetanide. Bumetanide was discovered by screening ∼5000 3-amino-5-sulfamoylbenzoic acid derivatives, long before NKCC2 was identified in the kidney. Therefore, structure–activity studies on effects of bumetanide derivatives on NKCC2 are not available. EXPERIMENTAL APPROACH: In this study, the effect of a series of diuretically active bumetanide derivatives was investigated on human NKCC2 variant A (hNKCC2A) expressed in Xenopus laevis oocytes. KEY RESULTS: Bumetanide blocked hNKCC2A transport with an IC(50) of 4 μM. There was good correlation between the diuretic potency of bumetanide and its derivatives in dogs and their inhibition of hNKCC2A (r(2) = 0.817; P < 0.01). Replacement of the carboxylic group of bumetanide by a non-ionic residue, for example, an anilinomethyl group, decreased inhibition of hNKCC2A, indicating that an acidic group was required for transporter inhibition. Exchange of the phenoxy group of bumetanide for a 4-chloroanilino group or the sulfamoyl group by a methylsulfonyl group resulted in compounds with higher potency to inhibit hNKCC2A than bumetanide. CONCLUSIONS AND IMPLICATIONS: The X. laevis oocyte expression system used in these experiments allowed analysis of the structural requirements that determine relative potency of loop diuretics on human NKCC2 splice variants, and may lead to the discovery of novel high-ceiling diuretics

Influence of 24-Nor-Ursodeoxycholic Acid on Hepatic Disposition of [(18)F]Ciprofloxacin, a Positron Emission Tomography Study in Mice

24-nor-ursodeoxycholic acid (norUDCA) is a novel therapeutic approach to cholestatic liver diseases. In mouse models of cholestasis, norUDCA induces basolateral multidrug resistance-associated proteins 4 (Mrp4) and 3 in hepatocytes, which provide alternative escape routes for bile acids accumulating during cholestasis but could also result in altered hepatic disposition of concomitantly administered substrate drugs. We used positron emission tomography imaging to study the influence of norUDCA on hepatic disposition of the model Mrp4 substrate [(18)F]ciprofloxacin in wild-type and Mdr2((-/-)) mice, a model of cholestasis. Animals underwent [(18)F]ciprofloxacin positron emission tomography at baseline and after norUDCA treatment. After norUDCA treatment, liver-to-blood area under the curve ratio of [(18)F]ciprofloxacin was significantly decreased compared to baseline, both in wild-type (-34.0 ± 2.1%) and Mdr2((-/-)) mice (-20.5 ± 6.0%). [(18)F]Ciprofloxacin uptake clearance from blood into liver was reduced by -17.1 ± 9.0% in wild-type and by -20.1 ± 7.3% in Mdr2((-/-)) mice. Real-time PCR analysis showed significant increases in hepatic Mrp4 and multidrug resistance-associated protein 3 mRNA after norUDCA. Transport experiments in organic anion transporting polypeptide (OATP)1B1-, OATP1B3-, and OATP2B1-transfected cells revealed weak transport of [(14)C]ciprofloxacin by OATP1B3 and OATP2B1 and no inhibition by norUDCA. In conclusion, our data suggest that changes in hepatic [(18)F]ciprofloxacin disposition in mice after norUDCA treatment were caused by induction of basolateral Mrp4 in hepatocytes

Effect of mitomycin C (MMC) on Pgp-EGFP functionality studied with the Rho123 uptake assay.

<p>MMC significantly increased the functionality of Pgp-EGFP fusion protein. “A”: hCMEC/D3-Pgp-EGFP (doxycycline-off/-on) cells were treated with MMC in Opti-MEM medium (0.1 µM, 1 µM, 10 µM, 50 µM and 100 µM) for 4 hours after which Opti-MEM medium was replaced by complete medium again. Control cells were treated with Opti-MEM medium. After 23 h the cells were incubated with 10 µM Rho123 for 1 h and intracellular accumulation was measured, resulting in a dose-dependent increase in Pgp-EGFP functionality. Experiments were performed in triplicates and values are shown as mean ± SEM. Significant effects of MMC during doxycycline-off conditions are indicated by circles, while significant effects of MMC during doxycycline-on conditions are indicated by rhombs (P<0.05). Significant differences between doxycycline-on and –off conditions are indicated by asterisk (P<0.05). “B” shows data (shown in percent control) in which rhodamine uptake was measured directly after 2 h of MMC (1 µM) exposure or at 4, 8, and 20 hrs following 4 h of MMC (1 µM) exposure in hCMEC/D3-Pgp-EGFP (doxycyline-on) cells, indicating a lack of MMC exposure on Pgp function in the 2, 8 and 12, but not the 24 h experiment. The 24 h experiment was also performed with 10 µM MMC, resulting in similar results (not illustrated). However, when lipid rafts were disrupted by MβCD, MMC (1 µM) exposure for 2 h significantly increased the functionality of Pgp-EGFP fusion protein, which is illustrated by the right columns in “B”. As shown in the inset, this was correlated with a decrease of Pgp in the DRM fraction and an increase of Pgp in the detergent soluble fraction. Significant differences between controls and MMC-treated cells are indicated by asterisk (P<0.05).</p