ORIGINAL ARTICLE

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Heterogeneous brain distribution of bumetanide following systemic administration in rats

Corresponding Author

Wolfgang Löscher

[email protected]

orcid.org/0000-0002-9648-8973

Translational Neuropharmacology Laboratory, NIFE, Department of Experimental Otology of the ENT Clinics, Hannover Medical School, Hannover, Germany

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany

Center for Systems Neuroscience Hannover, Hannover, Germany

Correspondence

Wolfgang Löscher, Translational Neuropharmacology Laboratory, NIFE, Department of Experimental Otology of the ENT Clinics, Hannover Medical School, Stadtfelddamm 34, Hannover 30625, Germany.

Email: [email protected]

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Martina Gramer,

Martina Gramer

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany

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Kerstin Römermann,

Kerstin Römermann

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany

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Wolfgang Löscher,

Corresponding Author

Wolfgang Löscher

[email protected]

orcid.org/0000-0002-9648-8973

Translational Neuropharmacology Laboratory, NIFE, Department of Experimental Otology of the ENT Clinics, Hannover Medical School, Hannover, Germany

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany

Center for Systems Neuroscience Hannover, Hannover, Germany

Correspondence

Wolfgang Löscher, Translational Neuropharmacology Laboratory, NIFE, Department of Experimental Otology of the ENT Clinics, Hannover Medical School, Stadtfelddamm 34, Hannover 30625, Germany.

Email: [email protected]

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Martina Gramer,

Martina Gramer

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany

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Kerstin Römermann,

Kerstin Römermann

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany

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First published: 01 June 2024

https://doi.org/10.1002/bdd.2390

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Abstract

Bumetanide is used widely as a tool and off-label treatment to inhibit the Na-K-2Cl cotransporter NKCC1 in the brain and thereby to normalize intra-neuronal chloride levels in several brain disorders. However, following systemic administration, bumetanide only poorly penetrates into the brain parenchyma and does not reach levels sufficient to inhibit NKCC1. The low brain penetration is a consequence of both the high ionization rate and plasma protein binding, which restrict brain entry by passive diffusion, and of brain efflux transport. In previous studies, bumetanide was determined in the whole brain or a few brain regions, such as the hippocampus. However, the blood–brain barrier and its efflux transporters are heterogeneous across brain regions, so it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete brain areas. Here, bumetanide was determined in 14 brain regions following i.v. administration of 10 mg/kg in rats. Because bumetanide is much more rapidly eliminated by rats than humans, its metabolism was reduced by pretreatment with piperonyl butoxide. Significant, up to 5-fold differences in regional bumetanide levels were determined with the highest levels in the midbrain and olfactory bulb and the lowest levels in the striatum and amygdala. Brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Regional brain levels were significantly correlated with local cerebral blood flow. However, regional bumetanide levels were far below the IC₅₀ (2.4 μM) determined previously for rat NKCC1. Thus, these data further substantiate that the reported effects of bumetanide in rodent models of brain disorders are not related to NKCC1 inhibition in the brain.

1 INTRODUCTION

The potent loop diuretic, bumetanide, which acts by inhibiting the Na-K-2Cl cotransporter NKCC2 on the apical membrane of the thick ascending limb of Henle epithelial cells, is used widely for palliative treatment of renal insufficiency, heart failure, nephrotic syndrome, and hypertension (Oh & Han, 2015; Roush et al., 2014; Ward & Heel, 1984; Wargo & Banta, 2009). In addition to NKCC2, which is mainly expressed in the kidney, bumetanide inhibits NKCC1, which is expressed in many cell types throughout the body, including the brain, where it mediates cell volume regulation and participates in Cl⁻ homeostasis (Delpire & Gagnon, 2018). In brain neurons, NKCC1 and the neuron-specific K-Cl cotransporter KCC2 mediate neuronal Cl⁻ uptake and extrusion, respectively, which is important for shaping GABAergic signaling (Kaila et al., 2014). Altered neuronal chloride regulation and the consequent effects on GABAergic signaling have been implicated in numerous CNS disorders, making NKCC1 and KCC2 attractive drug targets (Kahle et al., 2008; Kharod et al., 2019; Löscher & Kaila, 2022; Schulte et al., 2018). While KCC2 modulators are not yet clinically available, bumetanide is used widely as a tool for NKCC1 inhibition in preclinical models and has been used, off-label, with conflicting results in clinical studies to treat brain disorders, such as neonatal seizures, Alzheimer’s disease, and autism (Kahle et al., 2008; Kharod et al., 2019; Löscher & Kaila, 2022; Schulte et al., 2018).

However, we and others have shown previously that bumetanide only poorly penetrates the blood–brain barrier (BBB), so the drug levels needed to inhibit NKCC1 in the brain parenchyma are not reached after systemic administration of clinically used or supra-therapeutic doses (cf., Löscher & Kaila, 2022). In respective rodent studies, bumetanide was either determined in the whole brain of mice or rats (Cleary et al., 2013; Hampel et al., 2021; Johne et al., 2021a, 2021b; Li et al., 2011; Römermann et al., 2017; Savardi et al., 2020; Töllner et al., 2014; Töpfer et al., 2014; Wang et al., 2015;) or in a few rat brain regions, that is, the hippocampus, amygdala, and piriform cortex (Brandt et al., 2010; Donovan et al., 2015; Töllner et al., 2014; Töpfer et al., 2014). Brain:plasma ratios in whole brain or brain regions ranged from 0.0043 to 0.06, indicating that less than 10% of the plasma concentration enters the brain. In a study in dogs, low bumetanide levels were determined in the brain (choroid plexus; tissue:plasma ratio 0.11) and cerebrospinal fluid (CSF; CSF:plasma ratio 0.0012–0.0038) during i.v. infusion with bumetanide, demonstrating that the poor brain penetration of bumetanide is not restricted to rodents (Javaheri et al., 1993). Seizures or status epilepticus hardly affected the low brain levels of bumetanide (Cleary et al., 2013; Töllner et al., 2014; Wang et al., 2015). Furthermore, the neonatal BBB seems not to be more permeable to bumetanide than the adult BBB (Löscher & Kaila, 2022). Studies of our group have indicated that both restricted passive diffusion of the highly ionized and plasma protein-bound drug and active efflux transport at the BBB explain the extremely low brain and CSF concentrations that are achieved after systemic administration of bumetanide (Römermann et al., 2017).

The poor brain penetration of bumetanide seems to indicate that the effects of this drug that have been reported for CNS disorders both experimentally and clinically are not mediated by inhibition of NKCC1 in brain parenchyma (Löscher & Kaila, 2022). However, the BBB is not uniform throughout the brain and, as a consequence, heterogeneous brain distribution has been reported for several drugs (Luptáková et al., 2021; Wilhelm et al., 2016; Zhao & Pollack, 2009). Thus, it cannot be excluded that bumetanide reaches sufficiently high brain levels for NKCC1 inhibition in some discrete areas of the brain.

This prompted us to study the distribution of bumetanide across 14 brain regions following i.v. administration in rats. Because bumetanide is much more rapidly eliminated by adult rats (t_0.5 ∼10 min) than by humans (t_0.5 ∼60–90 min), we pretreated the rats with the cytochrome P450 inhibitor, piperonyl butoxide (PBO), which inhibits the N-butyl side chain oxidation of bumetanide, thereby increasing the elimination half-life and diuretic potency of bumetanide in rats (Halladay et al., 1978; Töpfer et al., 2014). Based on these findings, we suggested previously that coadministration of piperonyl butoxide may be an easy method for enhancing the translational value of rodent experiments with bumetanide (Töpfer et al., 2014).

2 MATERIALS AND METHODS

2.1 Animals

Female Sprague-Dawley rats were purchased at a body weight of 200–220 g from Envigo (Horst, Netherlands). Experiments were performed according to the EU council directive 210/63/EU and the German Law on Animal Protection (“Tierschutzgesetz”). Ethical approval for the study was granted by an ethical committee (according to §15 of the Tierschutzgesetz) and the government agency (Lower Saxony State Office for Consumer Protection and Food Safety; LAVES) responsible for approval of animal experiments in Lower Saxony (reference numbers for this project: 14/1659 and 15/1825).

Rats were housed in groups of a maximum of 10 under controlled conditions (temperature: 23 ± 1°C; humidity: 50%–60%), under a 12 h light-dark cycle (lights on at 6:00 h). Standard laboratory chow (Altromin 1324 standard diet, Altromin Spezialfutter GmbH) and tap water were provided ad libitum. Female rats were used to allow comparisons with our previous pharmacokinetic studies on bumetanide and its derivatives (Brandt et al., 2010; Römermann et al., 2017; Töllner et al., 2014; Töpfer et al., 2014). Rats were housed in rooms without males to keep them acyclic or asynchronous concerning their estrous cycle (Kücker et al., 2010). All efforts were made to minimize both the suffering and the number of animals.

2.2 Drug treatment

As in most of our previous studies with bumetanide in rodents, the drug (which was purchased from Sigma; Munich, Germany) was administered i.v. at a dose of 10 mg/kg. Drug administration was by slow infusion into a tail vein. A group of six rats received an alkaline aqueous solution of bumetanide, for which bumetanide was dissolved in a small amount of 0.1 M NaOH and water was then added to reach an injection volume of 3 mL/kg. In another group of six rats, bumetanide was dissolved in a vehicle containing 5% ethanol, 5% Solutol® HS 15 (a non-ionic solubilizer consisting of ethoxylated 12-hydroxystearic acid; BASF, Ludwigshafen, Germany), and 90% distilled water as described previously (Töllner et al., 2014). The injection volume was 3 mL/kg. Both types of bumetanide solutions have been used previously in studies on brain penetration of this drug (Brandt et al., 2010; Hampel et al., 2021; Römermann et al., 2017; Töllner et al., 2014; Töpfer et al., 2014; Wang et al., 2015) and we wanted to exclude that the type of solution affects bumetanide’s brain penetration. A direct comparison of the brain penetration of bumetanide dissolved either in alkaline water or ethanol/solutol/water indicated that both solutions resulted in similar brain levels, so data from both drug solutions were combined for the final analysis.

Based on the studies of Halladay et al. (1978) and Töpfer et al. (2014) with piperonyl butoxide and bumetanide in rats, the rats were pretreated with piperonyl butoxide at two i.p. doses of 150 mg/kg at 30 and 10 min before bumetanide as shown in Figure 1. Piperonyl butoxide (Merck Schuchhardt; Hohenbrunn, Germany) was dissolved in 3 mL/kg germ oil (Mazola; Unilever, Hamburg, Germany). Following the i.v. administration of bumetanide, the rats were killed 15 min later for analysis of drug in plasma and brain, because at this time maximal brain levels of bumetanide were determined in our previous studies (Töllner et al., 2014; Töpfer et al., 2014).

Details are in the caption following the image — **FIGURE 1**
Open in figure viewer PowerPoint

Schematic presentation of the experimental protocol used in this study. The mixed-function oxidase system (cytochrome P-450) inhibitor piperonyl butoxide (PBO) was used to reduce the oxidation of the N-butyl side chain (indicated by the stippled box) of bumetanide, thereby increasing the elimination half-life in rats (Halladay et al., 1978). As shown by us previously (Töpfer et al., 2014), this can be used to enhance the translational value of experiments with bumetanide in rats and mice, which otherwise eliminate bumetanide much more rapidly than humans.

2.3 Analysis of bumetanide in plasma and brain

For analysis of bumetanide in the brain, animals were decapitated 15 min after i.v. drug administration, blood was sampled, and the brain was removed and dissected into 14 regions on a cold plate at −13°C as described in detail previously (Löscher et al., 1984, 1989, 1991). The following regions were dissected: olfactory bulb, frontal cortex, piriform cortex, amygdala, hippocampus, thalamus, hypothalamus, nucleus accumbens, striatum (caudate/putamen), tectum (superior and inferior colliculus), substantia nigra, cerebellum, pons, and medulla oblongata. Regions were dissected from both hemispheres and pooled for drug analysis. As described previously (Löscher et al., 1984), during the development of the dissection technique, the different brain areas were confirmed by histological examination of sections of the resected tissue samples.

For the present study, brain samples were immediately homogenized (about 50 mg brain tissue in 1 mL distilled water), centrifuged for 20 min at 15,000 rpm at 4°C, and the supernatant was purified by solid phase extraction, using a Chromabond HR-X column (Macherey-Nagel; Düren; Germany) as described previously (Brandt et al., 2010; Töllner et al., 2014). Bumetanide was extracted from the column by methanol, the extract was evaporated to dryness by nitrogen, the residue was dissolved in 100 μL buffer, and 20 μL was used for analysis by high-performance liquid chromatography (HPLC) as described previously in detail (Brandt et al., 2010).

For analysis of bumetanide in plasma, the blood that was sampled after decapitation was centrifuged and plasma samples were stored deep-frozen until HPLC analysis as described previously (Brandt et al., 2010; Töllner et al., 2014). As a standard for HPLC analysis, we used a commercial solution (0.5 mg/mL) of the sodium salt of bumetanide (Burinex). The detection limit for bumetanide was about 12 ng/mL in plasma and 50 ng/g in brain tissue.

The analysis of compounds in the brain did not consider the blood volume in the brain, because previous experiments, in which brain bumetanide levels in mice with and without brain perfusion were compared, did not indicate any significant difference in these levels (Römermann et al., 2017). For these experiments, the mice were perfused via the aorta with 0.01 M phosphate buffer under anesthesia with chloral hydrate 30 min after i.v. administration of 10 mg/kg bumetanide. Brain levels were compared with those of mice receiving the same treatment but decapitated without perfusion. Average brain levels of bumetanide were 0.14 μg/g with perfusion versus 0.15 μg/g without perfusion, respectively, indicating that most of the blood in the brain was removed by decapitation and the subsequent dissection and preparation of brain samples. Therefore, all experiments described here were performed without perfusion.

A preliminary analysis of some of the data has been published in a recent review (Löscher & Kaila, 2022) but for the present study, the data were completed and reanalyzed.

2.4 Statistics

For comparison of two independent groups, the Mann–Whitney U-test was used. For the comparison of paired regional brain levels, a mixed effects model approach for repeated data was used; posthoc analyses were performed by Dunnett’s multiple comparisons test. Correlation analyses were performed either by the nonparametric Spearman’s rank correlation coefficient method or by Pearson’s correlation coefficient method. All analyses were performed by GraphPad Prims (vs. 9). A value of p < 0.05 was considered significant.

3 RESULTS

Fifteen min after i.v. administration of 10 mg/kg bumetanide in 12 rats, the median plasma level was 4.8 μg/mL (range 1.1–10.8 μg/mL). As shown in Figure 2a, the brain levels of bumetanide were not homogeneously distributed across the 14 brain regions examined. The lowest brain levels were determined in the striatum (median 0.029 μg/g), while 3–5-fold higher median drug levels were determined in the olfactory bulb (0.081), hippocampus (0.087), nucleus accumbens (0.095), tectum (0.128), and substantia nigra (0.153). Similarly low median levels as in the striatum were determined in the amygdala (0.024 μg/g). In most brain regions, marked inter-individual variation in drug levels was observed. At least in part, the large inter-individual variation observed in both plasma and brain levels of bumetanide may be a consequence of the two types of drug solutions used in these experiments, although (as pointed out in Methods), the data obtained in the two subgroups did not differ significantly.

In Figure 2b, the brain levels of bumetanide are illustrated in μM. For comparison, the IC₅₀ (2.4 μM) of bumetanide for inhibition of NKCC1 expressed in rat HEK-293 cells (Dehaye et al., 2003) is indicated in Figure 2b. This demonstrates that – despite the marked variation in regional brain levels of bumetanide – these levels are far below those needed to inhibit NKCC1, although a high dose (10 mg/kg) of bumetanide was administered.

Figure 2c illustrates the brain:plasma ratio of bumetanide calculated for the 14 brain regions. The median brain:plasma ratios ranged between 0.004 (amygdala) and 0.022 (olfactory bulb). Statistical analysis substantiated that brain:plasma ratios of bumetanide significantly differed across brain regions. The highest median ratios were determined for the olfactory bulb (0.022), tectum (0.0217), medulla (0.0195), hippocampus (0.0182), substantia nigra (0.0181), and nucleus accumbens (0.0176). Except for a few regions, inter-individual variation in brain:plasma ratios was lower than the inter-individual variation in the brain levels, indicating that the variation in brain levels was, at least in part, a consequence of the variation in plasma levels following i.v. administration of bumetanide.

Indeed, when the brain and plasma levels of bumetanide determined in the 12 rats were subjected to correlation analysis, significant positive correlations were obtained for all regions, except for the piriform cortex and substantia nigra (Table 1). One of these correlations (olfactory bulb) is illustrated in Figure 2d.

TABLE 1. Correlation between plasma and brain concentrations following i.v. administration of bumetanide (10 mg/kg) in 12 rats.

Brain region	Correlation between plasma and brain levels (r)	Significance
Olfactory bulb	0.8811	p = 0.0003
Frontal cortex	0.9301	p < 0.0001
Piriform cortex	0.4795	p = 0.1374
Amygdala	0.7345	p = 0.0089
Hippocampus	0.6364	p = 0.0299
Thalamus	0.9231	p < 0.0001
Hypothalamus	0.8811	p = 0.0003
Nucleus accumbens	0.7273	p = 0.0144
Striatum (caudate/putamen)	0.9371	p < 0.0001
Tectum	0.6993	p = 0.0142
Substantia nigra	0.2870	p = 0.3893
Cerebellum	0.9107	p < 0.0001
Pons	0.8231	p = 0.0016
Medulla oblongata	0.8741	p = 0.0004

Note: The correlation coefficient r was calculated by Spearman’s rank correlation coefficient method. See Figure 2 for individual plasma and brain region levels.

We also evaluated whether the differences in regional brain levels of bumetanide are related to the heterogeneous local cerebral blood flow (LCBF) in the rat brain. As shown in Figure 3, the LCBF values, which were determined by Beck and Krieglstein (1987) using the ^l4C-iodoantipyrine (IAP) technique and quantitative autoradiography, correlated significantly with the median bumetanide levels.

4 DISCUSSION

To our knowledge, this is the first study that examines the regional distribution of bumetanide in the brain after systemic administration. Although up to 5-fold differences in bumetanide levels across brain regions were found, drug concentrations did not reach levels that would inhibit NKCC1 in any of the 14 regions examined, at least when using the IC₅₀ (2.4 μM) for rat NKCC1 (determined by Dehaye et al. (2003) in HEK-293 cells) as a reference value. The K_i/IC₅₀ of bumetanide for NKCC1 is often cited to be around 100–300 nM (Puskarjov et al., 2014), although the sensitivity of mammalian NKCC1 to bumetanide may vary over almost two orders of magnitude, depending on animal species, cell type, the activation state of NKCC1, and whether measurements are done in native/endogenous NKCC1 or heterologous expression systems (Alvarez-Leefmans, 2012; Löscher & Kaila, 2022; Russell, 2000). For a selective block of NKCC1 by bumetanide, concentrations of 1–10 μM are often used (Blaesse et al., 2009; Russell, 2000), and 1 μM has been used as a cutoff for pharmacokinetic considerations in mouse studies (Brandt et al., 2018). The highest regional bumetanide levels determined in the brains of some rats of the present study were in the range of 600–800 nM. However, our study does not exclude that bumetanide reaches higher levels in brain regions that we did not examine here. For example, at the level of circumventricular organs (CVOs such as the area postrema, posterior pituitary, intermediate lobe of the pituitary gland, median eminence, subcommissural organ, pineal gland, subfornical organ, and the organum vasculosum laminae terminalis) capillaries are more permeable, containing fenestrations and discontinuous tight junctions (Wilhelm et al., 2016). Although the exchange of circulating substances is relatively free in the CVOs, they do not provide direct passage of blood-borne substances to the rest of the brain due to the presence of diffusion barriers. Thus, even if higher levels of bumetanide exist in CVOs, which is not known, this is unlikely to explain the effects of this drug that have been reported for a variety of brain disorders (Löscher & Kaila, 2022).

4.1 Brain tissue binding of bumetanide

It is important to note that the brain levels of bumetanide reported here were not corrected by the high brain tissue binding of this drug. Previous experiments with in vitro equilibrium dialysis showed that brain tissue binding of bumetanide ranges from 77% in the rat to 83% in the mouse (Brandt et al., 2018; Donovan et al., 2016). This, in addition to its poor brain penetration, further reduces the functionally relevant brain concentrations of bumetanide, because only the free (unbound) drug can interact with NKCC1. By the method used for brain extraction of bumetanide in the present study, both free and tissue-bound bumetanide is extracted (Töllner et al., 2014). Thus, the brain levels shown in Figure 2 represent free and tissue-bound levels of bumetanide, so only about 20% of these levels may be functionally relevant. However, in vivo, Donovan et al. (2015) detected only 9.6% of the bumetanide levels in hippocampal tissue in hippocampal dialyzates of rats, indicating that the extracellular (functionally relevant) concentration of bumetanide in the brain is lower than expected from in vitro dialysis studies. While nonspecific drug–protein interactions occur in the brain parenchyma, membrane partitioning, and hence drug–lipid interactions, has been suggested to dominate nonspecific drug binding in brain tissue, resulting in a strong correlation between drug lipophilicity and brain tissue binding (Loryan et al., 2016; Nagar & Korzekwa, 2012). Because of the regional differences in brain lipid content, brain tissue binding of drugs is heterogeneous across brain regions in both rats and humans (Gustafsson et al., 2019). For instance, the highly lipophilic drug diazepam exhibited the lowest tissue binding in the cerebellum compared with all other regions, whereas other regions (frontal cortex, parietal cortex, basal ganglia, hippocampus) did not differ significantly (Gustafsson et al., 2019).

4.2 Heterogeneous local cerebral blood flow

The regional differences in bumetanide brain levels reported here are likely related to the regional heterogeneity of the BBB (Löscher & Friedman, 2020; Villabona-Rueda et al., 2019; Wilhelm et al., 2016), regional differences in drug transporters (Alnaqbi et al., 2023; Villabona-Rueda et al., 2019; Zhao & Pollack, 2009), and regional differences in capillary density and perfusion rate (Beck & Krieglstein, 1987; Horton et al., 1980; Zhao & Pollack, 2009). Zhao and Pollack (2009) reported that the regional perfusion flow rate varies 7.5-fold, and capillary density (based on vascular volume) varies 3.7-fold, across 13 brain regions (olfactory bulb, striatum, hippocampus, frontal cortex, parietal cortex, occipital cortex, superior and inferior colliculi, hypothalamus, thalamus, midbrain, pons, medulla, and cerebellum) examined in the mouse. The highest vascular volume and cerebral flow rate were determined in the midbrain, that is, the part of the brain for which we determined the highest median bumetanide levels in rats. Similar to mice, in rats the highest LCBF was determined in the tectum, whereas much lower capillary blood flows were observed in the striatum, in which we found the lowest bumetanide levels (Beck & Krieglstein, 1987; Horton et al., 1980). When correlating the median regional bumetanide brain levels determined here with the LCBFs reported by Beck and Krieglstein (1987), a highly significant correlation was obtained.

4.3 Active transport of bumetanide at the blood–brain barrier

However, the heterogeneity of LCBF alone is not sufficient to explain the large inter-regional differences in bumetanide levels in the brain. Because of its high degree of ionization (>99%) and protein binding (97%–98%) in plasma, bumetanide only poorly penetrates the BBB by passive diffusion (Löscher et al., 2013; Löscher & Kaila, 2022). Because bumetanide is a monocarboxylic acid, it is actively transported by the monocarboxylic acid transport system, particularly MCT6 (Murakami et al., 2005), which is expressed in the brain as an uptake carrier (Morris & Felmlee, 2008). However, several transporters actively extrude bumetanide out of the brain parenchyma, including organic anion transporter 3 (Oat3), organic anion-transporting polypeptide 1a4 (OATP1a4; previously termed OATP2), multidrug resistance protein 4 (MRP4; ABCC4), and breast cancer resistance protein (BCRP; ABCG2), explaining the extremely low brain concentrations that are achieved after systemic administration of bumetanide, despite active uptake by MCT6 (Donovan et al., 2015, 2016; Römermann et al., 2017; Töllner et al., 2015). As shown by the latter studies, inhibiting efflux transporters by probenecid markedly increases bumetanide brain levels. Transporters involved in the uptake and efflux of drugs at the BBB are not homogeneously expressed at the BBB of different brain regions (Alnaqbi et al., 2023; Villabona-Rueda et al., 2019; Zhao & Pollack, 2009), which thus likely add to the heterogeneous brain distribution of bumetanide following systemic administration in rats observed here.

Both regional brain and plasma levels of bumetanide exhibited relatively large inter-individual variation, whereas lower variation was observed for most brain:plasma ratios, indicating that the variation in brain levels was, at least in part, a consequence of the variation in plasma levels following i.v. administration of bumetanide. Indeed, when individual brain and plasma levels of bumetanide were subjected to correlation analysis, significant positive correlations were obtained for all regions except for the piriform cortex and substantia nigra. We can currently not explain why no correlation was found for the latter regions.

4.4 Effect of piperonyl butoxide on the pharmacokinetics of bumetanide in rats

Bumetanide is a potent diuretic in humans but is relatively ineffective in rats and mice even at very high doses (Olsen, 1977; Ostergaard et al., 1972). This striking species variation is a result of the rapid and extensive metabolism of bumetanide in rodents. In humans, bumetanide is largely excreted in the urine as unchanged drug, whereas rodents excrete bumetanide less than 10% unchanged (Olsen, 1977). The major metabolites in mice and rats result from cleavage of the N-butyl sidechain of bumetanide by hepatic monooxygenases and thus metabolic inactivation of bumetanide (Kolis et al., 1976; Magnussen & Eilertsen, 1974; Schwartz, 1981). As a consequence, the elimination half-life of bumetanide is only about 10 min in rats compared with 60–90 min in humans (Töpfer et al., 2014; Ward & Heel, 1984). The rapid metabolic inactivation of bumetanide in rodents not only markedly restricts its diuretic activity but also all other pharmacodynamic effects. This, however, is largely ignored in studies using bumetanide as a tool for blocking NKCCs in rodents in vivo (Löscher & Kaila, 2022).

Halladay et al. (1978) first described that piperonyl butoxide, an inhibitor of cytochrome P-450 (CYP) enzymes in the liver, significantly increases the plasma half-life of bumetanide in rats, accompanied by bumetanide-induced diuresis and saluresis. The diuretic response correlated with an increase in unchanged bumetanide excreted in the urine. We subsequently confirmed that pretreatment of rats with piperonyl butoxide significantly increases plasma and brain levels of bumetanide and its diuretic activity (Töpfer et al., 2014). Thus, coadministration of piperonyl butoxide is a simple tool for enhancing the translational value of rodent experiments with bumetanide (Töpfer et al., 2014).

Piperonyl butoxide enters the brain (Kimura et al., 1983) and therefore may inhibit bumetanide metabolism also in the brain parenchyma, although the total CYP levels in the brain are much lower than those in the liver (Ferguson & Tyndale, 2011). In general, the distribution of drug metabolizing CYPs in the brain is heterogeneous, with expression levels varying among different brain regions (Ferguson & Tyndale, 2011). This raises the possibility that the heterogeneous brain distribution of bumetanide may be due to the heterogeneous metabolism, which, however, would be reduced by CYP inhibition in piperonyl butoxide-pretreated rats.

In theory, piperonyl butoxide may also interact with the protein binding and transporter recognition properties of bumetanide, although, to our knowledge, this has never been examined. Piperonyl butoxide is used widely as an insecticide synergist by inhibiting CYP enzymes of the insects (Gleave et al., 2021). In rabbits, piperonyl butoxide had only minor effects on organic anion and cation transport in the kidney (Kuo et al., 1983).

4.5 Similarities in the brain distribution of bumetanide and valproate

Concerning brain distribution, there are interesting similarities between bumetanide and the antiseizure medication valproate which, as bumetanide, is a monocarboxylic acid (Löscher, 2002). Similar to bumetanide, valproate is highly ionized at physiological pH and highly bound to plasma proteins, which restricts its brain penetration by passive diffusion (Löscher, 1999). We (Frey & Löscher, 1978) were the first to describe that valproate enters and leaves the brain by active, carrier-mediated transport. Subsequent studies indicated that, again similar to bumetanide, different drug transporters are involved, including MCTs, MRPs, OATs, OATPs, organic cation transporters novel (OCTN), and others, most of which can be inhibited by probenecid (Karlgren et al., 2012; Löscher, 1999; Pochini et al., 2019; Shen, 1999; VanWert et al., 2010; Vijay & Morris, 2014). Brain distribution studies using i.v. administration of ¹⁴C-labeled valproate in mice, rats, and monkeys have shown a heterogeneous distribution of valproate in the brain with the highest levels in the olfactory bulb (Hoeppner, 1990; Schobben et al., 1980), which was also the region with the highest brain:plasma ratio in the present study. Following bilateral olfactory bulb ablation in mice, the antiseizure effect of valproate was markedly decreased (Ueki et al., 1977). Thus, in contrast to bumetanide, the brain levels of valproate are sufficient to explain its neuropharmacological effects (Löscher, 2002). The average brain:plasma ratio of valproate in different species, including humans, is about 0.2 (Löscher, 1999), which is considerably higher than the brain:plasma ratio of bumetanide.

4.6 How to explain bumetanide’s neuropharmacological effects in the absence of NKCC1-inhibiting drug levels in the brain parenchyma?

The finding that bumetanide does not reach NKCC1-inhibiting concentrations in any brain region after systemic administration, even when using very high, supratherapeutic doses as administered here, indicates that the neuropharmacological effects of this drug are mediated by other mechanisms (Löscher & Kaila, 2022). One potential mechanism is the inhibition of NKCC1 at the luminal (blood-facing) membrane of brain capillary endothelial cells of the BBB (Löscher & Kaila, 2022). Here, NKCC1 is exposed to the high blood levels of bumetanide, which are sufficient to completely block the transporter. NKCC1 in brain endothelial cells contributes to the ionic composition and volume of brain interstitial fluid and seems to be critically involved in ionic edema and swelling of the parenchyma following brain insults (Kahle et al., 2009; Löscher & Kaila, 2022; Mokgokong et al., 2014). A reduction of the intracerebral volume (and pressure) via NKCC1 inhibition at the BBB might contribute to the seizure-reducing effect of bumetanide and several other of its reported neuropharmacological effects (Löscher & Kaila, 2022; Puskarjov et al., 2014). In the periphery, NKCC1 is expressed in numerous cells involved in chronic inflammation and the immune system, which, in turn, are known to be in tight bidirectional communication with the CNS (Löscher & Kaila, 2022). Thus, systemically applied bumetanide might have actions on brain disorders mediated by these pathways. Furthermore, via inhibition of NKCC2 in the kidney, the potent diuretic bumetanide leads to volume depletion, including reduction of the extracellular space in the brain and elsewhere, electrolyte depletion, and hypokalemia, all of which could be involved in the amelioration of symptoms in CNS disorders, both experimentally and clinically (Löscher & Kaila, 2022). Also, bumetanide exerts several off-target effects that could play a role.

4.7 Limitations of the present study

Based on previous experiments in rats (Töllner et al., 2014; Töpfer et al., 2014), we examined the brain distribution of bumetanide only at one time point (15 min) after i.v. administration, because maximal brain levels of bumetanide were determined at this time point in rats.

Single time point analysis is used widely for understanding brain uptake of drugs after i.v. bolus doses (Chowdhury et al., 2021). As a rule of thumb, this technique can be useful at time points where the blood concentrations are many folds higher than the brain concentrations of the drug. In future studies, the blood-to-brain influx transport rate of bumetanide should be examined to clarify whether the heterogeneity can be produced by the regional differences in the BBB transport.

As discussed above, vascular distribution in the brain is not homogeneous, so the calculation of drug concentrations using brain homogenates is often corrected by the vascular volume determined with radiolabeled markers such as ¹⁴C-sucrose or ³H-inulin (Chowdhury et al., 2021). When evaluating low drug concentrations, drug remaining in the vascular volume may lead to overestimation of brain levels. Thus, at least in part, the results of this study may depend on the vascular volume in each region of the brain. This, however, is unlikely because, as described in Methods, previous experiments, in which brain bumetanide levels in rodents with and without transcardial perfusion (to clear the vascular volume from the brain tissue) were compared, did not indicate any significant difference in these levels (Römermann et al., 2017).

5 CONCLUSIONS

The present data further substantiate that systemic administration of bumetanide is not suited to target NKCC1 in the brain parenchyma. Although the brain distribution of bumetanide is heterogeneous, regional brain levels are far below those needed to inhibit NKCC1, at least under the experimental conditions used here. Furthermore, even with intracerebral administration of bumetanide, it is not possible to target neuronal NKCC1, because the transporter is expressed by practically all cells in the brain, including different types of glial cells (Löscher & Kaila, 2022). Indeed, the neuronal expression of NKCC1 mRNA in the brain is a very small fraction, several orders of magnitude lower than the overall brain expression of this transporter (Zeisel et al., 2018). Based on the different roles of NKCC1 in different cell types in the brain, direct application of bumetanide into brain tissue has, indeed, been shown to exert both beneficial and adverse effects (Löscher & Kaila, 2022). In line with this, Nguyen et al. (2023) have recently reported that astrocytic and neuronal NKCC1 exert opposing effects on GABA-mediated excitatory action during seizures. Further complicating the issue, Kurki et al. (2023) observed a differential expression of NKCC1 splice variants, with NKCC1a predominating in non-neuronal cells and NKCC1b in neurons. Bumetanide and other known NKCC1 inhibitors inhibit both splice variants with similar potency (Löscher & Kaila, 2022). Theoretically, a drug that is selective for the NKCC1b splice variant would mainly target neuronal NKCC1. By screening various structurally different loop diuretics, we recently failed to identify an NKCC1b selective compound (Hampel et al., 2018). However, as pointed out by Kurki et al. (2023), the recent breakthroughs in the de novo design of protein-binding proteins (Cao et al., 2022; Wang et al., 2022) provide much hope for the next generation of neuron-selective NKCC1-blockers. A major challenge in this regard, however, will be the delivery of such novel proteins over the BBB (Pardridge, 2020). Future advances in BBB delivery technology in parallel with new drug discovery are likely to resolve these issues.

ACKNOWLEDGMENTS

We thank Doris Möller and Edith Kaczmarek for skillful technical assistance. The study was supported in part by a grant (Lo 274/15) from the Deutsche Forschungsgemeinschaft (Bonn, Germany).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Open Research

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Volume45, Issue3

June 2024

Pages 138-148

Heterogeneous brain distribution of bumetanide following systemic administration in rats

Abstract

1 INTRODUCTION