Na+-K+-Cl+ cotransporter (NKCC); hNKCC1A (IC50 = 0.68 μM); hNKCC2A (IC50 = 4.0 μM)

The two main splice forms of human NKCC, hNKCC1A and hNKCC2A, are inhibited by bumetanide [1]. In NKCC1A-expressing oocytes, bumetanide (0.03-100 μM; 5 minutes) suppresses 86Rb+ in a dose-regulated manner [1]. In HEK-293 cells, bumetanide inhibits NKCC2 isoform B with an IC50 value of 0.54 μM[2].

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The Na(+)-K(+)-Cl(-) cotransporter NKCC1 plays a major role in the regulation of intraneuronal Cl(-) concentration. Abnormal functionality of NKCC1 has been implicated in several brain disorders, including epilepsy. Bumetanide is the only available selective NKCC1 inhibitor, but also inhibits NKCC2, which can cause severe adverse effects during treatment of brain disorders. A NKCC1-selective bumetanide derivative would therefore be a desirable option. In the present study, we used the Xenopus oocyte heterologous expression system to compare the effects of bumetanide and several derivatives on the two major human splice variants of NKCCs, hNKCC1A and hNKCC2A. The derivatives were selected from a series of ~5000 3-amino-5-sulfamoylbenzoic acid derivatives, covering a wide range of structural modifications and diuretic potencies. To our knowledge, such structure-function relationships have not been performed before for NKCC1. Half maximal inhibitory concentrations (IC50s) of bumetanide were 0.68 (hNKCC1A) and 4.0μM (hNKCC2A), respectively, indicating that this drug is 6-times more potent to inhibit hNKCC1A than hNKCC2A. Side chain substitutions in the bumetanide molecule variably affected the potency to inhibit hNKCC1A. This allowed defining the minimal structural requirements necessary for ligand interaction. Unexpectedly, only a few of the bumetanide derivatives examined were more potent than bumetanide to inhibit hNKCC1A, and most of them also inhibited hNKCC2A, with a highly significant correlation between IC50s for the two NKCC isoforms. These data indicate that the structural requirements for inhibition of NKCC1 and NKCC2 are similar, which complicates development of bumetanide-related compounds with high selectivity for NKCC1. [1]

The administration of bumetanide (7.6-30.4 mg/kg) intravenously prevented a 40–67% reduction in the cutaneous and striatal apparent diffusion coefficient (ADC), a sign of decreased edema development [3]. Additionally, intravenous reductions of bumetanide at doses of 2 mg/kg, 8 mg/kg, and 20 mg/kg are possible [4].

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Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia. [3]

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Bumetanide, 2, 8, and 20 mg/kg, was administered both intravenously and orally to determine the pharmacokinetics and pharmacodynamics of bumetanide in rats (n = 10-12). The absorption of bumetanide from various segments of GI tract and the reasons for the appearance of multiple peaks in plasma concentrations of bumetanide after oral administration were also investigated. After i.v. dose, the pharmacokinetic parameters of bumetanide, such as t1/2 (21.4, 53.8 vs. 127 min), CL (35.8, 19.1 vs. 13.4 ml/min per kg), CLNR (35.2, 17.8 vs. 12.6 ml/min per kg) and VSS (392, 250 vs. 274 ml/kg) were dose-dependent at the dose range studied. It may be due to the saturable metabolism of bumetanide in rats. After i.v. dose, 8-hr urine output per 100 g body weight increased significantly with increasing doses and it could be due to significantly increased amounts of bumetanide excreted in 8-hr urine with increasing doses. The total amount of sodium and chloride excreted in 8-hr urine per 100 g body weight also increased significantly after i.v. dose of 8 mg/kg, however, the corresponding values for potassium were dose-independent. After oral administration, the percentages of the dose excreted in 24-hr urine as unchanged bumetanide were dose-independent. Bumetanide was absorbed from all regions of GI tract studied and approximately 43.7, 50.0, and 38.4% of the orally administered dose were absorbed between 1 and 24 hr after oral doses of 2, 8, and 20 mg/kg, respectively. Therefore, the appearance of multiple peaks after oral administration could be mainly due to the gastric emptying patterns. [4]

NKCC1A activity assay [1]
To activate NKCC1A prior to the uptake experiment, hNKCC1A-expressing oocytes or uninjected control oocytes (5–15 oocytes per well) were preincubated for 30 min at room temperature in a hyperosmolar K+-free solution (containing in mM: 5 choline chloride, 95 NaCl, 1 MgCl2, 1 CaCl2, 10 Hepes; pH 7.4, 207 mOsm), which causes shrinkage of the oocyte and, thus, activation of NKCC1A. To measure K+ influx, oocytes were exposed to an isosmotic test solution in which KCl (5 mM) was substituted for choline chloride and 2–3 μCi/mL 86Rb+ included as a tracer for K+. Osmolarities of the test media were verified by using an automatic osmometer Type 15. Bumetanide (0.03–100 μM), its derivatives (1–100 μM), or control vehicle (≤ 1%, ensuring equal exposure to relevant drug solvent of all tested oocytes in the given experiment) was added to the test solution. The uptake assay was performed at room temperature with mild agitation for 5 min, which we have demonstrated to be within the linear phase of K+ uptake. The influx experiments were terminated by 3 times rapid wash in ice-cold 86Rb+-free assay solution after which the oocytes were individually dissolved in 200 μL 10% sodium dodecyl sulfate in scintillation vials. The radioactivity present was determined by liquid scintillation β-counting with Ultima Gold XR scintillation liquid using a Tri-Carb 2900TR Liquid Scintillation Analyzer. Human NKCC1 splice variant A-mediated K+ uptake was assessed as ([fluxNKCC1-expressing oocytes in the presence of x μM drug] − [fluxuninjected oocytes in the presence of x μM drug]), in order to correct for endogenous NKCC activity. All experiments were repeated at least three times (range: 3–6).

Animal/Disease Models: Normotensive SD (SD (Sprague-Dawley)) rats (250-300 g) [3]
Doses: 7.6 mg/kg, 15.2 mg/kg, 30.4 mg/kg
Route of Administration: intravenous (iv) (iv)injection
Experimental Results: diminished middle cerebral artery occlusion (MCAO) ) caused a decrease in ADC values in all four ipsilateral regions (L1-L4).

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Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat (220-300 g) [4]
Doses: 2 mg/kg, 8 mg/kg, 20 mg/kg (pharmacokinetic/PK/PK analysis)
Route of Administration: intravenous (iv) (iv)administration
Experimental Results: T1/2 (21.4 minutes, 53.8 minutes and 137 minutes for 2 mg/kg, 8 mg/kg and 20 mg/kg respectively)

Absorption, Distribution and Excretion
Bumetanide is completely absorbed (80%), and the absorption is not altered when taken with food. Bioavailability is almost complete.
Oral administration of carbon-14 labeled Bumex to human volunteers revealed that 81% of the administered radioactivity was excreted in the urine, 45% of it as unchanged drug. Biliary excretion of Bumex amounted to only 2% of the administered dose.
0.2 - 1.1 mL/min/kg [preterm and full-term neonates with respiratory disorders]
2.17 mL/min/kg [neonates receiving bumetanide for volume overload]
1.8 +/- 0.3 mL/min/kg [geriatric subjects]
2.9 +/- 0.2 mL/min/kg [younger subjects]

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Metabolism / Metabolites
45% is secreted unchanged. Urinary and biliary metabolites are formed by oxidation of the N-butyl side chain.

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Biological Half-Life
60-90 minutes

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
It is unknown if bumetanide is excreted into breastmilk. It should be avoided while breastfeeding a newborn because it may decrease milk flow or completely suppress lactation. Low doses in mothers whose lactation is well established are unlikely to suppress lactation. In general, alternate drugs are preferred.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information on bumetanide was not found as of the revision date. Intense diuresis, fluid restriction and breast binding have been used to suppress lactation immediately postpartum. The added contribution of the diuretic to the other measures, which are effective in suppressing lactation, has not been studied. No data exist on the effects of loop diuretics on established, ongoing lactation.

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Protein Binding
97%

[1]. The search for NKCC1-selective drugs for the treatment of epilepsy: Structure-function relationship of bumetanide and various bumetanide derivatives in inhibiting the human cation-chloride cotransporter NKCC1A. Epilepsy Behav. 2016 Jun;59:42-9.

[2]. Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways. J Cell Sci. 2011 Mar 1;124(Pt 5):789-800.

[3]. Bumetanide inhibition of the blood-brain barrier Na-K-Cl cotransporter reduces edema formation in the rat middle cerebral artery occlusion model of stroke. J Cereb Blood Flow Metab. 2004 Sep;24(9):1046-56.

[4]. Pharmacokinetics and pharmacodynamics of bumetanide after intravenous and oral administration to rats: absorption from various GI segments. J Pharmacokinet Biopharm. 1994 Feb;22(1):1-17.6.

Bumetanide is a member of the class of benzoic acids that is 4-phenoxybenzoic acid in which the hydrogens ortho to the phenoxy group are substituted by butylamino and sulfamoyl groups. Bumetanide is a diuretic, and is used for treatment of oedema associated with congestive heart failure, hepatic and renal disease. It has a role as a diuretic and an EC 3.6.3.49 (channel-conductance-controlling ATPase) inhibitor. It is a sulfonamide, an amino acid and a member of benzoic acids.
Bumetanide is a sulfamyl diuretic.
Bumetanide is a Loop Diuretic. The physiologic effect of bumetanide is by means of Increased Diuresis at Loop of Henle.
Bumetanide is a potent sulfamoylanthranilic acid derivative belonging to the class of loop diuretics. In the brain, bumetanide may prevent seizures in neonates by blocking the bumetanide-sensitive sodium-potassium-chloride cotransporter (NKCC1), thereby inhibiting chloride uptake thus, decreasing the internal chloride concentration in neurons and may block the excitatory effect of GABA in neonates.
A sulfamyl diuretic.

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Drug Indication
For the treatment of edema associated with congestive heart failure, hepatic and renal disease including the nephrotic syndrome.
FDA Label
Treatment of autism spectrum disorder

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Mechanism of Action
Bumetanide interferes with renal cAMP and/or inhibits the sodium-potassium ATPase pump. Bumetanide appears to block the active reabsorption of chloride and possibly sodium in the ascending loop of Henle, altering electrolyte transfer in the proximal tubule. This results in excretion of sodium, chloride, and water and, hence, diuresis.

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Pharmacodynamics
Bumetanide is a loop diuretic of the sulfamyl category to treat heart failure. It is often used in patients in whom high doses of furosemide are ineffective. There is however no reason not to use bumetanide as a first choice drug. The main difference between the two substances is in bioavailability. Bumetanide has more predictable pharmacokinetic properties as well as clinical effect. In patients with normal renal function, bumetanide is 40 times more effective than furosemide.

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Increased transport of Na+ across an intact blood-brain barrier (BBB) participates in edema formation during the early hours of cerebral ischemia. In previous studies, the authors showed that the BBB Na-K-Cl cotransporter is stimulated by factors present during ischemia, suggesting that the cotransporter may contribute to the increased brain Na+ uptake in edema. The present study was conducted to determine (1) whether the Na-K-Cl cotransporter is located in the luminal membrane of the BBB, and (2) whether inhibition of the BBB cotransporter reduces brain edema formation. Perfusion-fixed rat brains were examined for cotransporter distribution by immunoelectron microscopy. Cerebral edema was evaluated in rats subjected to permanent middle cerebral artery occlusion (MCAO) by magnetic resonance diffusion-weighted imaging and calculation of apparent diffusion coefficients (ADC). The immunoelectron microscopy studies revealed a predominant (80%) luminal membrane distribution of the cotransporter. Magnetic resonance imaging studies showed ADC ratios (ipsilateral MCAO/contralateral control) ranging from 0.577 to 0.637 in cortex and striatum, indicating substantial edema formation. Intravenous bumetanide (7.6-30.4 mg/kg) given immediately before occlusion attenuated the decrease in ADC ratios for both cortex and striatum (by 40-67%), indicating reduced edema formation. Bumetanide also reduced infarct size, determined by TTC staining. These findings suggest that a luminal BBB Na-K-Cl cotransporter contributes to edema formation during cerebral ischemia.[3]

C17H20N2O5S

364.4161

364.109

C, 56.03; H, 5.53; N, 7.69; O, 21.95; S, 8.80

28395-03-1

Bumetanide-d5;1216739-35-3;Bumetanide sodium;28434-74-4;Bumetanide-d5 Butyl Ester;1216685-32-3

2471

White to off-white solid powder

1.3±0.1 g/cm3

571.2±60.0 °C at 760 mmHg

230-2310C

299.3±32.9 °C

0.0±1.7 mmHg at 25°C

1.612

2.78

3

7

8

25

528

0

MAEIEVLCKWDQJH-UHFFFAOYSA-N

InChI=1S/C17H20N2O5S/c1-2-3-9-19-14-10-12(17(20)21)11-15(25(18,22)23)16(14)24-13-7-5-4-6-8-13/h4-8,10-11,19H,2-3,9H2,1H3,(H,20,21)(H2,18,22,23)

3-(butylamino)-4-phenoxy-5-sulfamoylbenzoic acid

PF 1593, PF-1593, PF1593; Ro 10-6338; bumetanide; 28395-03-1; 3-(Butylamino)-4-phenoxy-5-sulfamoylbenzoic acid; Bumex; Burinex; Fordiuran; Lunetoron; Fontego; Ro-10-6338; Ro 10 6338; Trade names: Bumex or Burinex;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~274.41 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.86 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)