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].

---

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].

---

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]

---

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).

---

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
Bumex is completely absorbed (80%), and its absorption is not affected by co-administration with food. Bioavailability is almost 100%. In human volunteers, after oral administration of carbon-14 labeled bumex, 81% of the radioactive material was excreted in the urine, of which 45% was excreted unchanged. Bile excretion of bumex is only 2% of the administered dose. 0.2 - 1.1 mL/min/kg [Respiratory disorders in preterm and full-term newborns] 2.17 mL/min/kg [Neonatals with volume overload treated with bumex] 1.8 ± 0.3 mL/min/kg [Elderly patients] 2.9 ± 0.2 mL/min/kg [Younger patients] Metabolisms/Metabolites 45% is excreted unchanged. Urinary and bile metabolites are formed by the oxidation of the N-butyl side chain.

---

Biological half-life
60-90 minutes

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
It is unclear whether bumetanib is excreted into breast milk. It should be avoided during the nursing period for newborns, as it may reduce milk production or completely suppress lactation. For mothers with established lactation, low doses are unlikely to suppress lactation. Generally, other medications should be preferred.
◉ Effects on Breastfed Infants
No relevant published information was found as of the revision date.
◉ Effects on Lactation and Breast Milk
No relevant published information was found as of the revision date. Lactation is suppressed immediately postpartum using methods such as potent diuretics, fluid restriction, and chest binding. The additional effects of diuretics on other effective lactation suppression measures have not been studied. There are currently no data on the effects of loop diuretics on established, sustained lactation.

---

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 belongs to the benzoic acid class of compounds, with the structure 4-phenoxybenzoic acid, where the hydrogen atom at the ortho-position of the phenoxy group is replaced by a butylamino group and a sulfonyl group. Bumetanide is a diuretic used to treat edema caused by congestive heart failure, liver disease, and kidney disease. It is both a diuretic and an EC 3.6.3.49 (channel conductance-controlled ATPase) inhibitor. It is a sulfonamide, amino acid, and benzoic acid compound. Bumetanide is a sulfonyl diuretic. Bumetanide is a loop diuretic. The physiological effect of bumetanide is achieved by increasing the diuretic effect of the loop of Henle. Bumetanide is a potent sulfonyl-an-aminobenzoic acid derivative, belonging to the loop diuretic class. In the brain, bumetanide may prevent neonatal seizures by blocking the bumetanide-sensitive sodium-potassium-chloride cotransporter (NKCC1), thereby inhibiting chloride uptake, reducing chloride concentration within neurons, and possibly blocking the excitatory effects of GABA in newborns.
A sulfonamide diuretic.

---

Drug Indications
For the treatment of edema associated with congestive heart failure, liver and kidney disease (including nephrotic syndrome).
FDA Label
Treatment of autism spectrum disorders

---

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 limb of the loop of Henle, thereby altering electrolyte transport in the proximal tubule. This leads to the excretion of sodium, chloride, and water, resulting in a diuretic effect.

---

Pharmacodynamics
Bumetanib is a sulfonamide loop diuretic used to treat heart failure. It is often used in patients who do not respond to high doses of furosemide. However, there is no reason not to use bumetanib as a first-line drug. The main difference between the two substances lies in bioavailability. Bumetanib has more predictable pharmacokinetic properties and clinical efficacy. In patients with normal renal function, bumetanib is 40 times more effective than furosemide.

---

In the early stages of cerebral ischemia, increased sodium ion (Na+) transport across the intact blood-brain barrier (BBB) is involved in the formation of cerebral edema.
In previous studies, the authors found that the sodium-potassium-chloride cotransporter of the blood-brain barrier (BBB) is activated by certain factors during ischemia, suggesting that this cotransporter may be involved in the increased uptake of sodium ions (Na+) in the brain during cerebral edema. This study aims to determine: (1) whether the sodium-potassium-chloride cotransporter is located on the luminal membrane of the blood-brain barrier; and (2) whether inhibiting the blood-brain barrier cotransporter can reduce the formation of cerebral edema. The distribution of this cotransporter in perfused and fixed rat brain tissue was detected by immunoelectron microscopy. Cerebral edema in rats with permanent middle cerebral artery occlusion (MCAO) was assessed by magnetic resonance diffusion-weighted imaging and apparent diffusion coefficient (ADC). Immunoelectron microscopy showed that the cotransporter was mainly (80%) distributed on the luminal membrane. Magnetic resonance imaging showed that the ADC ratio of the cortex and striatum (ipsilateral MCAO/contralateral control) ranged from 0.577 to 0.637, indicating significant edema formation. Immediate intravenous injection of bumetanide (7.6-30.4 mg/kg) before occlusion reduced the decrease in the cortex-striatum ADC ratio (by 40-67%), indicating reduced edema formation. Bumetanide also reduced the infarct area, which was determined by TTC staining. These findings suggest that the Na-K-Cl cotransporter in the blood-brain barrier lumen is involved in 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

CCCCNC1=C(C(=CC(=C1)C(=O)O)S(=O)(=O)N)OC2=CC=CC=C2

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.

---

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.

---

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