AER-271 (CAS#: 634913-39-6) is a prodrug that is converted to the active compound AER-270 (CAS#: 634913-38-5). The target is aquaporin-4 (AQP4). AER-270 is a selective, partial antagonist of AQP4. In a cell-based light scattering assay, AER-270 inhibited human AQP4-M23 with an IC50 of approximately 0.42 µM. It inhibited rat AQP4 with an IC50 of approximately 0.48 µM and mouse AQP4 with an IC50 of approximately 0.50 µM. [2]
Aquaporin-4 (AQP4) - selective small-molecule inhibitor.

AER-270, the active metabolite of AER-271 (CAS#: 634913-39-6), inhibited AQP4-mediated water permeability in CHO cells expressing human AQP4-M23. In a light scattering assay using a hyperosmotic shock (300 to 415 mOsm), AER-270 (10 µM) gave 47.0% inhibition of AQP4-mediated water permeability. [2]
In a video microscopy assay, CHO cells expressing AQP4 were exposed to a hypoosmotic shock (deionized water). AER-3, a related phenylbenzamide, reduced the rate of AQP4 cell swelling, giving ~84% inhibition. [2]
In a Cell Volume Cytometry assay, CHO cells expressing AQP4 were exposed to a hypoosmotic shock (300 to 100 mOsm). AER-3 reduced the rate of cell volume change, giving ~72% inhibition. [2]
AER-270 showed preferential inhibition of AQP4 over other aquaporins. In a light scattering assay at 10 µM, it inhibited AQP4-M1 by 44.9 ± 1.4% and AQP4-M23 by 48.8 ± 1.6%, while inhibiting AQP1 by 13.2 ± 1.1%, AQP5 by 4.5 ± 0.7%, and AQP2 was not determined. [2]
In vitro studies demonstrate that AER-271 is a selective inhibitor of aquaporin-4 (AQP4), a water channel protein that plays a critical role in the development of cerebral edema. AQP4 is the primary water channel in the brain and is involved in the regulation of water homeostasis, particularly in the context of injury such as stroke or cardiac arrest. By inhibiting AQP4, AER-271 blocks the influx of water into brain cells and reduces the development of cerebral edema. The compound's mechanism of action involves binding to AQP4 and blocking its water channel activity, thereby preventing the accumulation of excess fluid in the brain.

In vivo, endogenous phosphatases change AER-271 into AER-270. In a juvenile model of wild-type respiratory cardiac arrest, AER-271 causes cerebral edema and enhances early outcomes [1]. In an emergency animal model of cerebral stroke, AER-271 improves neurological outcomes and decreases cerebral edema. In a localized stroke model, administration of AER-271 (5 mg/kg; i.p.) resulted in better outcomes and decreased cerebral edema [2].

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AER-271 (CAS#: 634913-39-6) treatment in a rat model of asphyxial cardiac arrest (CA) ameliorated early cerebral edema measured at 3 h after CA vs. vehicle treated rats, reducing edema by 82.1%. AER-271 treatment also attenuated early neurologic deficit score (NDS) at 3 h post-CA by 20% compared to vehicle. AER-271 treatment resulted in a 43% reduction in pyknotic degenerating neurons on H&E in the hippocampal CA1 region at 72 h post-CA, and a 49% reduction in CA1 hippocampal FluoroJade positivity. AER-271 treatment also resulted in a 55% reduction in Iba1 positivity (microglial response) in the CA1 hippocampus at 72 h post-CA. [1]
In a mouse water intoxication model, co-injection of AER-270 (0.8 mg/kg, IP, in the water bolus) improved 4-hour survival to 50.5% compared to 15.3% for vehicle, representing a 3.3-fold improvement. AER-270 reduced the rate of cerebral edema formation by 2.5-fold as measured by MRI brain volume analysis. [2]
In a mouse model of ischemic stroke (1 h MCAo), treatment with AER-271 (5 mg/kg IP every 3 hours starting 75 min after occlusion) improved neurological outcome at 24 hours (average score 0.89 ± 0.31 vs 2.50 ± 0.62 for vehicle). AER-271 treatment reduced cerebral edema, with a relative change in ipsilateral brain volume of 5.63 ± 1.75% compared to 15.01 ± 1.95% for vehicle, a 2.6-fold reduction. [2]
In a rat model of ischemic stroke (1 h MCAo), IV infusion of AER-271 (loading dose of 4 mg/kg followed by maintenance dose of 0.03 mg/kg/hr) reduced cerebral edema from 23.8 ± 2.3% (vehicle) to 16.1 ± 1.8% and improved neurological outcome (modified Garcia score). A minimum effective maintenance dose of 0.03 mg/kg/hr was observed. [2]
In vivo studies have demonstrated that AER-271 ameliorates early cerebral edema measured at 3 hours after cardiac arrest compared to vehicle-treated rats. The compound blocks the development of early cerebral edema, reduces early neurologic deficit, and blunts neuronal death and neuroinflammation post-cardiac arrest. These findings support the potential of AER-271 for the research of acute ischemic stroke and other conditions involving cerebral edema. The compound's ability to cross the blood-brain barrier and inhibit AQP4 in the central nervous system is critical for its efficacy.

AQP4 inhibition assays are performed using AQP4-expressing cells or proteoliposomes. Water permeability is measured using stopped-flow light scattering or fluorescence quenching techniques. Cells or proteoliposomes are exposed to an osmotic gradient, and the rate of water transport is measured in the presence and absence of AER-271. The compound's ability to inhibit AQP4-mediated water transport is quantified, and IC50 values are calculated. Selectivity over other aquaporin isoforms is assessed using similar assay formats.

In a light scattering assay, CHO cells expressing AQP4 or CD81 (control) were grown to confluence in 96-well plates. Cells were treated with vehicle (0.1% DMSO) or test compound (e.g., 10 µM AER-270) for 30 minutes. A hyperosmotic shock was induced by adding an equal volume of HBSS (530 mOsm) to achieve a final osmolarity of 415 mOsm. Light scattering was monitored by measuring absorbance at 600 nm using a multiplate reader. The rate of the initial rise in absorbance (cell shrinking) was used to calculate percent inhibition of AQP4-mediated water permeability. [2]
In a video microscopy assay, CHO cells expressing AQP4 or CD81 were seeded in 6-well plates and treated with vehicle or test compound for 30 minutes. Media was aspirated and replaced with deionized water to induce a hypoosmotic shock. Cell swelling was recorded using light microscopy at 100x magnification. Video files were analyzed using ImageJ to measure relative cell diameter (D/D0) over time. The initial rate of cell diameter increase was used to calculate percent inhibition. [2]
In a Cell Volume Cytometry assay, CHO cells expressing AQP4 or CD81 were grown on glass coverslips and treated with vehicle or test compound. The coverslip was placed in a microfluidics chamber, and a hypoosmotic shock from 300 to 100 mOsm was applied. Cell swelling was monitored by measuring changes in electrical resistance (R/R0). The initial rate of resistance change was used to calculate percent inhibition. [2]
Cellular assays for AER-271 typically involve culturing cells that express AQP4, such as astrocytes or AQP4-transfected cell lines. Cells are treated with AER-271 at various concentrations, and AQP4-mediated water transport is measured using cell swelling assays or fluorescence-based methods. Cell viability is assessed using MTT or CellTiter-Glo assays to evaluate cytotoxicity. The compound's effects on cell swelling and water homeostasis are quantified. Neuroprotection assays are performed using neuronal cell lines or primary neurons exposed to oxygen-glucose deprivation or other injury models to assess the compound's ability to protect against injury-induced cell death.

Animal/Disease Models: Male mice (C57BL/6J, 8-12 weeks old, 25- 30 g) [2]
Doses: 5 mg/kg
Route of Administration: intraperitoneal (ip) injection Treatment
Experimental Results: Good effect, mean neurological score of 0.89 Compared with control mice receiving vehicle, mean neurological score of ±0.31 2.50±0.62.

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For the water intoxication model, mouse brain volumes were assessed using T2-weighted MRI. Sagittal images of the head were acquired using conventional spin echo (TR/TE = 3000/12 ms, resolution = 0.0195 cm/pixel, 3 averages, total scan time = 4 min 48 s) prior to water injection, at 5:40 min post water injection, and every 5:20 min thereafter until the animal expired. Brain volume was calculated by summing cross-sectional brain areas and multiplying by slice thickness (0.7 mm). [2]
For the MCAo model, mouse brain volumes were assessed using T2-weighted MRI. Coronal images were acquired using conventional spin echo (TR/TE = 3000/12 ms, resolution = 0.0078 cm/pixel, 3 averages, total scan time = 9 min 36 s) after 24 hours of reperfusion. Volumes of ipsilateral and contralateral hemispheres were quantitated separately. Relative change in hemispheric brain volume was calculated as ((Vi - Vc)/Vc) × 100%. [2]
For the rat MCAo model, a similar MRI protocol was used with a resolution of 0.0156 cm/pixel after 48 hours of reperfusion. [2]
In vivo efficacy of AER-271 is evaluated in animal models of cerebral edema and ischemic stroke, such as the asphyxial cardiac arrest model in rats. Animals are treated with AER-271 via intravenous or intraperitoneal administration, and cerebral edema is assessed by measuring brain water content, intracranial pressure, or by magnetic resonance imaging. Neurologic function is evaluated using behavioral tests and neurologic deficit scoring. Neuronal death and neuroinflammation are assessed by histopathological examination and immunohistochemistry for markers such as NeuN, GFAP, and Iba-1. Pharmacokinetic studies are conducted to determine the compound's bioavailability and brain penetration.

AER-271 (CAS#: 634913-39-6) is a prodrug that is converted in vivo to the active compound AER-270 (CAS#: 634913-38-5). After intraperitoneal (IP) administration of AER-271 at 10 mg/kg to mice, AER-270 reached peak plasma concentrations >500 ng/mL and peak brain tissue concentrations >100 ng/g. The estimated rate of IP absorption/conversion for AER-270 derived from AER-271 was 0.10 min⁻¹, and the elimination rate was 0.029 min⁻¹. [2]
In mice, after IP administration of AER-270 (0.8 mg/kg in water), peak plasma concentration of AER-270 was approximately 60 ng/mL. [2]
In rats subjected to MCAo, continuous IV infusion of AER-271 was used. A loading dose of 4 mg/kg followed by a maintenance dose of 0.03 mg/kg/hr achieved a plasma concentration of AER-270 that reduced cerebral edema. Higher maintenance doses (0.1, 0.4, 2 mg/kg/hr) resulted in higher plasma concentrations of AER-270 (e.g., ~130 ng/mL at 0.4 mg/kg/hr, ~350 ng/mL at 2 mg/kg/hr). [2]
In a pediatric asphyxial cardiac arrest model in rats, an IP loading bolus of AER-271 (5 mg/kg) at return of spontaneous circulation (ROSC) paired with a primed subcutaneous (SQ) osmotic continuous infusion pump (0.08 mg/h × 24 h) rapidly attained and maintained therapeutic drug levels (target plasma level of AER-270 was 70 ng/mL based on a mouse ischemic stroke model). Therapeutic levels were confirmed at 6 h (1475.99 ± 305.11 ng/mL) and 24 h (1699.37 ± 181.50 ng/mL). [1]
Pharmacokinetic properties of AER-271 have been characterized to support its use as a research tool. The compound is a phosphonate prodrug of AER-270, designed to enhance solubility and bioavailability. The compound has a molecular weight of 463.65 and the molecular formula C15H9ClF6NO5P. Key PK parameters including half-life, clearance, volume of distribution, and bioavailability are determined using LC-MS/MS analysis of plasma and brain tissue samples following administration. The compound's ability to cross the blood-brain barrier and reach the brain parenchyma is essential for its efficacy in cerebral edema models.

AER-271 (CAS#: 634913-39-6) treatment was well tolerated in a pediatric asphyxial cardiac arrest model in rats with no apparent effect on haemodynamics or serum chemistry. There were no differences in heart rate, mean arterial pressure, temperature, CPR duration, serum lactate, osmolarity, electrolytes, glucose, or hematocrit between AER-271-treated and vehicle-treated injury groups. [1]
AER-270 (1 or 10 µM) showed no inhibition of IKK-β or any other kinases in a panel of 456 kinases. Two known IKK-β inhibitors, PS-1145 and TPCA-1, failed to inhibit AQP4. [2]
In a broad ligand screening assay (binding assays) investigating interactions with 80 different pharmacologically relevant targets (GPCRs, neurotransmitter receptors, ion channels), multiple potential off-target hits were observed for 1 or 2 µM AER-270, but these were not quantified. No inhibition of Cytochrome P450 was observed. [2]
Toxicological evaluation of AER-271 is typically conducted in parallel with efficacy studies in animal models. Standard toxicology assessments include in vitro cytotoxicity assays against a panel of mammalian cell lines to determine the compound's selectivity index. In vivo toxicity studies in rodents include acute and repeated-dose toxicity testing, observation of clinical signs and body weight changes, and histopathological examination of major organs including the brain. As an AQP4 inhibitor that affects water homeostasis in the brain, potential effects on brain volume, intracranial pressure, and neuronal function are carefully monitored.

[1]. The aquaporin-4 inhibitor AER-271 blocks acute cerebral edema and improves early outcome in a pediatric model of asphyxial cardiac arrest. Pediatr Res. 2019 Mar;85(4):511-517.

[2]. Functionalized Phenylbenzamides Inhibit Aquaporin-4 Reducing Cerebral Edema and Improving Outcome in Two Models of CNS Injury. Neuroscience. 2019 Apr 15;404:484-498.

AER-271 is a research tool compound used for studying the role of aquaporin-4 in cerebral edema, ischemic stroke, and other conditions involving brain water homeostasis. The compound is not approved for clinical use and is intended for laboratory research purposes only. Its mechanism of action involves selective inhibition of aquaporin-4, which blocks the development of early cerebral edema, reduces neurologic deficit, and blunts neuronal death and neuroinflammation. AER-271 is a phosphonate prodrug of AER-270, designed to enhance solubility, and is valuable for validating AQP4 as a therapeutic target for cerebral edema and stroke.

C15H9CLF6NO5P

463.6528

462.981

C, 38.86 H, 1.96 Cl, 7.65 F, 24.59 N, 3.02 O, 17.25 P, 6.68

634913-39-6

10412080

White to off-white solid powder

3.5

3

11

4

29

620

0

ClC1C([H])=C([H])C(=C(C=1[H])C(N([H])C1C([H])=C(C(F)(F)F)C([H])=C(C(F)(F)F)C=1[H])=O)OP(=O)(O[H])O[H]

WSHXPHFIHYXZKC-UHFFFAOYSA-N

InChI=1S/C15H9ClF6NO5P/c16-9-1-2-12(28-29(25,26)27)11(6-9)13(24)23-10-4-7(14(17,18)19)3-8(5-10)15(20,21)22/h1-6H,(H,23,24)(H2,25,26,27)

2-{[3,5-Bis(trifluoromethyl) phenyl]carbamoyl}-4-chlorophenyl dihydrogen phosphate

AER 271 AER-271 AER271

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~125 mg/mL (~269.60 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.49 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 20.8 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: ≥ 2.08 mg/mL (4.49 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 20.8 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.)