human V2 receptor ( IC50 = 1.2 nM ); rat V2 receptor ( IC50 = 2.3 nM )

Lixivaptan exhibits competitive antagonist activity on V2 absorption [1].

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Vasopressin V2 receptor (V2R) antagonists (vaptans) are a new generation of diuretics. Compared with classical diuretics, vaptans promote the excretion of retained body water in disorders in which plasma vasopressin concentrations are inappropriately high for any given plasma osmolality. Under these conditions, an aquaretic drug would be preferable over a conventional diuretic. The clinical efficacy of vaptans is in principle due to impaired vasopressin-regulated water reabsorption via the water channel aquaporin-2 (AQP2). Here, the effect of Lixivaptan-a novel selective V2R antagonist-on the vasopressin-cAMP/PKA signaling cascade was investigated in mouse renal collecting duct cells expressing AQP2 (MCD4) and the human V2R. Compared to tolvaptan-a selective V2R antagonist indicated for the treatment of clinically significant hypervolemic and euvolemic hyponatremia-Lixivaptan has been predicted to be less likely to cause liver injury. In MCD4 cells, clinically relevant concentrations of Lixivaptan (100 nM for 1 h) prevented dDAVP-induced increase of cytosolic cAMP levels and AQP2 phosphorylation at ser-256. Consistent with this finding, real-time fluorescence kinetic measurements demonstrated that Lixivaptan prevented dDAVP-induced increase in osmotic water permeability. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of lixivaptan and suggest that lixivaptan has the potential to become a safe and effective therapy for the treatment of disorders characterized by high plasma vasopressin concentrations and water retention. [3]

In conscious dogs, lixivaptan (1, 3, and 10 mg/kg po) was administered with 30 mL/kg (po) and arginine vasopressin (AVP)-treated water (0.4 μg/kg in oil, subcutaneously) ) ) relative to the AVP-treated vehicle group increased Uvol by 438, 1018, and 1133%, respectively, while Uosm decreased from 1222 mOsm/kg (water-loaded and AVP-treated vehicle) to 307, 221, and 175 mOsm/kg, respectively. In AVP-deficient homozygous Brattleboro, lixivaptan at 10 mg/kg po (i.e., 10 times the dose that produced V2 multiantagonist activity) bid for 5 days showed sustained multiantagonist effects without evidence of agonist effects. In a randomized, double-blind, gradient-controlled ascending single-dose study, patients (fasted overnight before formulation) and mice received 30, 75, or 150 mg of lixivaptan. All of these increased doses increase urine flow and serum sodium concentrations and produce significant dose-related decreases in urine osmolality [1]. Phase II clinical trials in patients with congestive heart failure, liver tumor ascites, or inappropriate antidiuretic metabolic syndrome showed that lixivaptan increased water clearance rather than affecting renal sodium excretion or activating the neurohormonal system [2].

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Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations of PKD1 or PKD2 genes, is characterized by development and growth of cysts causing progressive kidney enlargement. Reduced resting cytosolic calcium and increased cAMP levels associated with the tonic action of vasopressin are two central biochemical defects in ADPKD. Here we show that co-targeting two GPCRs, the vasopressin V2 receptor (V2R) and the calcium sensing receptor, using the novel V2R antagonist Lixivaptan in combination with the calcimimetic R-568, reduced cyst progression in two animal models of human PKD. Lixivaptan is expected to have a safer liver profile compared to tolvaptan, the only drug approved to delay PKD progression, based on computational model results and initial clinical evidence. PCK rat and Pkd1RC/RC mouse littermates were fed without or with lixivaptan (0.5%) and R-568 (0.025% for rats and 0.04% for mice), alone or in combination, for 7 (rats) or 13 (mice) weeks. In PCK rats, the combined treatment strongly decreased kidney weight, cyst and fibrosis volumes by 20%, 49%, and 73%, respectively, compared to untreated animals. In Pkd1RC/RC mice, the same parameters were reduced by 20%, 56%, and 69%, respectively. In both cases the combined treatment appeared nominally more effective than the individual drugs used alone. These data point to an intriguing new application for two existing drugs in PKD treatment. The potential for synergy between these two compounds suggested in these animal studies, if confirmed in appropriate clinical investigations, would represent a welcome advancement in the treatment of ADPKD.[4]

Cell Preparations [3]
MCD4 cells were seeded onto 60-mm dishes and were left under basal condition or stimulated with 100 nM dDAVP for 1 h and/or treated with 100 nM Lixivaptan for 1h. Subsequently, cells were homogenized in cell fractionation buffer (20 mM NaCl, 130 mM KCl, 1 mM MgCl2, 10 mM HEPES, pH 7.5) in the presence of proteases (1 mM PMSF, 2 mg/mL leupeptin and 2 mg/mL pepstatin A) and phosphatases (10 mM NaF and 1 mM sodium orthovanadate) inhibitors. The resulting homogenates were sonicated at 80% amplitude for 10 s. Cellular debris was removed by centrifugation at 12,000× g for 10 min at 4 °C. The supernatants were collected and used for immunoblotting experiments.

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Fluorescence Resonance Energy Transfer Measurements [3]
To evaluate intracellular cAMP levels, fluorescence resonance energy transfer (FRET) experiments were performed. Briefly, MCD4 cells were seeded onto 20-mm glass coverslips at 37 °C, 5% CO2 and transiently transfected with a plasmid encoding the H96 probe containing the cAMP binding sequence of Epac1 between cyan fluorescent protein (CFP) and cp173Venus-Venus. Experiments were performed 48 h after transfection. Cells were left under basal condition or stimulated with 100 nM dDAVP for 1 h and/or treated with 100 nM Lixivaptan for 1 h.

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Water Permeability Assay [3]
Osmotic water permeability was measured by Video Imaging experiments. MCD4 cells were grown on 40 mm glass coverslips and loaded with 10 µM membrane permeable calcein green-AM for 45 min at 37 °C, 5% CO2 in DMEM. Cells were left under basal condition or stimulated with 100 nM dDAVP for 1h and/or treated with 100 nM Lixivaptan for 1h. The coverslips with dye-loaded cells were mounted in a perfusion chamber and measurements were performed using an inverted microscope, equipped for single cell fluorescence measurements and imaging analysis. The sample was illuminated through a 40× oil immersion objective (numerical aperture NA = 1.30). The calcein green-AM loaded sample was excited at 490 nm. Emitted fluorescence was passed through a dichroic mirror, filtered at 515 nm and captured by a cooled ECCD camera. Fluorescence measurements, following iso- (290 mOsm; 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM Hepes, 5 mM Glucose) or hyperosmotic (460 mOsm; isosmotic solution added with 135 mM Mannitol) solutions, were carried out using Metafluor® software v7.8.1.0. Calcein-AM is a nonfluorescent membrane-permeable dye, capable of quenching, which is converted to a green-fluorescent dye after acetoxymethyl ester hydrolysis, elicited by intracellular esterases. The exposure to a hyperosmotic solution leads to water efflux causing cell shrinkage with a consequent increase in calcein concentration, quenching, and ultimate decrease of fluorescence intensity. The best-fit tau values of the fluorescence intensity curve is proportional to the speed of water efflux and represents an indirect indication of the water permeability through AQP2. The time course of cell shrinkage was measured as time constant (Ki, s−1).

The animals were fed ground rodent chow ad libitum. At the 4th week of age, they were divided into four groups on a control diet or a diet containing Lixivaptan and/or R568. Rats (n = 80, 10 animals per group and gender) received ground rodent chow containing 0.5% Lixivaptan, 0.025% R568, 0.5% Lixivaptan and 0.025% R568 together, or rodent chow without drugs (control group) for 7 weeks. Mice (n = 80, 10 animals per treatment group and gender) were fed with ground rodent chow containing 0.5% Lixivaptan, 0.04% R568, 0.5% Lixivaptan and 0.04% R568 together, or rodent chow without drugs (control group) for 13 weeks. One week before the scheduled sacrifice, animals were housed in metabolic cages to collect 24‐h urine outputs. At 10 weeks (rats) or 16 weeks (mice) of age, animals were weighed and anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Blood was obtained by cardiac puncture and was used for plasma calcium, creatinine and urea levels determination. The right kidney was placed into pre‐weighed vials containing 10% formaldehyde/phosphate buffer saline (pH 7.4). Tissues were embedded in paraffin for histological and histomorphometric analysis. The left kidneys were immediately frozen in liquid nitrogen for cAMP or PKA activity measurements. [4]

[1]. 5-Fluoro-2-methyl-N-[4-(5H-pyrrolo[2,1-c]-[1, 4]benzodiazepin-10(11H)-ylcarbonyl)-3-chlorophenyl]benzamide (VPA-985): an orally active arginine vasopressin antagonist with selectivity for V2 receptors. J Med Chem. 1998 Jul 2;41(14):2442-4.

[2]. Lixivaptan, a non-peptide vasopressin V2 receptor antagonist for the potential oral treatment of hyponatremia. IDrugs. 2010 Nov;13(11):782-92.

[3]. Lixivaptan, a New Generation Diuretic, Counteracts Vasopressin-Induced Aquaporin-2 Trafficking and Function in Renal Collecting Duct Cells. Int J Mol Sci . 2019 Dec 26;21(1):183.

[4]. Pre-clinical evaluation of dual targeting of the GPCRs CaSR and V2R as therapeutic strategy for autosomal dominant polycystic kidney disease. FASEB J. 2021 Oct;35(10):e21874.

Drug Indication
Investigated for use/treatment in hyponatremia and congestive heart failure.
Treatment of hyponatraemia.

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Lixivaptan (VPA-985), being developed by Biogen Idec and Cardiokine, under license from Wyeth (now part of Pfizer), is a non-peptide, selective vasopressin V2 receptor antagonist for the potential oral treatment of hyponatremia associated with heart failure. Arginine vasopressin, the native V2 receptor ligand, stimulates water reabsorption via activation of V2 receptors that are expressed in the collecting ducts of the kidney. In preclinical studies, lixivaptan displayed competitive antagonist activity at V2 receptors in vitro, and increased urine volume and decreased urine osmolality in rats and dogs. The therapeutic benefits of lixivaptan are being evaluated in patients with conditions that are associated with water excess and hyponatremia. Phase II clinical trials in patients with congestive heart failure, liver cirrhosis with ascites or syndrome of inappropriate antidiuretic hormone have demonstrated that, unlike traditional diuretics, lixivaptan increases water clearance without affecting renal sodium excretion or activating the neurohormonal system. Administration of lixivaptan in combination with the diuretic furosemide has been tested in rats as well as in trials in healthy volunteers, in which the two agents were well tolerated. Ongoing phase III trials will determine the role of lixivaptan in the management of hyponatremia, especially when associated with heart failure.[2]

C27H21CLFN3O2

473.92594

473.13

C, 68.43; H, 4.47; Cl, 7.48; F, 4.01; N, 8.87; O, 6.75

168079-32-1

172997

White to off-white solid powder

1.3±0.1 g/cm3

626.5±55.0 °C at 760 mmHg

332.7±31.5 °C

0.0±1.8 mmHg at 25°C

1.658

7.23

1

3

3

34

753

0

O=C(C1=C(Cl)C=C(NC(C2=CC(F)=CC=C2C)=O)C=C1)N(C3)C4=CC=CC=C4CN5C3=CC=C5

PPHTXRNHTVLQED-UHFFFAOYSA-N

InChI=1S/C27H21ClFN3O2/c1-17-8-9-19(29)13-23(17)26(33)30-20-10-11-22(24(28)14-20)27(34)32-16-21-6-4-12-31(21)15-18-5-2-3-7-25(18)32/h2-14H,15-16H2,1H3,(H,30,33)

N-[3-chloro-4-(6,11-dihydropyrrolo[2,1-c][1,4]benzodiazepine-5-carbonyl)phenyl]-5-fluoro-2-methylbenzamide

VPA-985; VPA985; VPA 985; WAY-VPA-985; Lixivaptan; 168079-32-1; VPA-985; Lixar; WAY-VPA-985; VPA985; CRTX-080; Lixivaptan [USAN:INN]; WAY VPA-985; WAY-VPA 985; WAY VPA 985; CRTX-080; CRTX080; CRTX 080; Lixivaptan

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: ~95 mg/mL (~200.5 mM)
Ethanol: ~7 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.39 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 (4.39 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.)