human V2 receptor ( IC50 = 1.2 nM ); rat V2 receptor ( IC50 = 2.3 nM ); Lixivaptan is a highly potent and selective vasopressin V2 receptor (V2R) antagonist. It exhibits IC₅₀ values of 1.2 nM for the human V2 receptor and 2.3 nM for the rat V2 receptor . The compound demonstrates a V2:V1a binding affinity ratio of at least 100:1 in human cells, indicating high selectivity for the V2 subtype over the V1a receptor . This selective antagonism prevents arginine vasopressin (AVP) from binding to V2 receptors in the renal collecting ducts, thereby blocking the downstream signaling cascade that leads to water reabsorption .

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]

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In preclinical studies, lixivaptan displayed competitive antagonist activity at vasopressin V2 receptors in vitro . Detailed characterization of its mechanism revealed that lixivaptan effectively blocks the vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in renal collecting duct cells. This blockade of AQP2 is central to its aquaretic effect, as it prevents the insertion of these water channels into the apical membrane, thereby inhibiting water reabsorption independently of sodium handling .

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]

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The in vivo efficacy of lixivaptan has been demonstrated in several animal models and clinical studies. In rats and dogs, oral administration of lixivaptan significantly increased urine volume and decreased urine osmolality . In a key study using the PCK rat model of polycystic kidney disease, rats fed chow containing 0.5% lixivaptan for 8 weeks showed a 26% reduction in kidney weight-to-body weight ratio (p < 0.01), a 54% reduction in kidney cystic score (p < 0.001), a 23% reduction in renal cAMP levels (p < 0.05), and a 13% reduction in plasma creatinine (p < 0.001) compared to controls. These effects were associated with a 3-fold increase in 24-hour urine output . In human Phase II clinical trials, lixivaptan effectively increased water clearance without affecting renal sodium excretion or activating the neurohormonal system in patients with congestive heart failure, liver cirrhosis with ascites, or SIADH . Unlike traditional diuretics, lixivaptan specifically promotes aquaresis, making it particularly valuable for treating conditions of water retention with euvolemic or hypervolemic hyponatremia .

The binding affinity and selectivity of lixivaptan for the vasopressin V2 receptor were determined using competitive radioligand binding assays. In these cell-based assays, membranes expressing human or rat V2 receptors were incubated with radiolabeled arginine vasopressin in the presence of varying concentrations of lixivaptan. The concentration required to displace 50% of the bound ligand (IC₅₀) was calculated to be 1.2 nM for the human V2 receptor and 2.3 nM for the rat V2 receptor . The selectivity ratio for V2 versus V1a receptors was determined to be at least 100-fold in human cells, confirming its specificity for the V2 subtype . These assays demonstrate that lixivaptan acts as a competitive antagonist by binding directly to the orthosteric site of the V2 receptor, thereby preventing vasopressin from activating the receptor and its downstream G-protein signaling cascade .

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).

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Although detailed primary cell assay protocols for lixivaptan are not fully detailed in the available abstracts, its mechanism has been studied in renal collecting duct cell models. In these systems, lixivaptan was shown to block the vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels to the apical membrane. This was measured by assessing the cellular localization of AQP2 using immunofluorescence microscopy or by quantifying AQP2 expression in the plasma membrane fraction following vasopressin stimulation. The blockade of AQP2 translocation by lixivaptan prevents water reabsorption, which is the cellular basis for its aquaretic effect . No additional detailed cell-based assay protocols (e.g., viability, proliferation, or cytotoxicity assays) were described in the provided literature.

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]

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In the PCK rat model of polycystic kidney disease, lixivaptan was administered orally via chow admixture. Four-week old PCK rats were fed standard rodent chow containing either 0.5% lixivaptan (low dose) or 1% lixivaptan (high dose) for a duration of 8 weeks. A control group received chow only. Urine output was measured over 24 hours using metabolic cages at weeks 7 and 10 of age. At 12 weeks of age, animals were euthanized, and kidneys and livers were collected, weighed, and processed for analysis of cAMP levels, cystic burden (histomorphometric scoring), and fibrosis. Blood samples were collected for measurement of serum creatinine, sodium, and other markers of renal function . In other animal models, such as vasopressin-treated conscious dogs, lixivaptan (1, 3, and 10 mg/kg) was administered orally to evaluate its effects on urine output and osmolality following water loading .

Lixivaptan is rapidly absorbed after oral administration, with peak plasma concentrations achieved within 1 hour. The drug exhibits dose-proportional pharmacokinetics across doses ranging from 25 mg to 300 mg, with Cmax values increasing from 183 ng/mL to 1,877 ng/mL and AUC from 499 ng·h/mL to 14,884 ng·h/mL. The elimination half-life ranges from approximately 9 to 23 hours, depending on dose. Lixivaptan is extensively metabolized, primarily through the cytochrome P450 system (CYP3A4, with contributions from CYP2C8 and CYP3A5). After oral administration, nine metabolites were detected in human systemic circulation, eight of which exceeded 10% of parent drug exposure. Co-administration with strong CYP3A4 inhibitors such as ketoconazole increases lixivaptan exposure by approximately 3-fold, while CYP3A4 inducers decrease exposure by 30%. Grapefruit juice also significantly increases absorption when taken under fasted conditions.

Lixivaptan has demonstrated a favorable liver safety profile compared to the related V2 antagonist tolvaptan. Quantitative systems toxicology modeling predicted that lixivaptan has a markedly lower risk of hepatotoxicity, with no ALT elevations predicted at proposed clinical doses. This has been supported by clinical observations: a patient who had previously experienced clinically significant ALT elevations on three separate attempts with tolvaptan successfully completed 12 months of lixivaptan treatment without any liver chemistry abnormalities. The most common adverse effects reported in clinical trials include increased thirst, dehydration, nausea, headache, and constipation. The recommended starting dose for hospitalized patients is 50 mg once daily (25 mg for outpatients), with titration up to 100 mg once daily for SIADH or 100 mg twice daily for heart failure patients, depending on response.

[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
Ricivatan (VPA-985) has been investigated for the treatment of hyponatremia and congestive heart failure. Treatment of Hyponatremia. Ricivatan (VPA-985) was developed by Biogen Idic and Cardiokin and licensed by Wyeth (now part of Pfizer). It is a non-peptide selective angiotensin V2 receptor antagonist for the potential oral treatment of hyponatremia associated with heart failure. Arginine angiotensin is a naturally occurring V2 receptor ligand that stimulates water reabsorption by activating V2 receptors expressed in the collecting ducts of the kidneys. In preclinical studies, ricivatan demonstrated competitive antagonistic activity against the V2 receptor in vitro and increased urine volume and decreased urine osmolality in rats and dogs. The therapeutic benefit of ricivatan in patients with both water excess and hyponatremia is currently being evaluated. Phase II clinical trials in patients with congestive heart failure, cirrhosis with ascites, or syndrome of inappropriate antidiuretic hormone secretion showed that, unlike conventional diuretics, ribivatan can increase water clearance without affecting renal sodium excretion or activating the neurohormonal system. Ricivartan has been tested in combination with the diuretic furosemide in rats and healthy volunteers, and both drugs were well tolerated. An ongoing Phase III clinical trial will determine the role of ribivatan in the treatment of hyponatremia, particularly in patients 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; Lixivaptan; 168079-32-1 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.)