sGC/Soluble Guanylate Cyclase; Soluble guanylate cyclase (sGC) (EC50 = 14 nM for stimulation of sGC in the presence of the heme cofactor; EC50 = 1.4 μM for stimulation of heme-free sGC) [1]

Vericiguat (0.01 μM to 100 μM) increases the concentration of recombinant sGC in a dependent manner, from 1.7 to 57.6 times. Vericiguat and the NO donor diethylamine/nitric oxide complex (DEA/NO) work in concert to enhance enzyme activity across a broad concentration range. The highest concentrations of DEA/NO (100 nM) and vericiguat (100 μM) result in 341.6-fold increase in specific activity of sGC compared to baseline. With an EC50 of 1005±145 nM, vericiguat stimulates the sGC reporter cell line concentration-dependently. With IC50 values of 798, 692, and 3072 nM, respectively, vericiguat inhibits the contractions of rabbit saphenous artery rings, rabbit aortic rings, and canine femoral vein rings induced by phenylephrine in a concentration-dependent manner. Vericiguat, with an IC50 of 956 nM, inhibits the concentration-dependent contractions of pig coronary artery rings induced by U46619[1].

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Vericiguat (BAY 1021189) stimulates sGC in a dose-dependent manner. In the presence of the heme cofactor, it shows potent stimulation with an EC50 of 14 nM, and even in the absence of heme (heme-free sGC), it retains stimulatory activity with an EC50 of 1.4 μM. It enhances cGMP production in human umbilical vein endothelial cells (HUVECs) and rat aortic smooth muscle cells, with maximal effects observed at concentrations of 10 μM and 1 μM, respectively. The compound does not inhibit phosphodiesterases (PDEs) 1-5 at concentrations up to 10 μM, indicating selectivity for sGC [1]

Vericiguat (compound 24) (oral administration; 3 mg/kg, 10 mg/kg; once daily; 21 days) protects the kidneys and heart in a model of end-organ damage caused by hypertension in renin-transgenic rats treated with L-NAME. Comparing the Vericiguat-treated group to the control group, the former significantly lowers overall mortality[1].

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In anesthetized dogs, intravenous administration of Vericiguat (BAY 1021189) (0.3, 1, and 3 mg/kg) dose-dependently reduced mean arterial pressure (MAP) by 8, 15, and 24 mmHg, respectively, without affecting heart rate. In conscious spontaneously hypertensive rats (SHRs), oral administration (3, 10, and 30 mg/kg) decreased systolic blood pressure (SBP) by 14, 22, and 31 mmHg, respectively, with effects lasting for at least 8 hours. In a rat model of heart failure induced by myocardial infarction, chronic oral treatment (10 mg/kg/day for 4 weeks) improved left ventricular ejection fraction (LVEF) and reduced left ventricular end-diastolic volume (LVEDV) compared to vehicle controls [1]

Highly Purified sGC[1]
Enzyme activity was measured by the formation of [32P]-cGMP from α-[32P]-GTP, modified according to Hoenicka et al. and Schermuly et al. The modifications included using GTP, Mn2+/Mg2+, and cGMP at concentrations of 200 μM, 3 mM, and 1 mM, respectively. Enzyme concentrations were chosen carefully to achieve a substrate turnover of less than 10%, thus avoiding substrate or cofactor depletion. The characterization of the purified enzyme was performed at a protein concentration of 0.2 μg/mL. All measurements were performed in duplicate and were repeated five times. For enzyme characterization, the specific activity of sGC was expressed as x-fold stimulation vs specific basal activity. The highest DMSO concentration in the assay was 1% (v/v) and did not elicit any effect per se on cGMP production.[1]

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CYP Inhibition Assay[1]
The inhibitory potency of 24 was assessed in vitro by means on formation of metabolites from standard probes mediated by CYP isoforms (for details, please refer to the Supporting Information) based on assay conditions described. To investigate time-dependency, preincubation experiments on CYP3A4 were performed.

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Recombinant human sGC (with heme) was incubated with varying concentrations of Vericiguat (BAY 1021189) in the presence of GTP. cGMP production was measured using a competitive immunoassay to determine the EC50 for heme-containing sGC. For heme-free sGC, the enzyme was pretreated to remove heme, then incubated with the compound and GTP, and cGMP levels were quantified similarly to determine the EC50 for heme-free sGC. Additionally, PDE inhibition assays were performed by incubating PDEs 1-5 with the compound and measuring their activity using substrate conversion, confirming no significant inhibition at concentrations up to 10 μM [1]

Recombinant sGC-Overexpressing Cell Line[1]
The cellular activity of the test compounds was determined using a recombinant sGC-overexpressing cell line, as previously described. (34) Briefly, cells were plated in a volume of 25 μL on white 384-well Greiner Bio-One microplates and were cultured for 1 or 2 d in medium. Medium was removed, and cells were loaded for 3 h with calcium-free Tyrode-containing coelenterazine. Serial dilutions of the test compounds in a volume of 10 μL in calcium-free Tyrode were applied to the cells for 6 min. Thereafter, 35 μL of Tyrode-containing calcium (final concentration: 3 mM) was added to the cells and the emitted light was measured for 40 s using a CCD camera in a light-tight box. The minimal effective concentration (MEC) was determined as the concentration where a ≥3-fold increase in the basal luminescence value was observed.

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In Vitro Clearance Determinations with Rat and Human Hepatocytes[1]
Incubations with hepatocytes were performed at 37 °C, pH 7.4, in a total volume of 1.5 mL using a modified Janus robotic system (PerkinElmer). The incubation mixtures contained 1 × 106 cells/mL (corrected, according to the viability of the cells, determined via microscopy after staining with trypan blue), 1 μM substrate, and Williams’ medium E. The final MeCN concentration was ≤1%. Aliquots of 125 μL were withdrawn from the incubation mixture after 2, 10, 20, 30, 50, 70, and 90 min and dispensed in a 96-well plate, containing MeCN (250 μL) to stop the reaction. After centrifugation at 1000g, supernatants were analyzed by LC-MS/MS (AB Sciex Triple Quad 5500). The calculation of in vitro clearance values from half-life data using hepatocytes, reflecting substrate depletion, was performed using the following equations: CL′intrinsic [mL/(min·kg)] = (0.693/in vitro t1/2 [min]) (liver weight [g liver/kg body mass]) (cell no. [1.1 × 108]/liver weight [g])/(cell no. [1 × 106]/incubation volume [mL]). The CLblood was estimated using the nonrestricted well-stirred model: CLblood well-stirred [L/(h·kg)] = (QH [L/(h·kg)]·CL′intrinsic [L/(h·kg)])/(QH[L/(h·kg)] + CL′intrinsic [L/(h·kg)]). For calculations, the following values were used: human specific liver weight of 21 g/kg body mass, hepatic blood flow of 1.32 L/(h·kg), cell number in the liver was estimated to be 1.1 × 108 cells/g liver; rat specific liver weight of 32 g/kg body mass, hepatic blood flow of 4.2 L/(h·kg), cell number in the liver was estimated to be 1.1 × 108 cells/g liver.

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Caco-2 Permeability Assay[1]
The in vitro permeation of test compounds across a Caco-2 cell monolayer, a well-established in vitro system to predict the permeability from the gastrointestinal tract, was tested according to Artursson and Karlsson. Caco-2 cells were seeded on 24-well insert plates and were allowed to grow for 14–16 d. For permeability studies, the test compounds were dissolved in DMSO and diluted to the final test concentration of 2 μM with transport buffer [Hanks’ Buffered Salt Solution, further supplemented with glucose (final concentration 19.9 mM) and HEPES (final concentration 9.8 mM)]. For determination of the apical to basolateral permeability (Papp A–B), the test compound solution was added to the apical side of the cell monolayer and transport buffer to the basolateral side of the monolayer. For determination of the basolateral to apical permeability (Papp B–A), the test compound solution was added to the basolateral side of the cell monolayer and transport buffer to the apical side of the monolayer. Samples were taken from the donor compartment at the beginning of the experiment to confirm mass balance. After an incubation of 2 h at 37 °C, samples were taken from both compartments. Samples were analyzed by LC-MS, and the apparent permeability coefficients were calculated. The efflux ratio was calculated as Papp B–A/Papp A–B. Lucifer yellow permeability was assayed for each cell monolayer to ensure cell monolayer integrity, and the permeability of atenolol (low permeability marker) and sulfasalazine (marker for active excretion) was determined for each batch as a quality control.

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HUVECs and rat aortic smooth muscle cells were seeded in culture plates and grown to confluence. Cells were treated with Vericiguat (BAY 1021189) at concentrations ranging from 0.01 to 10 μM for 30 minutes. Intracellular cGMP levels were measured using a cGMP immunoassay kit. The results showed concentration-dependent increases in cGMP, with maximal responses at 10 μM (HUVECs) and 1 μM (smooth muscle cells) [1]

L-NAME-treated renin transgenic rats
3 mg/kg, 10 mg/kg
Oral administration; 3 mg/kg, 10 mg/kg; once daily; 21 days

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Rat Heart Langendorff Preparation[1]
Male Wistar rats (200–250 g) were anesthetized using Narcoren (100 mg/kg ip). The heart was rapidly excised and connected to a Langendorff perfusion system. The heart was perfused at a constant flow rate of 10 mL/min with Krebs–Henseleit buffer solution equilibrated with 95% O2 and 5% CO2. The perfusion solution contained (in mmol/L): NaCl 118, KCl 3, NaHCO3 22, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.8, glucose 10, and sodium pyruvate 2. A pressure transducer registered the perfusion pressure in the system. The left ventricular pressure was measured using a second pressure transducer connected to a water-filled balloon which was inserted into the left ventricle via the left atrium. The end diastolic pressure was initially set to 8–10 mmHg by adjusting the volume of the balloon. The hearts were spontaneously beating. The signals from the pressure transducer were amplified, registered, and used for the calculation of the heart frequency and + dP/dtmax by a personal computer. 24 was dissolved in a mixture of 10% DMSO and 90% saline and infused for 20 min with increasing concentration steps into the aortic cannula at a rate of 1% of the total flow rate. All values are presented as relative changes of baseline values before compound application.

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Chronic Treatment Study with L-NAME-Treated Renin Transgenic Rats[1]
Fifty male renin transgenic rats carrying an additional mouse renin gene [RenTG(mRRen2)27] at the age of 8 weeks were used. L-NAME was chronically administered via the drinking water (50 mg/L) in all study groups. Animals were randomly allocated to three study groups: placebo (control) (n = 20), 24 low dose, and 24 high dose (3 and 10 mg/kg per day, respectively, administered po by gavage qd, n = 15 per group). Blood pressure was measured via the tail-cuff method once before the start of the study (day 0) to exclude preexisting differences between the groups and on day 7, 14, and 21. Body weight and survival were assessed on day 1, 8, and 15 and at the study end. At the end of the study (day 22), all animals were anesthetized, blood was collected, and animals were sacrificed; blood was taken in order to assess plasma parameters, and the heart was dissected into the left and right ventricles and was weighed to assess potential heart hypertrophy. Creatinine, urea, and renin activity in plasma were determined after extraction, as previously described.

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Pharmacokinetic Parameters after Intravenous and Oral Application in Rats and Dogs[1]
For in vivo pharmacokinetic experiments, male Wistar rats and female beagle dogs were used. Intravenous application was carried out with a species-specific plasma/DMSO formulation in rats and with a H2O/PEG 400/EtOH formulation in dogs. Oral application in both species was by gavage with a H2O/PEG 400/EtOH formulation. For simplification of blood drawing in rats, a silicone catheter was implanted into the right vena jugularis externa. The surgery was performed at least 1 d before substance application under isoflurane anesthesia and additional administration of an analgetic (atropine/rimadyl 3:1, 0.1 mL sc). Blood drawing (usually more than 10 time points) was done in a time window that included at least two time points after 24 h (postsubstance application). Blood was passed into heparinized tubes. Afterward, blood plasma was obtained by centrifugation at 1000g. Where necessary, the plasma was stored at −20 °C until further analysis.
An internal standard was added to the sample, calibration, and qualifier solutions. The internal standard could also have been a compound from a different chemical class than the analyte of interest. Afterward, protein precipitation was performed by using an excess of MeCN. A buffer solution was added with a composition based on the mobile phases used in subsequent liquid chromatography. After centrifugation at 1000g, the supernatant was analyzed by LC-MS using different C18 reversed-phase columns and various mobile phase compositions. Quantification of the substance was conducted by using peak height or area calculated from extracted ion chromatograms of specific selected ion-monitoring experiments or high-resolution LC-MS experiments.
From the plasma concentration–time course, the pharmacokinetic parameters CL (clearance), t1/2 (terminal half-life), VSS (volume of distribution at steady state), and F (bioavailability after oral administration) were calculated by using a validated internal pharmacokinetic calculation software.
Because substance quantification was done in plasma, the blood/plasma distribution needed to be analyzed to calculate a blood clearance value. Therefore, a defined amount of the substance was added to blood in heparinized tubes and incubated for 20 min by gently swinging. The plasma was obtained by centrifugation at 1000g. The cblood/cplasma value was calculated after measurement of the substance concentration in plasma and blood by using peak height or area calculated from extracted ion chromatograms of specific selected ion-monitoring experiments or high-resolution LC-MS experiments.

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- For anesthetized dogs: Vericiguat (BAY 1021189) was dissolved in a vehicle (containing ethanol, polyethylene glycol 400, and water) and administered intravenously at doses of 0.3, 1, and 3 mg/kg. MAP and heart rate were monitored continuously for 2 hours post-administration.
- For conscious SHRs: The compound was administered orally via gavage at doses of 3, 10, and 30 mg/kg, dissolved in the same vehicle. SBP was measured using tail-cuff plethysmography at 1, 2, 4, 6, and 8 hours post-dose.
- For heart failure rats (post-myocardial infarction): The compound was administered orally once daily at 10 mg/kg/day for 4 weeks, starting 1 week after infarction. Cardiac function (LVEF, LVEDV) was assessed using echocardiography at the end of the treatment period [1]

Absorption, Distribution and Excretion
Following a once-daily oral administration of 10 mg veliciguat, the mean steady-state Cmax and AUC in patients with heart failure were 350 mcg/L and 6,680 mcg•h/L, respectively, with a Tmax of 1 hour. When taken with food, the absolute bioavailability of oral veliciguat is approximately 93%—co-administration with food reduces pharmacokinetic variability, prolongs Tmax to approximately 4 hours, and increases Cmax and AUC by 41% and 44%, respectively. After oral administration of radiolabeled veliciguat, approximately 53% of the radioactive material is recovered in urine and 45% in feces. A human mass balance study found that the drug recovered in urine contained approximately 40.8% N-glucuronide metabolites, 7.7% other metabolites, and 9% of the original drug, while the drug recovered in feces was almost entirely the original veliciguat.
In healthy subjects, the steady-state volume of distribution of veliciguat is approximately 44 liters.
Viliciguat is a low-clearance drug, with a plasma clearance of 1.6 L/h observed in healthy volunteers and 1.3 L/h observed in patients with systolic heart failure.
Metabolism/Metabolites
Viliciguat is primarily metabolized via II-binding reactions, with CYP-mediated oxidative metabolism accounting for a small proportion (<5%) of its overall biotransformation. The major inactive metabolite, veliciguat N-glucuronide (M1), is generated by UGT1A9, with a small amount generated by UGT1A1. Other identified metabolites include a debenzylated compound and an M15 metabolite, the latter believed to be a product of oxidative metabolism, but the characterization of these metabolites is not yet clear.
Biological Half-Life
In patients with heart failure, the half-life of veliciguat is 30 hours.

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- In rats, the oral bioavailability of veliciguat (BAY 1021189) was 73%. After intravenous injection (1 mg/kg), the clearance was 13 mL/min/kg, the steady-state volume of distribution (Vss) was 1.2 L/kg, and the terminal half-life was 3.6 hours.
In dogs, the oral bioavailability was 65%, the clearance was 9 mL/min/kg, the steady-state volume of distribution (Vss) was 0.9 L/kg, and the terminal half-life was 4.2 hours. This compound is not widely metabolized, and the main component in plasma is the unmetabolized parent drug [1].

Hepatotoxicity
In pre-registration trials, 2% of patients treated with veliciguat reported elevated serum transaminases with mild bilirubin elevation, but a similar incidence was reported in the placebo group. These abnormalities resolved spontaneously without dose adjustment or discontinuation. These abnormalities were considered a result of worsening heart failure and congestive liver injury. There are currently no published reports of clinically significant liver injury or jaundice caused by veliciguat treatment, but overall clinical experience with veliciguat is limited. Probability score: E (unlikely to be the cause of clinically significant liver injury). Protein Binding Veliciguat is extensively bound to proteins in plasma (approximately 98%), primarily serum albumin. In a two-week rat toxicity study, oral doses of up to 300 mg/kg/day of veliciguat (BAY 1021189) did not cause significant adverse reactions. No changes in body weight, food consumption, or clinical chemistry parameters (liver and kidney function) were observed. In both rat and human plasma, the plasma protein binding rate was high (93-95%) [1].

[1]. Discovery of the Soluble Guanylate Cyclase Stimulator Vericiguat (BAY 1021189) for the Treatment of Chronic Heart Failure. J Med Chem. 2017 Jun 22;60(12):5146-5161.

Pharmacodynamics
Vericiquigua induces vascular smooth muscle relaxation and vasodilation by directly stimulating the production of intracellular cyclic guanosine monophosphate (cGMP). Vericiquigua has a relatively long half-life (approximately 30 hours), allowing for once-daily dosing. Animal reproductive studies have shown that vericiquigua may have embryo-fetal toxicity in pregnant females—major vascular and cardiac developmental defects and spontaneous abortion/embryo resorption were observed in pregnant rabbits administered vericiquigua during organogenesis. Pregnancy should be ruled out before initiating vericiquigua treatment, and effective contraception should be used throughout treatment and for one month after discontinuation. Vericiquigua (BAY 1021189) is a first-in-class soluble guanylate cyclase stimulant designed for the treatment of chronic heart failure. It works by directly stimulating soluble guanylate cyclase (sGC), increasing cyclic guanosine monophosphate (cGMP) levels, thereby leading to vasodilation and improved cardiac function. The compound exhibits superior selectivity for sGC compared to other targets and possesses favorable pharmacokinetic properties, supporting oral administration [1]. Vericil is a pyrazolopyridine compound with the chemical name 5-fluoro-1H-pyrazolo[3,4-b]pyridine, wherein the 1-amino hydrogen is replaced by a 2-fluorobenzyl group and the 3-hydrogen is replaced by a 4,6-diamino-5-[(methoxycarbonyl)amino]pyrimidine-2-yl group. It is a soluble guanylate cyclase stimulator used to treat chronic heart failure. It has the effects of a soluble guanylate cyclase activator, vasodilator, and antihypertensive agent. It is an aminopyrimidine, pyrazolopyridine, carbamate, and organofluorine compound. Vericil is a direct stimulator of soluble guanylate cyclase (sGC) used to treat systolic heart failure to reduce mortality and hospitalization rates. The sGC enzyme is a key component of the NO-sGC-cGMP signaling pathway, which helps regulate the cardiovascular system. sGC enzymes are intracellular enzymes found in vascular smooth muscle cells (and other cell types) that catalyze the synthesis of cyclic guanosine monophosphate (cGMP) upon activation by NO (NO). cGMP acts as a second messenger, activating a series of downstream signaling cascades that trigger a variety of effects. These distinct cellular effects suggest that insufficient cGMP production (primarily due to inadequate NO bioavailability) is associated with the pathogenesis of various cardiovascular diseases. As a direct stimulator of soluble guanylate cyclase (sGC), veliciguat reduces the demand on the functional NO-sGC-cGMP axis, thereby helping to prevent myocardial and vascular dysfunction in heart failure caused by reduced sGC activity. Veliciguat, developed by Merck and marketed as Verquvo, received FDA approval in January 2021 for the treatment of certain patients with systolic heart failure. While veliciguat is not the first sGC stimulant to receive FDA approval (liociguat was approved in 2013 for the treatment of pulmonary hypertension), its unique feature lies in its structural modifications that significantly reduce its sensitivity to oxidative metabolism, thereby prolonging its half-life and allowing for once-daily dosing.
Viliciguat is a soluble guanylate cyclase stimulant. The mechanism of action of veliciguat is as a guanylate cyclase stimulant.
Viliciguat is an orally effective soluble guanylate cyclase (sGC) stimulant. sGC is a key enzyme in inducing vasodilatory nitric oxide action and is used to treat patients with chronic heart failure to reduce the risk of death and hospitalization. The incidence of elevated serum transaminases during veliciguat treatment is low, but it has not been found to be associated with clinically significant cases of acute liver injury.
Viliciguat is an orally bioavailable soluble guanylate cyclase (sGC) stimulant with vasodilatory activity. After oral administration, vericiguat directly stimulates the catalytic activity of sGC and increases the production of the intracellular second messenger cyclic guanosine monophosphate (cGMP), which is derived from guanosine triphosphate (GTP). This leads to smooth muscle relaxation and vasodilation. Soluble guanylate cyclase (sGC) is a heme-containing cytoplasmic signaling enzyme that catalyzes the formation of cyclic guanosine monophosphate (cGMP) from GTP upon the binding of nitric oxide (NO) to heme. Vericiguat stimulates sGC independently of NO and synergistically with NO. Vericiguat is a small molecule drug, currently in Phase IV clinical trials (covering all indications), and was first approved in 2021 for the treatment of cardiovascular disease and heart failure, with two investigational indications. The drug has been placed on a black box warning list by the U.S. Food and Drug Administration (FDA). A guanylate cyclase stimulator; FDA approved for the treatment of chronic heart failure.

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The first soluble guanylate cyclase (sGC) stimulant, riociguat, has recently been marketed as a novel treatment option for pulmonary hypertension. Despite its excellent pharmacological properties, riociguat's short half-life, requiring three-times daily dosing, has limited its use in other cardiovascular diseases. To further optimize such compounds, we discovered an interesting structure-activity relationship and significantly reduced oxidative metabolism. These studies ultimately led to the discovery of veliciguat (compound 24, BAY 1021189), an sGC stimulant that is administered once daily, and is currently in a Phase III clinical trial for the treatment of chronic heart failure. [1]

C19H16F2N8O2

426.3795

426.136

C, 53.52; H, 3.78; F, 8.91; N, 26.28; O, 7.50

1350653-20-1

54674461

Light yellow to brown solid powder

3.082

3

10

5

31

622

0

FC1C([H])=NC2=C(C=1[H])C(C1=NC(=C(C(N([H])[H])=N1)N([H])C(=O)OC([H])([H])[H])N([H])[H])=NN2C([H])([H])C1=C([H])C([H])=C([H])C([H])=C1F

QZFHIXARHDBPBY-UHFFFAOYSA-N

InChI=1S/C19H16F2N8O2/c1-31-19(30)25-14-15(22)26-17(27-16(14)23)13-11-6-10(20)7-24-18(11)29(28-13)8-9-4-2-3-5-12(9)21/h2-7H,8H2,1H3,(H,25,30)(H4,22,23,26,27)

methyl N-[4,6-diamino-2-[5-fluoro-1-[(2-fluorophenyl)methyl]pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl]carbamate

Vericiguat; BAY1021189; BAY-10-21189; BAY10-21189; BAY 1021189; BAY-1021189; Verquvo; Methyl (4,6-diamino-2-(5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)pyrimidin-5-yl)carbamate; MK-1242; BAY-1021189; Vericiguat [INN]; UNII-LV66ADM269; BAY 10-21189

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: 60~85 mg/mL (140.7~199.4 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)