sGC/Soluble Guanylate Cyclase

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].

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].

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.

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.

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.

Absorption, Distribution and Excretion
Following the administration of 10mg of vericiguat by mouth once daily, the average steady-state Cmax and AUC in patients with heart failure is 350 mcg/L and 6,680 mcg•h/L, respectively, with a Tmax of 1 hour. The absolute bioavailability of orally-administered vericiguat is approximately 93% when taken with food - co-administration with meals has been shown to reduce pharmacokinetic variability, increase Tmax to roughly 4 hours, and increase Cmax and AUC by 41% and 44%, respectively.
Following the oral administration of radiolabeled vericiguat, approximately 53% of the administered radioactivity was recovered in the urine and 45% in the feces. A human mass balance study found that the portion recovered in the urine comprised approximately 40.8% N-glucuronide metabolite, 7.7% other metabolites, and 9% unchanged parent drug, while virtually the entire portion recovered in the feces comprised unchanged vericiguat.
In healthy subjects the steady-state volume of distribution of vericiguat is approximately 44 liters.
Vericiguat is a low-clearance drug, with an observed plasma clearance of 1.6 L/h in healthy volunteers and 1.3 L/h in patients with systolic heart failure.

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Metabolism / Metabolites
Vericiguat is primarily metabolized via phase II conjugation reactions, with CYP-mediated oxidative metabolism comprising a small (<5%) portion of its overall biotransformation. The major inactive metabolite, vericiguat N-glucuronide (M1), is formed by UGT1A9 and, to a lesser extent, UGT1A1. Other identified metabolites include a denbenzylated compound and an M15 metabolite thought to be the result of oxidative metabolism, although these metabolites are poorly characterized.

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Biological Half-Life
In patients with heart failure, the half-life of vericiguat is 30 hours.

Hepatotoxicity
In preregistration trials, serum aminotransferase elevations with mild bilirubin elevations were reported to occur in 2% of patients on vericiguat, but similar rates were reported with placebo therapy and the abnormalities resolved without dose modification or discontinuation of therapy. These abnormalities were considered the result of exacerbations of heart failure and congestive liver injury. There have been no published reports of clinically apparent liver injury with symptoms or jaundice attributed to vericiguat therapy, but the overall clinical experience with vericiguat has been limited.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Vericiguat is extensively (~98%) protein-bound in plasma, primarily to serum albumin.

[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
By directly stimulating the increased production of intracellular cyclic guanosine monophosphate (cGMP), vericiguat causes the relaxation of vascular smooth muscle and vasodilation. Vericiguat has a relatively long half-life (~30h) that allows for once-daily dosing. Animal reproduction studies have demonstrated the potential for embryo-fetal toxicity when vericiguat is administered to pregnant females - defects in major vessel and heart formation, as well as spontaneous abortions/resorptions, were observed when vericiguat was administered to pregnant rabbits during organogenesis. The possibility of pregnancy should be excluded prior to beginning therapy with vericiguat, and adequate contraception should be used throughout therapy and for one month following cessation of treatment.

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.)