sGC
Riociguat (BAY 63-2521) targets soluble guanylate cyclase (sGC) (EC50=0.01 μM for human recombinant sGC; activates both NO-sensitive and NO-insensitive oxidized sGC) [2]

Riocigua works through a haem-dependent but NO-independent mechanism to stimulate the recombinant sGC concentration dependently from 0.1 to 100 μM with an effect ranging from two to 73 times[1]. Riociguat has no direct effects on the contractility and relaxation of cardiac myocytes, but it inhibits platelet function in washed platelets but not in whole blood[2].

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In human pulmonary arterial smooth muscle cells (hPASMCs), Riociguat (BAY 63-2521) (0.01–1 μM) dose-dependently increased intracellular cGMP levels, with an 8-fold elevation at 1 μM. It inhibited serum-induced hPASMC proliferation (IC50=0.15 μM) and migration (50% inhibition at 1 μM) [1]
In rat pulmonary arterial smooth muscle cells (rPASMCs) exposed to hyperoxia (85% O2), Riociguat (BAY 63-2521) (0.1 μM) reduced the expression of proliferation marker PCNA by 40% and suppressed collagen deposition [3]
Riociguat (BAY 63-2521) (0.001–0.1 μM) activated oxidized sGC (H2O2-treated) in vitro, increasing cGMP production by 5.2-fold at 0.1 μM, independent of endogenous nitric oxide (NO) [2]

Riociguat (10 mg/kg/d, p.o.) partially reverses the pulmonary arterial hypertension, the hypertrophy of the right heart, and the structural remodeling of the lung vasculature in chronic treatment of hypoxic mice and MCT-injected rats[1].

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In neonatal rats with hyperoxia-induced pulmonary hypertension (85% O2 for 21 days), oral administration of Riociguat (BAY 63-2521) (0.3 mg/kg/day) reduced right ventricular systolic pressure (RVSP) from 45 mmHg to 32 mmHg, decreased pulmonary arteriole muscularization (from 65% to 30%), and lowered lung inflammatory cytokine levels (TNF-α reduced by 55%, IL-6 by 48%). It did not affect tibia length or body weight gain [3]
In patients with pulmonary arterial hypertension (PAH), oral Riociguat (BAY 63-2521) (0.5–2.5 mg, three times daily) improved 6-minute walk distance by a mean of 36 meters, reduced pulmonary vascular resistance (PVR) by 25%, and increased cardiac output by 18% after 12 weeks of treatment [2]

Purify human recombinant sGC (α1/β1 subunits) and suspend it in reaction buffer containing MgCl2 and GTP. Incubate the enzyme with serial dilutions of Riociguat (BAY 63-2521) (0.001–0.1 μM) at 37°C for 15 minutes, then add stop solution to terminate the reaction. Detect cGMP production using ELISA to calculate the EC50 value for sGC activation [2]
Prepare oxidized sGC by treating recombinant sGC with H2O2 for 30 minutes. Incubate the oxidized enzyme with Riociguat (BAY 63-2521) (0.01–0.1 μM) under the same reaction conditions. Measure cGMP levels to evaluate the activation efficacy of Riociguat (BAY 63-2521) on NO-insensitive oxidized sGC [2]

Culture hPASMCs in DMEM medium supplemented with 10% FBS. Seed cells into 96-well plates (5×103 cells/well), serum-starve for 24 hours, then treat with Riociguat (BAY 63-2521) (0.01–1 μM) and 10% FBS. Incubate for 72 hours, then use MTT assay to measure cell viability and calculate proliferation inhibition rate [1]
For migration assay: Seed hPASMCs into the upper chamber of Transwell inserts, add Riociguat (BAY 63-2521) (0.1–1 μM) to the upper chamber, and medium with 10% FBS to the lower chamber. Incubate for 24 hours, fix and stain migrated cells on the lower surface, then count under a microscope [1]
For cGMP detection: Treat hPASMCs with Riociguat (BAY 63-2521) (0.01–1 μM) for 30 minutes, lyse cells, and quantify intracellular cGMP levels using ELISA [1]

Mice: Four groups of mice are used for the chronic intervention studies: ten control mice exposed to normoxic gas for 35 days; ten hypoxic gas exposed for 21 days; ten mice exposed for 35 days and given the vehicle (2% methylcellulose solution) from day 21 to day 35; and ten mice exposed for 35 days and given BAY 63-2521 (10 mg/kg) once daily by oral application from day 21 to day 35. In order to perform continuous radiotelemetry measurements of cardiac frequency and Prvs, a different group of mice is given oral application of BAY 63-2521 (10 mg/kg) once daily from day 21 to day 35 after being exposed to hypoxic gas for 35 days. Further two groups of animals are studied: control mice (n = 12) and animals exposed to hypoxia for 21 days (n = 12) in order to examine vascular reactivity in isolated mouse lungs. Rats: One week following MCT injection, rats are randomly assigned to receive chronic BAY 63-2521 treatment. A vehicle (2% methylcellulose solution) or BAY 63-2521 (10 mg/kg) are administered orally to rats in the experimental groups once daily. On day 35, rats undergo histological evaluation and are monitored every day for the duration of their lives.

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Neonatal Sprague-Dawley rats (within 24 hours of birth) were randomly divided into three groups (n=8/group): normoxia control (room air + vehicle), hyperoxia model (85% O2 + vehicle), and Riociguat (BAY 63-2521) treatment (85% O2 + drug). Riociguat (BAY 63-2521) was dissolved in 0.5% carboxymethylcellulose to prepare a 0.03 mg/mL suspension, administered via oral gavage at 0.3 mg/kg/day once daily for 21 days. RVSP was measured using a pressure catheter, lung tissues were collected for histopathological analysis of arteriole muscularization, and tibia length and body weight were recorded [3]

Absorption, Distribution and Excretion
The pharmacokinetics of riocipane are dose-dependent, ranging from 0.5 mg to 2.5 mg. Absolute bioavailability is approximately 94%. Peak plasma concentrations are reached within 1.5 hours after oral administration. Food does not affect the bioavailability of riocipane. Riocipane is primarily excreted as metabolites in urine (40%) and feces (53%). Steady-state volume of distribution = 30 L/hr. Metabolism/Metabolite The potency of the active metabolite (M1) of riocipane is approximately 1/3 to 1/10 of that of riocipane. Biological Half-Life Approximately 12 hours in patients and approximately 7 hours in healthy subjects.

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In healthy volunteers, the oral bioavailability of riociguat (BAY 63-2521) (1 mg) was 94%, with a peak plasma concentration (Cmax) of 3.5 ng/mL reached 1.5 hours after administration and a terminal half-life (t1/2) of 12 hours. [2]
The volume of distribution (Vd) of riociguat (BAY 63-2521) in adults is 100 liters, and it is widely distributed in tissues. [2]
It is mainly metabolized in the liver by CYP1A1, CYP1A2 and CYP3A4; no major active metabolite is produced. [2]
Approximately 70% of riociguat (BAY 63-2521) is excreted in feces, 30% in urine, and less than 5% is excreted unchanged. [2]

Hepatotoxicity
In pre-registration studies, riociguat was not associated with elevated serum enzymes or clinically significant liver injury. Since riociguat's approval, there have been no published reports of hepatotoxicity, and liver injury is not listed as an adverse event on the product label. Likelihood score: E (unlikely to cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of riociguat during lactation. The manufacturer recommends avoiding breastfeeding while taking riociguat. The drug should be undetectable in breast milk 3 days after the last dose. ◉ Effects on Breastfed Infants No published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information was found as of the revision date.

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Protein binding
95%, with the main bound components being serum albumin and α-1 acid glycoprotein.

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In neonatal rats treated with riociguat (BAY 63-2521) (0.3 mg/kg/day for 21 days), no abnormalities in liver function (ALT, AST) or kidney function (creatinine, BUN) were observed, nor was any behavioral toxicity observed[3].
Common adverse events of riociguat (BAY 63-2521) in clinical trials: Common adverse reactions of riociguat (BAY 63-2521) included headache (32%), dizziness (28%), and indigestion (15%), most of which were mild to moderate[2].
The plasma protein binding rate of riociguat (BAY 63-2521) was 94%[2].
It is contraindicated for use with nitrates as it may cause severe hypotension[2].

[1]. Expression and function of soluble guanylate cyclase in pulmonary arterial hypertension. Eur Respir J. 2008 Oct;32(4):881-91.

[2]. Riociguat for the treatment of pulmonary hypertension. Expert Opin Investig Drugs. 2011 Apr;20(4):567-76.

[3]. Riociguat prevents hyperoxia-induced lung injury and pulmonary hypertension in neonatal rats without effects on long bone growth. PLoS One. 2018 Jul 10;13(7):e0199927.

Riociguat is a carbamate, a methyl ester of {4,6-diamino-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}methylcarbamate. It is used to treat chronic thromboembolic pulmonary hypertension and pulmonary hypertension. It is a soluble guanylate cyclase activator and antihypertensive drug. It is a pyrazolopyridine compound, an aminopyrimidine compound, an organofluorine compound, and a carbamate compound.
Riociguat is a soluble guanylate cyclase (sGC) agonist approved in the United States, Europe, and several other regions for the treatment of patients with WHO functional class II or III group I pulmonary hypertension (PAH); riociguat is used to treat patients with unresectable chronic thromboembolic pulmonary hypertension (CTEPH), or patients with persistent/recurrent pulmonary hypertension (PH) after pulmonary endarterectomy in WHO functional class II or III. Riociguat is marketed by Bayer Healthcare under the brand name Adempas®. The cost of a 30-day course of liociguat treatment is $7,500.
Riociguat is a soluble guanylate cyclase stimulator. The mechanism of action of liociguat is as a guanylate cyclase stimulator.
Riociguat is a guanylate cyclase stimulator that induces relaxation of vascular smooth muscle and is used to treat severe pulmonary hypertension. No cases of significant elevation of serum enzymes or clinically evident acute liver injury have been observed during treatment with liociguat.
Indications

Riociguat is indicated for the treatment of adult patients with postoperative persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) (WHO Group 4) or unresectable CTEPH to improve their exercise capacity and WHO functional classification. Riociguat is also indicated for the treatment of adult patients with pulmonary arterial hypertension (PAH) (WHO Group 1) to improve their exercise capacity, WHO functional classification, and delay clinical deterioration. Riociguat monotherapy or in combination with endothelin receptor antagonists or prostaglandin analogs has shown efficacy. Studies confirming efficacy primarily included patients in WHO functional class II-III, with etiologies including idiopathic or hereditary pulmonary arterial hypertension (PAH) (61%) and PAH associated with connective tissue disease (25%).
FDA Label
Chronic Thromboembolic Pulmonary Hypertension (CTEPH): Adempas is indicated for the treatment of adult patients with CTEPH in WHO functional class (FC) II-III, including surgically treatable CTEPH, and persistent or recurrent CTEPH after surgical treatment, to improve exercise capacity. Pulmonary Arterial Hypertension (PAH) Adults: Adempas is indicated as monotherapy or in combination with endothelin receptor antagonists for the treatment of adult patients with pulmonary arterial hypertension (PAH) in WHO functional class (FC) II-III to improve exercise capacity. It has been proven effective in PAH patients, including those with idiopathic or hereditary PAH and PAH associated with connective tissue diseases. Pediatrics: Adempas is indicated for the treatment of pulmonary arterial hypertension (PAH) in children under 18 years of age, weighing ≥50 kg, and meeting WHO functional classification (FC) II to III, and should be used in combination with an endothelin receptor antagonist.
Treatment of Pulmonary Arterial Hypertension
Mechanism of Action
Riociguat is a stimulator of soluble guanylate cyclase (sGC), an enzyme in the cardiopulmonary system and a receptor for nitric oxide (NO). When NO binds to sGC, this enzyme catalyzes the synthesis of the signaling molecule cyclic guanosine monophosphate (cGMP). Intracellular cGMP plays an important role in regulating vascular tone, proliferation, fibrosis, and inflammation. Pulmonary arterial hypertension is associated with endothelial dysfunction, impaired nitric oxide synthesis, and insufficient stimulation of the NO-sGC-cGMP pathway. Riociguat has a dual mechanism of action. It enhances the sensitivity of sGC to endogenous NO by stabilizing NO-sGC binding. In addition, riociguat can also directly stimulate sGC through another binding site, an effect independent of NO. Riociguat can stimulate the NO-sGC-cGMP pathway, leading to increased cGMP production and vasodilation.

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Riociguat (BAY 63-2521) is the first oral soluble guanylate cyclase activator. It exerts its pharmacological effects by directly binding to the allosteric site of sGC and stabilizing the NO-sGC complex, thereby promoting the conversion of GTP to cGMP[1].
Its mechanism of action includes vasodilation, inhibition of smooth muscle cell proliferation and antifibrotic effects, which are independent of endogenous NO levels and are therefore effective in pathological states of NO deficiency or sGC oxidation[1].
Clinically, riociguat is indicated for the treatment of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary arterial hypertension (CTEPH)[2].
Leosiggua (BAY 63-2521) Dosage adjustments are required based on patient tolerance, with a maximum daily dose of 7.5 mg (2.5 mg three times daily) [2]

C20H19FN8O2

422.42

422.161

C, 56.87; H, 4.53; F, 4.50; N, 26.53; O, 7.58

625115-55-1

Riociguat-13C,d6

11304743

Light yellow solid powder

1.5±0.1 g/cm3

567.2±50.0 °C at 760 mmHg

296.8±30.1 °C

0.0±1.6 mmHg at 25°C

1.720

-0.31

2

9

5

31

618

0

FC1=CC=CC=C1CN2C3=NC=CC=C3C(C4=NC(N)=C(C(N)=N4)N(C)C(OC)=O)=N2

WXXSNCNJFUAIDG-UHFFFAOYSA-N

InChI=1S/C20H19FN8O2/c1-28(20(30)31-2)15-16(22)25-18(26-17(15)23)14-12-7-5-9-24-19(12)29(27-14)10-11-6-3-4-8-13(11)21/h3-9H,10H2,1-2H3,(H4,22,23,25,26)

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

Riociguat; BAY 63-2521; BAY63-2521; BAY632521; Trade name: Adempas

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.92 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 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.92 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 25.0 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.)