PDE3 (IC50 = 0.4 nM); PDE4 (IC50 = 1479 nM)
Ensifentrine is a dual inhibitor of phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4). It also acts as an activator of the cystic fibrosis transmembrane conductance regulator (CFTR). As a PDE3 inhibitor, it has an IC₅₀ of 0.4 nM, while its PDE4 inhibition has an IC₅₀ of 1,479 nM. The dual inhibition results in bronchodilation and anti-inflammatory properties, while CFTR activation reduces mucous viscosity and improves mucociliary clearance.

Ensifentrine (RPL-554) suppresses human monocytes' TNF-α release produced by lipopolysaccharide (IC50 of 0.52 μM) and their proliferation in response to phytohemagglutinin (IC50 of 0.46 μM) in a concentration-dependent manner[1]. Ensifentrine (10 μM) effectively inhibits the contraction of isolated tracheal preparations from superfused guinea pigs that is caused by electrical field stimulation. After superfusion ends, contractile responses are inhibited for up to 12 hours, with RPL-554 exhibiting a prolonged duration of action[1].
In vitro, ensifentrine is a potent PDE3 inhibitor (IC₅₀ = 0.4 nM) and is selective for PDE3 over PDE4 (IC₅₀ = 1,479 nM). It is a novel and effective PDE inhibitor with bronchoprotective and anti-inflammatory activity. Its ability to activate CFTR further contributes to its potential therapeutic effects by improving mucociliary clearance.

Ensifentrine (RPL-554; 10 mg/kg; oral dose; once) in ovalbumin-sensitized guinea pigs dramatically reduces eosinophil recruitment after antigen challenge[1]. The recruitment of eosinophils to the airways is dramatically inhibited when conscious guinea pigs inhale a dry powder containing ensefentrine (25% in micronized lactose) 1.5 hours prior to antigen exposure[1]. Over the course of 5.5 hours, conscious guinea pigs exposed to dry powder containing 2.5% ensefentrine in micronized lactose are shown to exhibit a considerable inhibition of histamine-induced bronchoconstriction and plasma protein extravasation in the trachea[1].
In vivo, ensifentrine is being investigated in clinical trials for the treatment of COPD, cystic fibrosis, asthma, and other respiratory diseases. It is currently in Phase 3 clinical trials (NCT04535986) to evaluate its safety and efficacy in patients with COPD. Its inhaled route of administration ensures delivery to the site of action in the lungs.

Non-cellular enzyme assays for ensifentrine involve assessing its inhibition of phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4) using purified enzyme preparations. The enzyme is incubated with a substrate (cAMP or cGMP) in the presence of varying concentrations of ensifentrine, and the rate of substrate hydrolysis is measured. IC₅₀ values for PDE3 and PDE4 inhibition are determined from dose-response curves.

In vitro cellular assays for ensifentrine involve treating bronchial epithelial cells or smooth muscle cells with the compound and measuring its effects on cAMP levels, cytokine production, and CFTR function. Cells are treated with ensifentrine, and intracellular cAMP levels are measured by ELISA. Anti-inflammatory activity is assessed by measuring the production of pro-inflammatory cytokines (e.g., TNF-α, IL-6) in stimulated cells. CFTR function is assessed by measuring chloride efflux.

Animal/Disease Models: Male Dunkin Hartley guinea pigs (200-300 g) injected with ovalbumin[1].
Doses: 10 mg/kg
Route of Administration: Oral administration; once
Experimental Results: Dramatically inhibits eosinophil recruitment following antigen challenge in ovalbumin-sensitized guinea pigs.
In vivo animal experiments with ensifentrine are conducted in models of COPD, asthma, and other respiratory diseases. Animals are exposed to cigarette smoke or allergens to induce airway inflammation and hyperresponsiveness, and ensifentrine is administered via inhalation. Airway resistance, inflammatory cell infiltration, and cytokine levels in bronchoalveolar lavage fluid are measured to assess the compound's efficacy.

Ensifentrine has a molecular weight of 477.56 and a molecular formula of C₂₆H₃₁N₅O₄. It is a light-yellow solid powder with a purity of >98%. The compound is typically stored at -20°C and has a shelf life of >3 years if stored properly. It is soluble in DMSO.

Important Safety Information
Indications
Ohtuvayre is a prescription medicine used to treat chronic obstructive pulmonary disease (COPD) in adults. COPD is a chronic (long-term) lung disease that includes chronic bronchitis, emphysema, or both.
What is the most important information I should know about Ohtuvayre?
Ohtuvayre may cause serious side effects, including:
Shortness of breath immediately after inhaling the medication. If you experience shortness of breath immediately after inhaling the medication, stop using Ohtuvayre right away and call your healthcare provider or go to the nearest hospital emergency room immediately. Mental health problems, including suicidal thoughts and behaviors. You may experience mood or behavioral changes while taking Ohtuvayre. If you experience any of the following symptoms, contact your healthcare provider right away, especially if these symptoms are new, worsening, or worrying: thoughts of suicide or death, suicide attempts, difficulty sleeping (insomnia), new or worsening anxiety, new or worsening depression, dangerous impulsive behavior, and/or other unusual changes in behavior or mood. Do not use Ohtuvayre to treat sudden difficulty breathing. Always carry an emergency inhaler with you.
Who should not use Ohtuvayre?
Do not use Ohtuvayre if you have an allergic reaction to entifentlin or any of the ingredients in Ohtuvayre.
What should I tell my healthcare provider before using Ohtuvayre?
Before using Ohtuvayre, tell your healthcare professional if you have or have had a history of mental health problems, including depression and suicidal behavior; if you have liver problems; if you are pregnant or planning to become pregnant; or if you are breastfeeding. It is currently unknown whether Ohtuvayre can harm an unborn baby. It is currently unknown whether any of the drug ingredients in Ohtuvayre pass into breast milk and whether this can harm the baby.
Inform your healthcare provider about any medications you are taking, including prescription drugs, over-the-counter drugs, vitamins, and herbal supplements.
What are the most common side effects of Ohtuvayre?
The most common side effects of Ohtuvayre include back pain, high blood pressure, bladder infection, and diarrhea.
https://www.veronapharma.com/news/verona-pharma-announces-us-fda-approval-of-ohtuvayre-ensifentrine/
Ensifentrine is a research compound for laboratory use only and is not yet approved for human therapeutic use. In preclinical and clinical studies, it has been generally well-tolerated. As a PDE3/4 inhibitor and CFTR activator, it may have effects on the cardiovascular system and other organ systems. Standard laboratory safety precautions should be followed when handling the compound.

[1]. The pharmacology of two novel long-acting phosphodiesterase 3/4 inhibitors, RPL554 [9,10-dimethoxy-2(2,4,6-trimethylphenylimino)-3-(n-carbamoyl-2-aminoethyl)-3,4,6,7-tetrahydro-2H-pyrimido[6,1-a]isoquinolin-4-one] and RPL565 [6,7-dihydro-2-(2,6-diisopropylphenoxy)-9,10-dimethoxy-4H-pyrimido[6,1-a]isoquinolin-4-one]. J Pharmacol Exp Ther. 2006 Aug;318(2):840-8.

[2]. Symptom Improvement Following Treatment with the Inhaled Dual Phosphodiesterase 3 and 4 Inhibitor Ensifentrine in Patients with Moderate to Severe COPD - A Detailed Analysis. Int J Chron Obstruct Pulmon Dis. 2020 Sep 16;15:2199-2206.

Ensifentrine is currently undergoing clinical trial NCT04535986 (a Phase III clinical trial evaluating the safety and efficacy of ensifentrine in COPD patients). Ensifentrine is an inhaled phosphodiesterase (PDE) inhibitor that inhibits type 3 (PDE3) and type 4 (PDE4) phosphodiesterases, possessing potential anti-inflammatory and bronchodilatory effects. After administration, ensifentrine targets, binds to, and inhibits PDE3 and PDE4, thereby increasing intracellular cyclic adenosine monophosphate (cAMP) levels and cAMP-mediated signaling. This may lead to bronchial smooth muscle relaxation and modulate the inflammatory response. PDE3 and PDE4 are members of the PDE superfamily that hydrolyze cAMP and 3',5'-cyclic guanosine monophosphate (cGMP) to inactive 5' monophosphate. PDE3 is the most widely expressed PDE isoenzyme in bronchial smooth muscle. PDE4 is expressed in immune cells including T cells, monocytes, macrophages, neutrophils, dendritic cells, and eosinophils. It plays an important role in inflammation, especially in inflammatory airway diseases.

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Mechanism of Action
Ensifentelin is a dual inhibitor of phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4); it is also an activator of cystic fibrosis transmembrane conduction regulator (CFTR). Due to its dual inhibitory effect, the drug has bronchodilatory and anti-inflammatory properties; due to CFTR activation, it also reduces mucus viscosity and improves mucociliary clearance. Ensifentelin is currently being investigated for chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, other respiratory diseases, and COVID-19.
Ensifentrine (RPL-554; CAS 1884461-72-6) is a first-in-class, inhaled dual inhibitor of PDE3 and PDE4. It has an IC₅₀ of 0.4 nM for PDE3 and 1,479 nM for PDE4. Ensifentrine is being developed for COPD, cystic fibrosis, and asthma and is currently in Phase 3 clinical trials.

C26H31N5O4

477.5554

477.237

C, 65.39; H, 6.54; N, 14.67; O, 13.40

1884461-72-6

(E/Z)-Ensifentrine;298680-25-8; 3098314-20-3; 1884461-72-6

9934746

Light yellow to green yellow solid powder

2.7

2

5

6

35

849

0

O=C1N(C([H])([H])C([H])([H])N([H])C(N([H])[H])=O)/C(/C([H])=C2C3=C([H])C(=C(C([H])=C3C([H])([H])C([H])([H])N21)OC([H])([H])[H])OC([H])([H])[H])=N/C1C(C([H])([H])[H])=C([H])C(C([H])([H])[H])=C([H])C=1C([H])([H])[H]

CSOBIBXVIYAXFM-BYNJWEBRSA-N

InChI=1S/C26H31N5O4/c1-15-10-16(2)24(17(3)11-15)29-23-14-20-19-13-22(35-5)21(34-4)12-18(19)6-8-30(20)26(33)31(23)9-7-28-25(27)32/h10-14H,6-9H2,1-5H3,(H3,27,28,32)/b29-23+

N-(2-{(2E)-9,10-dimethoxy-4-oxo-2-[(2,4,6- trimethylphenyl)imino]-6,7-dihydro-2H-pyrimido[6,1- a]isoquinolin-3(4H)-yl}ethyl)urea

Ensifentrine; RPL-554; 298680-25-8; N-[2-[6,7-Dihydro-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-2H-pyrimido[6,1-a]isoquinolin-3(4H)-yl]ethyl]urea; N-(2-(6,7-Dihydro-9,10-dimethoxy-4-oxo-2-((2,4,6-trimethylphenyl)imino)-2H-pyrimido(6,1-a)isoquinolin-3(4H)-yl)ethyl)urea; RefChem:360374; ...; 1884461-72-6; Ensifentrina; RPL554; Ensifentrinum

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 : ~25 mg/mL (~52.35 mM)

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.62 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 12.5 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: 1.25 mg/mL (2.62 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 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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 (Please use freshly prepared in vivo formulations for optimal results.)