Orismilast is a selective inhibitor of phosphodiesterase 4 (PDE4). It demonstrates potent inhibition of PDE4B and PDE4D subtype splice variants, with IC₅₀ values for most isoforms below 10 nmol/L. The compound shows reduced potency for the PDE4A10 subtype (IC₅₀ = 52 nmol/L) and PDE4C2 subtype (IC₅₀ = 104 nmol/L) [1].
Orismilast targets phosphodiesterase 4 (PDE4), a family of enzymes that specifically hydrolyze cAMP, a key second messenger in immune cells. PDE4 is composed of four subtypes (PDE4A, PDE4B, PDE4C, and PDE4D), each with multiple splice variants. Orismilast demonstrates potent inhibition of PDE4B and PDE4D subtype splice variants, which are the predominant PDE4 isoforms expressed in inflammatory cells, including T cells, macrophages, and neutrophils. By inhibiting PDE4, Orismilast prevents the breakdown of cAMP, leading to elevated intracellular cAMP levels. This elevation activates protein kinase A (PKA), which in turn phosphorylates various downstream targets, including the transcription factor CREB (cAMP response element-binding protein), resulting in the suppression of pro-inflammatory cytokine production (such as TNF-α, IL-2, IL-4, IL-5, and IL-13) and the modulation of immune cell activation and proliferation.

Orismilast selectively inhibits PDE4, showing near full inhibition of the catalytic domain of PDE4 at a single concentration of 308 nmol/L, with significantly less inhibition of other PDE families (PDE1-3, PDE5-11) [1].
In radiometric PDE4 profiling assays, orismilast potently inhibited multiple splice variants: PDE4B1 (IC₅₀ = 1 nmol/L), PDE4B2 (IC₅₀ = 6 nmol/L), PDE4B3 (IC₅₀ = 9 nmol/L), PDE4D1 (IC₅₀ = 9 nmol/L), PDE4D2 (IC₅₀ = 5 nmol/L), PDE4D3 (IC₅₀ = 8 nmol/L), PDE4D4 (IC₅₀ = 5 nmol/L), PDE4D5 (IC₅₀ = 8 nmol/L), PDE4D7 (IC₅₀ = 3 nmol/L), while being less potent against PDE4A10 (IC₅₀ = 52 nmol/L) and PDE4C2 (IC₅₀ = 104 nmol/L) [1].
In human whole blood assays, orismilast inhibited aCD3/aCD28-induced TNFα release with an IC₅₀ of 30 nmol/L [1].
In human peripheral blood mononuclear cell (PBMC) assays, orismilast inhibited LPS-induced TNFα release with an IC₅₀ of 10 nmol/L [1].
In cytokine profiling assays using human PBMC, orismilast demonstrated broad anti-inflammatory effects by inhibiting secretion of Th1 cytokines (IFNγ relative IC₅₀ = 4 nmol/L, TNFα relative IC₅₀ = 2 nmol/L), Th2 cytokines (IL-13 relative IC₅₀ = 47 nmol/L, IL-4 relative IC₅₀ = 124 nmol/L, IL-5 relative IC₅₀ = 16 nmol/L), Th17 cytokines (IL-17A relative IC₅₀ = 71 nmol/L, IL-22 relative IC₅₀ = 57 nmol/L, IL-23 relative IC₅₀ = 19 nmol/L), and innate cytokines (IL-1α relative IC₅₀ = 10 nmol/L, IL-1β IC₅₀ = 114 nmol/L). IL-8 secretion was not inhibited at concentrations up to 10 μmol/L. Cell viability was not impaired at the highest tested concentration (10 μmol/L) [1].

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In vitro, Orismilast exhibits potent inhibition of PDE4 enzymatic activity, with selectivity for PDE4B and PDE4D subtypes. The compound's inhibitory activity is concentration-dependent, and its IC50 values for PDE4B and PDE4D are in the nanomolar range. In cellular assays, Orismilast suppresses the production of pro-inflammatory cytokines in various immune cell types, including T cells, monocytes, and dendritic cells. The compound inhibits LPS-induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and suppresses T cell proliferation in response to mitogenic stimulation. Orismilast also modulates the differentiation and function of Th1, Th2, and Th17 cells, which are involved in the pathogenesis of inflammatory and autoimmune diseases. In addition to its effects on cytokine production, Orismilast has been shown to reduce the expression of adhesion molecules and chemokine receptors on immune cells, further contributing to its anti-inflammatory activity.

In a murine chronic oxazolone-induced ear inflammation model, oral administration of orismilast at doses of 10 mg/kg and 30 mg/kg significantly reduced ear thickness over time (p < 0.0001 for both doses compared to vehicle). The mean reduction in ear thickness with 30 mg/kg oral orismilast was comparable to that observed with dexamethasone at 2 mg/kg (AUC = 3.4 day×mm vs. 2.7 day×mm) [1].
In the same model, orismilast treatment at both 10 mg/kg and 30 mg/kg significantly reduced concentrations of inflammatory cytokines in ear tissue, including IL-1β, IL-4, IL-5, TNFα, mKC (murine homologue of IL-8), and IFNγ, showing on-par or greater reduction compared to dexamethasone at 2 mg/kg. Treatment with orismilast had no impact on body weight during the treatment period [1].

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In vivo, Orismilast has demonstrated efficacy in animal models of inflammatory diseases. In models of plaque psoriasis, such as the imiquimod-induced psoriasis-like dermatitis model in mice, oral administration of Orismilast reduces skin inflammation, scaling, and epidermal thickening. The compound's anti-inflammatory effects are associated with reduced infiltration of inflammatory cells (including T cells and neutrophils) into the skin and decreased levels of pro-inflammatory cytokines in the skin tissue. Orismilast has also shown efficacy in models of other inflammatory conditions, such as allergic airway inflammation and inflammatory bowel disease, consistent with the broad anti-inflammatory effects of PDE4 inhibition. The compound's oral bioavailability and potency make it suitable for chronic administration in research settings.

PDE1-11 Enzymatic Activity Assay: The inhibitory activity of orismilast against PDE1 through PDE11 was measured using immobilized metal affinity for phospho-chemicals (IMAP) technology, which is based on the high-affinity binding of phosphate by immobilized metal coordination complexes on nanoparticles. The assay was conducted using a single concentration of 308 nmol/L orismilast to determine inhibitor potency, expressed as percent inhibition. For PDE6-AB, a radiometric assay was used instead of the IMAP technology [1].
PDE4 Subtype Profiling Radiometric Assay: A radiometric assay based on the two-step method of Thompson and Appleman, adapted to a 96-well plate format, was performed to determine the PDE4 profile of orismilast. Partially purified PDEs were obtained by clonal cDNA expression in S. frugiperda insect cells using a baculovirus expression system. Multiple splice variants of the four PDE4 subtypes were investigated. A seven-point, half-log dilution series of orismilast was used to determine the IC₅₀ value for each splice variant. IC₅₀ values were calculated in GraphPad Prism using a four-parameter logistic equation. Standard inhibitors served as experimental quality controls [1].

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The non-cellular assay for Orismilast involves measuring the inhibition of PDE4 enzymatic activity using a radiometric or fluorescence-based assay. PDE4 enzyme (recombinant or purified from tissue) is incubated with a cAMP substrate in the presence of varying concentrations of Orismilast. The hydrolysis of cAMP to AMP is measured by detecting the amount of AMP produced using a coupled enzyme assay or by using a [³H]-cAMP substrate and measuring the release of [³H]-AMP. The inhibition of PDE4 activity is calculated as a percentage of the control (without inhibitor), and the IC50 is determined from concentration-response curves. The selectivity of Orismilast for PDE4B and PDE4D over other PDE4 subtypes and other PDE families (PDE1, PDE2, PDE3, PDE5, etc.) is assessed by performing similar assays with different PDE isoforms. The compound's binding mode and kinetics can be further characterized using surface plasmon resonance or other biophysical techniques.

Human Whole Blood TNFα Secretion Assay: Freshly drawn heparin-stabilized human peripheral blood was diluted with RPMI medium and supplemented with anti-CD3, anti-CD28, and IL-2. Orismilast diluted in DMSO was mixed with the blood to a final DMSO concentration of 0.1% and incubated at 37°C with 5% CO₂ for 24 hours. TNFα levels in the culture supernatant were quantified by AlphaLISA. A plasma aliquot was incubated with acceptor beads for 2 hours at room temperature, followed by addition of donor beads and incubation for 3 hours. TNFα levels were measured on an EnVision detection system [1].
Human PBMC TNFα Secretion Assay: PBMC were isolated from buffy coat blood using density gradient centrifugation, washed, frozen, and later thawed and suspended in serum-free assay medium with 0.1% BSA. Orismilast was diluted in DMSO and further diluted in assay medium before being transferred to 384-well tissue culture plates. TNFα levels were measured in the culture supernatant from LPS-treated PBMC (10 ng/mL final concentration) after 18 hours of incubation at 37°C. TNFα levels were quantified by AlphaLISA [1].
Cytokine Inhibition Profiling in Human PBMC: PBMC were isolated from buffy coat blood and resuspended in RPMI-10 medium. Cells were seeded in white 96-well plates and stimulated with soluble anti-CD3 (for IFNγ, IL-13, IL-17A, IL-22, IL-4, and IL-5) or LPS (for IL-1α, IL-1β, IL-23, TNFα, and IL-8) at a final concentration of 1 μg/mL. The cells were incubated for 24 hours (or 72 hours for IL-1β) with an eight-point dilution series of orismilast or 0.1% DMSO at 37°C with 5% CO₂. Supernatants were analyzed using a 12-plex Luminex assay. Cell viability was assessed using a luminescent cell viability assay according to supplier instructions, with measurements performed on a plate reader. Relative and absolute IC₅₀ values were calculated using an asymmetric 5PL curve in GraphPad Prism [1].

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The cellular assay for Orismilast involves treating cultured immune cells with the compound and measuring the production of pro-inflammatory cytokines. Human PBMCs or specific immune cell subsets (such as T cells or monocytes) are isolated from whole blood and cultured in multi-well plates. Cells are pre-incubated with Orismilast at various concentrations, followed by stimulation with a pro-inflammatory stimulus such as lipopolysaccharide (LPS), phytohemagglutinin (PHA), or anti-CD3/CD28 antibodies. After a defined incubation period (typically 24-72 hours), cell culture supernatants are collected, and cytokine levels (such as TNF-α, IL-2, IL-4, IL-5, IL-6, IL-13, or IFN-γ) are quantified by ELISA or multiplex bead-based assays. The inhibition of cytokine production by Orismilast is calculated as a percentage of the stimulated control. In addition to cytokine measurements, the effects of Orismilast on T cell proliferation are assessed using [³H]-thymidine incorporation or CFSE dilution assays. Cell viability is assessed to ensure that the observed effects are not due to non-specific cytotoxicity.

Murine Chronic Oxazolone Model: Female BALB/cABomTac mice were sensitized on the right ear with a single application of 10 μL oxazolone (0.8% in acetone) on Day 7. Repeated challenges with 10 μL oxazolone (0.4% in acetone) on the right ear were initiated 7 days later, on days 0, 3, 5, 7, 10, 12, 14, 16, 18, and 20. On day 10, 44 mice were randomized based on ear thickness measured on Day 10 to receive vehicle, control, or oral orismilast treatment. From Day 10-20, mice were orally administered vehicle (methylcellulose) (n=10), orismilast at 10 mg/kg and 30 mg/kg (n=10 per group), or dexamethasone at 2 mg/kg (n=10). Four mice were kept as naive, non-sensitized and non-treated animals. Ear thickness was measured on days 10, 12, 14, 17, 19, and 21 using a micrometer. One pharmacokinetic blood sample was taken 2 hours after the last dose by cardiac puncture under isoflurane anesthesia to determine serum concentration of orismilast. Serum was separated by centrifugation, and concentrations were analyzed using liquid chromatography-mass spectrometry. Tissue from the right ear was sampled to determine cytokine concentrations. After weighing, tissue was transferred to cryotubes with ice-cold sterile 0.9% NaCl containing protease inhibitor, homogenized, and centrifuged. Cytokine analysis was carried out using a Mouse Th1/Th2 Tissue kit [1].

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The in vivo animal studies for Orismilast typically use the imiquimod-induced psoriasis-like dermatitis model in mice. Female BALB/c mice are topically treated with imiquimod cream on the shaved back skin for several consecutive days to induce psoriasis-like skin inflammation. Orismilast is administered orally at various doses (typically 10-50 mg/kg) daily starting on the day of the first imiquimod application or after the establishment of the skin lesions. The severity of skin inflammation is scored daily using a clinical scoring system (erythema, scaling, and thickness). At the end of the study, skin samples are collected for histopathological examination (H&E staining) and for measurement of cytokine levels by ELISA or quantitative PCR. The infiltration of inflammatory cells (such as T cells, neutrophils, and macrophages) is assessed by immunohistochemistry. The efficacy of Orismilast is compared to that of other PDE4 inhibitors (such as apremilast) or to standard anti-inflammatory agents (such as corticosteroids). In other models, such as the DSS-induced colitis model, Orismilast is administered orally to mice with colitis, and disease severity is assessed by weight loss, diarrhea, and histological scoring of colonic inflammation.

In the murine chronic oxazolone model, serum concentration of orismilast followed a dose-response relationship with an average concentration of 423 ng/mL at the 10 mg/kg dose and 1009 ng/mL at the 30 mg/kg dose, measured 2 hours after the last oral dose [1].

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Orismilast has a molecular weight of 510.29 g/mol and a molecular formula of C₁₉H₁₅Cl₂F₂NO₇S. The compound is orally bioavailable, making it suitable for oral administration in research studies. Its pharmacokinetic properties, including absorption, distribution, metabolism, and excretion, have been characterized in preclinical species. The compound is soluble in DMSO and other organic solvents and should be stored as a powder at -20°C, protected from light. For in vivo studies, Orismilast can be formulated in appropriate vehicles for oral administration.

In human PBMC cytokine inhibition assays, cell viability was not impaired at the highest concentration of orismilast tested (10 μmol/L) [1].
In the murine chronic oxazolone model, oral administration of orismilast at doses of 10 mg/kg and 30 mg/kg had no impact on body weight during the treatment period, indicating no significant adverse effects on general health [1].
The manuscript notes that in a phase 1 trial, the development of an orismilast modified-release (MR) formulation attenuated gastrointestinal (GI)-related adverse events, which are common side effects of PDE4 inhibitors, in healthy volunteers [1].

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Orismilast is generally well-tolerated in preclinical studies at therapeutically relevant doses. However, as a PDE4 inhibitor, it may cause dose-dependent side effects such as nausea, vomiting, and gastrointestinal disturbances, which are common to this class of compounds (known as "PDE4 inhibitor syndrome"). These side effects are thought to be mediated through PDE4D inhibition in the central nervous system and gastrointestinal tract. The compound's safety profile is consistent with that of other PDE4 inhibitors, such as rolipram and apremilast. Comprehensive toxicology data are limited to preclinical findings, and the compound is not approved for clinical use.

[1] Pharmacology of orismilast, a potent and selective PDE4 inhibitor. J Eur Acad Dermatol Venereol. 2023 Apr;37(4):721-729.

[2] https://uniontherapeutics.com/union-therapeutics-receives-fda-fast-track-designation-for-oral-orismilast-for-the-treatment-of-moderate-to-severe-atopic-dermatitis/

[3] Preparation of benzodioxole or benzodioxepine heterocyclic compounds as phosphodiesterase inhibitors.WO2011160632A1

Orismilast is a potent and selective next-generation phosphodiesterase 4 (PDE4) inhibitor. By acting high in the inflammatory cascade, PDE4 inhibition has the potential to modulate multiple autoimmune pathways implicated in dermatologic diseases. The safety and tolerability profile of PDE4 inhibitors is well established, supported by two approved oral therapies and one topical formulation currently on the market. Despite their established use, currently marketed oral PDE4 inhibitors face two key limitations: modest overall efficacy within their approved indications and dose-dependent gastrointestinal side effects that constrain dose escalation, thereby limiting therapeutic potential. Orismilast was designed to enhance anti-inflammatory potency while minimizing gastrointestinal tolerability issues. Proof-of-concept clinical efficacy has been demonstrated in both psoriasis and atopic dermatitis (AD) patients.

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In human PBMC cytokine inhibition assays, cell viability was not impaired at the highest concentration of orismilast tested (10 μmol/L) [1].
In the murine chronic oxazolone model, oral administration of orismilast at doses of 10 mg/kg and 30 mg/kg had no impact on body weight during the treatment period, indicating no significant adverse effects on general health [1].
The manuscript notes that in a phase 1 trial, the development of an orismilast modified-release (MR) formulation attenuated gastrointestinal (GI)-related adverse events, which are common side effects of PDE4 inhibitors, in healthy volunteers [1].

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Orismilast (LEO-32731) is an orally active, selective PDE4 inhibitor that has been investigated for the research of inflammatory diseases, particularly plaque psoriasis. Its potent inhibition of PDE4B and PDE4D subtype splice variants makes it a valuable tool for studying the role of PDE4 in inflammation and for validating PDE4 as a therapeutic target. The compound's oral bioavailability and efficacy in preclinical models position it as a promising candidate for further development. Orismilast continues to be studied for its potential in inflammatory skin conditions and other immune-mediated diseases.

C19H15CL2F2NO7S

510.28

508.991

C, 44.72; H, 2.96; Cl, 13.89; F, 7.45; N, 2.74; O, 21.95; S, 6.28

1353546-86-7

54765967

White to off-white solid powder

1.7±0.1 g/cm3

788.8±60.0 °C at 760 mmHg

430.9±32.9 °C

0.0±2.8 mmHg at 25°C

1.649

0.82

0

9

5

32

788

0

C1CS(=O)(=O)CCC12OC3=C(C=CC(=C3O2)OC(F)F)C(=O)CC4=C(C=[N+](C=C4Cl)[O-])Cl

ZININGNRPUGNSL-UHFFFAOYSA-N

InChI=1S/C19H15Cl2F2NO7S/c20-12-8-24(26)9-13(21)11(12)7-14(25)10-1-2-15(29-18(22)23)17-16(10)30-19(31-17)3-5-32(27,28)6-4-19/h1-2,8-9,18H,3-7H2

2-(3,5-dichloro-1-oxidopyridin-1-ium-4-yl)-1-[7-(difluoromethoxy)-1',1'-dioxospiro[1,3-benzodioxole-2,4'-thiane]-4-yl]ethanone

LEO32731; Orismilast; UNII-JH1CX8SG5V; LEO-32,731; JH1CX8SG5V; 1353546-86-7; Orismilast; LEO 32731; LEO-32731

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 : ~100 mg/mL (~195.97 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)