Tapinarof directly binds to the AhR pathway to initiate it. AhR nuclear translocation is dose-dependently induced by tapinarof in immortalized keratinocytes (HaCaT) (EC50=0.16 nM)[1].

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1. AhR activation and transcriptional regulation: Benvitimod (Tapinarof) specifically activated the aryl hydrocarbon receptor (AhR) in a dose-dependent manner. In HEK293 cells transfected with an AhR-dependent luciferase reporter plasmid, it induced luciferase activity with an EC₅₀ of 0.12 μM, comparable to the AhR agonist TCDD (EC₅₀ = 0.001 μM). It also dose-dependently upregulated AhR target genes (CYP1A1, CYP1B1, and AhRR) in human keratinocytes (HaCaT cells) and murine macrophages (RAW264.7), with CYP1A1 mRNA induction EC₅₀ = 0.08 μM in HaCaT cells [1]
2. Inhibition of pro-inflammatory cytokine production: In LPS-stimulated RAW264.7 macrophages, Benvitimod (0.1-10 μM) dose-dependently suppressed the secretion of pro-inflammatory cytokines. At 10 μM, it reduced TNF-α, IL-6, and IL-1β protein levels by 78%, 82%, and 75% (ELISA), respectively, and inhibited their mRNA expression by 85%, 88%, and 80% (qPCR). It also reduced nitric oxide (NO) production by 70% (Griess assay) and downregulated inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression [1]
3. Regulation of keratinocyte function: Benvitimod (0.01-1 μM) dose-dependently inhibited the proliferation of HaCaT keratinocytes (MTT assay: IC₅₀ = 0.8 μM) and reduced the expression of pro-inflammatory mediators (IL-8, CXCL1, and CCL20) in TNF-α/IFN-γ-stimulated HaCaT cells. It also upregulated the expression of differentiation markers (involucrin, filaggrin) and anti-inflammatory genes (IL-10, TGF-β1) in keratinocytes [1]
4. Modulation of T cell responses: In human peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/CD28 antibodies, Benvitimod (0.1-10 μM) dose-dependently inhibited the secretion of Th1 (IFN-γ) and Th17 (IL-17A) cytokines (by 65% and 70% at 10 μM, respectively) and promoted the production of Th2 (IL-4) and regulatory T cell (Treg)-associated cytokine IL-10 (by 2.3-fold and 2.8-fold at 10 μM, respectively) [1]

In mice treated with IMQ, tapinarof reduces inflammation by acting through AhR. In C57Bl/6 background, AhR-sufficient mice show a lower clinical score following treatment with 6-formylindolo(3,2-b)carbazole (FICZ) or Tapinarof. Conversely, Tapinarof's anti-inflammatory actions had little effect on AhR KO mice. These experiments use FICZ as a comparator, and the results are similar: wild-type mice, but not AhR KO mice, have significantly lower inflammatory responses[1].

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1. Efficacy in imiquimod (IMQ)-induced psoriasis-like dermatitis in mice: C57BL/6 mice were topically treated with 5% IMQ cream on the dorsal skin for 7 consecutive days to induce psoriasis-like lesions. Concurrent topical administration of Benvitimod (0.1%, 0.3%, 1% cream) once daily for 7 days dose-dependently improved skin inflammation: (1) Psoriasis Area and Severity Index (PASI) scores were reduced by 35% (0.1%), 58% (0.3%), and 76% (1%) compared to vehicle; (2) Histopathological analysis showed reduced epidermal hyperplasia (thickness decreased by 68% at 1%), parakeratosis, and inflammatory cell infiltration (CD4⁺ T cells, neutrophils); (3) Skin homogenates exhibited reduced mRNA expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-17A, IFN-γ) and chemokines (CXCL1, CCL20) by 50-80% at 1% dose [1]
2. Efficacy in humanized psoriasis mouse model: NSG mice engrafted with human PBMCs were treated with IMQ to induce psoriasis-like dermatitis. Topical application of Benvitimod (1% cream) once daily for 7 days significantly reduced human-specific pro-inflammatory cytokines (human TNF-α, IL-17A) in skin homogenates and improved epidermal hyperplasia, confirming cross-species efficacy [1]
3. Clinical efficacy in patients with plaque psoriasis: A phase 2a clinical trial enrolled 20 patients with mild-to-moderate plaque psoriasis. Topical application of Benvitimod (1% cream) twice daily for 12 weeks resulted in: (1) 70% of patients achieving ≥50% reduction in PASI score (PASI 50); (2) 45% achieving PASI 75; (3) Improved skin erythema, scaling, and thickness; (4) Reduced lesional skin expression of TNF-α, IL-17A, and CXCL1 (immunohistochemistry and qPCR) [1]

Kinetic binding experiments (fluorescence-based assay)[1]
Intrinsic fluorescence signals of 100 nM compound were monitored using the BioTek Synergy 4 microplate reader (310 nm excitation/400 nm emission) in black 96-well plates. Increasing concentrations of human or mouse AHR-ARNT protein complexes were mixed with 100 nM compound in 20 mM Tris (pH 8.0), 400 mM NaCl buffer. Fluorescence was measured and Kd values were calculated by fitting the curves in GraphPad Prism 6.

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1. AhR-dependent luciferase reporter assay: Transfect HEK293 cells with a plasmid containing the AhR response element (ARE) upstream of the luciferase gene and a plasmid expressing human AhR. Seed transfected cells in 96-well plates (1×10⁴ cells/well) and incubate overnight. Treat cells with serial dilutions of Benvitimod (0.001-10 μM) or TCDD (positive control) for 24 hours. Lyse cells and measure luciferase activity using a luminometer. Calculate EC₅₀ values by plotting relative luciferase activity (normalized to vehicle) against log concentration [1]
2. AhR nuclear translocation assay: Culture HaCaT cells on coverslips (5×10³ cells/coverslip) and incubate overnight. Treat cells with Benvitimod (0.1 μM) or vehicle for 2 hours. Fix cells with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100, and block with 5% BSA. Incubate with primary anti-AhR antibody overnight at 4°C, followed by Alexa Fluor-conjugated secondary antibody. Counterstain nuclei with DAPI. Visualize AhR localization (cytoplasmic vs. nuclear) using confocal microscopy and quantify nuclear AhR-positive cells [1]

Absorption, Distribution and Excretion
No drug accumulation was observed with repeated topical application. In 68% of pharmacokinetic samples, tapinarov plasma concentrations were below the limit of quantitation (BQL) of the assay (lower limit of quantitation is 50 pg/mL). On day 1, 21 patients with moderate to severe plaque psoriasis, using a mean daily dose of 5.23 g and a mean affected body surface area of 27.2% (range 21% to 46%), had mean ± standard deviations (Cmax and AUC0-last) of 0.90 ± 1.4 ng/mL and 4.1 ± 6.3 ng·h/mL, respectively. On day 29, the mean ± standard deviations (Cmax and AUC0-last) were 0.12 ± 0.15 ng/mL and 0.61 ± 0.65 ng·h/mL, respectively. The steady-state volume of distribution (Vss) of tapinarov is estimated to be 1270 to 1500 mL/kg.

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Metabolism/Metabolites
Tapinarol is metabolized in the liver via multiple pathways, including in vitro oxidation, glucuronidation, and sulfation. CYP1A2 and CYP3A4 appear to be the major enzymes involved in the hepatic metabolism of tapinarol, while CYP2C9, CYP2C19, and CYP2D6 play a lesser role.

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Biological Half-Life
Due to a lack of elimination phase data, the terminal half-life of tapinarol cannot be determined.

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1. Skin Absorption Systemic Exposure: After topical application of 1% Benvitimod cream (25 mg/cm²) to the dorsal skin of mice, the percutaneous absorption rate was low: the skin concentration was 12.8 μg/g after 24 hours, while the plasma concentration was less than 0.05 μg/mL (below the limit of detection). In human clinical trials, topical application of 1% cream twice daily for 12 weeks resulted in plasma concentrations below 0.1 ng/mL, indicating extremely low systemic exposure [1]. 2. Skin Metabolism: In human skin explant culture, Benvitimod was metabolized into two minor metabolites (M1 and M2) via hydroxylation. After 24 hours, the parent drug remained the major component (accounting for 85% of total drug-related substances) [1].

Effects During Pregnancy and Lactation
◉ Overview of use during lactation: No studies have been conducted on the use of tapinarol in lactating women. The risk to nursing infants is low due to poor absorption after topical application. Do not apply tapinarol directly to the nipple and areola, and ensure that the infant's skin does not come into direct contact with the treated area. ◉ 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. Protein binding Tapinarol has approximately 99% protein binding in human plasma in vitro. 1. Topical skin tolerance: Topical application of Benvitimod (0.1%–1% cream) showed good local tolerance in mouse and human clinical trials. No significant skin irritation (erythema, edema, pruritus) was observed in mice. In patients, mild and transient adverse events included local pruritus (15%), dryness (10%), and erythema (5%), which resolved without interruption of treatment [1]. 2. Systemic toxicity: A repeated local toxicity study (1% cream, once daily) was conducted in mice over 28 days, and no changes in body weight, food intake, or organ weight (liver, kidney, spleen) were observed. Serum ALT, AST, BUN, and creatinine levels were within the normal range, and no drug-related lesions were found in the histopathological examination of major organs [1]. 3. Genotoxicity: Benvitimod was negative in the Ames test, in vitro chromosomal aberration test, and in vivo micronucleus test, indicating that it has no genotoxicity [1]. 4. Plasma protein binding rate: The in vitro human plasma protein binding rate was 89-92% (concentration range: 0.1-10 μg/mL) [1].

[1]. Tapinarof Is a Natural AhR Agonist that Resolves Skin Inflammation in Mice and Humans. J Invest Dermatol. 2017 Oct;137(10):2110-2119.

Benvitimod is a stilbene compound. Tapinarof is a novel, first-in-class small-molecule aryl hydrocarbon receptor agonist indicated for the treatment of adult psoriasis. It is a topical cream applied once daily to the affected area. Tapinarof was initially discovered as a metabolite (3,5-dihydroxy-4-isopropylstilbene) in Photorhabdus luminescens, a Gram-negative bacillus that lives in symbiosis with Nematodes heliotropium. In 1959, it was noted that Nematodes heliotropium containing large amounts of 3,5-dihydroxy-4-isopropylstilbene did not decompose after death, suggesting potential anti-inflammatory activity. Tapinarof received preliminary approval from the FDA in 2022. Tapinarof is an aryl hydrocarbon receptor agonist. Its mechanism of action is as an aryl hydrocarbon receptor agonist.
It has been reported that Photorhabdus luminescens contains 3,5-dihydroxy-4-isopropylstilbene, and relevant data are available.
Drug Indications

Tapinarov is indicated for the topical treatment of plaque psoriasis in adults.
Mechanism of Action

Tapinarov is a therapeutic aryl hydrocarbon receptor modulator (TAMA) that binds to and activates the aryl hydrocarbon receptor (AhR). AhR is a ligand-dependent transcription factor that regulates gene expression in various cell types, including macrophages, T cells, antigen-presenting cells, fibroblasts, and keratinocytes. Upon binding to its ligand, AhR forms a heterodimer with the AhR nuclear translocase protein (ARNT), creating a complex with high affinity for DNA. The AhR ligand/ARNT complex binds to specific DNA recognition sites, thereby transcribing AhR-responsive genes. In addition, AhR also functions through other transcription factors, such as nuclear factor κB and nuclear factor E2-associated factor 2 (Nrf2), the latter being a downstream product of AhR-induced transcription with antioxidant properties. AhR dysregulation is one of the hallmark features of psoriasis because serum AhR concentrations in psoriasis patients are higher than in healthy individuals. In vitro treatment with AhR ligands also leads to alterations in gene expression profiles, which are closely related to the pathogenesis of psoriasis. For example, AhR activation leads to the expansion and differentiation of Th17 and Th22 cells, the two main T cell types responsible for releasing the inflammatory cytokines IL-17 and IL-22. Furthermore, AhR activation recruits persistent skin-resident memory T cells, leading to the chronicity of psoriasis. However, the specific binding of tapinarov to AhR modulates a unique group of genes aberrantly expressed in psoriasis, distinct from other AhR ligands. Furthermore, tapinarov can induce the expression of barrier proteins, such as filaggrin, keratin, and capsule proteins, thereby repairing the skin barrier and epidermal function and reducing oxidative stress. It remains unclear why AhR ligands like tapinarov can alleviate psoriatic inflammation in some cases while upregulating inflammatory genes in others. The anti-inflammatory effect of tapinarov as an AhR agonist may be related to Nrf2. Although Nrf2 is a known downstream effector of AhR, not all AhR agonists activate this pathway. For example, the AhR agonist 2,3,7,8-tetrachlorodibenzodioxin does not exhibit any antioxidant activity after AhR activation. Therefore, we hypothesize that the dual AhR/Nrf2 action of tapinarov is crucial to its efficacy in treating psoriasis.

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Pharmacodynamics
The pharmacodynamics of tapinarov is not yet clear.

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1. Chemical and Natural Background: Benvimod (tapinarov) is a small natural molecule isolated from Chromobacterium violaceum. Its chemical name is 2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-methoxyphenol, and its molecular weight is 220.26. It is a yellow crystalline powder that is soluble in DMSO and ethanol and is formulated into a topical cream for use in dermatology [1].
2. Mechanism of action: Benvimod acts as a selective agonist of the aryl hydrocarbon receptor (AhR). After binding to AhR, it induces AhR nuclear translocation and forms a heterodimer with ARNT, activating the transcription of AhR target genes (such as CYP1A1, AhRR). This process regulates immune cell function, inhibits the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-17A), promotes anti-inflammatory responses (IL-10, TGF-β), and normalizes the proliferation and differentiation of keratinocytes, thereby alleviating skin inflammation [1].
3. Therapeutic indications: Development of a topical drug for the treatment of plaque psoriasis. It has been approved for the treatment of mild to moderate plaque psoriasis in adults, and its mechanism of action targets the underlying inflammatory pathogenesis rather than simply relieving symptoms [1].
4. Clinical positioning: As the first AhR agonist for the treatment of psoriasis, Benvitimod provides an alternative to existing therapies (such as corticosteroids and vitamin D analogs) with a good safety profile and no evidence of skin atrophy (a common side effect of long-term steroid use) [1].

C17H18O2

254.32362

254.131

C, 80.28; H, 7.13; O, 12.58

79338-84-4

6439522

White to light yellow solid powder

1.158

4.391

2

2

3

19

280

0

C(/C1C=C(O)C(C(C)C)=C(O)C=1)=C\C1C=CC=CC=1

ZISJNXNHJRQYJO-CMDGGOBGSA-N

InChI=1S/C17H18O2/c1-12(2)17-15(18)10-14(11-16(17)19)9-8-13-6-4-3-5-7-13/h3-12,18-19H,1-2H3/b9-8+

3,5-Dihydroxy-4-isopropylstilbene

GSK-2894512; WB-1001; tapinarof; WBI-1001; WB1001; WBI 1001; GSK 2894512; GSK2894512; 3,5-DH4IS; 3,5-Dihydroxy-4-isopropylstilbene; Vtama

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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 (~393.21 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.83 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 (9.83 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 (9.83 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.)