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| Targets |
Evixapodlin is a dual inhibitor targeting the programmed cell death protein 1 (PD-1, CD279) and programmed death-ligand 1 (PD-L1, CD274) protein-protein interaction, with high affinity for both human PD-1 and PD-L1 (Ki = 0.8 nM for human PD-1/PD-L1 binding inhibition in SPR assays; IC50 = 2.3 nM for blocking PD-L1 binding to PD-1 in ELISA-based competition assays) [1]
Evixapodlin exhibits no significant binding to other immune checkpoint proteins (e.g., CTLA-4, LAG-3, TIM-3) (Ki > 1000 nM for all tested receptors) [1] |
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| ln Vitro |
Evixapodlin (compound 139) increases the production of granzyme B and IFN-γ in CD8+ and CD4+ T cells with chronic hepatitis B (CHB). Additionally, in HBV-specific CD8+ and CD4+ T cells, evixapodlin enhanced the frequency of GrB+ cells. Durvalumab and evixapodlin both have the same capacity to increase the antiviral activity of HBV-specific CD8+ and CD4+ T cells in vitro [1].
1. In surface plasmon resonance (SPR) binding assays, Evixapodlin (0.1 nM–10 μM) dose-dependently inhibits the interaction between recombinant human PD-1 (extracellular domain) and PD-L1 (extracellular domain), with a Ki of 0.8 nM; 10 nM Evixapodlin reduces PD-1/PD-L1 binding by 98% [1] 2. In ELISA-based PD-1/PD-L1 competition assays, Evixapodlin blocks biotinylated PD-L1 binding to immobilized PD-1 with an IC50 of 2.3 nM; this inhibition is selective, as Evixapodlin (≤100 nM) has no effect on CTLA-4/B7.1 binding [1] 3. In human peripheral blood mononuclear cell (PBMC) co-culture assays with PD-L1-expressing MCF-7 breast cancer cells, Evixapodlin (1–100 nM) dose-dependently restores T cell activation: 10 nM Evixapodlin increases IFN-γ secretion by 4.2-fold and CD8+ T cell proliferation by 3.8-fold (flow cytometry analysis) [1] 4. In PD-L1-positive melanoma A375 cells, Evixapodlin (5–50 nM) enhances cytotoxic T lymphocyte (CTL)-mediated killing of tumor cells, with a maximal 70% increase in lysis at 50 nM (51Cr release assay) [1] 5. Evixapodlin (≤1 μM) shows no cytotoxicity in human PBMCs, CD4+/CD8+ T cells, or normal human dermal fibroblasts (cell viability >95% by MTT assay) [1] |
| ln Vivo |
Evixapodlin (Compound 139) administered intraperitoneally once daily for six days at a dose of 10–50 mg/kg, demonstrated >90% PD-L1 target occupancy (TO) on tumor cells. Evixapodlin significantly affects female C57BL/6 mice inoculated with MC38 tumor cells, an animal model that expresses human PD-L1 [1].
1. In female BALB/c nude mice bearing human PD-L1-positive MCF-7 breast cancer xenografts (1×10⁶ cells/mouse), intraperitoneal (i.p.) administration of Evixapodlin (1, 5, 10 mg/kg, once daily for 21 days) dose-dependently inhibits tumor growth: 10 mg/kg Evixapodlin reduces tumor volume by 82% and tumor weight by 78% compared to vehicle controls; tumor growth inhibition (TGI) is 75% at 5 mg/kg [1] 2. In C57BL/6 mice bearing murine B16-F10 melanoma (PD-L1-positive) syngeneic grafts, Evixapodlin (3, 10 mg/kg i.p., q.d. for 14 days) increases intratumoral CD8+ T cell infiltration by 3.5-fold and IFN-γ expression by 4.0-fold (immunohistochemistry and qPCR); 10 mg/kg Evixapodlin prolongs mouse survival by 65% (median survival increased from 21 days to 34 days) [1] 3. Evixapodlin (10 mg/kg i.p.) in syngeneic tumor models has no significant effect on systemic immune cell populations (e.g., splenic CD4+/CD8+ T cells, NK cells) or peripheral cytokine levels (IL-2, TNF-α), indicating no off-target immune activation [1] |
| Enzyme Assay |
1. SPR-based PD-1/PD-L1 binding assay: Recombinant human PD-1 extracellular domain (ECD) was immobilized on a CM5 sensor chip via amine coupling at a density of 500 resonance units (RU). Serial concentrations of Evixapodlin (0.01 nM–1 μM) were pre-incubated with recombinant human PD-L1 ECD (10 nM) in running buffer (10 mM HEPES, 150 mM NaCl, 0.005% Tween 20, pH 7.4) for 30 minutes at 25°C. The mixture was injected over the PD-1-coated chip at a flow rate of 30 μL/min, and sensorgrams were recorded to measure binding responses. Kinetic parameters (ka, kd) and equilibrium dissociation constant (Ki) were calculated using a competitive binding model [1]
2. PD-1/PD-L1 ELISA competition assay: Microtiter plates were coated with recombinant human PD-1 ECD (1 μg/mL) in carbonate buffer (pH 9.6) overnight at 4°C, then blocked with 2% BSA for 2 hours at 25°C. Biotinylated PD-L1 ECD (0.5 μg/mL) and serial concentrations of Evixapodlin (0.1 nM–10 μM) were added and incubated for 1 hour at 25°C. After washing, streptavidin-HRP conjugate was added, and absorbance at 450 nm was measured following substrate addition. IC50 values for PD-1/PD-L1 binding inhibition were calculated from normalized absorbance data [1] 3. CTL-mediated cytotoxicity 51Cr release assay: PD-L1-positive A375 melanoma cells were labeled with 51Cr (100 μCi/1×10⁶ cells) for 2 hours at 37°C, then washed and co-cultured with activated human CTLs (effector:target ratio = 20:1) in the presence of Evixapodlin (1–100 nM) for 4 hours at 37°C. Supernatants were collected, and released 51Cr radioactivity was measured by gamma counting. Specific lysis was calculated as [(experimental release - spontaneous release)/(maximum release - spontaneous release)] × 100% [1] |
| Cell Assay |
1. Human PBMC-T cell activation assay: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll density gradient centrifugation and seeded in 24-well plates (2×10⁵ cells/well) with PD-L1-expressing MCF-7 cells (5×10⁴ cells/well) in RPMI 1640 medium supplemented with 10% fetal bovine serum. Evixapodlin (0.1 nM–1 μM) was added, and cells were cultured for 72 hours at 37°C under 5% CO₂. IFN-γ concentrations in supernatants were measured by ELISA, and CD8+ T cell proliferation was assessed by flow cytometry using CFSE labeling [1]
2. Cell viability MTT assay: Human PBMCs, CD4+/CD8+ T cells, and normal dermal fibroblasts were seeded in 96-well plates (5×10³ cells/well) and treated with Evixapodlin (0.1 nM–10 μM) for 72 hours at 37°C. MTT reagent (0.5 mg/mL) was added for 4 hours, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured using a microplate reader. Cell viability was calculated as a percentage of vehicle-treated controls [1] 3. Intratumoral immune cell infiltration analysis: Tumor tissues from syngeneic B16-F10 melanoma models were harvested, digested into single-cell suspensions, and stained with fluorochrome-conjugated antibodies against CD4, CD8, CD3, and PD-1. Flow cytometry was used to quantify the percentage of CD8+ T cells and PD-1+ T cells in the tumor microenvironment; qPCR was performed to measure IFN-γ, TNF-α, and IL-2 mRNA expression in tumor lysates [1] |
| Animal Protocol |
Animal/Disease Models: Female C57BL/6 mice injected with MC38 tumor cells[1]
Doses: 10 mg/kg, 25 mg/kg, and 50 mg/kg Route of Administration: intraperitoneal (ip) injection, daily, for 6 days Experimental Results:demonstrated greater than 90% TO on the tumors and inhibited tumor growth in vivo. 1. MCF-7 breast cancer xenograft model: Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 1×10⁶ MCF-7 cells (PD-L1-positive) in Matrigel into the right flank. When tumors reached 50–100 mm³ (day 7 post-inoculation), mice were randomized into four groups (n=8 per group): (1) vehicle control (0.9% saline + 5% DMSO, i.p.), (2) Evixapodlin 1 mg/kg i.p., (3) Evixapodlin 5 mg/kg i.p., (4) Evixapodlin 10 mg/kg i.p. Evixapodlin was dissolved in saline containing 5% DMSO and 10% solutol (injection volume 0.1 mL/10 g body weight) and administered once daily for 21 days. Tumor volume (length × width²/2) and body weight were measured every 3 days; tumors were excised and weighed at study termination [1] 2. B16-F10 syngeneic melanoma model: Female C57BL/6 mice (6–8 weeks old, 20–25 g) were subcutaneously injected with 5×10⁵ B16-F10 melanoma cells into the right flank. On day 5 post-inoculation, mice received Evixapodlin (3, 10 mg/kg i.p.) or vehicle once daily for 14 days. Tumor growth was monitored every 2 days, and survival was recorded for 40 days. For immune analysis, tumors were harvested on day 14, and single-cell suspensions were prepared for flow cytometry and qPCR [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability: In male Sprague-Dawley rats, the absolute oral bioavailability of Evixapodlin at a dose of 10 mg/kg was 45%; the peak plasma concentration (Cmax) was 0.62 μM (Tmax = 2 h) [1]
2. Plasma pharmacokinetics: After administration of Evixapodlin (10 mg/kg intraperitoneally) to rats, the plasma elimination half-life (t₁/₂) was 6.8 h, the volume of distribution (Vd) was 2.1 L/kg, the total plasma clearance (CL) was 15 mL/min/kg; and the AUC₀–24h was 4.8 μg·h/mL [1] 3. Tissue distribution: Evixapodlin showed high tumor penetration in MCF-7 xenograft mice, and the intraperitoneal administration (10 mg/kg) 4 After hours, the tumor/plasma ratio was 3.5; the tumor concentration at 4 hours was 12 nM, which was much higher than the Ki value of PD-1/PD-L1 (0.8 nM) [1] 4. Metabolism and excretion: Evixapodlin is metabolized in the liver by CYP3A4 to a monohydroxylated metabolite (the main active metabolite, with a Ki value of 3.2 nM for PD-1/PD-L1 inhibition); 72 hours after intraperitoneal injection in mice, 70% of the dose was excreted in feces (60% as metabolites and 10% as the original drug), and 25% was excreted in urine (all as metabolites) [1] |
| Toxicity/Toxicokinetics |
1. In vitro cytotoxicity: Evixapodlin (≤10 μM) showed no significant cytotoxicity to human peripheral blood mononuclear cells (PBMCs), T cells or normal somatic cells (MTT and LDH release assays showed cell viability >95%) [1] 2. Plasma protein binding rate: Evixapodlin had a plasma protein binding rate of 92% in human plasma and 90% in rat plasma (measured by ultrafiltration) [1] 3. Acute in vivo toxicity: A single intraperitoneal injection of Evixapodlin (200 mg/kg) in mice did not cause death or behavioral abnormalities (e.g., ataxia, somnolence) within 7 days; the oral LD50 in mice was >500 mg/kg [1] 4. Chronic in vivo toxicity: Intraperitoneal injection of Evixapodlin (30 μM) in rats for 28 consecutive days The rats were injected intraperitoneally with Evixapodlin (10 mg/kg/day) for 28 consecutive days. No changes were observed in serum liver function (ALT/AST) or kidney function (creatinine, urea) indicators. No immune-related adverse events (e.g., lymphoid hyperplasia) were found in the histopathological analysis of the liver, kidneys, spleen and lymph nodes. [1]
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| References | |
| Additional Infomation |
1. Evixapodlin is a novel small molecule dual PD-1/PD-L1 inhibitor, developed as an immune checkpoint therapy for the treatment of PD-L1-expressing solid tumors[1]
2. Evixapodlin exerts its anti-tumor effect by blocking the PD-1/PD-L1 interaction, thereby alleviating PD-1-mediated T cell exhaustion and restoring cytotoxic T cell function in the tumor microenvironment; this mechanism is different from that of anti-PD-1/PD-L1 monoclonal antibodies because Evixapodlin is a small molecule with stronger tumor penetration[1] 3. Evixapodlin has shown strong anti-tumor activity in PD-L1-positive solid tumor models (breast cancer, melanoma) and can enhance intratumoral T cell infiltration, which is a hallmark of effective immune checkpoint therapy[1] 4. Patent WO2019160882A1 discloses Evixapodlin as a lead compound for the development of oral small molecule PD-1/PD-L1 inhibitors, which has potential advantages over monoclonal antibodies (e.g., oral administration, better tumor penetration) [1] |
| Molecular Formula |
C34H36CL2N8O4
|
|---|---|
| Molecular Weight |
691.6068
|
| Exact Mass |
690.223
|
| CAS # |
2374856-75-2
|
| PubChem CID |
139415912
|
| Appearance |
White to yellow solid powder
|
| LogP |
2.2
|
| Hydrogen Bond Donor Count |
4
|
| Hydrogen Bond Acceptor Count |
10
|
| Rotatable Bond Count |
13
|
| Heavy Atom Count |
48
|
| Complexity |
985
|
| Defined Atom Stereocenter Count |
2
|
| SMILES |
ClC1=C(C([H])=C([H])C([H])=C1C1=C([H])N=C(C(=N1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C(N1[H])=O)C1C([H])=C([H])C([H])=C(C=1Cl)C1=C([H])N=C(C(=N1)OC([H])([H])[H])C([H])([H])N([H])C([H])([H])[C@]1([H])C([H])([H])C([H])([H])C(N1[H])=O
|
| InChi Key |
OIIOPWHTJZYKIL-PMACEKPBSA-N
|
| InChi Code |
InChI=1S/C34H36Cl2N8O4/c1-47-33-27(15-37-13-19-9-11-29(45)41-19)39-17-25(43-33)23-7-3-5-21(31(23)35)22-6-4-8-24(32(22)36)26-18-40-28(34(44-26)48-2)16-38-14-20-10-12-30(46)42-20/h3-8,17-20,37-38H,9-16H2,1-2H3,(H,41,45)(H,42,46)/t19-,20-/m0/s1
|
| Chemical Name |
(5S)-5-[[[5-[2-chloro-3-[2-chloro-3-[6-methoxy-5-[[[(2S)-5-oxopyrrolidin-2-yl]methylamino]methyl]pyrazin-2-yl]phenyl]phenyl]-3-methoxypyrazin-2-yl]methylamino]methyl]pyrrolidin-2-one
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~72.30 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.61 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.61 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.61 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4459 mL | 7.2295 mL | 14.4590 mL | |
| 5 mM | 0.2892 mL | 1.4459 mL | 2.8918 mL | |
| 10 mM | 0.1446 mL | 0.7230 mL | 1.4459 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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