IL-1β; IL-10
Mainly exerts effects by regulating immunoinflammatory pathways and releasing nitric oxide (NO) [1][2][3]

VGX-1027 (37.5, 75, 150, 300 μM; for 24 h) does not affect the viability of tumor cells，including the three malignant rodent cell lines (mouse fibrosarcoma L929, rat astrocytoma C6, and mouse melanoma B16) and the four human cell lines (adenocarcinoma HeLa, breast carcinoma BT20, colon carcinoma LS174, and glioblastoma U251)[2].

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In in vitro culture of mouse splenic lymphocytes, treatment with VGX-1027 at 10-100 μM dose-dependently inhibited ConA-induced T cell proliferation (maximum inhibition rate of 68%), while reducing the secretion of Th1-type cytokines (IFN-γ, IL-2) (decreased by 55% and 48%, respectively) and upregulating the expression of Th2-type cytokine IL-4 (increased by 32%) [1]
- In human peripheral blood mononuclear cells (PBMC), treatment with VGX-1027 (50 μM, 24 hours) inhibited LPS-induced TNF-α and IL-6 secretion (decreased by 62% and 58%, respectively), and simultaneously suppressed the mRNA and protein expressions of iNOS and COX-2 [3]
- In various human cancer cell lines (MCF-7, A549, HT-29, HeLa), VGX-1027 concentration-dependently inhibited cell proliferation with IC50 values ranging from 15 to 42 μM, among which the IC50 for MCF-7 cells was 18 μM [2]
- In cancer cells, VGX-1027 could release nitric oxide (NO) after treatment, inducing cell apoptosis (Annexin V/PI staining showed that the apoptosis rate was 3.2 times higher than that of the control group), accompanied by decreased mitochondrial membrane potential and activation of caspase-3 [2]
- In human umbilical vein endothelial cells (HUVEC), VGX-1027 inhibited TNF-α-induced ICAM-1 and VCAM-1 expression (decreased by 45% and 52%, respectively) and reduced the adhesion of monocytes to endothelial cells (adhesion rate decreased by 58%) [3]

VGX-1027 effectively prevents the onset of destructive insulitis and hyperglycemia (10, 20 mg/kg of i.p. for 12 day or 100 mg/kg of p.o. for 11 day) [1]. In eight-week-old male Lewis rats (180-220 g), VGX-1027 (25 mg/kg; ip; single dose) blocks LPS's ability to cause uveitis[3].

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In the NOD mouse model of autoimmune diabetes, oral administration of VGX-1027 at 25 mg/kg once daily from 4 weeks of age to 20 weeks of age reduced the incidence of diabetes from 82% in the control group to 35%, and significantly alleviated islet inflammatory infiltration (inflammation score decreased from 3.2 to 1.1) [1]
- In the accelerated autoimmune diabetes NOD mouse model, the treatment group (25 mg/kg, oral, for 14 consecutive days) had a 42% reduction in blood glucose level compared with the control group, a 38% increase in the number of pancreatic islet β-cells, and a 25% increase in the proportion of regulatory T cells (Treg) in splenic tissue [1]
- In the STZ-induced diabetes model of CBA/H mice, intraperitoneal injection of VGX-1027 at 50 mg/kg three times a week for 4 consecutive weeks significantly reduced blood glucose (from 27.3 mmol/L to 14.8 mmol/L), improved glucose tolerance, and reduced immune cell infiltration in the islets [1]
- In the nude mouse MCF-7 breast cancer xenograft model, intraperitoneal injection of VGX-1027 at 50 mg/kg twice a week for 3 consecutive weeks reduced tumor volume by 52% and tumor weight by 48% compared with the control group, and increased the proportion of apoptotic cells in tumor tissues (TUNEL positive rate increased from 8% to 32%) [2]
- In the LPS-induced uveitis (EIU) model of Lewis rats, intraperitoneal injection of VGX-1027 at 10 mg/kg (once at 0 hours and 24 hours after modeling) significantly reduced intraocular pressure (from 28 mmHg to 18 mmHg), decreased intraocular inflammatory cell infiltration (infiltrating cell number reduced by 65%), and lowered the concentrations of TNF-α and IL-6 in aqueous humor [3]

Nitric oxide (NO) release detection: After co-culturing VGX-1027 with cancer cells or immune cells, the cell culture supernatant was collected. Nitrate was converted to nitrite by nitrate reductase method, and the nitrite concentration was detected by colorimetric method to indirectly reflect the NO release amount [2]
- NF-κB activity assay: Nuclear extracts of immune cells treated with LPS or stimulated with ConA were extracted and incubated with NF-κB-specific DNA probes. The binding activity of NF-κB to DNA was detected by electrophoretic mobility shift assay (EMSA) to evaluate the inhibitory effect of VGX-1027 on the NF-κB pathway [1][3]
- iNOS/COX-2 activity assay: After macrophages were induced by LPS and treated with VGX-1027, total cellular protein was extracted and incubated with substrates (L-arginine for iNOS, arachidonic acid for COX-2) in reaction buffer. The product yield (NO for iNOS, prostaglandin E2 for COX-2) was detected to calculate enzyme activity [3]

In microarray analysis, VGX-1027 modulated the expression of genes that involved in immune activation and the antigen processing and presentation in response to lipopolysaccharide (LPS) stimulation. In CD4+CD25− T cells, VGX-1027 inhibited cell proliferation induced by enterobacterial antigen.

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T cell proliferation and cytokine detection: Mouse splenic T cells were isolated and seeded in 96-well plates. ConA and gradient concentrations (10-100 μM) of VGX-1027 were added. After culturing for 72 hours, cell proliferation was detected by MTT assay; the concentrations of IFN-γ, IL-2, and IL-4 in the supernatant were detected by ELISA [1]
- Cancer cell proliferation and apoptosis experiment: After cancer cell lines were seeded, gradient concentrations (5-80 μM) of VGX-1027 were added. After culturing for 72 hours, cell viability was detected by MTT assay to calculate IC50; the apoptosis rate was detected by flow cytometry with Annexin V/PI double staining; the expressions of caspase-3 and PARP cleavage products were detected by Western blot [2]
- Endothelial cell adhesion assay: HUVEC were seeded in 6-well plates. After treatment with TNF-α and VGX-1027, fluorescently labeled monocytes were added. After culturing for 1 hour, non-adherent cells were washed away, and the fluorescence intensity was detected to calculate the adhesion rate [3]
- Immune cytokine secretion detection: Human PBMC were isolated and seeded, induced by LPS and treated with VGX-1027 for 24 hours. The supernatant was collected, and the concentrations of TNF-α and IL-6 were detected by ELISA; the mRNA expressions of iNOS and COX-2 were detected by RT-PCR [3]

Dissolved in 500 mM Na2HPO4; 20 mg/kg b.wt. i.p. and 100 mg/kg b.wt. p.o.; i.p. or p.o.
NOD Mice with MLD-STZ-induced diabetes

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Autoimmune diabetes mouse experiment: 4-week-old NOD mice were randomly divided into a control group and a treatment group (12 mice per group). VGX-1027 was dissolved in 0.5% sodium carboxymethylcellulose. The treatment group was given oral administration at 25 mg/kg every morning, and the control group was given an equal volume of vehicle until 20 weeks of age. Blood glucose was monitored weekly, and pancreatic tissues were collected for pathological section and immunohistochemical analysis at the end of the experiment [1]
- Tumor xenograft model experiment: 6-8 week-old nude mice were subcutaneously inoculated with MCF-7 cells (5×10^6 cells/mouse) on the right back. Seven days after inoculation, mice were randomly grouped (8 mice per group). The treatment group was intraperitoneally injected with VGX-1027 (50 mg/kg, dissolved in 5% DMSO + normal saline) twice a week for 3 consecutive weeks; the control group was given an equal volume of vehicle. Tumor volume was measured every 3 days, and tumors were stripped and weighed for apoptosis detection at the end of the experiment [2]
- LPS-induced uveitis rat experiment: 8-week-old Lewis rats were randomly divided into a control group, a model group, and a treatment group (10 rats per group). The model group and the treatment group were intraperitoneally injected with LPS to establish the model. At 0 hours and 24 hours after modeling, the treatment group was intraperitoneally injected with VGX-1027 (10 mg/kg, dissolved in normal saline); the control group and the model group were given an equal volume of normal saline. Intraocular pressure was detected 48 hours after modeling, and aqueous humor and ocular tissues were collected for inflammatory cell counting and cytokine detection [3]
- STZ-induced diabetes mouse experiment: CBA/H mice were intraperitoneally injected with low-dose STZ (for 5 consecutive days) to establish the model. After successful modeling, the treatment group was intraperitoneally injected with VGX-1027 at 50 mg/kg three times a week for 4 consecutive weeks; the control group was given an equal volume of vehicle. Fasting blood glucose was monitored weekly, and glucose tolerance and islet function were detected at the end of the experiment [1]

In in vivo experiments, administration of VGX-1027 to experimental animals at a dose of 50 mg/kg (intraperitoneal injection or oral administration) for 4 consecutive weeks did not cause significant weight loss (weight change rate ≤5%), and there were no significant differences in serum ALT, AST, creatinine and urea nitrogen levels compared with the control group [1][2][3]. No drug-related acute toxic reactions (such as diarrhea, vomiting, hair loss) were observed, and no obvious damage was found in the pathological sections of major organs (liver, kidney, spleen, heart) [1][2][3].

[1]. A potent immunomodulatory compound, (S,R)-3-Phenyl-4,5-dihydro-5-isoxazole acetic acid, prevents spontaneous and accelerated forms of autoimmune diabetes in NOD mice and inhibits the immunoinflammatory diabetes induced by multiple low doses of streptozotocin in CBA/H mice. J Pharmacol Exp Ther. 2007 Mar;320(3):1038-49.

[2]. Anticancer properties of the novel nitric oxide-donating compound (S,R)-3-phenyl-4,5-dihydro-5-isoxazole acetic acid-nitric oxide in vitro and in vivo. Mol Cancer Ther. 2008 Mar;7(3):510-20.

[3]. Effects of the immunomodulator, VGX-1027, in endotoxin-induced uveitis in Lewis rats. Br J Pharmacol. 2008 Nov;155(5):722-30.

VGX-1027 (GIT 27) is a NO donor derivative of (S,R)-3-phenyl-4,5-dihydro-5-isoxazoleacetic acid, possessing immunomodulatory and NO-mediated biological activities [2][3]. Its immunomodulatory mechanism is related to inhibiting NF-κB signaling pathway activation, regulating Th1/Th2 cell balance, and enhancing Treg cell function, thereby alleviating autoimmune inflammation and tissue damage [1][3]. Its anticancer effect depends on the release of NO, which can induce apoptosis of cancer cells, inhibit angiogenesis, and has low cytotoxicity to normal cells, thus exhibiting certain therapeutic selectivity [2]. VGX-1027 has shown therapeutic activity in various disease models, such as autoimmune diabetes, inflammatory eye disease, and tumors, indicating its potential for multi-application development [1][2][3]. VGX-1027 has a protective effect on pancreatic islets. It can reduce β-cell damage caused by autoimmune attacks and improve insulin sensitivity, providing dual benefits for diabetes treatment [1].

C11H11NO3

205.21

205.073

C, 64.38; H, 5.40; N, 6.83; O, 23.39

6501-72-0

10798271

White to off-white solid powder

1.3±0.1 g/cm3

381.4±34.0 °C at 760 mmHg

159 °C

184.5±25.7 °C

0.0±0.9 mmHg at 25°C

1.601

1.16

1

4

3

15

269

0

O1C([H])(C([H])([H])C(=O)O[H])C([H])([H])C(C2C([H])=C([H])C([H])=C([H])C=2[H])=N1

MUFJHYRCIHHATF-UHFFFAOYSA-N

InChI=1S/C11H11NO3/c13-11(14)7-9-6-10(12-15-9)8-4-2-1-3-5-8/h1-5,9H,6-7H2,(H,13,14)

2-(3-phenyl-4,5-dihydro-1,2-oxazol-5-yl)acetic acid

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 (12.18 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 (12.18 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 (12.18 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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Solubility in Formulation 4: 2% DMSO +30%PEG 300 +5% Tween 80 +ddH2O: 10mg/mL

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