NLRP3 inflammasome inhibitor. [1]
Farnesyltransferase inhibitor. [1]

By blocking farnesyl transferase, which activates the RAS proto-oncogene and is assumed to be a major factor in 20–30% of human cancers, arglabin exhibits its anti-tumor effect. In fact, it prevents farnesyl transferase (FTase) from incorporating farnesyl pyrophosphate into the human H-ras protein [2].

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Arglabin inhibited IL-1β and IL-18 secretion in lipopolysaccharide (LPS)-primed and cholesterol crystal (CC)-activated cultured mouse peritoneal macrophages in a concentration-dependent manner. The maximum effect was observed at ≈50 nmol/L, with EC50 values for both cytokines of ≈10 nmol/L. Cell viability remained >95% throughout the treatments. [1]
Arglabin did not significantly affect the production of IL-6 and IL-12 in LPS/arglabin-treated macrophages, indicating no general inhibition of NF-κB-dependent cytokines but rather specific inhibition of those depending on NLRP3 activation. [1]
Arglabin did not show a significant inhibitory effect at 50 nmol/L on the activity of NLRP1, AIM2, and NLRC4 inflammasomes, which were specifically activated by anthrax, double-stranded DNA, or flagellin, respectively. [1]
Arglabin reduced the secretion of IL-1α from LPS-primed and CC-activated mouse peritoneal macrophages in a dose-dependent manner (tested at 10, 25, and 50 nmol/L). [1]
In LPS-primed macrophages activated with ATP (5 mmol/L), a known NLRP3 activator, arglabin inhibited the increase in IL-1β production in a dose-dependent manner. [1]
The inhibitory effect of arglabin (50 nmol/L) on IL-1β and IL-18 production in LPS/CC-activated macrophages was reversible. Maximum inhibition was observed 72 hours after exposure, with a gradual loss of the inhibitory effect thereafter. [1]
Western blot analysis of whole-cell lysates from LPS-primed, CC-activated macrophages showed that treatment with arglabin (50 nmol/L) led to a complete abolition of mature caspase-1. Concomitantly, arglabin reduced the expression of NLRP3 and pro-IL-1β. Analysis of supernatants confirmed a reduction in the secretion of the active 17-kDa form of IL-1β. [1]
Arglabin induced autophagy in mouse macrophages. It strongly induced the accumulation of the LC3 type II protein, both in the absence and presence of inflammasome activators (LPS and CCs). Confocal microscopy showed that arglabin induced the clustering of LC3-II at autophagosomal membranes, evidenced by a punctuated pattern. This effect was similar to that of known autophagy inducers like amino acid-deficient media (EBSS) and bafilomycin A1. [1]

In ApoE2.Ki animals fed a high-fat diet, Arglabin decreased inflammation and plasma lipids, enhanced autophagy, and shifted tissue macrophages toward an anti-inflammatory phenotype [1].

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In female ApoE2.Ki mice fed a high-fat diet (HFD) for 13 weeks, intraperitoneal injection of arglabin (2.5 ng/g body weight, twice daily) significantly reduced the plasma level of IL-1β compared to vehicle-treated mice (5.2 ± 1.0 pg/mL vs. 11.7 ± 1.1 pg/mL). [1]
In the same mouse model, arglabin treatment significantly increased the plasma level of the anti-inflammatory cytokine IL-10 (29.7 ± 1.1 pg/mL vs. 12.1 ± 0.3 pg/mL in controls). The plasma level of TNF-α remained largely unaffected. [1]
Arglabin treatment significantly reduced plasma total cholesterol by 59% and triglycerides by 42% in ApoE2.Ki mice fed an HFD. This reduction was observed across all different fractions of lipoprotein particles (LDL, IDL, and VLDL). No effect on plasma lipids was observed in ApoE2.Ki/Nlrp3-/- mice fed an HFD and treated with arglabin. [1]
Arglabin treatment significantly reduced the plasma concentration of autoantibodies directed against oxidized LDL (oxLDL) by approximately 44% in ApoE2.Ki mice fed an HFD. In ApoE2.Ki/Nlrp3-/- mice, which had low baseline levels of anti-oxLDL antibodies even when fed an HFD, arglabin did not affect these levels. [1]
In ApoE2.Ki mice fed an HFD, arglabin treatment decreased the number of proinflammatory Ly-6C\(^{hi}\) monocytes/macrophages and increased the number of Ly-6C\(^{lo}\) cells in the spleen. This effect was not observed in similarly treated ApoE2.Ki/Nlrp3-/- mice. [1]
Immunohistochemical analysis of atherosclerotic lesions from ApoE2.Ki mice fed an HFD showed that arglabin treatment directed lesional macrophages from a proinflammatory M1 phenotype (F4/80+ IL-1β+) toward an anti-inflammatory M2 phenotype (F4/80+ CD206+). [1]
Arglabin treatment significantly reduced the median atherosclerotic lesion area in the aortic sinus by 54% (P = 0.02) and in the whole aorta (en face) by 41% (P = 0.02) in ApoE2.Ki mice fed an HFD. This inhibitory effect was comparable to the lesion size observed in untreated ApoE2.Ki/Nlrp3-/- mice fed an HFD. Treatment of ApoE2.Ki/Nlrp3-/- mice with arglabin did not further reduce the lesion area. [1]

IL-1β, IL-18, IL-6, IL-12 Cytokine Assay: Mouse peritoneal macrophages were isolated from C57Bl/6 mice, seeded at a density of 0.5 x 10\(^6\) cells/mL, and primed with LPS (10 ng/mL) for 4 hours. They were then treated with various concentrations of arglabin for 1 hour, followed by activation with cholesterol crystals (1 mg/mL). After 24 hours, the levels of IL-1β, IL-18, IL-6, and IL-12 in the culture supernatants were quantified by ELISA. [1]
NLRP3 Dependency Assay: Peritoneal macrophages isolated from Nlrp3+/+, Nlrp3+/−, or Nlrp3−/− mice (C57BL/6 background) were primed with LPS (10 ng/mL) and activated with cholesterol crystals (1 mg/mL). The levels of IL-1β and IL-18 in the supernatants were then measured by ELISA to confirm the specific role of NLRP3. [1]
IL-1α Cytokine Assay: Mouse peritoneal macrophages were primed with LPS (10 ng/mL) and activated with cholesterol crystals (1 mg/mL) in the presence of different doses of arglabin (10, 25, and 50 nmol/L). The levels of IL-1α secreted into the supernatants were measured. [1]
ATP-induced NLRP3 Activation Assay: Cells were primed with LPS (10 ng/mL) for 4 hours. Arglabin or vehicle was then added at various concentrations. One hour later, cells were activated with ATP (5 mmol/L) for 24 hours. IL-1β production was measured by ELISA. [1]
Inflammasome Specificity Assay: Cultured macrophages were stimulated with specific activators: anthrax to activate NLRP1, double-stranded DNA to activate AIM2, or flagellin to activate NLRC4. The effect of arglabin at 50 nmol/L on the activity of these inflammasomes was then assessed. [1]
Reversibility Assay: Peritoneal macrophages were first treated with LPS (10 ng/mL). Arglabin (50 nmol/L) was added 1 hour before activation with cholesterol crystals (1 mg/mL). The levels of IL-1β and IL-18 in the supernatants were evaluated by ELISA at different time points post-activation. [1]
Western Blot Analysis (Cellular Proteins): Cells were pretreated for 2 hours with LPS (10 ng/mL) and then treated with arglabin (50 nmol/L) or left untreated. One hour later, cholesterol crystals (1 mg/mL) were added to all samples, and cells were incubated for an additional 6 hours. Whole-cell lysates were then analyzed by Western immunoblotting for the expression of NLRP3, pro-IL-1β, procaspase-1, and active caspase-1, with actin as a loading control. [1]
Western Blot Analysis (Secreted IL-1β): Cells were treated as described above for 24 hours. The supernatants were collected and analyzed by Western immunoblotting using an antibody that specifically recognizes the cleaved, active, 17-kDa form of IL-1β. [1]
Autophagy Assay (LC3-II Accumulation): Cells were treated with arglabin for 24 hours. Bafilomycin A1 (30 nmol/L), which blocks autophagosome-lysosome fusion, was used as a positive control. Whole-cell lysates were analyzed by Western immunoblotting using an antibody against LC3 type II. In a separate experiment, cells were pretreated with LPS for 2 hours, treated with arglabin (50 nmol/L) or left untreated for 1 hour, and then activated with cholesterol crystals for an additional 6 hours. Cells incubated in amino acid-deficient medium (EBSS), known to induce autophagy, served as a positive control. Lysates were again analyzed for LC3-II formation. [1]
Confocal Microscopy for Autophagy: Cells were incubated with arglabin for 24 hours. They were then permeabilized, stained with an LC3-II antibody (green fluorescence) and DAPI for nuclei (blue fluorescence), and analyzed by confocal microscopy at an original magnification of x630 to visualize the formation of cellular autophagosome punctae containing LC3-II. [1]

In Vivo Efficacy in ApoE2.Ki Mice:** Female ApoE2.Ki mice, 6 weeks of age, were fed a high-fat diet for 13 weeks. They were randomly divided into two groups. The first group (control, n=4) received intraperitoneal injections of 5 µL dimethyl sulfoxide (vehicle) twice daily. The second group (arglabin, n=4) received intraperitoneal injections of arglabin at a dose of 2.5 ng/g body weight, also twice daily. After 13 weeks, under isoflurane anesthesia, blood was collected in EDTA tubes for plasma analysis of cytokines, lipids, and anti-oxLDL antibodies. Hearts and descending aortas were excised for histological and immunohistochemical studies. [1]
* **In Vivo Studies with Nlrp3-/- Mice:** Female ApoE2.Ki/Nlrp3-/- mice, 6 weeks of age, were also fed a high-fat diet for 13 weeks. One group was treated with vehicle, and another group was treated with arglabin (2.5 ng/g body weight, twice daily) following the same protocol as above, to assess the dependency of arglabin's effects on NLRP3. [1]
* **Atherosclerotic Lesion Analysis:** After euthanasia, hearts were cut directly under and parallel to the leaflets. The upper portions were embedded in optimal cutting temperature medium. One hundred serial cryosections, each 10 µm thick, were prepared from the top of the left ventricle. Ten sections, each separated by 90 µm, were stained for lipids with Oil Red O and counterstained with Harris hematoxylin. The mean lesion size in these 10 sections was used to evaluate the lesion size in the aortic sinus for each animal. For the whole aorta (en face), the descending thoracic and abdominal aorta were cleaned, processed, and stained with Oil Red O. The extent of atherosclerosis was assessed with computer-assisted histomorphometry. [1]
* **Immunohistochemistry for Macrophage Phenotyping:** Frozen serial sections (10 µm) of proximal aortas from control and arglabin-treated ApoE2.Ki mice fed an HFD were analyzed by immunohistochemistry. This involved double immunostaining with antibodies against the macrophage marker F4/80 combined with either the M1 marker IL-1β or the M2 marker CD206. The number of positive cells within a high-powered field was quantified. [1]

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In Vivo Efficacy in ApoE2.Ki Mice: Female ApoE2.Ki mice, 6 weeks of age, were fed a high-fat diet for 13 weeks. They were randomly divided into two groups. The first group (control, n=4) received intraperitoneal injections of 5 µL dimethyl sulfoxide (vehicle) twice daily. The second group (arglabin, n=4) received intraperitoneal injections of arglabin at a dose of 2.5 ng/g body weight, also twice daily. After 13 weeks, under isoflurane anesthesia, blood was collected in EDTA tubes for plasma analysis of cytokines, lipids, and anti-oxLDL antibodies. Hearts and descending aortas were excised for histological and immunohistochemical studies. [1]
In Vivo Studies with Nlrp3-/- Mice: Female ApoE2.Ki/Nlrp3-/- mice, 6 weeks of age, were also fed a high-fat diet for 13 weeks. One group was treated with vehicle, and another group was treated with arglabin (2.5 ng/g body weight, twice daily) following the same protocol as above, to assess the dependency of arglabin's effects on NLRP3. [1]
Atherosclerotic Lesion Analysis: After euthanasia, hearts were cut directly under and parallel to the leaflets. The upper portions were embedded in optimal cutting temperature medium. One hundred serial cryosections, each 10 µm thick, were prepared from the top of the left ventricle. Ten sections, each separated by 90 µm, were stained for lipids with Oil Red O and counterstained with Harris hematoxylin. The mean lesion size in these 10 sections was used to evaluate the lesion size in the aortic sinus for each animal. For the whole aorta (en face), the descending thoracic and abdominal aorta were cleaned, processed, and stained with Oil Red O. The extent of atherosclerosis was assessed with computer-assisted histomorphometry. [1]
Immunohistochemistry for Macrophage Phenotyping: Frozen serial sections (10 µm) of proximal aortas from control and arglabin-treated ApoE2.Ki mice fed an HFD were analyzed by immunohistochemistry. This involved double immunostaining with antibodies against the macrophage marker F4/80 combined with either the M1 marker IL-1β or the M2 marker CD206. The number of positive cells within a high-powered field was quantified. [1]

In cultured mouse peritoneal macrophages treated with arglabin (up to 50 nmol/L), cell viability remained greater than 95% throughout the duration of the experiments, indicating no acute cytotoxicity at the concentrations used. [1]
Arglabin treatment did not result in any observable difference in hepatic steatosis between treated and untreated ApoE2.Ki mice fed an HFD. [1]
Arglabin did not show an inhibitory effect on intracellular cholesterol biosynthesis in hepatic cells, nor did it affect the expression of LDL receptors in these cells. [1]
The study notes that arglabin, similar to helenalin, has a γ-butyrolactone ring that can be opened by nucleophiles, suggesting a mode of action that involves forming adducts with biological nucleophiles. This chemical reactivity is considered integral to its biological activity. [1]

[1]. Anti-inflammatory and antiatherogenic effects of the NLRP3 inflammasome inhibitor arglabin in ApoE2.Ki mice fed a high-fat diet. Circulation. 2015;131(12):1061-1070.

[2]. Arglabin: From isolation to antitumor evaluation. Chem Biol Interact. 2015;240:180-198.

Arglabin is an organic heterotetracyclic compound belonging to the guaiacene sesquiterpene lactone family. Its structure involves the substitution of acrylic acid at the 2-position with a 4-hydroxy-3,8-dimethyl-1,3a,4,5,6,7-hexahydroazine-5-yl group, where the double bond on the seven-membered ring is epoxidized, and the hydroxyl and carboxyl groups undergo a condensation reaction to generate the corresponding γ-lactone. It is found in plants of the genus Artemisia glabella. Arglabin-DMA HCl is the hydrochloride salt of an adduct of dimethylamine and an enyl lactone, and has been successfully used in Kazakhstan for the treatment of breast cancer, colon cancer, ovarian cancer, and lung cancer. It is both an antitumor drug and a metabolite. It is an organic heterotetracyclic compound, a γ-lactone, an epoxide, and a sesquiterpene lactone. Arglabin has been reported in Artemisia argyi and Pentzia eenii, and available data are available.

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Arglabin is a natural guaianolide sesquiterpene lactone primarily synthesized by the plant Artemisia glabella, which grows exclusively in Kazakhstan. [1]
Its chemical structure consists of a cyclopentane ring with 5 contiguous stereocenters to which 5-membered rings are transannulated. [1]
It exhibits anti-inflammatory and antitumor activity. A dimethylamino hydrochloride derivative of arglabin is being explored in Kazakhstan for the treatment of breast, colon, ovarian, and lung cancer. [1]
In this study, arglabin is identified as a promising new drug candidate to treat inflammation and atherosclerosis by inhibiting the NLRP3 inflammasome, reducing plasma lipids, inducing autophagy, and polarizing macrophages towards an anti-inflammatory M2 phenotype. [1]
The anti-atherogenic effect of arglabin in ApoE2.Ki mice was found to be comparable to the effect observed with Nlrp3 gene deficiency, suggesting its primary action is through NLRP3 inflammasome inhibition. [1]
The reduction in plasma cholesterol and triglycerides by arglabin was an unexpected finding. The authors hypothesize that it might result from inhibition of caspase-1, which has been linked to intestinal lipid absorption and hepatic lipid metabolism. [1]

C15H18O3

246.31

246.125

84692-91-1

5574924

White to light yellow solid powder

1.2±0.1 g/cm3

404.6±45.0 °C at 760 mmHg

102 °C

171.3±23.3 °C

0.0±0.9 mmHg at 25°C

1.571

1.74

0

3

0

18

506

5

CC1=CC[C@@]23[C@H]1[C@@H]4[C@@H](CC[C@@]2(O3)C)C(=C)C(=O)O4

UVJYAKBJSGRTHA-CUZKYEQNSA-N

InChI=1S/C15H18O3/c1-8-4-7-15-11(8)12-10(9(2)13(16)17-12)5-6-14(15,3)18-15/h4,10-12H,2,5-7H2,1,3H3/t10-,11+,12-,14-,15+/m0/s1

(1R,3S,6S,10S,11R)-3,12-dimethyl-7-methylidene-2,9-dioxatetracyclo[9.3.0.01,3.06,10]tetradec-12-en-8-one

Arglabin (+)-Arglabin

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 (~406.01 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.15 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 (10.15 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 (10.15 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.)