PC-PLC (competitive inhibitor; no IC50/Ki/EC50/DC50 values reported in this study)
SMS1 and SMS2 (inhibitor; no IC50/Ki/EC50/DC50 values reported in this study) [2]

D609 at 100 μM for two hours greatly reduces edema in several cell lines [2]. After activating caspase-3 at 200 μM for two hours, D609 (100 μM; two hours) at 50, 100 μM significantly inhibited BrdU in BV-2 astrocytes. It occurs annually and results in a decrease in the number of cells in the S phase and an accumulation of cells in the G1 phase [2]. 100 μM; for two hours, then for two more hours or for twenty-two hours without D609) raises ceramide levels, boosts p21 expression, and causes phosphorylated Rb to drop [2].

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D609 (100 μM, 2 h treatment + 22 h chase) significantly attenuated proliferation of RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC1 astrocytes without affecting cell viability (>90% by trypan blue exclusion). [2]
D609 (100 μM, 2 h treatment + 2 h BrdU chase) significantly inhibited BrdU incorporation in BV-2 microglia, indicating fewer cells progressing into S-phase. [2]
Flow cytometric analysis: D609 (100 μM, 2 h treatment + 2 h chase) caused accumulation of BV-2 cells in G1 phase (54.3±1.5% vs control 43.4±3.9%) and decreased cells in S phase (39.9±3.3% vs control 48.8±2.4%). [2]
D609 (100 μM, 2 h) significantly increased ceramide levels in BV-2 microglia (primarily palmitic C16:0, lignoceric C24:0, and nervonic C24:1 ceramides), which remained elevated 2 h after removal and returned to control levels 22 h later. [2]
Western blot: D609 (100 μM, 2 h) increased expression of Cdk inhibitor p21 and down-regulated phospho-retinoblastoma (Rb) in BV-2 microglia; both returned to basal levels 22 h after D609 removal. [2]
D609 did not induce caspase-3 cleavage at 50 or 100 μM for 2 h, but 200 μM for 2 h induced apoptosis. 100 μM D609 for 8 h or longer induced caspase-3 activation. [2]
D609 treatment significantly increased DAG levels in BV-2 cells (control 1.77±0.17 nmol/mg protein vs 2 h D609 2.14±0.08 nmol/mg protein, p<0.05). [2]
Exogenous C8-ceramide (30 μM, 24 h) significantly inhibited BV-2 proliferation without inducing cell death (viability: control 94±0.2% vs C8-ceramide 93±0.5%); 20 μM had no effect, 40 μM caused significant cell death. [2]
C8-ceramide (30 μM, 6 h + 2 h BrdU chase) significantly inhibited BrdU incorporation; 2 h or 4 h treatment had no significant effect. [2]

In apoE-/-mice, D609 (2.5, 10 mg/kg/day; i.p.; for 6 weeks) inhibits the progression of preexisting atherosclerotic lesions, changing the event component to a more stable phenotype [3 LPS administered intratracheally (30 mg/kg; i.p.; single dose) 30 minutes prior to LPS (3 mg/kg) prevents LPS-induced pulmonary hypertension in Island Wistar [4]. C57BL/6 WT and apoE−/− 26-week-old mice [3]
Previous studies from the same group showed that D609 treatment decreased cerebral infarct volume in a rat middle cerebral artery occlusion stroke model, attributed to cell cycle regulation via up-regulation of p21. The protective effect was associated with inhibition of cell cycle progression. (Detailed in vivo experimental protocol not described in this paper; refer to Adibhatla and Hatcher 2010 Mol Neurobiol 41:206-217) [2]

PC-PLC enzymatic activity was measured in serum samples from WT and apoE-/- mice. The mean value of serum PC-PLC activity of WT mice was normalized to 1.0; apoE-/- mice showed 2.6±0.56-fold higher activity (P<0.05). [3]
PC-PLC enzymatic activity was measured in total cell lysates of HUVEC. oxLDL-mediated PC-PLC enzymatic activation was completely abrogated in HUVEC pretreated with D609. [3]
PC-PLD enzymatic activity was detected using the Amplex Red assay to confirm specificity of D609 inhibition. PC-PLD activity did not increase after oxLDL treatment and was not affected by D609. [3]

Cell Proliferation Assay[2]
Cell Types: RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC1 astrocytes
Tested Concentrations: 100 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Dramatically attenuated RAW 264.7 macrophages , N9 and BV-2 microglia, and DITNC1 astrocytes without affecting cell viability.

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Apoptosis analysis[2]
Cell Types: BV-2 Cell
Tested Concentrations: 50, 100 and 200 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Activation of caspase-3 in a dose- and time-dependent manner.

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Cell cycle analysis [2]
Cell Types: BV-2 Cell
Tested Concentrations: 100 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Dramatically inhibited the incorporation of BrdU in BV-2 microglia, resulting in G1 phase cell aggregation and S phase cell number reduction phase.

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Western Blot Analysis[2]
Cell Types: BV-2 Cell
Tested Concentrations: 100 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Increased ceramide levels, upregulation of p21 expression and resulting reduction in phospho-Rb.

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Cell culture: All cell lines (BV-2 microglia, N9 microglia, RAW 264.7 macrophages, DITNC1 astrocytes) were maintained in DMEM/high glucose containing 10% FBS with 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were plated, allowed to attach overnight, and treatments were given the next day. D609 was dissolved in sterile saline and added to cultures to desired concentration. [2]
Cell proliferation assay: Cells were treated with 100 μM D609 for 2 h, followed by media change. The media was centrifuged to recover non-adherent cells, which were returned to respective dishes. Cells were counted using a hemocytometer and viability was determined by trypan blue exclusion following 22 h incubation without D609. [2]
BrdU labeling assay: BV-2 microglia were plated in 96-well plates at 2×10⁴ cells/well. Cells were treated with 100 μM D609 for 2 h or 30 μM C8-ceramide for 2 h, 4 h, and 6 h. Chase was done with 5 μM BrdU at 37°C for 2 h followed by fixation in ice-cold methanol for 10 min. DNA was denatured with 2 N HCl for 30 min. Cells were washed four times with PBS after each step. Non-specific sites were blocked with 10% normal donkey serum in 0.5% BSA and 0.4% Triton X-100 for 45 min at RT. Cells were incubated with anti-BrdU primary antibody (1:50) overnight at 4°C. After four washes, cells were probed with Alexafluor-488 conjugated secondary antibody (1:500) for 1 h. Nuclei were counter-stained with DAPI (10 μg/mL). Images were acquired with epifluorescence microscope and analyzed using ImageJ; results expressed as percentage of BrdU-positive nuclei. [2]
Cell cycle analysis: BV-2 cells were treated with D609, washed with PBS and trypsinized. Cells were pelleted and adjusted to 1-2×10⁶ cells/mL. Cells were fixed by adding 3 mL of 100% ethanol drop-wise into 1 mL of cell suspension with continuous vortexing. After fixation overnight at -20°C, cells were pelleted and resuspended in PBS with 1% BSA. After 2 washes, cells were suspended in propidium iodide staining solution (50 μg/mL PI, 1 mg/mL RNase, 0.5% Triton X-100) for 30 min at 37°C in the dark and analyzed using flow cytometer. Data were analyzed using MODFIT cell cycle analysis program. [2]
Western blotting: Cells were lysed in protein extraction buffer (20 mM Na₂HPO₄, 50 mM NaF, 10 mM Na₄P₂O₇, 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 2% Triton X-100, 0.5% deoxycholate, 1 mM Na₃VO₄, and protease inhibitor cocktail). Cell lysates were sonicated and centrifuged at 13,000 rpm for 10 min at 4°C. Supernatant was used for protein estimation by Lowry's method. Fifty μg of protein were loaded per well in 10% or 12% polyacrylamide gels and subjected to SDS-PAGE at 150 V. Proteins were transferred to nitrocellulose at 100 V for 1 h. Nonspecific binding sites were blocked with 5% non-fat dry milk in TBST at RT for 1 h. Blots were incubated with primary antibodies (diluted in 5% BSA or 5% non-fat dry milk in TBST) overnight at 4°C, washed with TBST, then incubated with appropriate secondary antibodies for 1 h at RT. Protein bands were visualized with chemiluminescent reagent. Blots were stripped and re-probed for β-actin as loading control. [2]
Lipid analysis: Cell pellets were resuspended in 0.5 mL saline and an aliquot taken for protein measurement. Cell suspension was transferred into 3 mL CHCl₃:MeOH 1:2, followed by additional saline and CHCl₃. The CHCl₃ layer containing lipid extract was evaporated under nitrogen and redissolved in CHCl₃:MeOH 9:1. Ceramide was separated on silica gel H plates developed in CHCl₃:MeOH:CH₃COOH (94:2:5). SM was separated on silica gel plates using CHCl₃:EtOH:EtN:H₂O (30:35:35:7). Lipids were identified using authentic standards. DAG, ceramide, and SM bands were scraped into 1.5 mL MeOH containing 30 μL H₂SO₄ and 10 nmol heptadecanoic acid (17:0) as internal standard. SM and ceramide were methylated by heating at 100°C for 2 h; DAG was methylated by heating at 70°C for 30 min. Fatty acid methyl esters were extracted into hexane and analyzed by gas chromatography. For C8-ceramide analysis in media, 0.5 mL aliquots of media were extracted and separated on silica gel H plates and analyzed by GC. [2]
C8-ceramide treatment: C8-ceramide dissolved in 100% EtOH was first dispersed in a small volume of media by gentle vortexing, then added to BV-2 cultures. [2]

Animal/Disease Models: 26weeks old apoE−/− and C57BL/6 WT mice[3]
Doses: 2.5, 10 mg/kg
Route of Administration: IP; per day for 6 weeks
Experimental Results:Inhibited the progression of preexisting atherosclerotic lesions in apoE−/− mice and changed the lesion composition into a more stable phenotype.
Dramatically diminished the aortic endothelial expression of the vascular cell adhesion molecule-1 and the intercellular adhesion molecule-1.

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ApoE-/- mice (6 weeks old) were weaned and fed an atherogenic diet. At 20 weeks of age, 4 groups of study were established. The first group was euthanized to determine baseline established lesions. The second and third groups received intraperitoneal injections of D609 at 10 mg/kg/day (D609-HD) or 2.5 mg/kg/day (D609-LD). The fourth control group was injected with the same volume (100 μL) of phosphate-buffered saline. After 6 weeks of treatment, blood and tissue were collected for further analysis. All mice appeared healthy and survived during the 6-week treatment period. [3]
Tissue collection and analysis: Aortic roots and brachiocephalic arteries were collected. Sections (4-5 sections per tissue, at least 3 sites of analysis per slide) were prepared for histology and immunofluorescence staining. Aortae were stained with oil red O for en face analysis of lesion area. Aortic sinus plaque volume was determined. Hematoxylin and eosin (H&E) staining was performed on aortic root and brachiocephalic artery sections. Immunostaining was performed for Mac-3 (macrophages), α-smooth muscle actin (smooth muscle cells), LOX-1, VCAM-1, ICAM-1, MMP-3, MMP-9, and MMP-13. Masson trichrome staining was used for collagen detection. In situ zymography was performed to detect MMP-2/MMP-9 activity. [3]

D609 was stable in saline and cell culture media as measured by absorption maximum at 300 nm, with <10% decrease in absorption observed after 24 h; no absorption at 350 nm indicative of disulfide formation was observed over 24 h. [2]
D609 (100 μM, 2 h treatment) caused a transient, reversible increase in ceramide levels in BV-2 cells, which returned to control levels 22 h after removal of the agent. [2]
C8-ceramide (30 μM) was stable in culture media at 37°C for up to 24 h with no decomposition. However, C8-ceramide levels in media from BV-2 cells declined rapidly, decreasing to <1 μM by 24 h, indicating near-complete metabolism by the cells. Cellular C8-ceramide levels reached 6529±245 pmol/mg protein after 2 h, declining to <10% of 2 h levels by 24 h. Endogenous ceramide forms also declined to less than half the 2 h levels by 24 h. [2]

D609-induced apoptosis was dependent on dose and duration of exposure. BV-2 microglia treated with 50 or 100 μM D609 for 2 h followed by 22 h chase appeared morphologically normal with no significant trypan blue staining (>90% viability), whereas 200 μM caused cell shrinkage and staining. Western blot showed no caspase-3 cleavage at 50 or 100 μM, but 200 μM D609 for 2 h resulted in caspase-3 activation indicating apoptosis. [2]
100 μM D609 for 2 h did not result in caspase-3 cleavage immediately or after 22 h chase; exposure for 8 h or longer induced caspase-3 activation. [2]
Exogenous C8-ceramide: 30 μM for 24 h inhibited proliferation without inducing cell death (viability 93±0.5% vs control 94±0.2%); 40 μM resulted in significant cell death. [2]

[1]. The antiviral, antitumoural xanthate D609 is a competitive inhibitor of phosphatidylcholine-specific phospholipase C. Drugs Exp Clin Res. 1996;22(6):287-94.

[2]. Anti-proliferative effects of tricyclodecan-9-yl-xanthogenate (D609) involve ceramide and cell cycle inhibition.Mol Neurobiol. 2012 Jun;45(3):455-64. Epub 2012 Mar 14.

[3]. D609 inhibits progression of preexisting atheroma and promotes lesion stability in apolipoprotein e-/- mice: a role of phosphatidylcholine-specific phospholipase in atherosclerosis. Arterioscler Thromb Vasc Biol. 2010 Mar;30(3):411-8.

[4]. Role of acid sphingomyelinase and IL-6 as mediators of endotoxin-induced pulmonary vascular dysfunction. Thorax. 2017 May;72(5):460-471.

[5]. D609 inhibits the proliferation of neural progenitor cells.Neuroreport. 2010 Jul 14;21(10):700-3.

D609 is a xanthate compound originally developed as a broad-spectrum antiviral agent. It possesses antioxidant properties due to the thiol function. [2]
D609 inhibits both PC-PLC and SMS, linking two lipid second messengers with opposing actions: ceramide (growth arrest) and DAG (proliferation). [2]
After cerebral ischemia, proliferation of astrocytes and microglia contributes to glial scar formation and release of inflammatory factors. D609 may offer benefit after stroke through ceramide-mediated cell cycle arrest, restricting glial cell proliferation. [2]
D609 may prevent neuronal death by blocking aberrant induction of the cell cycle at early reperfusion in mature neurons (24 h reperfusion), while not interfering with microglia/macrophage proliferation (72 h reperfusion) that are sources of trophic factors during the repair phase of stroke. [2]
Ceramide functions are chain length-dependent (e.g., C16:0, C24:0, C24:1) which may ultimately decide cellular events such as apoptosis, proliferation, or cell cycle arrest. [2]
D609 treatment upregulated p21 expression in an in vivo stroke model (Adibhatla and Hatcher 2010). [2]

C11H15KOS2

266.4583

266.02

83373-60-8

83373-60-8 (K+); 145764-52-9 (free);

4234241

White to off-white solid powder

1.24 g/cm3

303.8ºC at 760 mmHg

137.5ºC

3.31

0

3

2

15

271

0

[K].S=C(OC1C2C3C(C(C2)C1)CCC3)S

IGULCCCBGBDZKQ-UHFFFAOYSA-M

InChI=1S/C11H16OS2.K/c13-11(14)12-10-5-6-4-9(10)8-3-1-2-7(6)8;/h6-10H,1-5H2,(H,13,14);/q;+1/p-1

potassium;8-tricyclo[5.2.1.02,6]decanyloxymethanedithioate

D609D-609 D609

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 (~375.29 mM)
H2O : ~2 mg/mL (~7.51 mM)

Solubility in Formulation 1: ≥ 3 mg/mL (11.26 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 30.0 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: ≥ 3 mg/mL (11.26 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 30.0 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: ≥ 3 mg/mL (11.26 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 30.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (93.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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