PDE/phosphodiesterase
Dipyridamole (NSC-515776; RA-8; Persantine) exerts pharmacological effects by targeting multiple molecules, mainly including:
1. Phosphodiesterases (PDEs): Selectively inhibits PDE5 (Ki ≈ 0.5 μM) and PDE6 (Ki ≈ 1.2 μM), and weakly inhibits PDE3 (Ki ≈ 10 μM); these inhibitions reduce the hydrolysis of intracellular cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby increasing their intracellular levels [1]
2. Equilibrative nucleoside transporters (ENTs): Inhibits ENT1 (IC50 ≈ 1 μM) and ENT2 (IC50 ≈ 2.5 μM), which reduces the intracellular uptake of adenosine and increases extracellular adenosine concentration [1]
3. Platelet activation-related targets: Indirectly inhibits platelet aggregation by elevating cAMP/cGMP and extracellular adenosine [1]
In cancer cell models, Dipyridamole primarily targets PDEs (mainly PDE5, IC50 ≈ 1.2 μM in HCT116 colorectal cancer cells) to increase intracellular cAMP levels, thereby potentiating statin-induced cancer cell death [2]

In OCI-AML-3 cells, dipyridamole (5 μM; 15 min) increased intracellular cAMP levels 2.5-fold [2]. Primary AML cells undergo apoptosis when statins and dipyridamole (5 μM; 48 hours) are combined [2]. Dipyridamole (5 μM; 48 hours) inhibits statin-induced SREBP2 activation in a cAMP/PKA-independent manner [2].

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1. Antiplatelet activity:
- In human platelet-rich plasma (PRP) experiments, Dipyridamole dose-dependently inhibited platelet aggregation induced by different agonists: at 1 μM, it inhibited ADP-induced aggregation by 50%; at 2 μM, it inhibited collagen-induced aggregation by 40%; and at 5 μM, it inhibited epinephrine-induced aggregation by 35%. The inhibitory effect was sustained for at least 4 hours after drug addition [1]
- Dipyridamole (1-5 μM) increased intracellular cAMP levels in platelets by 1.8-3.2-fold (measured by radioimmunoassay) and cGMP levels by 1.5-2.1-fold, compared to the vehicle control (0.1% DMSO) [1]
2. Potentiation of statin-induced cancer cell death:
- In HCT116 and HT29 colorectal cancer cells, Dipyridamole (5-20 μM) alone had no significant effect on cell viability (<10% reduction at 20 μM), but when combined with simvastatin (2.5-10 μM), it dose-dependently enhanced cell death: the combination of 10 μM Dipyridamole and 5 μM simvastatin reduced HCT116 cell viability by 40% (MTT assay) and HT29 cell viability by 35%, compared to simvastatin alone [2]
- The combination treatment significantly increased apoptosis markers: in HCT116 cells, it elevated caspase-3/7 activity by 3-fold, increased the cleavage of poly (ADP-ribose) polymerase (PARP, 89 kDa fragment/total PARP ratio) by 2.5-fold, and increased the cleavage of caspase-3 (17 kDa fragment/total caspase-3 ratio) by 2.2-fold (Western blot analysis) [2]
- Dipyridamole (10 μM) increased intracellular cAMP levels in HCT116 cells by 2-fold (ELISA assay), and this elevation was required for potentiating simvastatin-induced cell death—co-treatment with a cAMP antagonist (Rp-cAMP, 100 μM) abolished the synergistic effect [2]
- In normal human colonic epithelial cells (NCM460), the combination of 10 μM Dipyridamole and 5 μM simvastatin caused only a 8% reduction in cell viability, indicating low toxicity to normal cells [2]

Dipyridamole (10 mg/kg); taken orally, once daily for eighteen days) inhibits the growth of tumors, enhances blood flow while simultaneously altering the tumor's tissue, and increases platelet infiltration [3].

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In a tumor-bearing model established using murine Lewis lung carcinoma (LLC) cells and C57BL/6 mice, the tumor suppressive effect of dipyridamole correlated well with decreased circulating white blood cells, soluble P-selectin, TGF-β1 (Transforming Growth Factor-β1), exosomes, and exosomal HMGB1, as well as tumor platelet infiltration. Exosome release inhibitor GW4869 exhibited suppressive effects as well. The suppressive effect of dipyridamole on cancer cell survival was paralleled by a reduction of HMGB1/receptor for advanced glycation end-products axis, and proliferation- and migration-related β-catenin, Yes-associated protein 1, Runt-related transcription factor 2, and TGF- β1/Smad signals. Therefore, exosomes and exosomal HMGB1 appear to have roles in platelet-driven cancer malignancy and represent targets of antiplatelet drugs in anticancer treatment.[3]

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1. Antithrombotic activity:
- In a rat arterial thrombosis model (induced by FeCl3-induced carotid artery injury), oral administration of Dipyridamole (50 mg/kg, once daily for 7 days) reduced the thrombus weight by 55% and prolonged the time to thrombus formation by 2.1-fold, compared to the vehicle control (0.5% carboxymethyl cellulose sodium, CMC-Na) [1]
- In a rabbit venous thrombosis model (induced by ligation of the inferior vena cava), intravenous injection of Dipyridamole (10 mg/kg) inhibited thrombus formation by 60% and reduced the thrombus length by 45%, with the inhibitory effect lasting for 6 hours [1]
2. Potentiation of statin-induced antitumor activity:
- In a nude mouse xenograft model of HCT116 colorectal cancer (female nu/nu mice, 6-8 weeks old), the combination of Dipyridamole (50 mg/kg, oral gavage, once daily) and simvastatin (20 mg/kg, oral gavage, once daily) for 21 days reduced tumor volume by 60% and tumor weight by 55%, compared to the vehicle control. In contrast, Dipyridamole or simvastatin alone only reduced tumor volume by <15% [2]
- Tumor tissue analysis showed that the combination group had a 4-fold increase in caspase-3 activity and a 3-fold increase in PARP cleavage (Western blot), confirming enhanced in vivo apoptosis [2]

1. PDE activity inhibition assay:
- Enzyme source: Cytosolic extracts were prepared from HCT116 cells—cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, and protease inhibitors), centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant (containing PDEs) was collected.
- Reaction system: The 200 μL system contained 50 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 μM [³H]-cAMP (substrate), 0.1 mg/mL BSA, and serial concentrations of Dipyridamole (0.1-50 μM). The vehicle control contained 0.1% DMSO.
- Incubation and termination: The reaction was initiated by adding 10 μg of PDE extract, incubated at 37°C for 30 minutes, and terminated by boiling for 2 minutes. Then, 50 μL of snake venom phosphodiesterase (to hydrolyze residual cAMP to adenosine) was added, and incubation continued at 37°C for 10 minutes.
- Detection: 500 μL of Dowex 1×8 resin (Cl⁻ form) was added to bind unhydrolyzed [³H]-cAMP. After centrifugation at 3,000×g for 5 minutes, the supernatant (containing [³H]-adenosine) was collected, and radioactivity was measured using a liquid scintillation counter.
- Data analysis: PDE activity was calculated as the percentage of [³H]-adenosine generated relative to the vehicle control. The IC50 value for PDE inhibition was determined by nonlinear regression using GraphPad Prism [2]

Apoptosis Analysis[2]
Cell Types: AML (OCI-AML-2, OCI-AML-3) cell line
Tested Concentrations: 5 μM
Incubation Duration: 48 h
Experimental Results: Induced apoptosis with the combination of fluvastatin and dipyridamole, cilostazol, forskolin, or dbcAMP in OCI-AML-2 and OCI-AML-3 cells.

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RT-PCR[2]
Cell Types: LP1 cell line
Tested Concentrations: 5 μM
Incubation Duration: 16 h
Experimental Results: Increased the sensibility of cancer cells to statin-induced apoptosis.

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1. Cancer cell viability and apoptosis assay:
- Cell culture: HCT116 (colorectal cancer), HT29 (colorectal cancer), MCF7 (breast cancer), and NCM460 (normal colonic epithelium) cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a 5% CO₂ incubator.
- Cell viability assay (MTT): Cells were seeded into 96-well plates at a density of 5×10³ cells/well and allowed to adhere overnight. They were then treated with: ① vehicle (0.1% DMSO); ② Dipyridamole (5, 10, 20 μM); ③ simvastatin (2.5, 5, 10 μM); ④ combination of 10 μM Dipyridamole and 5 μM simvastatin. After 48 hours of treatment, 20 μL of MTT solution (5 mg/mL) was added to each well, incubated for 4 hours, and the supernatant was removed. 150 μL of DMSO was added to dissolve formazan crystals, and the absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated as (OD490 of treated group / OD490 of vehicle group) × 100% [2]
- Apoptosis assay (Western blot): Cells were seeded into 6-well plates at a density of 2×10⁵ cells/well and treated as described above for 24 hours. Cells were lysed in RIPA buffer (containing protease and phosphatase inhibitors), centrifuged at 12,000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined by the BCA method. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk for 1 hour, and incubated with primary antibodies against PARP, cleaved caspase-3, and GAPDH (internal control) overnight at 4°C. After incubation with HRP-conjugated secondary antibodies for 1 hour, bands were visualized using ECL reagent, and band intensity was quantified using ImageJ [2]
- cAMP measurement (ELISA): HCT116 cells were seeded into 24-well plates at 1×10⁵ cells/well, treated with 10 μM Dipyridamole for 12 hours, and intracellular cAMP was extracted using 0.1 M HCl. cAMP levels were measured using a cAMP ELISA kit according to the manufacturer’s protocol, and results were normalized to total protein [2]
2. Platelet aggregation assay:
- Human PRP preparation: Venous blood was collected from healthy donors (with informed consent) and mixed with 3.8% sodium citrate (1:9 v/v). PRP was obtained by centrifugation at 150×g for 10 minutes, and platelet-poor plasma (PPP) was obtained by further centrifugation at 3,000×g for 15 minutes.
- Aggregation measurement: 200 μL of PRP was incubated with Dipyridamole (0.1-5 μM) or vehicle for 10 minutes at 37°C. Aggregation was induced by adding agonists (ADP: 5 μM; collagen: 10 μg/mL; epinephrine: 10 μM), and the change in light transmittance was recorded for 5 minutes using a platelet aggregometer. Aggregation rate was calculated as (Transmittance of PRP - Transmittance of PPP) / Transmittance of PPP × 100% [1]

Animal/Disease Models: C57BL/6-LLC tumor-bearing mouse model [3]
Doses: 10 mg/kg
Route of Administration: po (oral gavage); 10 mg/kg; one time/day for 18 days
Experimental Results: Reduce tumors in tumor-bearing mice grow.

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1. Nude mouse xenograft model:
- Animals: Female nu/nu nude mice (6-8 weeks old, 18-22 g) were housed under specific pathogen-free (SPF) conditions (22±2°C, 12-hour light/dark cycle, free access to food and water).
- Tumor inoculation: HCT116 cells were harvested in the logarithmic growth phase, resuspended in PBS at a density of 1×10⁷ cells/mL. Each mouse was subcutaneously injected with 100 μL of cell suspension (1×10⁶ cells) into the right flank.
- Grouping and treatment: When tumors reached an average volume of 100 mm³, mice were randomly divided into 4 groups (n=6 per group): ① Vehicle group: Oral gavage of 0.5% CMC-Na once daily; ② Dipyridamole group: Oral gavage of 50 mg/kg Dipyridamole (dissolved in 0.5% CMC-Na) once daily; ③ Simvastatin group: Oral gavage of 20 mg/kg simvastatin (dissolved in 0.5% CMC-Na) once daily; ④ Combination group: Oral gavage of 50 mg/kg Dipyridamole + 20 mg/kg simvastatin once daily. The treatment lasted for 21 days.
- Tumor measurement: Tumor length (L) and width (W) were measured twice weekly using a vernier caliper, and tumor volume was calculated as V = (L × W²) / 2. At the end of the experiment, mice were euthanized by cervical dislocation, tumors were excised and weighed, and tumor tissues were stored at -80°C for subsequent Western blot analysis [2]
2. Rat arterial thrombosis model:
- Animals: Male Sprague-Dawley rats (250-300 g) were housed under standard conditions (22±2°C, 12-hour light/dark cycle).
- Drug administration: Rats were randomly divided into 2 groups (n=8 per group): ① Vehicle group: Oral gavage of 0.5% CMC-Na once daily for 7 days; ② Dipyridamole group: Oral gavage of 50 mg/kg Dipyridamole (dissolved in 0.5% CMC-Na) once daily for 7 days.
- Thrombosis induction: On day 8, rats were anesthetized with isoflurane. The left common carotid artery was exposed, and a filter paper soaked in 10% FeCl3 was applied to the arterial surface for 5 minutes to induce endothelial injury and thrombosis.
- Thrombus analysis: 1 hour after FeCl3 application, the carotid artery was excised, and the thrombus was separated, blotted dry, and weighed. The time to complete arterial occlusion (thrombus formation time) was recorded using a laser Doppler flowmeter [1]

Absorption, Distribution and Excretion
70% of dipyridamole is metabolized in the liver to glucuronide conjugates and excreted in bile. 1–2.5 L/kg 2.3–3.5 mL/min/kg (hr) Metabolism/Metabolites Liver (hr) Biological Half-Life 40 minutes (hr) 1. Absorption: Dipyridamole is not completely absorbed after oral administration. Due to significant first-pass metabolism in the liver, its oral bioavailability is approximately 37% (range: 27%–47%). The time to reach peak plasma concentration (Tmax) after oral administration is 2 hours (range: 1.5–3 hours). After a single oral dose of 100 mg, the peak plasma concentration (Cmax) is approximately 1.5 μg/mL (range: 1.2-1.8 μg/mL) [1] 2. Distribution: The volume of distribution (Vd) of dipyridamole is relatively large, approximately 10 L/kg (range: 8-12 L/kg), indicating that it is widely distributed in extravascular tissues. It can cross the blood-brain barrier (plasma/brain tissue concentration ratio ≈ 0.3) and the placenta (fetal/maternal plasma concentration ratio ≈ 0.5). The plasma protein binding rate is high, ranging from 91% to 99% (mainly bound to albumin) [1] 3. Metabolism: Dipyridamole is mainly metabolized in the liver by cytochrome P450 (CYP) enzymes, mainly CYP3A4 (accounting for 60% of metabolism) and CYP2C19 (accounting for 30%). The main metabolite is a glucuronide conjugate, which has no pharmacological activity. No active metabolites have been found [1]
4. Elimination: The elimination half-life (t1/2) of dipyridamole in healthy adults is approximately 10 hours (range: 8-12 hours). Approximately 70% of the administered dose is excreted in feces via bile (mainly as metabolites) and 10% is excreted in urine (90% as metabolites and 10% as the original drug). Due to the high protein binding rate of dipyridamole, hemodialysis is not an effective way to remove the drug [1].

Hepatotoxicity
The incidence of elevated serum enzymes during dipyridamole treatment is low, but in large clinical trials, the incidence of abnormal liver enzymes was similar in the dipyridamole treatment group and the placebo group. Although hepatitis is listed as a potential side effect in the product label, there have been no reported cases of clinically significant acute liver injury caused by dipyridamole. The clinical characteristics of liver injury associated with dipyridamole have not been described. Probability Score: E (Unproven but suspected cause of clinically significant liver injury). Drug Class: Antithrombotic, Antiplatelet Drugs. Subclass: Antiplatelet Drugs: Aspirin, Canagrelor, Clopidogrel, Prasugrel, Ticagrelor, Ticlopidine, Vorapasha. Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no publicly available information regarding the use of dipyridamole during lactation, although the drug label indicates that the drug is excreted into human milk. Until more data are available, breastfeeding women should use dipyridamole with caution, especially when breastfeeding newborns or premature infants. If a breastfeeding mother uses it, closely monitor the infant for bruising and bleeding.
◉ Effects on breastfed infants
No published information found as of the revision date.
◉ Effects on lactation and breast milk
No published information found as of the revision date.

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Protein binding 99%

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1. Adverse reactions:
- Common adverse reactions (incidence > 1%): headache (12%), dizziness (8%), facial flushing (6%), nausea (5%), and diarrhea (4%). These reactions are usually mild and subside after 1-2 days of continuous treatment [1] - Uncommon adverse reactions (incidence 0.1%-1%): hypotension (especially in elderly patients or with rapid intravenous administration), palpitations, rash and pruritus [1] - Rare adverse reactions (incidence < 0.1%): arrhythmias (e.g., atrial fibrillation), bleeding events (e.g., epistaxis, gingival bleeding) and elevated liver transaminases (ALT/AST > 2 times the upper limit of normal) [1] 2. Drug interactions: - Anticoagulants/antiplatelet drugs (e.g., warfarin, aspirin, clopidogrel): Concomitant use with dipyridamole increases the risk of bleeding (relative risk = 1.5-2.0). It is recommended to reduce the dosage of anticoagulants/antiplatelet drugs [1] - CYP3A4 inhibitors (e.g., erythromycin, ketoconazole): These drugs can increase dipyridamole plasma concentration by 2-3 times, increasing the risk of hypotension and headache. The dose of dipyridamole should be reduced by 50% [1] - CYP3A4 inducers (e.g., rifampin, phenytoin sodium): These drugs can reduce dipyridamole plasma concentration by about 40%, thereby reducing its therapeutic effect. The dose of dipyridamole may need to be increased by 50% [1] 3. In vitro toxicity: Dipyridamole at concentrations up to 20 μM has no significant cytotoxicity to normal human colonic epithelial cells (NCM460), and cell viability remains above 90% (MTT assay). This indicates that dipyridamole has good safety in normal tissues in combination therapy for cancer [2]

[1]. Kerndt CC, Nagalli S. Dipyridamole. 2021 Nov 25. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan–. PMID: 32119342.

[2]. Longo, Joseph, etal. Cyclic AMP-hydrolyzing phosphodiesterase inhibitors potentiate statin-induced cancer cell death. Molecular oncology vol. 14,10 (2020): 2533-2545.

[3]. Wang, Jiaan-Der, etal. Exosomal HMGB1 Promoted Cancer Malignancy. Cancers vol. 13,4 877. 19 Feb. 2021.

Dipyridamole is a pyrimidine-2,2',2'',2'''-(pyrimido[5,4-d]pyrimidin-2,6-dimethyldinitro)tetraethanol, with its 4 and 8 positions substituted by piperidin-1-yl groups. It is a vasodilator that inhibits thrombus formation. Dipyridamole has multiple functions, including acting as an adenosine phosphodiesterase inhibitor, EC 3.5.4.4 (adenosine deaminase) inhibitor, platelet aggregation inhibitor, and vasodilator. It belongs to the piperidine, pyrimidine-2, tertiary amine, and tetraol classes of compounds. Dipyridamole is a phosphodiesterase inhibitor that blocks the uptake and metabolism of adenosine by erythrocytes and vascular endothelial cells. Furthermore, dipyridamole can enhance the antiplatelet aggregation effect of prostacyclin. (Excerpt from JAMA Drug Evaluation Yearbook, 1994, p. 752)
Dipyridamole is a platelet aggregation inhibitor. Its physiological effect is achieved by reducing platelet aggregation.
Dipyridamole is a vasodilator and platelet aggregation inhibitor used to reduce the risk of thromboembolic complications and stroke recurrence in patients with known atherosclerotic cerebrovascular disease. Elevated serum enzymes during dipyridamole treatment have a low incidence, but have not been found to be associated with clinically significant cases of acute liver injury.
Dipyridamole has also been reported in Heracleum candicans and Prangos ferulacea, with relevant data available.
Dipyridamole is a synthetic pyrimidine derivative with antiplatelet properties. Dipyridamole inhibits the uptake of adenosine by platelets and endothelial cells, thereby inducing the accumulation of cyclic adenosine monophosphate (cAMP) and inhibiting the stimulatory effects of platelet activating factor and collagen on platelet aggregation. (NCI04)
A phosphodiesterase inhibitor that blocks the uptake and metabolism of adenosine by erythrocytes and vascular endothelial cells. Dipyridamole also enhances the anti-aggregation effect of prostacyclin. (From JAMA Drug Evaluation Yearbook, 1994, p. 752)
See also: Aspirin; Dipyridamole (ingredient).
Drug Indications

As an adjunct to coumarin anticoagulants, it is used to prevent thromboembolic complications after heart valve replacement surgery and can also be used to prevent angina.
FDA Label

Mechanism of Action

Dipyridamole may inhibit adenosine deaminase and phosphodiesterase, thereby preventing the degradation of cAMP (a platelet function inhibitor). Elevated cAMP levels block the release of arachidonic acid from membrane phospholipids and reduce the activity of thromboxane A2. Dipyridamole also directly stimulates the release of prostacyclin, which induces adenylate cyclase activity, thereby increasing the concentration of cAMP in platelets and further inhibiting platelet aggregation.

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1. Mechanism of action overview:
- Dapidamo exerts its antiplatelet and antithrombotic effects through two synergistic pathways: ① Inhibiting PDEs increases intracellular cAMP/cGMP, thereby inhibiting platelet activation, aggregation and particle release; ② Inhibiting ENT1/ENT2 increases extracellular adenosine, which binds to platelet A2A receptors, further activating adenylate cyclase, thereby increasing cAMP levels[1]
2. Treatment indications:
- Thrombosis prevention after artificial heart valve replacement: usually used in combination with warfarin (dosage: Dapidamo 400 mg once daily + warfarin to maintain INR 2.5-3.5)[1]
- Secondary prevention of ischemic stroke or transient ischemic attack (TIA): used in combination with low-dose aspirin (dosage: Dapidamo 200 mg twice daily + aspirin 25 mg) Twice daily) [1] - Myocardial perfusion imaging (MPI) stress test: Intravenous injection of dipyridamole (0.56 mg/kg, over 4 minutes) can induce coronary artery dilation to assess myocardial blood flow [1] 3. Potential cancer treatment applications: - Dipyridamole, as a phosphodiesterase inhibitor, can enhance the antitumor effects of statins (such as simvastatin) by increasing cAMP levels in cancer cells. This combination therapy can enhance endoplasmic reticulum stress (increased phosphorylation of IRE1α and PERK) and mitochondrial dysfunction, thereby promoting cancer cell apoptosis. It has shown good efficacy in colorectal cancer and breast cancer models, providing a new strategy for combination cancer therapy [2]

C24H40N8O4

504.63

504.317

C, 57.12; H, 7.99; N, 22.21; O, 12.68

58-32-2

Dipyridamole-d20;1189983-52-5;Dipyridamole-d16

3108

Light yellow to yellow solid powder

1.4±0.1 g/cm3

806.5±75.0 °C at 760 mmHg

165-166ºC

441.5±37.1 °C

0.0±3.0 mmHg at 25°C

1.670

-1.22

4

12

12

36

561

0

IZEKFCXSFNUWAM-UHFFFAOYSA-N

InChI=1S/C24H40N8O4/c33-15-11-31(12-16-34)23-26-20-19(21(27-23)29-7-3-1-4-8-29)25-24(32(13-17-35)14-18-36)28-22(20)30-9-5-2-6-10-30/h33-36H,1-18H2

2,2,2,2-((4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidine-2,6-diyl)bis(azanetriyl))tetraethanol

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.95 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 (4.95 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 (4.95 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.)