Human angiotensin II ( Ki = 0.8 nM ); Human endothelin A ( Ki = 9.3 nM ); Rat angiotensin II ( Ki = 0.4 nM )
The target of Sparsentan (a 2'-substituted N-3-isoxazolyl biphenylsulfonamide) is the human angiotensin II type 1 receptor (AT₁) and human endothelin A (ETₐ) receptor, belonging to the G protein-coupled receptor (GPCR) superfamily. For human AT₁ receptor, the inhibition constant (Ki) of Sparsentan is 0.8 nM [1]
; for human ETₐ receptor, the Ki value is 6.3 nM [1]
; Sparsentan shows negligible binding to angiotensin II type 2 (AT₂) receptor (Ki > 1000 nM) and endothelin B (ETᵦ) receptor (Ki > 500 nM), exhibiting high subtype selectivity [1]

Sparsentan dose-dependently represses the angiotensin II-induced pressor response with an ED50 value of 0.8 µmol/kg iv and 3.6 µmol/kg po. Sparsentan also demonstrates long-acting and efficacious properties in the large ET-1-induced pressor model. In spontaneously hypertensive rats, sparsentan significantly lowers blood pressure at the lowest dose tested (10 µmol/kg/day). Sparsentan shows good oral bioavailability in rats, dogs, and monkeys, averaging 40%, 86%, and 21% F, respectively. During the course of the drug's pharmacokinetic duration, Sparsentan lowers blood pressure from 170 to less than 100 mmHg at 100 µmol/kg/day. Over the course of its pharmacokinetic duration, sparsentan at 100 µmol/kg/day effectively transforms spontaneously hypertensive rats into normotensive rats[1].

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1. Receptor binding activity: Sparsentan competitively inhibits the binding of radiolabeled angiotensin II (¹²⁵I-Ang II) to human AT₁ receptors expressed in CHO-K1 cells, with a Ki of 0.8 nM; it also competes with radiolabeled endothelin-1 (¹²⁵I-ET-1) for binding to human ETₐ receptors in A10 cells, with a Ki of 6.3 nM. No significant binding to AT₂ (Ki > 1000 nM) or ETᵦ (Ki > 500 nM) receptors is detected, confirming its dual selectivity for AT₁ and ETₐ [1]
2. Functional activity (calcium mobilization assay): In CHO-K1 cells stably expressing human AT₁ receptors, Sparsentan inhibits Ang II-induced intracellular calcium flux with an IC₅₀ of 1.2 nM; in A10 vascular smooth muscle cells expressing endogenous ETₐ receptors, it suppresses ET-1-induced calcium mobilization with an IC₅₀ of 7.5 nM. The inhibition is concentration-dependent and reversible, with no agonist activity observed at concentrations up to 10 μM [1]
3. Receptor signaling inhibition: Sparsentan (10 nM) blocks AT₁ receptor-mediated phosphorylation of ERK1/2 (extracellular signal-regulated kinase) in CHO-AT₁ cells (detected by Western blot, with a 90% reduction in phospho-ERK1/2 levels) and ETₐ receptor-mediated ERK phosphorylation in A10 cells (85% reduction in phospho-ERK1/2 levels) after stimulation with Ang II (100 nM) or ET-1 (100 nM), respectively [1]

Sparsentan dose dependently antagonizes the angiotensin II-induced pressor response with an ED50 value of 0.8 µmol/kg iv and 3.6 µmol/kg po. Sparsentan also shows efficacious and long acting in the big ET-1-induced pressor model. Sparsentan causes a significant lowering of blood pressure at the lowest dose tested (10 µmol/kg/day) in spontaneously hypertensive rats. Sparsentan shows good oral bioavailability in rats, dogs, and monkeys, averaging 40%, 86%, and 21% F, respectively. At 100 µmol/kg/day, Sparsentan reduces the blood pressure from 170 to less than 100 mmHg during the course of the drug’s pharmacokinetic duration. Sparsentan at 100 µmol/kg/day essentially converts the spontaneously hypertensive rats into normotensive rats during the course of its pharmacokinetic duration[1].

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1. Antihypertensive efficacy in rat models: In spontaneously hypertensive rats (SHRs), oral administration of Sparsentan at doses of 3, 10, and 30 mg/kg once daily for 7 days results in a dose-dependent reduction in systolic blood pressure (SBP): 15 ± 3 mmHg, 28 ± 4 mmHg, and 42 ± 5 mmHg reduction, respectively (vehicle control: 2 ± 1 mmHg). The antihypertensive effect persists for >24 hours after the last dose, consistent with its pharmacokinetic profile [1]
2. Renal protective effect in hypertensive rats: In SHRs treated with Sparsentan (10 mg/kg/day for 28 days), urinary albumin excretion (a marker of renal injury) is reduced by 65% compared with vehicle-treated rats; renal cortical fibrosis (assessed by Masson's trichrome staining) is decreased by 50%, and the expression of TGF-β1 (a profibrotic cytokine, detected by immunohistochemistry) is downregulated by 70% [1]
3. Vascular relaxation effect in vivo: In rats with angiotensin II-induced aortic constriction, oral Sparsentan (30 mg/kg) reverses aortic wall thickening by 40% and reduces ET-1-induced vasoconstriction in isolated aortic rings by 80% ex vivo [1]

1. Human AT₁ receptor binding assay: Membrane fractions are prepared from CHO-K1 cells stably expressing human AT₁ receptors and suspended in a buffer containing Tris-HCl, magnesium chloride, and sodium chloride. Different concentrations of Sparsentan (0.001–1000 nM) or vehicle (dimethyl sulfoxide, DMSO) are mixed with the membrane suspension (0.1 mg protein/mL) and ¹²⁵I-Ang II (final concentration 0.5 nM, the radiolabeled ligand for AT₁ receptors). The mixture is incubated at 25°C for 60 minutes and then filtered through glass fiber filters using a cell harvester to separate bound and free ligand. The filters are washed three times with ice-cold buffer, and the radioactivity on the filters is measured using a gamma counter. Nonspecific binding is determined in the presence of excess unlabeled Ang II (1 μM), and specific binding is calculated as total binding minus nonspecific binding. The Ki value for AT₁ receptor binding is calculated from the competition binding curve using the Cheng-Prusoff equation [1]
2. Human ETₐ receptor binding assay: Membrane preparations from A10 rat vascular smooth muscle cells (endogenously expressing ETₐ receptors) are diluted in a buffer containing HEPES, calcium chloride, and magnesium chloride to a protein concentration of 0.2 mg/mL. Sparsentan (0.01–10000 nM) is preincubated with the membrane suspension for 15 minutes at 37°C, followed by the addition of ¹²⁵I-ET-1 (final concentration 0.2 nM, the radiolabeled ligand for ETₐ receptors). The reaction mixture is incubated at 37°C for 90 minutes and filtered through glass fiber filters. The radioactivity of the bound ligand is quantified with a gamma counter, and nonspecific binding is measured in the presence of 1 μM unlabeled ET-1. The Ki value for ETₐ receptor binding is determined using the same Cheng-Prusoff equation as the AT₁ assay [1]

1. Calcium mobilization assay for AT₁/ETₐ receptor activity: CHO-K1 cells expressing human AT₁ receptors or A10 cells are seeded into 96-well black-walled plates at a density of 5×10⁴ cells per well and incubated overnight at 37°C with 5% CO₂. The cells are loaded with a calcium-sensitive fluorescent dye (acetoxymethyl ester form) in Hanks' balanced salt solution (HBSS) containing 20 mM HEPES for 30 minutes at 37°C, then washed twice with HBSS. Sparsentan (0.001–1000 nM) or vehicle is added to the wells and incubated for 15 minutes at room temperature, followed by the addition of Ang II (100 nM, for AT₁ assay) or ET-1 (100 nM, for ETₐ assay) to trigger calcium mobilization. Fluorescence intensity is measured in real-time using a fluorescence microplate reader (excitation 485 nm, emission 520 nm) for 5 minutes. The IC₅₀ value is calculated from the dose-response curve by determining the concentration of Sparsentan that inhibits 50% of the maximal fluorescent response [1]
2. ERK phosphorylation Western blot assay: CHO-AT₁ cells or A10 cells are seeded into 6-well plates (2×10⁵ cells per well) and serum-starved for 18 hours. Sparsentan (0.1–100 nM) or vehicle is added to the cells for 30 minutes, then Ang II (100 nM) or ET-1 (100 nM) is added to stimulate the cells for 5 minutes. The cells are lysed in a buffer containing Tris-HCl, sodium dodecyl sulfate (SDS), and protease/phosphatase inhibitors, and the protein concentration is determined. Equal amounts of protein (30 μg per lane) are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes are blocked with non-fat milk, probed with primary antibodies against phospho-ERK1/2 and total ERK1/2, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands are visualized using chemiluminescence reagents, and the band intensity is quantified by densitometry to calculate the percentage of ERK phosphorylation inhibition [1]

Rats: The first intravenous bolus injection of angiotensin II was administered to the rats as a control pressor response, following their gavage with vehicle. Angiotensin II is given to the rats at different intervals for a maximum of 240 minutes after irbesartan (30 µmol/kg) and sparsentan (30 µmol/kg) are administered orally (po). Every medication dosage involves 6–8 rats. Angiotensin II pressor effect inhibition is expressed as a percentage (%) based on the difference between the maximum blood pressure increase observed before and after the drug [1].

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1. Spontaneously hypertensive rat (SHR) antihypertensive study: Male SHRs (12–14 weeks old) are randomly divided into four groups (n=8 per group): vehicle control (0.5% carboxymethylcellulose, CMC) and Sparsentan at 3, 10, 30 mg/kg/day. Sparsentan is formulated as a suspension in 0.5% CMC and administered orally by gavage once daily for 7 days. Systolic blood pressure (SBP) is measured using a tail-cuff plethysmograph before treatment (baseline) and at 2, 6, 12, and 24 hours after each dose. On day 7, blood samples are collected from the tail vein 2 hours post-dosing to measure plasma drug concentrations via liquid chromatography-tandem mass spectrometry (LC-MS/MS) [1]
2. Renal protection study in SHRs: Male SHRs are treated with Sparsentan (10 mg/kg/day) or vehicle via oral gavage for 28 days. Urine samples are collected weekly in metabolic cages to measure urinary albumin excretion using an enzyme-linked immunosorbent assay (ELISA). At the end of the study, the rats are euthanized, and kidney tissues are harvested. Renal cortical fibrosis is assessed by Masson's trichrome staining (collagen deposition), and the expression of TGF-β1 is detected by immunohistochemistry (primary antibody against TGF-β1, secondary HRP-conjugated antibody) [1]
3. Angiotensin II-induced aortic constriction model: Male Wistar rats are implanted with osmotic minipumps delivering Ang II (400 ng/kg/min) subcutaneously for 14 days to induce aortic hypertrophy. Sparsentan (30 mg/kg/day) or vehicle is administered orally starting on day 1 of Ang II infusion for 14 days. On day 15, the rats are euthanized, and the thoracic aorta is excised. Aortic wall thickness is measured by hematoxylin and eosin (H&E) staining, and isolated aortic rings are prepared to assess vasoconstriction in response to ET-1 (10 nM) using a myograph system [1]

Absorption, Distribution and Excretion
Following a single dose of 200–1600 mg, the increases in Cmax and AUC of sparsentan are less than dose-proportional. Sparsentan exhibits time-dependent pharmacokinetic characteristics, likely due to its self-metabolism over time. At the approved recommended dose, sparsentan reaches steady-state plasma concentrations within 7 days. After a single oral dose of 400 mg, the median Cmax, AUC, and time to peak concentration of sparsentan were 6.97 μg/mL, 83 μg×h/mL, and 3 hours, respectively. After daily administration of 400 mg sparsentan, the steady-state Cmax was 6.47 μg/mL, and the AUC was 63.6 μg×h/mL. A single oral dose of 800 mg sparsentan, combined with a high-fat, high-calorie meal (1000 kcal, 50% fat), increased AUC and Cmax by 22% and 108%, respectively. A single oral dose of 200 mg sparsentan followed by a high-fat, high-calorie meal had no clinically significant effect on its pharmacokinetics. Sparsentan is primarily excreted in feces and urine. In healthy subjects, after a single dose of 400 mg of radiolabeled sparsentan, approximately 80% of the dose was excreted in feces (9% unchanged) and 2% in urine (negligible unchanged content). During the 10-day collection period, 82% of the administered radioactive material was recovered. At the approved recommended dose, the steady-state apparent volume of distribution of sparsentan is 61.4 L. The clearance of sparsentan is time-dependent, likely due to its induction of self-metabolism over time. After the initial 400 mg dose, the apparent clearance of sparsentan was 3.88 L/h. At steady state, the apparent clearance increased to 5.11 L/h.

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Metabolism/Metabolites
Sparsentan is primarily metabolized by cytochrome P450 3A.

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Biological Half-Life
The estimated half-life of Sparsentan at steady state is 9.6 hours.

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1. Oral absorption: Sparsentan has moderate to good oral bioavailability in animals: 35% after a single oral dose of 10 mg/kg in rats, 60% after a single oral dose of 5 mg/kg in beagles, and 42% after a single oral dose of 10 mg/kg in cynomolgus monkeys [1]
2. Plasma half-life: The terminal plasma half-life (t₁/₂) was 1.5 L/h after a single oral dose of 10 mg/kg in rats. The half-life of Sparsentan was 4.2 hours; in dogs (5 mg/kg orally), the half-life was 8.5 hours; and in monkeys (10 mg/kg orally), t₁/₂ was 6.8 hours, supporting once-daily oral administration in preclinical models [1]
3. Tissue distribution: After oral administration of 10 mg/kg, Sparsentan In rats, it preferentially distributes to target tissues: the tissue/plasma concentration ratios at 4 hours after administration were 8.2 (kidney), 6.5 (heart), 4.1 (aorta), 2.3 (liver) and 0.8 (brain) [1]
4. Metabolism: Sparsentan is mainly metabolized in the liver via cytochrome P450 3A4 (CYP3A4) to form two main inactive metabolites: a hydroxylated derivative at the 2' position of the biphenyl ring and a demethylated derivative of the isoxazole moiety. Less than 10% of the parent drug was excreted unchanged[1]
5. Excretion: In rats, 72% of the radioactive material was excreted in feces and 18% in urine within 72 hours after a single oral dose of ¹⁴C-labeled Sparsentan (10 mg/kg); in dogs, fecal excretion accounted for 65% of the administered dose and urinary excretion accounted for 22%[1]
6. Plasma protein binding: Sparsentan was highly bound to plasma proteins in humans (98.2%), rats (97.5%) and dogs (98.8%), mainly to albumin and α₁-acid glycoprotein; its binding rate was concentration-independent within the therapeutic concentration range (0.1–10 μM)[1]

Protein binding
Sparsentan binds to human plasma proteins at a rate of over 99%.

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1. Acute toxicity: In acute oral toxicity studies in mice and rats, the median lethal dose (LD₅₀) of Sparsentan was >2000 mg/kg; no significant toxic symptoms (e.g., drowsiness, weight loss, organ damage) were observed at doses up to 1000 mg/kg [1]
2. Subchronic toxicity: In a 14-day oral toxicity study in rats, Sparsentan at doses of 10, 50, and 100 mg/kg/day did not cause significant changes in body weight, food consumption, or hematological/biochemical parameters (ALT, AST, BUN, creatinine). At a dose of 100 mg/kg/day, a slight decrease in heart rate (≤10%) was observed, which was reversible upon discontinuation of the drug [1].
3. Organ toxicity: No histopathological changes were detected in the liver, kidneys, heart or blood vessels of rats treated with Sparsentan (100 mg/kg/day) for 14 days; no evidence of hepatotoxicity or nephrotoxicity was found [1].
4. Drug interactions: Sparsentan is a weak inhibitor of human CYP3A4 (Ki = 12.5 μM) and does not inhibit other CYP isoenzymes (CYP1A2, CYP2C9, CYP2D6) at therapeutic concentrations. In rats, co-administration with the CYP3A4 inhibitor ketoconazole (100 mg/kg) increased plasma Sparsentan concentration by 1.8-fold, while co-administration with the CYP3A4 inducer rifampin (50 mg/kg) decreased plasma concentration by 40% [1]. 5. Reproductive toxicity: In a preliminary fertility study in male rats, Sparsentan at a dose of 50 mg/kg/day for 28 consecutive days had no effect on sperm count, motility, or morphology; no teratogenic effects were observed in pregnant rats treated with doses up to 30 mg/kg/day during organogenesis (data limited to preclinical screening) [1].

[1]. Dual angiotensin II and endothelin A receptor antagonists: synthesis of 2'-substituted N-3-isoxazolyl biphenylsulfonamides with improved potency and pharmacokinetics. J Med Chem. 2005 Jan 13;48(1):171-9.

Sparsentan is a biphenyl with the structure 1,1'-biphenyl, substituted at the 2, 2', and 4' positions with (4,5-dimethyl-1,2-oxazol-3-yl)aminosulfonyl, ethoxymethyl, and (2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl groups, respectively. It is a dual antagonist of endothelin and angiotensin II receptors and is approved for reducing proteinuria in adult patients with rapidly progressing primary IgA nephropathy. It has the effects of angiotensin receptor antagonist, antihypertensive agent, endothelin A receptor antagonist, and renal protectant. It is an azaspirocyclic compound belonging to the biphenyl, sulfonamide, isoxazole, and benzyl ether classes. Sparsentan is a dual antagonist of endothelin A receptor (ETAR) and angiotensin II (Ang II) type 1 receptor (AT1R), exhibiting similar affinities for both (9.3 nM for ETAR and 0.8 nM for AT1R). Sparsentan is the first orally active drug in its class, composed of structural units from the AT1R antagonist irbesartan and the ETAR antagonist biphenylsulfonamide. In February 2023, the U.S. Food and Drug Administration (FDA) approved Sparsentan for reducing proteinuria in adult patients with rapidly progressing primary immunoglobulin A nephropathy (IgAN), an accelerated approval based on its reduction in proteinuria. Sparsentan was initially developed for the treatment of hypertension; however, it has been shown to effectively reduce proteinuria in patients with IgA nephropathy and focal segmental glomerulosclerosis (FSGS). Compared to irbesartan, Sparsentan significantly reduces proteinuria. Furthermore, it is the first non-immunosuppressant used to reduce proteinuria in IgA nephropathy. Use of sparsentan may result in hepatotoxicity and embryo-fetal toxicity. Sparsentan is an endothelin receptor antagonist and angiotensin II receptor blocker. Its mechanism of action is as an endothelin receptor antagonist, an angiotensin II type 1 receptor antagonist, a cytochrome P450 2B6 inducer, a cytochrome P450 2C9 inducer, a cytochrome P450 2C19 inducer, a P-glycoprotein inhibitor, and a breast cancer resistance protein inhibitor. Indications: Sparsentan is indicated for reducing proteinuria in adult patients with primary immunoglobulin A nephropathy (IgAN) at risk of rapid progression, typically in patients with a urine protein/creatinine ratio (UPCR) ≥1.5 g/g.
Treatment of Focal Segmental Glomerulosclerosis
Treatment of Primary IgA Nephropathy
Mechanism of Action
Spasentan is a dual antagonist molecule. This drug blocks the endothelin A receptor (ET_AR) and the angiotensin II (Ang II) 1 receptor (AT₁R). It has two clinically validated mechanisms of action, selectively blocking the effects of two potent vasoconstrictors and mitogens—Ang II and endothelin 1 (ET-1)—on their respective receptors. ET-1 and Ang II are involved in the pathogenesis of immunoglobulin A nephropathy (IgAN), characterized by increased production of galactose-deficient IgA1 (Gd-IgA1) antibodies. Gd-IgA1 antibodies lead to mesangial cell activation and proliferation, which in turn stimulates the production of ET-1 and Ang II, and is in turn stimulated by ET-1 and Ang II. The pathological cycle of IgAN ultimately leads to damage to the glomerular filtration barrier, resulting in secondary proteinuria and hematuria. Spasentan, acting as both an angiotensin receptor blocker (ARB) and an endothelin receptor antagonist (ERA), reduces proteinuria in patients with IgA nephropathy. Spasentan exhibits high affinity for both ETAR (Ki = 12.8 nM) and ATIR (Ki = 0.36 nM), and its selectivity for these two receptors is more than 500 times higher than that for endothelin B receptor and angiotensin II subtype 2 receptor.

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Pharmacodynamics
Spasentan is a dual endothelin and angiotensin II receptor antagonist.
At week 36, within the observed sparsentan exposure range, the exposure-response (ER) relationship between sparsentan exposure and the percentage decrease in the urine protein-to-creatinine ratio (UPCR) from baseline was not statistically significant. ER was not statistically associated with any level of hypotension or the most severe level of peripheral edema. In healthy subjects, sparsentan caused QTc interval prolongation, with a maximum mean effect of 8.8 ms at a dose of 800 mg and 8.1 ms at a dose of 1600 mg. The mechanism of the observed QTc interval prolongation is unclear, but it is unlikely to be mediated by direct inhibition of hERG channels. At the recommended dose, clinically significant QTc interval prolongation is not expected. Use of sparsentan may cause hepatotoxicity, embryo-fetal toxicity, hypotension, acute kidney injury, hyperkalemia, and fluid retention. Sparsentan is a novel dual angiotensin II type 1 (AT₁) and endothelin A (ETₐ) receptor antagonist developed from 2'-substituted N-3-isoxazolyl biphenylsulfonamide compounds. Compared with previous dual antagonists, its efficacy against both receptors has been optimized, and it has better pharmacokinetic properties (oral bioavailability, half-life)[1]. 2. Mechanism of action: Sparsentan competitively binds to AT₁ and ETₐ receptors, blocking the signaling pathways activated by their endogenous ligands (angiotensin II and endothelin-1, respectively). This dual inhibitory effect can inhibit vasoconstriction, renal fibrosis, vascular hypertrophy, and inflammation—pathological processes associated with hypertension, chronic kidney disease (CKD), and heart failure[1].
3. Theoretical basis of dual-target strategy: Single AT₁ or ETₐ receptor antagonists have limited efficacy in treating progressive nephropathy and refractory hypertension because activation of one receptor pathway can compensate for inhibition of the other. Sparsentan addresses this limitation by blocking both pathways simultaneously [1]
4. Clinical development background (2005): At the time of the study, Sparsentan was in the preclinical development stage for the treatment of hypertensive nephropathy and chronic nephropathy; subsequent clinical trials confirmed its efficacy in focal segmental glomerulosclerosis (FSGS) and IgA nephropathy, and it was eventually approved by the FDA for these indications (data after 2005 are not included due to study limitations) [1]

C32H40N4O5S

592.7488

592.272

C, 64.84; H, 6.80; N, 9.45; O, 13.50; S, 5.41

254740-64-2

Sparsentan-d5; 1801597-09-0

10257882

White to off-white solid powder

7.066

1

8

12

42

1060

0

O=S(C1=CC=CC=C1C2=CC=C(CN3C(CCCC)=NC4(CCCC4)C3=O)C=C2COCC)(NC5=NOC(C)=C5C)=O

WRFHGDPIDHPWIQ-UHFFFAOYSA-N

InChI=1S/C32H40N4O5S/c1-5-7-14-29-33-32(17-10-11-18-32)31(37)36(29)20-24-15-16-26(25(19-24)21-40-6-2)27-12-8-9-13-28(27)42(38,39)35-30-22(3)23(4)41-34-30/h8-9,12-13,15-16,19H,5-7,10-11,14,17-18,20-21H2,1-4H3,(H,34,35)

2-[4-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-2-(ethoxymethyl)phenyl]-N-(4,5-dimethyl-1,2-oxazol-3-yl)benzenesulfonamide

RE-021; BMS 346567; RE021; Filspari; PS-433540; BMS346567; RE 021; PS 433540; DARA-a; BMS-346567

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 (~168.7 mM)
Ethanol: ~40 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.51 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 20.8 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: 2.08 mg/mL (3.51 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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: ≥ 2.08 mg/mL (3.51 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 20.8 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.)