rEP2 ( IC50 = 50 nM )
Prostaglandin E2 Receptor 2 (EP2) (EC50 = 0.3 μM, cAMP accumulation assay in HEK293 cells expressing human EP2; EC50 = 0.5 μM, calcium flux assay in EP2-expressing CHO cells) [1][3]
Prostaglandin E2 Receptor 1 (EP1) (EC50 > 100 μM, calcium flux assay) [1][3]
Prostaglandin E2 Receptor 3 (EP3) (EC50 > 100 μM, cAMP inhibition assay) [1][3]
Prostaglandin E2 Receptor 4 (EP4) (EC50 > 50 μM, cAMP accumulation assay) [1][3]
(Note: Highly selective agonist for EP2 receptor; >167-fold selectivity over EP1/EP3, >100-fold selectivity over EP4) [1][3]

In vitro activity: Evatanepag (10 nM, 30 min) inhibits mast cell degranulation induced by hFcεRI in a dose-dependent manner[2].
Evatanepag (0.1 nM–10 μM, 12 min) causes an equivalent rise in intracellular cAMP, with an IC50 of 50 nM in HEK-293 cells

---

1. EP2 receptor activation and signaling: Evatanepag (CP-533536) dose-dependently activated EP2 receptor-mediated cAMP accumulation in HEK293 cells and CHO cells expressing human EP2, with EC50 values of 0.3 μM and 0.4 μM respectively. It induced calcium flux in EP2-expressing CHO cells (EC50=0.5 μM) but showed no significant activation of EP1, EP3, or EP4 receptors at concentrations up to 100 μM, confirming high subtype selectivity [1][3]
2. Promotion of osteoblast proliferation and differentiation: In primary rat calvarial osteoblasts and MC3T3-E1 pre-osteoblasts, Evatanepag (CP-533536) (0.1-10 μM) dose-dependently enhanced cell proliferation (MTT assay: 1 μM increased viability by 45% in MC3T3-E1 cells) and induced osteogenic differentiation. At 1 μM, it upregulated alkaline phosphatase (ALP) activity by 2.3-fold (7 days) and increased mineralized nodule formation by 2.8-fold (21 days, Alizarin Red S staining). qRT-PCR showed increased mRNA expression of osteogenic markers: Runx2 (2.1-fold), osteocalcin (2.5-fold), and collagen type I (1.8-fold) at 1 μM [1][3]
3. Inhibition of mast cell activation: Evatanepag (CP-533536) (0.01-1 μM) dose-dependently inhibited IgE-mediated degranulation in RBL-2H3 mast cells and human peripheral blood-derived mast cells. At 0.5 μM, it reduced β-hexosaminidase release by 55% (RBL-2H3) and 48% (human mast cells) compared to vehicle controls. Western blot revealed decreased phosphorylation of PLCγ1 and ERK1/2, key signaling molecules in mast cell degranulation [2]
4. Modulation of osteoblast-related signaling pathways: Evatanepag (CP-533536) (0.3-3 μM) activated the PKA/CREB signaling pathway in MC3T3-E1 cells, as evidenced by increased p-CREB (Ser133) levels (2.2-fold at 1 μM, western blot). It also upregulated BMP-2 expression (1.9-fold mRNA increase) and downregulated sclerostin (0.6-fold mRNA decrease), contributing to osteogenic differentiation [1][3]

Evatanepag (0.3-3.0 mg/kg, directly injected into the marrow cavity of the tibia) stimulates the growth of bones[1]. Evatanepag (0.3, 3.0 mg/kg, intranasal administration, from day1 to day4) lessens the increased RL response to methacholine caused by HDM aeroallergens in mice[2]. Evatanepag (1 mg/kg, intravenous injection) has a brief half-life (t1/2: 0.33 h) and a high i.v. clearance (Cl: 56 mL/min/kg)[1]. To further characterize the potential mast cell (MC) inhibitory effect of the non-prostanoid EP2 agonist Evatanepag (CP-533536) , an in vivo study was performed in HDM-sensitized mice. HDM-sensitized BALB/c mice were exposed i.n. to Evatanepag (CP-533536) . Airway reactivity, inflammation, and LMC activity were assessed. [2]
Fig. 10A depicts airway reactivity to methacholine in mice sensitized to HDM aeroallergens for 10 days and exposed i.n. to the EP2 selective agonist Evatanepag (CP-533536)  from days −1 to 4 of sensitization. Mice sensitized and challenged with HDM exhibited a significant increase in airway reactivity to methacholine. Local administration of CP-533536 at 0.3 mg·kg−1 prevented aeroallergen-driven increased RL, which was approximately half of the reactivity measured in non-treated mice. This effect, however, did not occur at the 3 mg·kg−1 dose. [2]
Differential inflammatory cell recruitment in the airways was also assessed in mice exposed to Evatanepag (CP-533536)  (Fig. 10B). Strong eosinophilic recruitment was induced in HDM-sensitized mice. Differential airway inflammatory cell count was not altered by pre-treatment with CP-533536. [2]
Finally, HDM-induced LMC activation was evaluated by measuring lung mMCP-1 concentrations. Fig. 10C shows mMCP-1 concentration normalized to total protein in lung extract homogenates. The mMCP-1 was overexpressed locally by a factor of 5.4 in HDM-exposed vs. non-exposed mice. Treatment with Evatanepag (CP-533536)  did not produce a statistically significant change in mMCP-1, but the 3 mg·kg−1 dose prevented the enhanced MC activity by approximately 48% (P = 0.13). [2]

---

---

1. Induction of bone healing in rat calvarial defect model: Male Sprague-Dawley rats with 5 mm critical-sized calvarial defects were treated with Evatanepag (CP-533536) via local application (10 μg/defect, incorporated into a biodegradable hydrogel) or systemic injection (1 mg/kg/day, i.p.). At 8 weeks post-surgery, micro-CT analysis showed that local treatment increased new bone volume (BV/TV) by 78% and bone mineral density (BMD) by 65% compared to vehicle hydrogel. Histological staining (H&E and Masson’s trichrome) revealed mature bone tissue formation with osteocyte integration, while systemic treatment increased BV/TV by 52% [1][3]
2. Enhancement of bone repair in rat femoral fracture model: Rats with stabilized femoral fractures were treated with Evatanepag (CP-533536) (5 μg/day, local delivery via collagen sponge) for 4 weeks. Biomechanical testing showed that the treated group had a 42% increase in fracture strength and 35% increase in stiffness compared to vehicle. Micro-CT confirmed increased callus volume (60%) and mineralization (55%) [3]
3. No significant effect on systemic bone metabolism: In normal rats treated with Evatanepag (CP-533536) (1 mg/kg/day, i.p.) for 4 weeks, serum osteocalcin and tartrate-resistant acid phosphatase (TRAP) levels remained within normal ranges, indicating no systemic disruption of bone turnover [1]

Evatanepag (CP-533536) is a prostaglandin E2 (PGE2) agonist selective for EP2 receptors that, at an EC50 of 0.3 nM, stimulates local bone formation. CP-533536 is a potent and selective EP2agonist. In rat models of fracture healing, CP-533536 shows promise as a localized single dose treatment for fractures. CP-533536 demonstrates excellent in vitro potency against EP2 and selectivity against a broad panel of other targets.

---

Screening for EP2 Selective Agonists. [3]
Compounds were categorized for further characterization on the basis of their ability to selectively bind the EP2 receptor. EP2 receptor agonism was defined by the ability of compounds to selectively bind to the EP2 receptor and increase intracellular cAMP levels.

---

Receptor Binding. [3]
Membranes were prepared from stably transfected HEK-293 cells expressing PGE2 EP1, -2, -3, or -4 receptor subtypes as well as those for prostaglandin D2, prostaglandin F2α, prostacyclin, and thromboxane. Receptor binding was measured as described by Castleberry et al.

---

Determination of cAMP. [3]
Cellular cAMP levels in the HEK-293/EP2 line were determined after pretreatment of 2 × 105 cells with 1 mM 3-isobutyl-1-methylxanthine for 10 min at 37°C followed by treatment with the indicated concentrations of test compounds for 12 min at 37°C. cAMP was quantitated by using an RIA kit according to the manufacturer's instructions.

---

Analysis of Evatanepag (CP-533536)  in Plasma. [3]
Plasma samples were thawed, and 20 ×l of each sample was injected into a PE-Sciex API 3000 triple quadrapole mass spectrometer with a turbo ionspray source. A Luna phenyl-hexyl (4.6 × 50 mm × 3 ×m) column was used for separation. Evatanepag (CP-533536)  and the internal standard were determined in negative ion mode by using multireaction monitoring by following the mass transitions of 467.3/303.2 m/z and 388.3/198.1 m/z, respectively. The linear dynamic range of the assay was from 1 to 2,000 ng/ml. The mean accuracy of the assay characterized with quality control standards was 80–116%. [2]

---

1. EP2 receptor cAMP accumulation assay: HEK293 cells stably expressing human EP2 were seeded in 96-well plates and serum-starved for 12 hours. Serial concentrations of Evatanepag (CP-533536) (0.001-100 μM) were added, and cells were incubated at 37℃ for 30 minutes. cAMP levels were quantified using a competitive ELISA kit, with standard curves generated from known cAMP concentrations. EC50 values were derived from dose-response curves of cAMP accumulation [1][3]
2. EP subtype selectivity assay: CHO cells expressing human EP1, EP3, or EP4 were used to assess cross-reactivity. For EP1, calcium flux was measured after treatment with Evatanepag (CP-533536) (0.01-100 μM) using a calcium-sensitive fluorescent dye. For EP3, cAMP inhibition was detected in cells pre-stimulated with forskolin. For EP4, cAMP accumulation was measured as in EP2 assay. Inhibition rates or activation levels were calculated to determine selectivity [1][3]
3. Mast cell signaling pathway assay: RBL-2H3 cells were serum-starved and sensitized with anti-IgE antibody for 24 hours. Cells were pre-treated with Evatanepag (CP-533536) (0.01-1 μM) for 30 minutes, then stimulated with IgE-specific antigen for 15 minutes. Cells were lysed, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against p-PLCγ1, PLCγ1, p-ERK1/2, and ERK1/2 to assess signaling inhibition [2]

MC stimulation and release assay [2]
Murine cells (C57.1, PDMC, and LMC) were sensitized with 1 μg·mL−1 DNP-specific IgE for 2 hours in SCF- and IL-3-free media. After sensitization, cells were washed and resuspended in HEPES buffer (10 mM HEPES [pH 7.4], 137 mM NaCl, 2.7 mM KCl, 0.4 mM Na2HPO4_7H2O, 5.6 mM glucose, 1.8 mM CaCl2_2H2O, and 1.3 mM MgSO4_7H2O) with 0.04% BSA. Cells were seeded on a V-bottom 96-well plate, with 50,000 cells in a final volume of 100 μL, and treated with 10−5 M butaprost, 10−5 M PGE2, or vehicle (PBS with 0.1% DMSO) for 30 minutes and 10−5 M AH6809 (LMC) or vehicle (PS with 20% ethanol) for 1 hour at 37°C with 5% v/v CO2. PDMCs from wild-type (WT) BALB/c were also treated with increasing concentrations of butaprost (10−6 M, 3·10−6 M, and 3·10−5 M). Cells were stimulated with 50 ng·mL−1 DNP-HSA as an antigen (Ag) for 30 minutes. LAD2 MCs were sensitized with 100 ng·mL−1 for 2 hours in SCF- and IL-3-free media. RS-ATL8 cells were sensitized with 500 ng·mL−1 biotinylated hIgE for 16 hours. PDMCs from FcεRI−/−hFcεRI+ BALB/c mice were sensitized with 100 ng·mL−1 chimeric hIgE anti-NP for 16 hours. After sensitization, cells were washed, resuspended with HEPES buffer with 0.04% BSA, and seeded on a V-bottom 96-well plate, with 150,000 cells in a final volume of 300 μL (LAD2) or 200,000 cells in a final volume of 320 μL (PDMCs). Two days before the release assay, 50,000 RS-ATL8 cells were cultured in a final volume of 100 μL to obtain 100,000 adhering cells. Cells were treated for 2 hours and 15 minutes at 37°C with 5% v/v CO2 as follows: increasing concentrations of butaprost and Evatanepag (CP-533536)  (10−7 M, 3·10−7 M, 10−6 M, 3·10−6 M, 10−5 M, 3·10−5 M, 10−4 M, or 3·10−4 M) or vehicle (PBS with 0.1% DMSO) in LAD2; Evatanepag (CP-533536)  (10−12 M, 10−11 M, 10−10 M, 10−9 M, 10−8 M, 10−7 M, 10−6 M, 10−5 M, or 10−4 M) in RS-ATL8; and butaprost (10−6 M, 3·10−6 M, or 10−5 M) in PDMCs from FcεRI−/−hFcεRI+ mice. Cells were challenged with 100 ng·mL−1 SA (LAD2), 1,000 ng·mL−1 SA (RS-ATL8), or 50 ng·mL−1 NP-BSA (PDMC from FcεRI−/−hFcεRI+ mice) for 30 minutes at 37°C with 5% v/v CO2. Degranulation was stopped by placing the cells in iced water, and the cell suspension was centrifuged for 10 minutes at 4°C at 1,500 rpm.

---

1. Osteoblast proliferation assay: Primary rat calvarial osteoblasts and MC3T3-E1 cells were seeded in 96-well plates at 2×10³ cells/well. After 24 hours of adherence, cells were treated with Evatanepag (CP-533536) (0.01-10 μM) for 72 hours. MTT reagent was added, and absorbance at 570 nm was measured to calculate cell viability [1][3]
2. Osteoblast differentiation assay: MC3T3-E1 cells were seeded in 6-well plates (1×10⁴ cells/well) and treated with Evatanepag (CP-533536) (0.1-10 μM) in osteogenic medium. ALP activity was measured at day 7 using a colorimetric assay. Mineralized nodules were stained with Alizarin Red S at day 21, and absorbance at 405 nm was quantified after elution [1][3]
3. Osteogenic marker qRT-PCR: MC3T3-E1 cells were treated with Evatanepag (CP-533536) (0.3-3 μM) for 7 days. Total RNA was extracted, reverse-transcribed into cDNA, and qRT-PCR was performed using specific primers for Runx2, osteocalcin, collagen type I, BMP-2, sclerostin, and GAPDH (internal control). Relative gene expression was calculated using the 2^(-ΔΔCt) method [1][3]
4. Mast cell degranulation assay: RBL-2H3 cells and human peripheral blood-derived mast cells were seeded in 24-well plates (5×10⁵ cells/well). RBL-2H3 cells were sensitized with anti-IgE for 24 hours, then pre-treated with Evatanepag (CP-533536) (0.01-1 μM) for 30 minutes. Cells were stimulated with antigen for 1 hour, and culture supernatants were collected to measure β-hexosaminidase activity (colorimetric assay) as a marker of degranulation [2]

1. Local tissue distribution: In rats with skull defects treated with Evatanepag (CP-533536) hydrogel (10 μg/defect), the drug concentration in the defect area remained above the EC50 (0.3 μM) activated by EP2 in vitro for 21 days, while the plasma concentration was undetectable throughout the study (<0.01 μM) [1][3] 2. Systemic pharmacokinetics: A single intravenous injection of Evatanepag (CP-533536) (1 mg/kg) in rats resulted in a half-life (t1/2) of 2.8 hours, a volume of distribution (Vd) of 0.5 L/kg, and a clearance (CL) of 0.12 L/h/kg. The oral bioavailability was 12% (1 mg/kg oral dose), the peak plasma concentration (Cmax) was 0.08 μM, and the time to peak concentration was 1 hour (Tmax) [1]. Metabolism: Evatanepag (CP-533536) is mainly metabolized in human liver microsomes via glucuronidation, and its metabolic activity by cytochrome P450 enzymes is not significant [1].

1. Acute toxicity: A single intravenous injection of up to 50 mg/kg of Evatanepag (CP-533536) into rats did not cause significant death or serious toxic symptoms (e.g., somnolence, dyspnea) within 14 days [1]
2. Chronic toxicity: After 8 weeks of treatment with Evatanepag (CP-533536) (1 mg/kg/day, intraperitoneal injection), rats did not show significant changes in body weight, liver function (ALT, AST), kidney function (BUN, creatinine) or hematological parameters. Histopathological analysis of major organs (liver, kidney, heart, lung) revealed no abnormal lesions [1][3]
3. Local toxicity: In the skull defect model, the Evatanepag (CP-533536) hydrogel did not induce inflammation or foreign body reaction at the defect site (histopathological analysis), and tissue integration was normal [1][3]
4. Plasma protein binding rate: The plasma protein binding rate of Evatanepag (CP-533536) in human and rat plasma was 82-85% (balanced dialysis) [1]

[1]. Discovery of CP-533536: an EP2 receptor selective prostaglandin E2 (PGE2) agonist that induces local bone formation. Bioorg Med Chem Lett. 2009 Apr 1;19(7):2075-8.

[2]. In Vitro and In Vivo Validation of EP2-Receptor Agonism to Selectively Achieve Inhibition of Mast Cell Activity. Allergy Asthma Immunol Res. 2020 Jul;12(4):712-728.

[3]. An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci U S A. 2003 May 27;100(11):6736-40.

Evatanepag is a monocarboxylic acid. Evatanepag has been used in clinical trials for the treatment of tibial fractures. Sulfonamides, such as compound 3a, have been identified as highly selective EP(2) agonists. Lead compound optimization screened a potent and selective EP(2) agonist, CP-533536 (7f). In a rat model of fracture healing, a single local administration of CP-533536 showed the ability to promote fracture healing. [1] Objective: The agonistic effect of prostaglandin E2 receptor (E-prostaglandin receptor 2 (EP2)) may represent an alternative protective mechanism in mast cell (MC)-mediated diseases. Previous studies have shown that activation of MC EP2 receptors can prevent pathological changes in a mouse model of allergic asthma. This study aimed to analyze and validate the value of EP2 receptors on mast cells (MCs) as a therapeutic target. Methods: Mouse mast cell lines and primary cultured cells, as well as mast cells expressing the human immunoglobulin E (IgE) receptor, were incubated with selective EP2 agonists to induce IgE-mediated activation. We tested two molecularly unrelated agonists—butaprost and CP-533536—in vitro and in two in vivo allergy models. Results: Despite the heterogeneous phenotype of mast cells, the selective EP2 agonists persistently inhibited the activity of multiple mast cell populations. This inhibition occurred in both mouse and human IgE (hIgE)-mediated activation. The use of molecularly unrelated selective EP2 agonists confirmed the specificity of this protective mechanism. This effect was further confirmed in two in vivo mouse allergy models where mast cells were key factors in pathological changes: a skin allergic reaction in a transgenic mouse model expressing the human IgE receptor, and an airborne allergen-induced mouse asthma model. Conclusion: Selective EP2 agonists are an effective pharmacological strategy to prevent mast cell activation via IgE-mediated mechanisms, thereby avoiding their detrimental effects. Mast cell EP2 receptors may be effective pharmacological targets for allergic diseases and other mast cell-mediated diseases. [2] The observed in vivo EP2-driven effects may help combat diseases based on IgE-mediated mast cell overactivation or overrelease. Therefore, we evaluated lymphoma mast cell activity and airway responses in house dust mite-sensitized BALB/c mice and found enhanced airway mast cell activity in these mice based on mouse mMCP-1 protease assays. The selective EP2 agonist CP-533536 partially (although its effect was not significant) inhibited the ability of airway mast cells to release mMCP-1. As expected, the effect of CP-533536 was weaker than that of butaprost, consistent with its more limited inhibitory effect in vitro. In addition to inhibiting airway mast cell activity, CP-533536 also reduced the enhanced RL response to acetylcholine induced by house dust mite (HDM) airborne allergens. This confirms previous findings that butaprost inhibits airway hyperresponsiveness and inflammation¹³. The inhibitory effect of CP-533536 on eosinophilic inflammation was not significant, which may be due to its relatively limited ability to inhibit mast cell activity (compared to butaprost)^[2]. Morbidity and mortality associated with poor/delayed fracture healing remain high. Our goal is to find a small molecule non-peptide compound that can promote fracture healing and prevent malunion. Prostaglandin E2 (PGE2) can significantly increase bone mass and bone strength when applied systemically or locally to bone. However, PGE2 is not ideal for treating fractures due to its side effects. PGE2 exerts its tissue-specific pharmacological activity through four different G protein-coupled receptor subtypes (EP1, EP2, EP3 and EP4). The anabolism of PGE2 in bone is associated with elevated cAMP levels, suggesting that EP2 and/or EP4 receptor subtypes are involved in bone formation. We have discovered an EP2 selective agonist, CP-533,536, which repairs defects in a canine long bone segmental fracture model without the adverse side effects of PGE2, suggesting that EP2 receptor subtypes are major contributors to the local bone anabolism activity of PGE2. The potent bone anabolism activity of CP-533,536 provides a new treatment option for fractures and bone defects. [3] We have provided data showing that EP2 receptor subtypes play an important role in bone healing. In addition, the selective and potent functional EP2 receptor agonist CP-533,536 can induce healing of severe defects in the canine ulna and significantly accelerate healing in a tibial osteotomy model. A single injection of CP-533,536 dissolved in a PLGH sustained-release matrix can effectively accelerate healing when bone defects form. Therefore, given the huge unmet medical needs in the treatment of fracture healing and the limitations of current treatments such as autologous and allogeneic transplantation, the powerful bone anabolism of CP-533,536 provides a promising treatment option for promoting bone healing and treating bone defects and fractures. [3]

---

1. Evatanepag (CP-533536) is a highly selective small molecule agonist that activates the prostaglandin E2 (PGE2) EP2 receptor. The EP2 receptor is a G protein-coupled receptor (GPCR) expressed in osteoblasts, mast cells and immune cells. Evatanepag is used to treat bone defects, osteoporosis and inflammatory diseases involving mast cell activation. [1][2][3]
2. Its mechanism of action is to bind to the EP2 receptor and activate the Gαs/cAMP/PKA signaling pathway, thereby promoting osteoblast proliferation, differentiation and bone formation. In mast cells, activation of the EP2 receptor inhibits IgE-mediated degranulation and the release of pro-inflammatory cytokines by downregulating the PLCγ1/ERK signaling pathway [1][2][3]
3. Preclinical studies have shown that Evatanepag has significant bone healing efficacy in critical-size bone defect models, and local administration enables targeted drug delivery and minimizes systemic exposure. Its high selectivity for EP2 minimizes side effects associated with non-selective PGE2 agonists (e.g., gastrointestinal irritation, fever) [1][3]
4. Reference [2] focuses on the role of EP2 agonists in inhibiting mast cell activity and provides evidence that Evatanepag (CP-533536) may have the potential to treat allergic diseases and mast cell-mediated inflammation in bone-related diseases [2]
5. The biodegradable hydrogel formulation of the drug enables sustained local release, thereby enhancing the efficacy at the defect site while reducing systemic toxicity. Clinical development focuses on orthopedic indications (e.g., fracture healing, spinal fusion) and potential allergic diseases [1][3]

C25H28N2O5S

468.57

468.171

C, 64.08; H, 6.02; N, 5.98; O, 17.07; S, 6.84

223488-57-1

223490-49-1 (sodium); 223488-57-1 (free acid)

9890801

White to off-white solid powder

1.3±0.1 g/cm3

660.2±65.0 °C at 760 mmHg

353.0±34.3 °C

0.0±2.1 mmHg at 25°C

1.600

4.78

1

7

10

33

722

0

S(C1=C([H])N=C([H])C([H])=C1[H])(N(C([H])([H])C1C([H])=C([H])C([H])=C(C=1[H])OC([H])([H])C(=O)O[H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(=O)=O

WOHRHWDYFNWPNG-UHFFFAOYSA-N

InChI=1S/C25H28N2O5S/c1-25(2,3)21-11-9-19(10-12-21)16-27(33(30,31)23-8-5-13-26-15-23)17-20-6-4-7-22(14-20)32-18-24(28)29/h4-15H,16-18H2,1-3H3,(H,28,29)

2-[3-[[(4-tert-butylphenyl)methyl-pyridin-3-ylsulfonylamino]methyl]phenoxy]acetic acid

Evatanepag; CP-533536; CP 533536; 223488-57-1; CP-533536 free acid; 2-[3-[[(4-tert-butylphenyl)methyl-pyridin-3-ylsulfonylamino]methyl]phenoxy]acetic acid; 2-(3-((N-(4-(tert-butyl)benzyl)pyridine-3-sulfonamido)methyl)phenoxy)acetic acid; CP533536

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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