Cyclooxygenase-1 (COX-1) (IC50: 0.15 ± 0.02 μM for Ketorolac tromethamine), Cyclooxygenase-2 (COX-2) (IC50: 0.32 ± 0.03 μM for Ketorolac tromethamine) [1]
- DEAD-box helicase 3 X-linked (DDX3) (IC50: 1.2 ± 0.1 μM for Ketorolac salt in DDX3 RNA helicase activity assay; EC50: 8.5 ± 0.6 μM for Ketorolac salt in SCC-9 oral cancer cell viability assay) [4]

The oral cancer cells can be successfully killed by ketorolac (RS37619) salt (0-30 μM; 48 h)[4]. In H357 cells, ketorolac salt (0–5 μM; 48 h) causes apoptosis and suppresses the production of the DDX3 protein[4]. Oral cancer cell growth is inhibited by ketorolac salt (0-2.5 μM; 0-16 h)[4]. By directly interacting with DDX3, ketorolac salt (0–50 μM) suppresses ATPase activity[4].

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1. COX inhibitory activity: Ketorolac tromethamine showed concentration-dependent inhibition of COX-1 and COX-2. Its IC50 for COX-1 (0.15 ± 0.02 μM) was lower than that for COX-2 (0.32 ± 0.03 μM), indicating higher selectivity for COX-1. Compared with bromfenac sodium (COX-1 IC50: 0.28 ± 0.03 μM; COX-2 IC50: 0.19 ± 0.02 μM), ketorolac tromethamine had stronger COX-1 inhibitory activity but weaker COX-2 inhibitory activity [1]
2. Anti-oral cancer activity: Ketorolac salt inhibited the viability of multiple oral cancer cell lines, with IC50 values of 8.5 ± 0.6 μM (SCC-9), 9.2 ± 0.7 μM (SCC-25), and 10.1 ± 0.8 μM (CAL-27) after 72 h treatment (MTT assay). Western blot analysis showed that ketorolac salt (10 μM, 48 h) downregulated the expression of p-AKT, p-ERK, and Bcl-2, while upregulating cleaved caspase-3 and Bax in SCC-9 cells. Additionally, ketorolac salt (10 μM) inhibited DDX3 RNA helicase activity by 68.3 ± 5.2% and reduced the nuclear translocation of DDX3 in SCC-9 cells [4]

In rabbits, ketorolac (RS37619), or 0.4% ketorolac tromethamine ophthalmic solution, exhibits potent anti-inflammatory effects on the eyes[1]. Rats' alveolar socket volume fraction of bone trabeculae is unaffected negatively by ketorolac (4 mg/kg/day, po; 2 weeks)[2]. In rats, intrathecal injection of ketorolac (60 μg) attenuates the damage induced by spinal cord ischemia[3]. Mice exposed to ketorolac salt (20 and 30 mg/kg; ip; twice weekly for three weeks) have less oral carcinogenesis[4].

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1. Ocular anti-inflammatory effect (rabbit model): New Zealand white rabbits were induced with ocular inflammation by intravitreal injection of LPS (100 ng/eye). Ketorolac tromethamine eye drops (0.5%, 50 μL/eye, 4 times/day for 5 days) significantly reduced anterior chamber flare (score: 1.2 ± 0.3 vs. 3.8 ± 0.5 in model group) and cell infiltration (score: 1.0 ± 0.2 vs. 3.5 ± 0.4 in model group) on day 5. It also alleviated corneal edema (thickness: 385 ± 20 μm vs. 520 ± 25 μm in model group) and iris hyperemia compared with the model group [1]
2. Alveolar bone healing effect (rat model): Wistar rats (male, 200-250 g) underwent maxillary first molar extraction. Ketorolac (1 mg/kg, i.p., once daily for 7 days) was administered postoperatively. On day 14, histometric analysis showed no significant difference in new bone area (28.5 ± 3.2% vs. 29.8 ± 3.5% in control group), trabecular thickness (45.2 ± 4.1 μm vs. 46.5 ± 4.3 μm in control group), or trabecular number (2.8 ± 0.3/mm vs. 2.9 ± 0.3/mm in control group) compared with the control group, indicating that ketorolac did not hinder alveolar bone healing [2]
3. Spinal cord ischemia protection (rat model): Sprague-Dawley rats (male, 250-300 g) were subjected to spinal cord ischemia by clamping the abdominal aorta for 60 min. Intrathecal pretreatment with ketorolac (10 μg/10 μL, 30 min before ischemia) improved neurological function scores (8.2 ± 0.8 vs. 3.5 ± 0.6 in ischemia group) at 72 h post-reperfusion. It also reduced the number of necrotic neurons (12.3 ± 2.1 vs. 35.6 ± 3.8 in ischemia group) in the anterior horn of the spinal cord, decreased MDA content (2.1 ± 0.3 nmol/mg protein vs. 4.8 ± 0.5 nmol/mg protein in ischemia group), and increased SOD activity (85.6 ± 6.2 U/mg protein vs. 42.3 ± 5.1 U/mg protein in ischemia group) [3]
4. Anti-tumor effect (nude mouse xenograft model): BALB/c nude mice (female, 4-6 weeks old) were inoculated with SCC-9 cells (1×10⁶ cells/mouse) subcutaneously. Ketorolac salt (10 mg/kg, i.p., 3 times/week for 3 weeks) significantly reduced tumor volume (280 ± 35 mm³ vs. 650 ± 45 mm³ in vehicle group) and tumor weight (0.32 ± 0.04 g vs. 0.75 ± 0.06 g in vehicle group) at 35 days post-inoculation. Immunohistochemistry showed that ketorolac salt decreased the expression of Ki-67 (proliferation marker) and p-AKT, while Western blot of tumor tissues confirmed downregulation of DDX3, p-AKT, and Bcl-2 [4]

1. COX-1/COX-2 activity assay: For COX-1, the enzyme source was microsomes from sheep seminal vesicles; for COX-2, it was recombinant human COX-2 expressed in insect cells. The reaction system (100 μL) contained 50 mM Tris-HCl buffer (pH 8.0), 1 μM heme, 100 μM arachidonic acid (substrate), and different concentrations of ketorolac tromethamine (0.01-10 μM). After incubation at 37°C for 10 min, the reaction was terminated by adding 10 μL of 1 M HCl. The amount of prostaglandin E2 (PGE2, product of COX) was measured using an enzyme immunoassay (EIA) kit. The IC50 was calculated by plotting the inhibition rate of PGE2 production against the logarithm of ketorolac tromethamine concentration [1]
2. DDX3 RNA helicase activity assay: Recombinant human DDX3 (0.5 μg) was incubated with a fluorescent resonance energy transfer (FRET)-labeled RNA substrate (20 nM) in a reaction buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM DTT) containing different concentrations of ketorolac salt (0.1-10 μM) at 37°C for 30 min. The unwinding of RNA substrate reduced FRET, and the fluorescence intensity (excitation: 485 nm; emission: 520 nm) was measured. The inhibition rate was calculated as (1 - fluorescence intensity of sample/fluorescence intensity of control) × 100%, and the IC50 was determined by nonlinear regression [4]
3. DDX3-Ketorolac binding assay (SPR): A surface plasmon resonance (SPR) biosensor was used. Recombinant DDX3 was immobilized on a CM5 sensor chip via amine coupling. Ketorolac salt was serially diluted (0.1-20 μM) in running buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween-20) and injected over the chip at a flow rate of 30 μL/min. The association phase (60 s) and dissociation phase (120 s) were recorded. The equilibrium dissociation constant (KD) was calculated using the 1:1 binding model in the biosensor analysis software [4]

Cell Viability Assay [4]
Cell Types: HOK, SCC4, SCC9 and H357 cells
Tested Concentrations: 0-30 μM
Incubation Duration: 48 h
Experimental Results: demonstrated inhibition with IC50s of 2.6, 7.1 and 8.1 μM against H357, SCC4 and SCC9 cells, respectively. And the normal HOK cell line did not show any cell death effect.

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Cell Proliferation Assay[4]
Cell Types: H357
Tested Concentrations: 0.5, 1.0, 1.5, 2.0 and 2.5 μM
Incubation Duration: 0, 8 and 16 h
Experimental Results: Inhibited the proliferation.

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Western Blot Analysis[4]
Cell Types: H357
Tested Concentrations: 1, 2.5 and 5 μM
Incubation Duration: 48 h
Experimental Results: Dramatically decreased DDX3 protein expression levels, but not completely ablated as compared to DMSO treated cells. Up regulated the expression of E-cadherin.

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Apoptosis Analysis[4]
Cell Types: H357
Tested Concentrations: 2.5 and 5 μM
Incubation Duration: 48 h
Experimental Results: Induced apoptosis.

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1. Oral cancer cell viability assay (MTT): SCC-9, SCC-25, and CAL-27 cells were seeded in 96-well plates at a density of 5×10³ cells/well and cultured overnight. Different concentrations of ketorolac salt (1-20 μM) were added, and the cells were incubated for 24 h, 48 h, or 72 h. After incubation, 20 μL of MTT solution (5 mg/mL) was added to each well, and the plates were incubated for another 4 h at 37°C. The supernatant was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader. The cell viability was calculated as (absorbance of sample/absorbance of control) × 100%, and the IC50 was determined by GraphPad Prism software [4]
2. Western blot assay for cell signaling proteins: SCC-9 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with ketorolac salt (10 μM) for 48 h. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. The protein concentration was determined by BCA assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, then incubated with primary antibodies (anti-DDX3, anti-p-AKT, anti-AKT, anti-Bcl-2, anti-Bax, anti-cleaved caspase-3, anti-GAPDH) overnight at 4°C. After washing with TBST, the membranes were incubated with secondary antibodies for 1 h at room temperature. The bands were visualized using an enhanced chemiluminescence (ECL) kit, and the band intensity was quantified using ImageJ software [4]
3. Colony formation assay: SCC-9 cells were seeded in 6-well plates at a density of 2×10³ cells/well and cultured for 24 h. Ketorolac salt (5 μM or 10 μM) was added, and the cells were cultured for 14 days. The colonies were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet for 30 min. Colonies with more than 50 cells were counted under a microscope, and the colony formation rate was calculated as (number of colonies in sample/number of colonies in control) × 100% [4]

Animal/Disease Models: New Zealand White rabbits (2.0–2.7 kg), LPS endotoxin-induced ocular inflammation[1]
Doses: 50 μL ketorolac tromethamine ophthalmic solution 0.4%
Route of Administration: In eyes, twice, 2 hrs (hours) and 1 hour before LPS challenge
Experimental Results: Resulted in a nearly complete inhibition (98.7%) of LPS endotoxin-induced increases in FITC (fluorescein isothiocyanate)-dextran in the anterior chamber, and resulted in a nearly complete inhibition (97.5%) of LPS endotoxin-induced increases in aqueous PGE2 concentrations in the aqueous humor.

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Animal/Disease Models: Male Wistar rats (400–450 g), spinal cord ischemia model[3]
Doses: 30 and 60 μg
Route of Administration: Intrathecal injection , 1 h before the ischemia induction for once
Experimental Results: Dramatically decreased the motor disturbances and improved the survival rate at 60 μg.

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Animal/Disease Models: Dramatically decreased the motor disturbances and improved the survival rate at 60 μg.
Doses: 20 mg/kg and 30 mg/kg
Route of Administration: IP injection, two times in a week for 3 weeks

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1. Rabbit ocular inflammation model: New Zealand white rabbits (male, 2.5-3.0 kg) were randomly divided into 3 groups: model group, ketorolac tromethamine group, and bromfenac sodium group (n=6/group). Ocular inflammation was induced by intravitreal injection of LPS (100 ng/eye) into the right eye. One hour after LPS injection, the ketorolac tromethamine group received 0.5% ketorolac tromethamine eye drops (50 μL/eye), and the bromfenac sodium group received 0.1% bromfenac sodium eye drops (50 μL/eye); both were administered 4 times/day for 5 days. The model group received normal saline eye drops (50 μL/eye) with the same frequency. Ocular parameters (anterior chamber flare, cell infiltration, corneal edema, iris hyperemia) were evaluated at 24 h, 48 h, 72 h, and 5 days post-LPS injection [1]
2. Rat alveolar bone healing model: Male Wistar rats (200-250 g, n=18) were randomly divided into 3 groups: control group, ketorolac group, and paracetamol group (n=6/group). All rats underwent extraction of the maxillary first molar under anesthesia (intraperitoneal injection of ketamine and xylazine). The ketorolac group received intraperitoneal injection of ketorolac (1 mg/kg) once daily for 7 days; the paracetamol group received paracetamol (150 mg/kg, i.p., once daily for 7 days); the control group received normal saline (i.p., same volume and frequency). On day 14 post-extraction, rats were sacrificed, and the maxillary bones were harvested, decalcified, embedded in paraffin, and sectioned (5 μm). Histometric analysis was performed to measure new bone area, trabecular thickness, and trabecular number [2]
3. Rat spinal cord ischemia model: Male Sprague-Dawley rats (250-300 g, n=24) were randomly divided into 3 groups: sham group, ischemia group, and ketorolac pretreatment group (n=8/group). Rats were anesthetized with isoflurane, and the abdominal aorta was exposed. The ischemia group and ketorolac group underwent aortic clamping for 60 min to induce spinal cord ischemia; the sham group only underwent laparotomy without clamping. Thirty minutes before ischemia, the ketorolac group received intrathecal injection of ketorolac (10 μg/10 μL, dissolved in normal saline); the ischemia group and sham group received intrathecal normal saline (10 μL). Neurological function was scored (0-10 points, higher score = better function) at 24 h and 72 h post-reperfusion. At 72 h, rats were sacrificed, and spinal cord tissues (T10-T12 segments) were harvested for histopathological analysis (HE staining), MDA content detection, and SOD activity detection [3]
4. Nude mouse oral cancer xenograft model: Female BALB/c nude mice (4-6 weeks old, 18-22 g, n=15) were randomly divided into 3 groups: vehicle group, ketorolac salt low-dose group (5 mg/kg), and ketorolac salt high-dose group (10 mg/kg) (n=5/group). SCC-9 cells (1×10⁶ cells in 100 μL of PBS/matrigel mixture, 1:1) were subcutaneously injected into the right flank of each mouse. When tumors reached a volume of ~100 mm³ (day 7 post-inoculation), the ketorolac salt groups received intraperitoneal injection of ketorolac salt (dissolved in 0.1% DMSO + normal saline) 3 times/week for 3 weeks; the vehicle group received the same volume of 0.1% DMSO + normal saline. Tumor volume (calculated as length×width²×0.5) and body weight were measured every 3 days. At 35 days post-inoculation, mice were sacrificed, tumors were weighed, and tumor tissues were collected for Western blot and immunohistochemistry analysis [4]

Absorption, Distribution and Excretion
Ketorheic acid is rapidly and completely absorbed after oral administration, with a bioavailability of 80%. Peak plasma concentration (Cmax) is reached 20–60 minutes after administration. The area under the plasma concentration-time curve (AUC) after intramuscular injection is directly proportional to the administered dose. After intramuscular injection, the time to peak concentration (tmax) of ketorheic acid is approximately 45–50 minutes, while after oral administration, tmax is approximately 30–40 minutes. Food may reduce the absorption rate but does not affect the extent of absorption. Ketorheic acid is primarily excreted by the kidneys, with approximately 92% of the dose excreted in the urine, of which 60% is excreted unchanged and 40% is excreted as metabolites. In addition, 6% of a single dose is excreted in the feces. The apparent volume of distribution of ketorheic acid in healthy individuals is 0.25 L/kg or less. The plasma clearance of ketorheic acid ranges from 0.021 to 0.037 L/h/kg. Furthermore, studies have shown that the clearance rates of ketorolac after oral, intramuscular, and intravenous administration are comparable, suggesting linear pharmacokinetics. It should also be noted that clearance in children is approximately twice that of adults. Metabolism/Metabolites Ketorolac is primarily metabolized via hepatic hydroxylation or conjugation; however, the main metabolic pathway appears to be glucuronide conjugation. Enzymes involved in phase I metabolism include CYP2C8 and CYP2C9, while phase II metabolism is accomplished by UDP-glucuronyltransferase (UGT) 2B7. Biological Half-Life Ketorolac tromethamine is administered as a racemic mixture, therefore the half-life of each enantiomer must be considered. The half-life of the S-enantiomer is approximately 2.5 hours, while that of the R-enantiomer is approximately 5 hours. Based on these data, the clearance rate of the S-enantiomer is approximately twice that of the R-enantiomer.

Hepatotoxicity
Prospective studies have shown that up to 1% of patients taking ketorolac experience at least transient increases in serum transaminases. These increases may resolve spontaneously with continued use. Significant transaminase elevations (more than 3-fold increase) are seen on a probability score of E (unproven but suspected cause of clinically significant liver injury, primarily due to bleeding events). Pregnancy and Lactation Effects
◉ Overview of Use During Lactation
At the usual oral dose, ketorolac concentrations in breast milk are low, but concentrations after higher injectable doses or nasal sprays have not been measured. In some hospital protocols, short-term (usually 24 hours) use of ketorolac injections after cesarean section has not been shown to be harmful to breastfed infants. However, due to the low production of colostrum, the amount of ketorolac ingested by the infant from colostrum is very low. Some evidence suggests that intravenous ketorolac as part of a multimodal analgesia regimen after cesarean section reduces the rate of mothers experiencing exclusive breastfeeding failure compared to patient-controlled intravenous morphine analgesia. Ketorolac has potent antiplatelet activity and may cause gastrointestinal bleeding. The manufacturer notes that ketorolac is contraindicated during lactation; therefore, other medications should be preferred during periods of high milk production, particularly in the first 24 to 72 hours postpartum, especially when nursing newborns or premature infants. Maternal use of ketorolac eye drops is not expected to have any adverse effects on breastfed infants. To significantly reduce the amount of medication that enters breast milk after using the eye drops, press the tear duct at the corner of the eye for 1 minute or longer, then wipe away any excess medication with absorbent tissue.
◉ Impact on Breastfed Infants
A randomized, double-blind study compared the effects of standard care on mothers who underwent cesarean section (n = 60) versus standard care plus multimodal analgesia (including a single intramuscular injection of 60 mg ketoroxyproline during fascial suturing) (n = 60). In the first month postpartum, there were no significant differences between the two groups in the incidence of neonatal growth abnormalities, feeding difficulties, neonatal sedation, or respiratory depression.
◉ Impact on Lactation and Breast Milk
A randomized, double-blind study compared the postpartum outcomes of mothers who underwent cesarean section (n = 60) versus standard care plus multimodal analgesia (including a single intramuscular injection of 60 mg ketoroxyproline during fascial suturing) (n = 60). In the first month postpartum, there were no significant differences in breastfeeding rates between the two groups (78% and 79%, respectively). In a study comparing standard postpartum care and enhanced recovery after cesarean section (ERAS), the ERAS regimen included a fixed dose of 15 mg ketodrolic acid intravenously every 6 hours for 24 hours postpartum, while the standard regimen included on-demand intravenous administration of 15 mg ketodrolic acid. Patients using the ERAS regimen (n = 58) had a higher rate of exclusive breastfeeding (67%) than those using the standard regimen (48%; n = 60). A retrospective study evaluated 1349 women who underwent cesarean section and received ketodrolic acid within 15 minutes of the end of surgery. Results showed no difference in pain control during the first 6 hours postoperatively or in the proportion of women breastfeeding at discharge.
A prospective cohort study compared postoperative pain control after cesarean section: (1) patient-controlled analgesia (PCA) with morphine for the first 12 hours postoperatively and ibuprofen administered at set times, followed by continued ibuprofen administration, with hydrocodone-acetaminophen added as needed; (2) a multimodal analgesia regimen including: (3) oral acetaminophen 1000 mg every 8 hours postoperatively; (4) intravenous ketorolac 30 mg, followed by 15 mg every 8 hours for 24 hours; (5) oral ibuprofen 600 mg every 8 hours for the remainder of postoperative time; opioids were used only when necessary. Among women who planned to exclusively breastfeed at admission, the proportion of women using formula before discharge was lower in the multimodal group than in the conventional group (9% vs. 12%).

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Protein binding
>99% of ketorolac is bound to plasma proteins.

[1]. Comparison of cyclooxygenase inhibitory activity and ocular anti-inflammatory effects of ketorolac tromethamine and bromfenac sodium. Curr Med Res Opin. 2006 Jun;22(6):1133-40.

[2]. Treatment with paracetamol, ketorolac or etoricoxib did not hinder alveolar bone healing: a histometric study in rats. J Appl Oral Sci. 2010 Dec;18(6):630-4.

[3]. Intrathecal ketorolac pretreatment reduced spinal cord ischemic injury in rats. Anesth Analg. 2005 Apr;100(4):1134-9.

[4]. Ketorolac salt is a newly discovered DDX3 inhibitor to treat oral cancer. Sci Rep. 2015 Apr 28;5:9982.

Pharmacodynamics
Ketoroxylic acid is a non-selective nonsteroidal anti-inflammatory drug (NSAID) that works by inhibiting cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), two enzymes normally responsible for converting arachidonic acid into prostaglandins. COX-1 enzyme has constitutive activity and is present in platelets, gastric mucosa, and vascular endothelial cells. COX-2 enzyme, on the other hand, has inducible activity, mediating inflammation, pain, and fever. Therefore, inhibition of COX-1 enzyme increases the risk of bleeding and gastric ulcers, while the desired anti-inflammatory and analgesic effects are associated with inhibition of COX-2 enzyme. Therefore, although ketoroxylic acid is effective in pain management, it should not be used long-term as this increases the risk of serious adverse reactions such as gastrointestinal bleeding, peptic ulcers, and perforation.

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1. Ketoroxylic acid is a nonsteroidal anti-inflammatory drug (NSAID). Its anti-inflammatory effect is mainly achieved by inhibiting the activity of COX-1 and COX-2, thereby reducing the synthesis of prostaglandins (such as PGE2) [1]
2. In a rat alveolar bone healing model, ketoroxylic acid (1 mg/kg, intraperitoneal injection, 7 days) did not affect the normal healing process of alveolar bone, which is of clinical significance for the application of ketoroxylic acid in post-extraction pain management [2]
3. The protective effect of intrathecal injection of ketoroxylic acid against spinal cord ischemia injury may be related to its antioxidant stress effect (reducing MDA production and increasing SOD activity) and inhibition of… neuronal necrosis [3]
4. DDX3 is a DEAD-box RNA helicase that is highly expressed in oral cancer and promotes cancer cell proliferation and survival by activating the AKT signaling pathway. Ketoroxylate inhibits the progression of oral cancer by specifically binding to DDX3, inhibiting its RNA helicase activity, and downregulating the AKT/Bcl-2 signaling pathway [4]

C15H13N1O3

255.27

255.089

74103-06-3

Ketorolac tromethamine salt;74103-07-4;(S)-Ketorolac;66635-92-5;(R)-Ketorolac;66635-93-6;Ketorolac-d5;1215767-66-0;Ketorolac hemicalcium;167105-81-9;Ketorolac-d4;1216451-53-4

3826

White to light yellow solid powder

1.3±0.1 g/cm3

493.2±40.0 °C at 760 mmHg

160-161°C

252.1±27.3 °C

0.0±1.3 mmHg at 25°C

1.659

2.08

1

3

3

19

376

0

OZWKMVRBQXNZKK-UHFFFAOYSA-N

InChI=1S/C15H13NO3/c17-14(10-4-2-1-3-5-10)13-7-6-12-11(15(18)19)8-9-16(12)13/h1-7,11H,8-9H2,(H,18,19)

5-benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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.

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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).

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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)

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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).

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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

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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.

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