Neural Stem Cell (NSC) proliferation and differentiation-related pathways ( Diclofenac Sodium (GP 45840) inhibited NSC proliferation at concentrations ≥10 μM and disrupted NSC differentiation at 20 μM) [3]

Diclofenac, with an IC50 of 7±3 nM, efficiently inhibits COX-1-mediated microsomal synthesis of prostaglandins in U937 cells[1]. Neural stem cells (NSCs) undergo concentration-dependent apoptosis when exposed to diclofenac sodium (1–60 μM; 1 day) [3]. Cloned (activated) caspase-3 is expressed more when exposed to Diclofenac Sodium (10–60 μM) for six hours [3].

---

1. Inhibition of NSC proliferation: Diclofenac Sodium (GP 45840) was tested on primary NSCs isolated from embryonic day 14 (E14) rat cerebral cortex. After 48 h of treatment, diclofenac sodium at 10 μM significantly reduced BrdU (5-bromo-2'-deoxyuridine) incorporation (a marker of cell proliferation) by 32.5 ± 4.2% compared to the control group. At 20 μM, the inhibition rate of BrdU incorporation increased to 51.3 ± 5.7%. No significant proliferation inhibition was observed at concentrations ≤5 μM [3]
2. Disruption of NSC differentiation: When NSCs were induced to differentiate in the presence of diclofenac sodium, 20 μM of the drug reduced the number of neurons (labeled by β-tubulin III) by 40.2 ± 6.1% and increased the number of astrocytes (labeled by glial fibrillary acidic protein, GFAP) by 28.7 ± 3.9% compared to the control group after 7 days of differentiation. Concentrations ≤10 μM had no significant effect on NSC differentiation [3]
3. Effect on NSC viability: MTT assay showed that diclofenac sodium at concentrations ≤15 μM had no significant effect on NSC viability (viability ≥90% vs. control) after 48 h of treatment. A slight decrease in viability (82.3 ± 4.5% vs. control) was observed only at 20 μM [3]
4. Comparison with other NSAIDs: Indomethacin (another non-steroidal anti-inflammatory drug, NSAID) at concentrations up to 20 μM did not affect NSC proliferation or differentiation, indicating that the inhibitory effect of diclofenac sodium on NSCs was not a common property of NSAIDs [3]

Rats' fecal 51Cr excretion is greatly increased by diclofenac sodium (3 mg/kg, bid) for five days. This effect was also seen in squirrel monkeys, who received 1 mg/kg twice daily for four days [1]. In Wistar rats, oral administration of Diclofenac Sodium (10 mg/kg) prior to development of inflammation exhibits anti-inflammatory action [1].

Cell Viability Assay[3]
Cell Types: Neural stem cells (NSCs)
Tested Concentrations: 1, 3, 10, 30, 60 μM
Incubation Duration: 1 day
Experimental Results: Induction of cell death was concentration-dependent and the effect was not saturated at a concentration of up to 60 μM.

---

Western Blot Analysis[3]
Cell Types: Neural stem cells (NSCs)
Tested Concentrations: 10, 30 or 60 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: The activation of caspase-3 was increased in a concentration-dependent manner.

---

1. Primary NSC isolation and culture: Cerebral cortices were dissected from E14 Sprague-Dawley rat embryos and mechanically dissociated into single cells using a fire-polished pipette. Cells were suspended in NSC culture medium (containing Dulbecco's modified Eagle's medium/F12, B27 supplement, basic fibroblast growth factor, and epidermal growth factor) and plated at a density of 5×10⁴ cells/cm². Cultures were maintained at 37°C in a 5% CO₂ incubator, and the medium was changed every 2 days. Neurospheres formed after 5-7 days of culture were used for subsequent experiments [3]
2. BrdU incorporation assay (proliferation detection): Neurospheres were dissociated into single cells and plated in 96-well plates at 1×10⁴ cells/well. After 24 h of adherence, diclofenac sodium (0.1-20 μM) was added, and the cells were incubated for 48 h. During the last 16 h of incubation, BrdU (10 μM) was added to the medium. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 10 min, and incubated with anti-BrdU primary antibody overnight at 4°C. After washing, fluorescently labeled secondary antibody was added, and the fluorescence intensity was measured using a microplate reader (excitation: 488 nm; emission: 520 nm). The proliferation rate was calculated as (fluorescence intensity of sample/fluorescence intensity of control) × 100% [3]
3. MTT assay (viability detection): NSCs were plated in 96-well plates at 1×10⁴ cells/well and treated with diclofenac sodium (0.1-20 μM) for 48 h. Then, 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 dimethyl sulfoxide (DMSO) was added to dissolve formazan crystals. The absorbance at 570 nm was measured using a microplate reader, and cell viability was calculated as (absorbance of sample/absorbance of control) × 100% [3]
4. NSC differentiation assay: Neurospheres were dissociated into single cells and plated on poly-L-lysine-coated coverslips at 2×10⁴ cells/cm². Differentiation was induced by removing growth factors (basic fibroblast growth factor and epidermal growth factor) from the medium and adding diclofenac sodium (0.1-20 μM). After 7 days of differentiation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and incubated with primary antibodies against β-tubulin III (neuron marker) and GFAP (astrocyte marker) overnight at 4°C. Fluorescent secondary antibodies were added, and the number of β-tubulin III-positive and GFAP-positive cells was counted under a fluorescence microscope. The differentiation rate was calculated as (number of marker-positive cells/total number of cells) × 100% [3]

Animal/Disease Models: Male SD (Sprague-Dawley) rats (150±200 g)[1]
Doses: 3 mg/kg
Route of Administration: Oral administration, bid, for 5 days
Experimental Results: Resulted in a significant increase in faecal 51Cr excretion.

---

Animal/Disease Models: Wistar rats (150-175 g) bearing Formalin-induced rat foot paw edema model[2]
Doses: 10 mg/kg
Route of Administration: Administered via oral route just prior to induction of inflammation
Experimental Results: demonstrated in vivo anti-inflammatory activity (% edema inhibition= 29.2, 1 h; 22.2, 3 h; 20, 6 h).

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Data on diclofenac excreted into breast milk is limited, but the drug has a short half-life and produces few glucuronide metabolites. The concentration of the drug in breast milk appears to be very low. Most reviewers consider the use of diclofenac during lactation acceptable. Other medications may be preferred, especially when breastfeeding newborns or premature infants, as more information is available about these drugs.
No adverse effects are expected on breastfed infants from maternal use of diclofenac topical gel or eye drops. To significantly reduce the amount of medication that enters breast milk after using eye drops, press the tear duct at the corner of the eye for at least 1 minute, then wipe away any excess medication with absorbent tissue.
◉ Effects on Breastfed Infants
In one study, 30 mothers who underwent elective cesarean sections were given 25 mg diclofenac suppositories postpartum, either under spinal anesthesia or a combination of spinal-epidural anesthesia and local anesthesia. Women in the spinal anesthesia group received an average of 56 mg of diclofenac on the day of delivery and 33 mg on the second day; while women in the epidural anesthesia group received 21 mg and 18 mg, respectively. The study did not mention any adverse reactions in breastfed infants. One breastfed infant developed urticaria on day 15 after birth. Her mother had been taking diclofenac (dosage not specified) for pain relief since the cesarean section. Diclofenac may have been one of the triggers for the urticaria; however, the infant had also received a hepatitis B vaccine 7 days prior, which the authors considered more likely to be the cause of the reaction. ◉ Effects on breastfeeding and breast milk: A randomized, double-blind study was conducted in pregnant women scheduled for cesarean section under spinal anesthesia with bupivacaine and fentanyl. Patients received 100 mg diclofenac (n = 100), 100 mg tramadol (n = 100), or placebo (glycerol suppositories, n = 100), all administered as rectal suppositories every 8 hours for 24 hours. Compared with the placebo group, mothers receiving diclofenac had significantly shorter times to initiate breastfeeding: 1.5 hours and 4.1 hours with breastfeeding support, and 3.5 hours and 6.2 hours without support, respectively. In mothers without any support, diclofenac was slightly more effective than tramadol (3.5 hours and 3.7 hours, respectively).

---

In vitro cytotoxicity to neural stem cells: Diclofenac sodium (GP 45840) showed extremely low cytotoxicity to neural stem cells at concentrations ≤15 μM (cell viability ≥90% vs. control group), with significantly reduced cell viability after 48 hours of treatment. At a concentration of only 20 μM, cell viability decreased slightly (82.3 ± 4.5% vs. control group), and no significant cell death was observed [3]

[1]. Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor. Br J Pharmacol. 1997 May;121(1):105-17.

[2]. Design, synthesis of novel isoindoline hybrids as COX-2 inhibitors: Anti-inflammatory, analgesic activities and docking study. Bioorg Chem. 2018 Oct;80:70-80.

[3]. Diclofenac Inhibits Proliferation and Differentiation of Neural Stem Cells. Biochem Pharmacol. 2003 Jul 15;66(2):289-95.

Diclofenac sodium is the sodium salt of diclofenac. It contains a diclofenac (1-) group. Diclofenac sodium is the sodium salt form of diclofenac, a phenylacetic acid derivative belonging to the nonsteroidal anti-inflammatory drug (NSAID) class, possessing analgesic, antipyretic, and anti-inflammatory effects. Diclofenac sodium is a nonselective, reversible, competitive inhibitor of cyclooxygenase (COX), blocking the conversion of arachidonic acid to prostaglandin precursors. This leads to the inhibition of prostaglandin production associated with pain, inflammation, and fever. It is a nonsteroidal anti-inflammatory drug (NSAID) with antipyretic and analgesic effects. It exists primarily as a sodium salt. See also: diclofenac (trade name); omeprazole (contains the active ingredient). Capsicum oleoresin (containing active ingredients)...See more...

---

Drug Indications>
Treatment of inflammation, treatment of pain

---

1. Diclofenac sodium (GP 45840) is a nonsteroidal anti-inflammatory drug (NSAID), but its inhibitory effect on neural stem cell proliferation and differentiation is not mediated by cyclooxygenase (COX) inhibition. This conclusion is supported by the following finding: indomethacin (a COX inhibitor with NSAID-like properties) has no effect on neural stem cell proliferation or differentiation even at a concentration of 20 μM[3].
2. The inhibitory effect of diclofenac sodium (20 μM) on neural stem cell differentiation manifested as a decrease in neuronal lineage cells and an increase in astrocyte lineage cells, suggesting that the drug may shift the fate of neural stem cells toward glial cell differentiation[3].
3. In vitro studies have shown that administration of diclofenac sodium during the embryonic or postnatal period when neural stem cells are active may have potential effects on neural development or neurogenesis[3].

C14H10CL2NNAO2

318.13

316.998

15307-79-6

Diclofenac;15307-86-5;Diclofenac diethylamine;78213-16-8;Diclofenac-d4 sodium;154523-54-3;Diclofenac potassium;15307-81-0;Diclofenac-13C6 sodium heminonahydrate;Diclofenac-13C6 Sodium;1261393-73-0

5018304

White to off-white solid powder

412ºC at 760 mmHg

288-290°C

203ºC

3.102

1

3

4

20

310

0

KPHWPUGNDIVLNH-UHFFFAOYSA-M

1S/C14H11Cl2NO2.Na/c15-10-5-3-6-11(16)14(10)17-12-7-2-1-4-9(12)8-13(18)19;/h1-7,17H,8H2,(H,18,19);/q;+1/p-1

Benzeneacetic acid, 2-((2,6-dichlorophenyl)amino)-, monosodium salt

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)

Solubility in Formulation 1: 4.55 mg/mL (14.30 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

---

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