Meclofenamate Sodium inhibits cyclooxygenase (COX) enzymes, specifically COX-1 and COX-2 [1].
It also inhibits mitochondrial Complex I (C-I) and Complex III (C-III) [1]. At 100 µM, MS reduced C-I activity by ~70% [1]. At 10 µM or higher, MS significantly decreased C-III activity [1].
MS inhibits the 20S and 26S proteasome, reducing β5 (chymotrypsin-like) activity [1]. 500 µM MS inhibited β5 activity by ~80% in mouse heart lysates, and 100 µM MS decreased purified 20S proteasome β5 activity by >20% [1].

Meclofenamate Sodium significantly inhibited proteasome activity in cardiac cells. In H9c2 rat cardiac cells, 100 µM MS for 24 h decreased β5 26S proteasome activity by >30% [1]. Long-term treatment (12 days) with 10 µM MS also decreased β5 activity [1]. In mouse neonatal cardiomyocytes, 10 µM MS for 5 days significantly reduced β5 proteasome activity [1]. MS increased reactive oxygen species (ROS) generation in H9c2 cells and neonatal cardiomyocytes at concentrations as low as 10 µM [1]. The rate of ROS production was greater in MS-treated cells than in naproxen-treated cells [1]. MS (100 µM) significantly decreased mitochondrial membrane potential (ΔΨm) in H9c2 cells by ~40% [1]. MS treatment (100 µM or 200 µM) increased oxidation of 26S proteasome subunits (particularly 19S subunits) and increased the relative proportion of 20S proteasome complexes while decreasing 26S complexes [1]. MS (500 µM) significantly increased levels of polyubiquitinated proteins and decreased free ubiquitin in H9c2 cells [1]. Caspase 3 activity was significantly increased at 200 or 500 µM MS, but not at 30 µM [1]. MS (200 or 500 µM) increased oxidized protein levels in H9c2 cells [1]. MS upregulated mRNA expression of heme oxygenase 1 (HO1), glutathione S-transferase omega (GSTO), and catalase 1 (CAT1) in H9c2 cells, but not thioredoxin reductase (TRXR1) [1]. MS inhibited purified ovine COX-1 activity by ~90% at 200 µM [1].
In cell viability assays, MS decreased H9c2 cell viability: 50 µM for 24 h caused >30% reduction; 10 µM for 48 h reduced viability; 10 µM and 30 µM for 6 days caused ~24% and ~37% reduction respectively; 250 µM and 500 µM caused greater cell death [1]. Neonatal cardiomyocytes treated with 10 µM MS for 48 h showed decreased cell viability [1]. Antioxidants (ascorbic acid and Tempol) prevented MS-induced cell death and restored proteasome activity [1]. Transfection of purified 26S or 20S proteasome into MS-treated H9c2 cells significantly improved cell viability [1].

Enzyme Assay for 20S Proteasome Activity: Purified murine 20S proteasome was incubated with different concentrations of Meclofenamate Sodium (MS) or naproxen for 30 minutes at room temperature. Proteasome activity was measured using fluorogenic substrates specific for each catalytic subunit: β1 (caspase-like), β2 (trypsin-like), and β5 (chymotrypsin-like). The β5 activity was assessed using substrate Suc-LLVY-AMC, β2 using Boc-LRR-AMC, and β1 using Z-LLE-AMC. Fluorescence was measured at excitation 360 nm and emission 460 nm [1].
Enzyme Assay for Mitochondrial Complex I (C-I) Activity: Fresh or freeze-thawed isolated mouse heart mitochondria were incubated with Meclofenamate Sodium (10 µM or 100 µM) or naproxen. Complex I activity was measured spectrophotometrically at absorbance 530 nm using a specific assay. Rotenone (10 µM) was used as a positive control inhibitor [1].
Enzyme Assay for Mitochondrial Complex II (C-II) Activity: Isolated mouse heart mitochondria were treated with Meclofenamate Sodium (10 µM or 100 µM) or naproxen. Complex II activity was measured at absorbance 595 nm. Thenoyltrifluoroacetone (TTFA, 500 µM) was used as a specific inhibitor [1].
Enzyme Assay for Mitochondrial Complex III (C-III) Activity: Isolated mouse heart mitochondria were incubated with Meclofenamate Sodium (10 µM, 30 µM, or 100 µM) or naproxen. Complex III activity was determined. Antimycin A (0.01 mg/ml) was used as an inhibitor control [1].
Enzyme Assay for Cyclooxygenase (COX) Activity: COX activity was measured using a fluorescent activity assay kit. Purified ovine COX-1 was pre-incubated with varying concentrations of antioxidants (ascorbic acid or Tempol) or vehicle, then Meclofenamate Sodium (200 µM) was added. COX activity was measured according to kit instructions [1].

Cell Assay for Proteasome Activity in H9c2 Cells: Rat cardiac H9c2 cells were treated with Meclofenamate Sodium (100 µM) or naproxen (500 µM) for 24 hours. For long-term treatment, cells were treated with 10 µM MS for 12 days, with media replaced every 48 h with fresh NSAID-containing media. Cells were then washed with PBS, lysed, and 26S proteasome activity was measured using fluorogenic substrates (Suc-LLVY-AMC for β5, Boc-LRR-AMC for β2, Z-LLE-AMC for β1). Fluorescence was measured at excitation 360 nm and emission 460 nm [1].
Cell Assay for Proteasome Activity in Neonatal Cardiomyocytes: Mouse neonatal cardiomyocytes were treated with Meclofenamate Sodium (10 µM) or naproxen (100 µM) for 5 days. Cells were then lysed and β5 proteasome activity was measured as described [1].
Cell Viability Assay (Alamar Blue): H9c2 cells or neonatal cardiomyocytes were plated in 96-well plates and treated with various concentrations of Meclofenamate Sodium (e.g., 10, 30, 50, 100, 250, 500 µM) for different time periods (4, 24, 48 h, or 6 days). Alamar Blue was added and cell viability was measured by absorbance at 550 nm. For treatments longer than 48 h, media was replaced every 48 h with fresh NSAID-containing media [1].
Cell Viability Assay (CCK-8): H9c2 cells were plated in 96-well plates and treated with Meclofenamate Sodium for different time intervals. CCK-8 reagent was added and absorbance was measured at 450 nm [1].
ROS Detection Assay (H2DCFDA): H9c2 cells, neonatal cardiomyocytes, CV-1 cells, CH3/10T1/2 cells, or equine skin cells were treated with different concentrations of Meclofenamate Sodium (10-100 µM) or naproxen. ROS generation was measured immediately after treatment using the cell-permeable fluorescent probe H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate). Fluorescence was measured at excitation 490 nm and emission 520 nm. For fluorescence microscopy, cells were incubated with H2DCFDA and imaged [1].
Mitochondrial Membrane Potential Assay (JC-1): H9c2 cells were treated with Meclofenamate Sodium (5 µM or 10 µM) for 24 h. Mitochondrial membrane potential (ΔΨm) was assessed using JC-1 dye (5 µg/ml). Fluorescence was measured at excitation 490 nm and emission 590 nm. Rotenone (10 µM) and DNPH (200 µM) were used as positive controls for membrane depolarization [1].
Western Blotting: H9c2 cells treated with Meclofenamate Sodium (100 or 500 µM) were lysed and proteins separated by SDS-PAGE. Antibodies against PSMA6, β1, Rpt1, NAG-1, HSP60, and ubiquitin (VU-1) were used. Polyubiquitinated protein levels and free ubiquitin levels were detected [1].
MV151 Proteasome Active Site Labeling: Rat heart lysates were incubated with different concentrations of Meclofenamate Sodium (100 or 500 µM) or MG-132 (10 µM, positive control) for 20 minutes, then incubated with 0.1 µM MV151 (fluorescent reversible proteasome inhibitor) for 15 minutes. Labeled proteasomes were subjected to SDS-PAGE and fluorescence was detected [1].
OxyBlot Analysis for Protein Oxidation: Purified 26S proteasomes from H9c2 cells treated with Meclofenamate Sodium (100 µM for 48 h) were derivatized with dinitrophenylhydrazine to label carbonyl groups. Oxidized subunits were detected by Western blotting using anti-DNP antibody [1].
Purification of 26S Proteasomes from MS-treated Cells: H9c2 cells were treated with Meclofenamate Sodium (100 µM) for 48 h. 26S proteasomes were purified using a Rapid 26S Proteasome Purification Kit with slight modifications. Purified proteasomes were assayed for β5 activity and subjected to OxyBlot and native gel electrophoresis [1].
Native Gel Electrophoresis and Western Blotting for Proteasome Complexes: Lysates from H9c2 cells treated with Meclofenamate Sodium (100 µM) or naproxen were separated on 3.5% native acrylamide gels for 3 hours, allowing detection of proteins >200 kDa. Gels were transferred to nitrocellulose membranes and probed with antibodies against PSMA6, β1, and Rpt6 to identify 20S and 26S proteasome complexes [1].
Caspase 3 Activity Assay: H9c2 cells treated with Meclofenamate Sodium (30, 200, or 500 µM) were lysed and caspase 3 activity was measured in the presence or absence of 10 µM caspase-3 inhibitor Ac-DEVD-CHO using a fluorogenic substrate [1].
Transfection of Purified Proteasomes into H9c2 Cells: H9c2 cells were treated with Meclofenamate Sodium (100 µM) or bortezomib (2 nM). Purified 26S or 20S proteasome (0.05 or 0.1 µg) was transfected using Saint PhD transfection reagent in serum-free media for 4 hours at 37°C, followed by overnight incubation in normal media. Cell viability and proteasome activity were then measured [1].
Immunocytochemistry for Transfected Proteasomes: H9c2 cells transfected with purified 26S or 20S proteasomes were fixed, permeabilized, and probed with anti-β1 polyclonal antibody, followed by Dylight 549-conjugated goat anti-rabbit secondary antibody. Nuclei were stained with DAPI. Fluorescence intensity was measured to quantify proteasome uptake [1].
RT-PCR for Gene Expression: H9c2 cells were treated with Meclofenamate Sodium (100 µM) for 24 h. RNA was extracted and RT-PCR was performed to measure mRNA levels of PSMB5 (β5), PSMB6 (β1), PSMB8 (β5i), HO1, GSTO, CAT1, and TRXR1. Results were compared to control [1].

Peak pharmacological plasma levels of Meclofenamate Sodium in humans range from 10 to 22 µM [1]. In horses given doses typically used for treating musculoskeletal disease, peak levels >337 µM have been reported [1].

Effects During Pregnancy and Lactation
◉ Overview of Medication Use During Lactation
Since there is currently no information regarding the use of meclofenamic acid during lactation, it is recommended to prioritize other medications, especially when breastfeeding newborns or premature infants. ◉ Effects on Breastfed Infants
As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk
As of the revision date, no relevant published information was found.

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Cardiac Cell Viability Reduction: Meclofenamate Sodium significantly decreased viability of H9c2 cardiac cells and neonatal cardiomyocytes at concentrations ≥10 µM after 48 h or longer, with higher concentrations (250-500 µM) causing greater cell death. Cell death was prevented by antioxidants (ascorbic acid and Tempol), suggesting a ROS-dependent mechanism [1].
Proteasome Inhibition: MS inhibited proteasome activity in cardiac cell lysates and purified proteasomes, leading to accumulation of polyubiquitinated proteins at high concentrations (500 µM) and reduced free ubiquitin levels [1].
Mitochondrial Dysfunction: MS impaired mitochondrial Complex I and III activities and decreased mitochondrial membrane potential (ΔΨm) at low concentrations (5-10 µM), indicating mitochondrial damage [1].
Increased ROS and Oxidative Stress: MS induced significant ROS generation in cardiac cells, increased oxidation of proteasome subunits (especially 19S subunits), and increased oxidized protein levels at 200 µM. The rate of ROS production was higher in MS-treated cells compared to naproxen [1].
No Apoptosis at Low Concentrations: At 30 µM MS, no caspase 3 activation was detected, suggesting that cell death at pharmacological levels is not due to apoptosis [1].
Upregulation of Antioxidant Enzymes: MS (100 µM) upregulated mRNA expression of HO1, GSTO, and CAT1 in H9c2 cells, suggesting a cellular antioxidant response [1].
Cardiovascular Disease Risk: The use of MS has been associated with an increased risk of cardiovascular disease (CVD), consistent with clinical data suggesting MS increases CVD risk while naproxen does not [1].

J Mol Cell Cardiol.2016May;94:131-44;

Anhydrous meclofenamic acid sodium is an organosodium salt. It contains meclofenamic acid (1-). It is a nonsteroidal anti-inflammatory drug with antipyretic and antigranulomatous effects. It also inhibits prostaglandin biosynthesis. See also: Meclofenamic acid sodium (note moved to).

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Meclofenamate Sodium is a nonsteroidal anti-inflammatory drug (NSAID) that exerts its anti-inflammatory and analgesic effects primarily through inhibition of cyclooxygenase (COX) enzymes, both COX-1 and COX-2 [1]. It has been used to treat autoimmune diseases such as chronic and acute osteoarthritis and rheumatoid arthritis [1]. Unlike naproxen, which has been associated with some cardioprotective effects, MS increases the risk of cardiovascular disease (CVD) and heart failure [1]. The adverse cardiac effects of MS are mediated through a ROS-dependent mechanism involving mitochondrial dysfunction (Complex I and III inhibition) and proteasome impairment, leading to reduced cell viability [1]. Antioxidants (ascorbic acid, Tempol) can prevent MS-induced cell death without interfering with its COX inhibitory activity, as they only partially inhibit COX-1 (~50%) compared to MS (~90%) [1].

C14H11CL2NO2.NA

318.13

335.009

6385-02-0

Meclofenamic acid;644-62-2

4038

White to off-white solid powder

399.4ºC at 760 mmHg

287 °C

195.3ºC

3.481

1

3

3

20

332

0

QHJLLDJTVQAFAN-UHFFFAOYSA-M

InChI=1S/C14H11Cl2NO2.Na.H2O/c1-8-6-7-10(15)13(12(8)16)17-11-5-3-2-4-9(11)14(18)19;;/h2-7,17H,1H3,(H,18,19);;1H2/q;+1;/p-1

sodium 2-((2,6-dichloro-3-methylphenyl)amino)benzoate hydrate

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 (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.54 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 (6.54 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 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 (6.54 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.)