NF-κB; COX-2; TGF-β; RelA; Autophagy
Sulfasalazine is a potent and specific inhibitor of NF-κB activation. Its metabolites, 5-aminosalicylic acid (5-ASA) and sulfapyridine, do not inhibit NF-κB activation at the doses tested.

Sulfasalazine treatment prevents NFκB activation brought on by TNFα, LPS, or phorbol ester in SW620 colon cells. Sulfasalazine inhibits NFκB-dependent transcription at micro- to millimolar concentrations. Through the inhibition of IB degradation, sulfasalazine prevents TNFα-induced nuclear translocation of NFκB[1]. All pro-inflammatory cytokines have their basal mRNA expression levels significantly increased by pre-incubation with 5 mM sulfasalazine alone, with IL-6 mRNA levels increasing by 80 times when compared to vehicle control[2]. Colonic bacteria break down sulfasalazine after digestion to produce sulfapyridine and 5-aminosalicylic acid, both of which have been shown to inhibit NF-kappaB activity[3].

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Treatment of SW620 human colonic epithelial cells with Sulfasalazine (micro- to millimolar concentrations) inhibited TNFα-, LPS-, or PMA-induced NF-κB activation, as assessed by electromobility shift assay (EMSA).
Sulfasalazine inhibited NF-κB-dependent transcription in a dose-dependent manner in SW620 cells transfected with a κB-dependent luciferase reporter construct (3xIgκBLuc). Treatment with 0.5 mM sulfasalazine resulted in ~50% inhibition, and 5 mM sulfasalazine caused complete inhibition of TNFα- or LPS-induced luciferase activity.
In contrast, its metabolites 5-ASA (up to 5 mM) and sulfapyridine showed almost no effect or only a marginal reduction on NF-κB-dependent transcription in SW620 cells.
Similar inhibitory effects of Sulfasalazine on TNFα-induced NF-κB-dependent transcription were observed in Jurkat T cells.
The inhibitory effect of Sulfasalazine was specific to NF-κB, as it did not inhibit DNA binding activity or transactivation by the transcription factor AP1 at concentrations up to 5 mM.
Sulfasalazine (5 mM) prevented TNFα-induced nuclear translocation of the RelA (p65) subunit of NF-κB in SW620 cells, as shown by immunofluorescence and Western blot analysis of nuclear extracts.
Sulfasalazine blocked TNFα-induced degradation of the inhibitory protein IκBα in the cytoplasm of SW620 cells, as determined by Western blot.
Sulfasalazine appeared to prevent TNFα-induced phosphorylation of IκBα. In the presence of the proteasome inhibitor MG132, a slower-migrating phosphorylated form of IκBα was detected in TNFα-stimulated cells, but this form was not detectable in cells pretreated with sulfasalazine.
Sulfasalazine (5 mM) pretreatment significantly decreased the steady-state mRNA levels of IκBα induced by TNFα in SW620 cells, as shown by slot blot analysis. It did not affect RelA (p65) mRNA levels.
The suppressive effect of Sulfasalazine on NF-κB activation was independent of de novo protein synthesis, as pretreatment with cycloheximide did not alter the effect.

The amount of leukocytes that accumulated in the inflamed (carrageenan, 2 mg/ml) air pouch in the murine air pouch model of inflammation is significantly reduced by sulfasalazine. It is consistent with the in vitro finding that sulfasalazine inhibits AICAR transformylase that sulfasalazine treatment leads to a significant increase in splenocyte 5-aminoimidazole-4-carboxamidoribonucleotide (AICAR) concentration.

Transcription factors of the NF-kappaB/Rel family are critical for inducible expression of multiple genes involved in inflammatory responses. Sulfasalazine and its salicylate moiety 5-aminosalicylic acid are among the most effective agents for treating inflammatory bowel disease and rheumatoid arthritis. However, the mode of action of these drugs remains unclear. Here we provide evidence that the transcription factor NF-kappaB is a target of sulfasalazine-mediated immunosuppression. Treatment of SW620 colon cells with sulfasalazine inhibited TNFalpha-, LPS-, or phorbol ester- induced NF-kappaB activation. NF-kappaB-dependent transcription was inhibited by sulfasalazine at micro- to millimolar concentrations. In contrast, 5-aminosalicylic acid or sulfapyridine did not block NF-kappaB activation at all doses tested. TNFalpha-induced nuclear translocation of NF-kappaB was prevented by sulfasalazine through inhibition of IkappaBalpha degradation. When blocking proteasome-mediated degradation of IkappaBalpha, we could demonstrate that sulfasalazine interfered with IkappaBalpha phosphorylation, suggesting a direct effect on an IkappaBalpha kinase or on an upstream signal. Inhibition of NF-kappaB activation seems to be specific since other DNA-binding activities such as AP1 were not affected. These results demonstrate that sulfasalazine is a potent and specific inhibitor of NF-kappaB activation, and thus may explain some of the known biological properties of sulfasalazine.[1]

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Preterm birth occurs in 10% of pregnancies and is a major cause of neonatal morbidity and mortality. The majority of cases of early preterm labour are associated with infection/inflammation, which places the fetal central nervous system at risk. Targeting immune activation is therefore an appealing therapeutic strategy for the prevention of preterm labour and neonatal brain injury. The expression of many labour-associated and inflammatory-response genes is controlled by the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which makes them therapeutic targets of interest. Sulfasalazine (SASP) has been shown to inhibit NF-κB and reduce lipopolysaccharide-induced cytokine concentrations in fetal membrane explants and reduce the rate of Escherichia coli-induced preterm labour in mice. Its effects upon AP-1 in the context of pregnancy are unknown. In this study the effect of SASP on interleukin-1β (IL-1β) -induced NF-κB and AP-1 activity, cytokine production and cyclo-oxygenase-2 (COX-2) expression was examined in amniocytes and myocytes. A supra-therapeutic concentration (5 mm) was required to inhibit IL-1β-induced NF-κB (P < 0·0001) in amniocytes and IL-1β-induced NF-κB (P < 0·01), AP-1 (P < 0·01) and COX-2 (P < 0·05) in myocytes. Despite inhibiting IL-1β-induced cytokines, a basal increase in IL-6 (P < 0·01), IL-8 (P < 0·0001) and tumour necrosis factor-α (TNF-α) (P < 0·001) was seen with 5 mm SASP in amniocytes, and significant cytotoxic effects were seen in myocytes. The therapeutic concentration of 0·015 mm had no inhibitory effects on pro-inflammatory mediators, but led to an augmented response to IL-1β-induced IL-6 (P < 0·01), IL-8 (P < 0·05) and TNF-α (P < 0·05) in amniocytes and IL-8 (P < 0·05) in myocytes. SASP is therefore an unlikely therapeutic candidate for the prevention of inflammation-induced preterm labour.[2]

In the culture medium, sulfasalazine is dissolved. In addition to glutamine, 10% heat-inactivated FCS, and 1% (wt/vol) penicillin/streptomycin, SW620 cells are grown in Dulbecco's modified Eagle medium. The 3xIgkBLuc reporter construct is transfected into SW620 cells. Prior to stimulation with TNFα, LPS, or PMA, cells are first allowed to rest for 18 hours with either medium alone or sulfasalazine (0.1, 0.2, 0.5, 1, 2, 5 mM). The luciferase assay is carried out[1].

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For EMSA, SW620 cells were incubated with or without inhibitors (sulfasalazine, 5-ASA, sulfapyridine) for 30 minutes before stimulation with TNFα, LPS, or PMA. After stimulation, nuclear extracts were prepared. Equal amounts of nuclear protein were incubated with a ³²P-labeled DNA oligonucleotide containing the κB motif or an AP1 site. Protein-DNA complexes were separated on native polyacrylamide gels and visualized by autoradiography.
For luciferase reporter assays, SW620 or Jurkat cells were transfected with the 3xIgκBLuc reporter construct or an AP1 reporter construct. After 18-24 hours, cells were pretreated with the drugs for 30 minutes, then stimulated with TNFα or LPS for an additional 24 hours. Cells were lysed, and luciferase activity was measured and normalized.
For immunofluorescence, SW620 cells were seeded on chamber slides. Cells were pretreated with or without sulfasalazine (5 mM) for 30 minutes, then stimulated with TNFα (150 U/ml) for 1 hour. Cells were fixed, permeabilized, and incubated with an anti-RelA (p65) primary antibody, followed by a Cy3-conjugated secondary antibody. Subcellular localization was visualized using a confocal laser scanning microscope.
For Western blot analysis, cytoplasmic and nuclear protein extracts were prepared from treated cells. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against RelA (p65), IκBα, NF-κB1 (p50), or NF-κB2 (p52). Signals were detected using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence.
For slot blot analysis of mRNA, SW620 cells were treated with sulfasalazine before TNFα stimulation. Total RNA was extracted at various time points, blotted onto nylon membranes, and hybridized with ³²P-labeled cDNA probes specific for RelA (p65) or IκBα. Membranes were stripped and reprobed with an 18S rRNA probe for normalization.
For the phosphorylation assay, SW620 cells were pretreated with the proteasome inhibitor MG132 with or without sulfasalazine, followed by TNFα stimulation. Cytoplasmic extracts were prepared and analyzed by Western blotting with an IκBα antibody to detect the phosphorylated and unphosphorylated forms.

Mice: Sulfasalazine is dissolved in 0.1 M NaOH and neutralized by titrating with 0.1 M HCl. A SCID mouse's head is implanted with U-87MG glioma cells. Animals are randomly divided into three groups of five after seven days. For three weeks, one group is given twice-daily 1 mL intraperitoneal saline injections. The two test groups are given 8 mg of sulfasalazine in 1 mL of saline twice daily for three weeks. Animal health and tumor growth were observed. Mouse brains were removed, rinsed, and put in 30% sucrose after being perfused with 4% paraformaldehyde[3].

In patients with inflammatory bowel disease, after an average daily oral administration of 3-6 grams of sulfasalazine, serum sulfasalazine concentrations have been reported to be 10-15 μg/ml (equivalent to 0.025-0.038 mM). Fecal sulfasalazine concentrations are approximately 1.25-2.0 mM. Interstitial sulfasalazine concentrations can reach as high as 0.5-1.0 mM. Approximately 30% of sulfasalazine is absorbed unchanged. The remainder is degraded by azoreduction by colonic bacteria into the metabolites sulfapyridine and 5-aminosalicylic acid (5-ASA). Approximately 70% of sulfasalazine is absorbed, while the majority of 5-ASA appears unchanged in feces.

[1]. Sulfasalazine: a potent and specific inhibitor of nuclear factor kappa B. J Clin Invest. 1998 Mar 1;101(5):1163-74.

[2]. Sulfasalazine augments a pro-inflammatory response in interleukin-1β-stimulated amniocytes and myocytes. Immunology. 2015 Dec;146(4):630-44.

[3]. Sulfasalazine inhibits the growth of primary brain tumors independent of nuclear factor-kappaB. J Neurochem. 2009 Jul;110(1):182-93.

[4]. DHODH-mediated ferroptosis defence is a targetable vulnerability in cancer. Nature. 2021;593(7860):586-590.

According to the National Toxicology Program (NTP), sulfasalazine (salicylate sulfasalazine) is potentially carcinogenic. Depending on state or federal labeling requirements, it may be toxic to the male reproductive system. Sulfasalazine is an azobenzene compound composed of diphenyldiazo groups with a carboxyl substituent at the 4-position, a hydroxyl substituent at the 3-position, and a 2-pyridylaminosulfonyl substituent at the 4'-position. It is a nonsteroidal anti-inflammatory drug, anti-infective, gastrointestinal agent, EC 2.5.1.18 (glutathione transferase) inhibitor, drug allergen, and ferroptosis inducer. It is a sulfonamide drug, belonging to the pyridine and azobenzene classes. Functionally, it is related to sulfonamides. Sulfasalazine is an aminosalicylate. It is a drug used to treat inflammatory bowel disease. Its activity is generally believed to lie in its metabolic breakdown product, 5-aminosalicylic acid (see mesalazine), which is released in the colon. (Excerpt from Martindale Pharmacopoeia, 30th edition, page 907)
See also: Sulfasalazine (note moved to).
Sulfasalazine is a drug synthesized in 1942, composed of sulfapyridine (an antibiotic) and 5-aminosalicylic acid (5-ASA, an anti-inflammatory drug). It is used to treat inflammatory bowel disease (ulcerative colitis, Crohn's disease) and rheumatoid arthritis.
This study proposes that the anti-inflammatory activity of sulfasalazine may depend on the inhibition of the transcription factor NF-κB, a key mediator of immune responses and the production of inflammatory cytokines.
Its mechanism involves inhibiting the phosphorylation of IκBα, thereby preventing its degradation, subsequent nuclear translocation of NF-κB (especially the RelA/p65 subunit), and NF-κB-dependent gene transcription. This NF-κB inhibition is unique to the parent compound sulfasalazine; its active metabolites, 5-aminosalicylic acid (5-ASA) and sulfapyridine, do not possess this effect, which may explain the difference in clinical efficacy between the compound and its metabolites. The concentration (5 mM) required for complete NF-κB inhibition in vitro falls within the range of drug concentrations in patient feces and estimated interstitial fluid, supporting the potential relevance of this mechanism in clinical applications.

C18H14N4O5S

398.3926

398.068

C, 54.27; H, 3.54; N, 14.06; O, 20.08; S, 8.05

599-79-1

Sulfasalazine-d4;1346606-50-5

5339

Light yellow to orange solid powder

1.5±0.1 g/cm3

689.3±65.0 °C at 760 mmHg

260-265 °C (dec.)(lit.)

370.7±34.3 °C

0.0±2.3 mmHg at 25°C

1.691

3.18

3

9

6

28

657

0

S(C1C([H])=C([H])C(=C([H])C=1[H])/N=N/C1C([H])=C([H])C(=C(C(=O)O[H])C=1[H])O[H])(N([H])C1=C([H])C([H])=C([H])C([H])=N1)(=O)=O

NCEXYHBECQHGNR-UHFFFAOYSA-N

InChI=1S/C18H14N4O5S/c23-16-9-6-13(11-15(16)18(24)25)21-20-12-4-7-14(8-5-12)28(26,27)22-17-3-1-2-10-19-17/h1-11,23H,(H,19,22)(H,24,25)

NCEXYHBECQHGNR-UHFFFAOYSA-N

NSC-667219; NSC 667219; NSC667219; NSC203730; NSC-203730; NSC 203730; Sulfasalazine; Reupirin; Rorasul; Salicylazosulfapyridine; Azulfidine; Salazosulfapyridine; Sulphasalazine; Salazopyrin; Asulfidine; Azopyrin

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: ~80 mg/mL(~200.8 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.28 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 25.0 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: ≥ 2.5 mg/mL (6.28 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 25.0 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: 10 mg/mL (25.10 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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