NF-κB; COX-2; TGF-β; RelA; Autophagy

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].

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].

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].

[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.

Sulfasalazine (salicylazosulfapyridine) can cause cancer according to The National Toxicology Program. It can cause male reproductive toxicity according to state or federal government labeling requirements.
Sulfasalazine is an azobenzene consisting of diphenyldiazene having a carboxy substituent at the 4-position, a hydroxy substituent at the 3-position and a 2-pyridylaminosulphonyl substituent at the 4'-position. It has a role as a non-steroidal anti-inflammatory drug, an antiinfective agent, a gastrointestinal drug, an EC 2.5.1.18 (glutathione transferase) inhibitor, a drug allergen and a ferroptosis inducer. It is a sulfonamide, a member of pyridines and a member of azobenzenes. It is functionally related to a sulfanilamide.
Sulfasalazine is an Aminosalicylate.
A drug that is used in the management of inflammatory bowel diseases. Its activity is generally considered to lie in its metabolic breakdown product, 5-aminosalicylic acid (see MESALAMINE) released in the colon. (From Martindale, The Extra Pharmacopoeia, 30th ed, p907)
See also: Sulfasalazine (annotation moved to).

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.)