CYP2C9
Sulfaphenazole (10 μM) reduces the number of light-induced necrotic and apoptotic cells by 44% and 33%, respectively, over the course of one hour[1].

Sulfaphenazole (10 μM) reduces the number of light-induced necrotic and apoptotic cells by 44% and 33%, respectively, over the course of one hour[1].

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Sulfaphenazole was identified as a leading cytoprotective hit from a high-throughput screen of an FDA-approved drug library, protecting mouse-derived photoreceptor (661W) cells from light-induced cell death.
It demonstrated dose-dependent cytoprotection, increasing cellular ATP levels in light-exposed 661W cells, and performed better than reference compounds minocycline and L-sulforaphane in rescuing cell viability.
Sulfaphenazole inhibited both light-induced necrosis (44% reduction) and apoptosis (33% reduction) in 661W cells, as determined by annexin V/propidium iodide flow cytometry.
It reduced the activity of pan-caspases in light-exposed cells, as measured by a fluorescent caspase activity assay.
Sulfaphenazole restored the loss of mitochondrial membrane potential (ΔΨm) induced by light exposure.
It significantly mitigated light-induced intracellular calcium influx in 661W cells.
Sulfaphenazole reduced the cellular superoxide anion (O2•−) level in light-exposed cells but did not significantly affect hydroxyl radical (•OH) levels.
Gene-targeted knockdown of the mouse CYP2C9 homolog, CYP2C55, using siRNA rescued 661W cells from light-induced death, mimicking the effect of Sulfaphenazole.
Stable expression of a functional human CYP2C9-GFP fusion protein in 661W cells increased their sensitivity to light-induced death, and pretreatment with Sulfaphenazole improved viability in these cells.
LC/MS analysis showed that light exposure increased the release of arachidonic acid (AA) from 661W cells. Sulfaphenazole treatment further increased the production of two non-epoxyeicosatrienoic acid (non-EET) metabolites of AA (with m/z 319/167 and 319/219), suggesting a metabolic shift upon CYP2C pathway inhibition. [1]

In diabetic mice, sulfaphenazole (5.13 mg/kg) administered intraperitoneally once a day for eight weeks restores endothelium-mediated relaxation. Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice[2].

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Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion[3].

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Effects of sulfaphenazole after collagenase-induced experimental intracerebral hemorrhage in rats[4].

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In a mouse model of grade 2 thermal injury, treatment with Sulfaphenazole (5.13 mg/kg, i.p., daily) reduced wound area from day 2 to 9 post-injury compared to vehicle-treated controls and increased wound tensile breaking force at day 15 post-injury. Tissue perfusion was improved in the treatment group, with the most pronounced effect from day 1 to 4 post-injury.[3]
In a mouse model of ischemia/reperfusion (I/R)-mediated pressure injury using ApoE-/- mice, daily administration of Sulfaphenazole (5.13 mg/kg, i.p.) reduced wound severity and area, and improved skin breaking force at day 14 post-injury compared to vehicle-treated controls. Histologically, it reduced wound area and wound gape at day 7 post-injury.[3]
Sulfaphenazole treatment restored tissue perfusion in and around the pressure injury wound rapidly to pre-injury levels from day 3 post-I/R onwards, and reduced tissue hypoxia (as indicated by reduced HIF-1α levels) at days 7 and 14.[3]
Sulfaphenazole reduced overall inflammatory cell infiltrate in pressure injury at day 7 and decreased the expression of numerous pro-inflammatory cytokines/chemokines (e.g., C5/5a, MIP-1α, MIP-2, IL-1ra, TIMP-1).[3]
Despite reducing overall inflammation, Sulfaphenazole increased F4/80-positive macrophage recruitment and iNOS expression in pressure injury granulation tissue at day 7, and reduced the number of Gram-positive bacterial colonies at the wound site.[3]
Sulfaphenazole reduced pro-fibrotic activity in pressure injury, evidenced by improved collagen maturation, reduced α-SMA (myofibroblast marker) expression, and mitigated TGF-β elevation at day 14.[3]

Enzymatic Assay of CYP2C9-GFP Fusion Protein [1]
A cell-based luminogenic P450-Glo assay was used to detect the enzymatic activity of CYP2C9-GFP fusion protein in pCMV6-CYP2C9-GFP stably transfected 661W cells. The assay is based on CYP2C9, which catalyzes a selective, cell-permeable proluciferin substrate (luciferin-H) to generate luminogenic signal for detection. Endogenous cellular NADPH is provided as the electron donor for the reaction. Essentially, 661W cells were seeded on 96-well plates (5 × 103 cells/well) overnight in regular culture medium. The next day, the cells in some wells were pretreated with sulfaphenazole (10 μm) for 2 h at 37 °C. Substrate incubation, enzymatic assay, and luminescence detection all followed the manufacturer's protocol. Mock pCMV6-GFP stably transfected 661W cells and wild-type 661W cells were normal controls. Cell-free empty wells with luciferin-H were blank controls. Enzymatic activity was expressed as luminescence (relative light units)/h/5 × 103 cells after subtraction of background luminescence from blank controls.

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Reactive Oxygen Species and Lipid Peroxidation Assays [1]
Cellular hydroxyl radical (•OH) and superoxide anion (O2⨪) were detected using the oxidation-sensitive fluorescent dyes chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate (488-nm excitation/535-nm emission) and dihydroethidium (495-nm excitation/595-nm emission) (Invitrogen), respectively. Light-exposed 661W cells on 96-well plates for different durations (15 min to 2 h) with/without sulfaphenazole pretreatment and control cells in the dark were washed with PBS. The fluorescent dyes (1 μm for each) predissolved in PBS were then added into the cells with incubation at 37 °C for 30 min. After incubation, the cells were washed again with PBS. Fluorescence was recorded with a microplate reader. In some experiments, the cells were also examined with a fluorescence microscope to evaluate consistency of the results.

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A cell-based luminogenic P450 assay was used to detect the enzymatic activity of a stably expressed CYP2C9-GFP fusion protein in 661W cells. The assay is based on CYP2C9-catalyzed conversion of a selective, cell-permeable proluciferin substrate (luciferin-H) into a luminogenic product. Endogenous cellular NADPH serves as the electron donor. Cells were seeded in culture plates overnight. Some wells were pretreated with Sulfaphenazole (10 µM) for 2 hours. Substrate incubation, enzymatic reaction, and luminescence detection were performed according to the standard protocol. Enzymatic activity was expressed as luminescence per hour per cell number after background subtraction. Sulfaphenazole dramatically inhibited the enzymatic activity of the CYP2C9-GFP fusion protein. [1]

Secondary Validation of Sulfaphenazole [1]
Confluent 661W cells on 96-well plates were treated with serially diluted (0.625–100 μm) sulfaphenazole at 37 °C for 2 h. Minocycline and l-sulforaphane were co-tested separately at equivalent concentrations at the same time. Cells were then exposed to light for 4–6 h after which cell viability was detected using CellTiter-Glo, which measures cellular ATP according to the manufacturer's protocol. Luminescence was detected with a microplate reader at an integration time of 0.25 s/well.

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For the primary high-throughput screen, confluent 661W cells on 384-well plates were pretreated overnight with the synthetic chromophore 9-cis-retinal (10 µM) to increase light sensitivity. The medium was replaced with serum-free medium, and compounds from the Prestwick library were added at a final concentration of 10 µM. After 2 hours of incubation, cells were exposed to intense white light for 4 hours. Cell viability was detected using a redox-sensitive fluorescent dye. [1]
For secondary validation and dose-response, 661W cells on 96-well plates were treated with serially diluted Sulfaphenazole (0.625–100 µM) for 2 hours, followed by light exposure for 4-6 hours. Cell viability was assessed by measuring cellular ATP levels using a luminescent assay. [1]
For siRNA-mediated gene silencing, 661W cells at 50% confluence were transfected with predesigned siRNAs targeting mouse CYP2C55 or CYP2C29, or scrambled control oligos, using a lipid-based transfection reagent. At 48-72 hours post-transfection, cells were used for viability assays or lysed for Western blot analysis of silencing efficiency. [1]
Apoptosis and necrosis were assessed by flow cytometry. 661W cells were treated with/without Sulfaphenazole, exposed to light, then harvested, stained with Alexa Fluor 488-annexin V and propidium iodide, and analyzed. [1]
Caspase activity was measured using a fluorescent-labeled caspase inhibitor (FLICA) that binds to active caspases. Treated cells were incubated with the FLICA reagent, washed, and fluorescence was analyzed by flow cytometry. [1]
Mitochondrial membrane potential (ΔΨm) was detected using the JC-1 dye. After treatment, cells were incubated with JC-1, and fluorescence of the monomeric form (indicative of low ΔΨm) was measured. [1]
Intracellular calcium was detected using the calcium-sensitive fluorescent dye Fluo-4 AM Direct. Cells were loaded with the dye, treated and exposed to light, and Ca2+ fluorescence was measured by flow cytometry or plate reader. [1]
Reactive oxygen species were detected using oxidation-sensitive fluorescent dyes: dihydroethidium for superoxide anion and CM-H2DCFDA for hydroxyl radical. Treated cells were incubated with the dyes, washed, and fluorescence was recorded. [1]
Lipid peroxidation was assessed by measuring malondialdehyde levels using a thiobarbituric acid reactive substances assay according to standard protocol. [1]
For lipidomics analysis, lipids were extracted from conditioned medium and cell pellets using chloroform and methanol. Arachidonic acid and its metabolites were analyzed by liquid chromatography-mass spectrometry (LC/MS) using deuterated internal standards for quantification. [1]

Animal Model: Diabetic male mice (db/db strain)[2]
Dosage: 5.13 mg/kg
Administration: Intraperitoneal injections; daily; for 8 weeks
Result: In diabetic mice, sulfaphenazole (5.13 mg/kg) administered intraperitoneally once a day for eight weeks restores endothelium-mediated relaxation[2].

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Sulfaphenazole (SPZ) has a different mechanism such as reactive oxygen species scavenging, in addition to the inhibition of superoxide production by cytochrome P450. The present study investigated the properties of SPZ in collagenase-induced intracerebral hemorrhage rat brain damage. The results show that systemic SPZ treatment after intracerebral hemorrhage reduces striatal dysfunction, the elevation of lipid peroxidation, and brain edema in the rat. These results suggest that SPZ is a potentially effective therapeutic approach for intracerebral hemorrhage as the effect of SPZ was initiated for either 1 h or 3 d post-intracerebral hemorrhage.[4]

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Thermal Injury Model: Grade 2 thermal injury was induced in female 8–10-week-old C57Bl/6 mice. Under anesthesia, a pre-heated metal rod (1 cm diameter) was lowered onto the shaved dorsum and maintained under its own weight for 5 seconds. Mice were treated daily via intraperitoneal injection with Sulfaphenazole (5.13 mg/kg, formulated in PBS, pH 7.2) or an equivalent volume of PBS vehicle. Wound photos were taken periodically. Animals were euthanized at day 15 for tissue analysis.[3]
Pressure Injury Model: Male, 15-week-old ApoE-/- mice were used. Under anesthesia, the dorsum was shaved. Skin was pinched between two round magnets (12 mm diameter, 5.0 mm thick) to produce a 5 mm skin bridge, creating cycles of ischemia (3 h magnet placement) and reperfusion (30 min or overnight rest). Mice were treated daily via intraperitoneal injection with Sulfaphenazole (5.13 mg/kg, in PBS, pH 7.2) or PBS vehicle. Wound photos were taken daily. Animals were euthanized at days 7 and 14 for tissue collection and analysis.[3]

Metabolism/Metabolites
Liver.

The subcutaneous LD50 in rats was 900 mg/kg, Nippon Yaku, 6(387), 1982; the intravenous LD50 in rats was 525 mg/kg, Nippon Yaku, 6(387), 1982; the oral LD50 in mice was 3016 mg/kg, Minerva Medica, 52(1789), 1961; the subcutaneous LD50 in mice was 660 mg/kg, Nippon Yaku, 6(387), 1982; and the intravenous LD50 in mice was 470 mg/kg, Nippon Yaku, 6(387), 1982. In the described mouse studies, daily intraperitoneal injection of sulfamethoxazole (5.13 mg/kg) was well tolerated, and no adverse events were observed. The mice lost less than 10% of their body weight after injury induction, and no difference was observed between the drug treatment group and the carrier treatment group. Specific toxicity parameters (e.g., LD50, organ toxicity, protein binding) were not reported. [3]

[1]. Cytochrome P450 2C epoxygenases mediate photochemical stress-induced death of photoreceptors. J Biol Chem. 2014 Mar 21;289(12):8337-52.

[2]. Sulfaphenazole treatment restores endothelium-dependent vasodilation in diabetic mice. Vascul Pharmacol. 2008 Jan;48(1):1-8.

[3]. Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion. Sci Rep. 2022 Jul 23;12(1):12622.

[4]. Effects of sulfaphenazole after collagenase-induced experimental intracerebral hemorrhage in rats. Biol Pharm Bull.2012;35(10):1849-53.

Sulfafenazole is a sulfonamide compound with the structure sulfanilamide, wherein the nitrogen atom of the sulfanilamide is replaced by a 1-phenyl-1H-pyrazol-5-yl group. It is a selective inhibitor of the cytochrome P450 (CYP) 2C9 isoenzyme and also an antibacterial agent. It exhibits antibacterial activity, is an EC 1.14.13.181 (13-deoxydaunorubicin hydroxylase) inhibitor, an EC 1.14.13.67 (quinine 3-monooxygenase) inhibitor, and a P450 inhibitor. It is a substituted aniline, sulfonamide, pyrazole, primary amino compound, and sulfonamide antibiotic. Sulfafenazole is a sulfonamide antibacterial drug. Sulfafenazole is a long-acting sulfonamide antibiotic used to treat leprosy. It is also a sulfonamide anti-infective drug. Drug Indications: Used to treat bacterial infections.

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Mechanism of Action
Sulfafenazole is a sulfonamide antibacterial drug. In bacteria, antibacterial sulfonamides act as competitive inhibitors of dihydropteroate synthase (DHPS), an enzyme involved in folate synthesis. As a result, microorganisms die due to folate deficiency.

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Sulfafenazole is an FDA-approved drug that was identified in this paper as a novel cytoprotective agent against photochemical stress-induced photoreceptor death.
Its mechanism of action involves inhibiting cytochrome P450 2C cyclooxygenases (particularly CYP2C9 and its mouse homologs), thereby inhibiting necrosis and apoptosis, reducing calcium ion influx, and altering arachidonic acid metabolism.
This study suggests that the CYP monooxygenase system is a risk factor for retinal light damage, and sulffafenazole shows promise as a potential candidate drug for treating light damage-related diseases such as retinal degeneration. [1]

C15H14N4O2S

314.36

314.083

C, 57.31; H, 4.49; N, 17.82; O, 10.18; S, 10.20

526-08-9

5335

White to yellow solid powder

1.4±0.1 g/cm3

541.9±56.0 °C at 760 mmHg

179-183ºC

281.5±31.8 °C

0.0±1.4 mmHg at 25°C

1.684

1.52

2

5

4

22

451

0

S(C1C([H])=C([H])C(=C([H])C=1[H])N([H])[H])(N([H])C1=C([H])C([H])=NN1C1C([H])=C([H])C([H])=C([H])C=1[H])(=O)=O

QWCJHSGMANYXCW-UHFFFAOYSA-N

InChI=1S/C15H14N4O2S/c16-12-6-8-14(9-7-12)22(20,21)18-15-10-11-17-19(15)13-4-2-1-3-5-13/h1-11,18H,16H2

4-amino-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide

BRN-0308518; BRN0308518; BRN 0308518; Sulfaphenazole; Firmazolo; sulfaphenazole; 526-08-9; Sulphaphenazole; Sulfabid; Sulfafenazol; Sulfaphenazon; 4-Amino-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide; Plisulfan; Inamil

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 : 63~100 mg/mL ( 200.4~318.11 mM )
Ethanol : ~20 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.62 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 (6.62 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 (6.62 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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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.08 mg/mL (6.62 mM)

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