Leukotriene A4 Hydrolase (LTA4H) (IC50: 0.87 ± 0.06 μM for Bufexamac (Bufexamic acid) against human recombinant LTA4H) [3]

In vitro activity: Bufexamac is a specific inhibitor of class IIB histone deacetylases (HDAC6 and HDAC10). Treatment of peripheral blood mononuclear cells with bufexamac inhibits the secretion of IFN-α. Bufexamac is a frequent and relevant contact sensitizer. Bufexamac is a non-steroidal anti-inflammatory drug.

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1. Inhibition of LTA4H activity and inflammatory mediator production (RAW264.7 cells):
- LTA4H enzyme inhibition: Bufexamac showed concentration-dependent inhibition of human recombinant LTA4H. At 1 μM, it inhibited LTA4H-mediated LTB4 formation by 89 ± 3%; at 0.5 μM, inhibition was 62 ± 4% [3]
- Anti-inflammatory activity in macrophages: Mouse RAW264.7 macrophages were pre-treated with bufexamac (0.1 μM, 0.5 μM, 1 μM, 5 μM) for 1 h, then stimulated with LPS (1 μg/mL) for 24 h. ELISA showed that 1 μM bufexamac reduced LPS-induced TNF-α secretion by 45 ± 5%, IL-6 secretion by 42 ± 4%, and LTB4 production by 58 ± 6% compared to the LPS-only group. Western blot revealed no significant effect on COX-2 or iNOS expression at concentrations ≤5 μM [3]

1. Amelioration of LPS-induced acute lung injury (mouse model): Female C57BL/6 mice (6-8 weeks old) were randomly divided into 4 groups: control, LPS, LPS + bufexamac 10 mg/kg, LPS + bufexamac 30 mg/kg (n=6/group). Acute lung injury was induced by intratracheal injection of LPS (5 mg/kg). Bufexamac was dissolved in 0.5% carboxymethyl cellulose (CMC-Na) and administered by oral gavage 1 h before and 6 h after LPS injection. On day 1 post-LPS:
- The 30 mg/kg group had a 35 ± 4% reduction in lung wet/dry weight ratio (edema marker) vs. LPS group [3]
- Bronchoalveolar lavage fluid (BALF) showed 42 ± 5% fewer total inflammatory cells, 48 ± 6% fewer neutrophils, and 39 ± 4% lower TNF-α levels in the 30 mg/kg group [3]
- Lung tissue LTB4 concentration was reduced by 52 ± 5% in the 30 mg/kg group vs. LPS group [3]
2. Effects on equine joints (healthy horse model): Six healthy adult horses (4-8 years old, 450-550 kg) received intra-articular injection of bufexamac suspension (20 mg/joint, dissolved in 2 mL sterile saline) into the radiocarpal joint. Contralateral joints received 2 mL sterile saline (control). At 24, 48, and 72 h post-injection:
- Synovial fluid white blood cell count was 38 ± 4% lower in bufexamac-treated joints vs. controls at 24 h [5]
- Synovial fluid prostaglandin E2 (PGE2) levels were reduced by 45 ± 5% (24 h) and 32 ± 4% (48 h) in bufexamac groups [5]
- Histological scoring of joint tissues showed no significant inflammation or tissue damage in bufexamac-treated joints [5]

1. Human recombinant LTA4H activity assay:
- Reaction system (100 μL): 50 mM Tris-HCl buffer (pH 7.5), 10 mM NaCl, 5 μM human recombinant LTA4H, 10 μM LTA4 (substrate), and serial dilutions of Bufexamac (Bufexamic acid) (0.01-10 μM).
- Incubation: Mixtures were incubated at 37°C for 15 min. The reaction was terminated by adding 20 μL of 1 M HCl.
- Detection: The product LTB4 was extracted with ethyl acetate, dried under nitrogen, and resuspended in methanol. LTB4 concentration was measured by high-performance liquid chromatography (HPLC) with a C18 column (mobile phase: methanol-water-acetic acid = 80:20:0.1, flow rate: 1 mL/min, detection wavelength: 270 nm). Inhibition rate = (1 - LTB4 concentration of sample/LTB4 concentration of control) × 100%, and IC50 was calculated via nonlinear regression [3]

1. RAW264.7 macrophage inflammation assay:
- Cell culture: Mouse RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in 5% CO₂.
- Drug treatment: Cells were plated in 24-well plates (1×10⁵ cells/well) and incubated overnight. Bufexamac (0.1 μM, 0.5 μM, 1 μM, 5 μM) was added for 1 h pre-treatment, followed by stimulation with LPS (1 μg/mL) for 24 h.
- Inflammatory mediator detection: Culture supernatant was collected. TNF-α and IL-6 concentrations were measured by commercial ELISA kits; LTB4 concentration was determined by HPLC (same conditions as enzyme assay).
- Protein detection: Cells were lysed with RIPA buffer containing protease inhibitors. COX-2 and iNOS protein levels were detected by Western blot using specific antibodies, with GAPDH as the loading control [3]

1. Mouse LPS-induced acute lung injury model:
- Animals: Female C57BL/6 mice (6-8 weeks old, 18-22 g), n=24, randomly divided into control, LPS, LPS + bufexamac 10 mg/kg, LPS + bufexamac 30 mg/kg groups (n=6/group).
- Model induction: Mice were anesthetized with isoflurane. LPS (5 mg/kg, dissolved in 50 μL sterile saline) was administered via intratracheal injection; control mice received 50 μL sterile saline.
- Drug administration: Bufexamac (Bufexamic acid) was dissolved in 0.5% CMC-Na to concentrations of 1 mg/mL and 3 mg/mL. Mice received oral gavage (10 μL/g body weight) 1 h before LPS injection and 6 h after LPS injection.
- Sample collection: On day 1 post-LPS, mice were sacrificed. Lungs were excised to measure wet/dry weight ratio; BALF was collected by lavaging the lungs with sterile saline (0.5 mL × 3 times) for cell counting and cytokine detection; lung tissue was homogenized for LTB4 measurement [3]
2. Equine intra-articular injection model:
- Animals: Six healthy adult horses (4-8 years old, 450-550 kg; 3 mares, 3 geldings), free of joint disease (confirmed by physical examination and radiography).
- Drug preparation: Bufexamac was ground into fine powder and suspended in sterile saline to a concentration of 10 mg/mL (20 mg/2 mL per joint).
- Administration: Horses were sedated with xylazine (0.5 mg/kg, i.v.). The radiocarpal joint was aseptically prepared, and 2 mL of bufexamac suspension was injected into one joint; the contralateral joint received 2 mL sterile saline (control).
- Sample collection: Synovial fluid was collected from each joint at 0 (baseline), 24, 48, and 72 h post-injection for white blood cell counting and PGE2 detection. At 72 h, horses were euthanized, and joint tissues were harvested for histological analysis [5]

Absorption, Distribution and Excretion
The method of administration affects the degree of skin absorption. Systemic absorption has been reported to be lower after rectal administration of suppositories. Following topical application of 5% ibuprofen, 3.5% of the administered dose is recovered in urine within 144 hours. Studies in healthy volunteers with oral doses ranging from 125 to 500 mg showed that, on average, 80% of the total dose was excreted in urine within 48 hours. No data available. No data available. Metabolism/Metabolites No data available. Biological Half-Life No data available.

Protein binding
No data available.

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1. Mouse toxicity: In a 1-day acute lung injury study, oral administration of 10 mg/kg and 30 mg/kg of bufenzamide (bufenzamide acid) had no significant effect on mouse body weight (final body weight was 20.5 ± 1.2 g and 19.8 ± 1.1 g, respectively, compared to 20.1 ± 1.3 g in the LPS group). Serum alanine aminotransferase (ALT) levels (46 ± 5 U/L vs. 43 ± 4 U/L in the control group) and creatinine levels (0.50 ± 0.04 mg/dL vs. 0.48 ± 0.03 mg/dL in the control group) were within the normal range [3]
2. Horse toxicity: In a 72-hour joint injection study, bufenzamide (20 mg/joint, intra-articular injection) did not cause adverse clinical symptoms (e.g., lameness, joint swelling) in horses. Serum AST (51 ± 6 U/L vs. baseline 49 ± 5 U/L), ALT (43 ± 4 U/L vs. baseline 41 ± 3 U/L), and creatinine (1.2 ± 0.1 mg/dL vs. baseline 1.1 ± 0.1 mg/dL) levels remained unchanged. No evidence of cytotoxicity (e.g., increased cell necrosis) was observed in the synovial fluid treated with ibufenamic acid. [5]

[1]. Chemoproteomics profiling of HDAC inhibitors reveals selective targeting of HDAC complexes. Nat Biotechnol. 2011 Mar;29(3):255-65.

[2]. Histone deacetylase 10 promotes autophagy-mediated cell survival. Proc Natl Acad Sci U S A. 2013 Jul 9;110(28):E2592-601.

[3]. Bufexamac ameliorates LPS-induced acute lung injury in mice by targeting LTA4H. Sci Rep. 2016 Apr 29;6:25298.

[4]. Acetylation site specificities of lysine deacetylase inhibitors in human cells. Nat Biotechnol. 2015 Apr;33(4):415-23.

[5]. Effects of intra-articular injections of bufexamac suspension in healthy horses. Am J Vet Res. 2001 Oct;62(10):1629-35.

Bufexamac is a hydroxyxamic acid derived from phenylacetamide, with a butoxy group substituted at the C-4 position of its benzene ring. It has anti-inflammatory, analgesic, and antipyretic effects. It is a non-narcotic analgesic, nonsteroidal anti-inflammatory drug (NSAID), and antipyretic. It is a hydroxyxamic acid and aromatic ether. Ibufenamic acid is a nonsteroidal anti-inflammatory drug (NSAID) marketed under the names Droxaryl, Malipuran, Paraderm, and Parfenac. It is commonly used topically to treat subacute and chronic eczema, including atopic eczema and other inflammatory skin conditions, as well as sunburn and other minor burns and itching. It has also been used in combination with local anesthetics in suppositories to treat hemorrhoids. Ibufenamic acid has been discontinued in Canada and the United States, likely due to its undetermined clinical efficacy and high rate of contact sensitization. The European Medicines Agency (EMA) also withdrew ibufenamic acid in April 2010. Ibufenamic acid is a phenylacetamide drug with anti-inflammatory, analgesic, and antipyretic effects. It can be administered topically, orally, or rectally. Indications It is indicated for the treatment of various skin conditions, such as atopic eczema and other inflammatory skin diseases. Mechanism of Action The complete mechanism of action of ibufenamic acid is not fully understood. Some studies suggest that the mechanism of action of ibufenamic acid is similar to other nonsteroidal anti-inflammatory drugs (NSAIDs), inhibiting prostaglandin biosynthesis by inhibiting cyclooxygenase (COX) (in vitro experiments). Systemic administration of ibufenamic acid may preferentially accumulate in the adrenal cortex of rats and may exert its effect upon adrenal stimulation; however, its local anti-inflammatory effect may be unrelated to this. Pharmacodynamics ibufenamic acid is a locally active anti-inflammatory drug that inhibits cyclooxygenase. Topical application of ibufenamic acid produces dose-dependent anti-inflammatory effects in experimental inflammation of the skin and deep tissues. In guinea pigs, ibufenamic acid is more effective than topical application of 5% acetylsalicylic acid or 5% phenylbutazone in delaying the increase in local temperature induced by ultraviolet radiation. Ibufenamic acid is unlikely to have any effect on wound healing.

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1. Ibufenamic acid (IBNA) is a nonsteroidal anti-inflammatory drug (NSAID) with a unique mechanism of action that targets LTA4H: it inhibits LTA4H-mediated LTB4 synthesis, thereby reducing LTB4-induced inflammatory cell recruitment and cytokine secretion—unlike conventional NSAIDs that target COX enzymes [3]
2. In veterinary medicine, Ibufenamic acid has shown potential for treating joint inflammation: intra-articular injection in healthy horses reduces the levels of synovial fluid inflammation markers (leukocytes, PGE2) without causing tissue damage, supporting its use in treating equine osteoarthritis or post-traumatic joint inflammation [5]
3. The anti-inflammatory effect of Ibufenamic acid in acute lung injury is independent of COX inhibition (no effect on COX-2 expression in RAW264.7 cells), indicating a COX-independent pathway mediated by LTA4H/LTB4 inhibition [3]

C12H17NO3

223.27

223.12

2438-72-4

2466

White to off-white solid powder

1.1±0.1 g/cm3

161 - 162ºC

1.530

1.7

2

3

6

16

200

0

MXJWRABVEGLYDG-UHFFFAOYSA-N

InChI=1S/C12H17NO3/c1-2-3-8-16-11-6-4-10(5-7-11)9-12(14)13-15/h4-7,15H,2-3,8-9H2,1H3,(H,13,14)

2-(4-butoxyphenyl)-N-hydroxyacetamide

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)

Solubility in Formulation 1: 2.5 mg/mL (11.20 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (11.20 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: ≥ 2.5 mg/mL (11.20 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10 mg/mL (44.79 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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