Bufexamac (formerly also known as CP-1044-J-3; CP1044J3; Droxaryl; Parfenac; Bufexamic acid), a non-steroidal anti-inflammatory drug/NSAID, is a potent COX enzyme inhibitor with potential anti-inflammatory activity. It inhibits IFN-α release with an EC50 of 8.9 μM. Bufexamac is also a specific inhibitor of class IIB HDAC (histone deacetylases) (HDAC6/10). Treatment of peripheral blood mononuclear cells with bufexamac blocks the secretion of the inflammatory cytokine IFN-α. Bufexamac is a frequent and relevant contact sensitizer and also used as an anti-inflammatory agent belong to the NSAID (non-steroidal anti-inflammatory drug) class.

Physicochemical Properties

Molecular Formula	C12H17NO3
Molecular Weight	223.27
Exact Mass	223.12
CAS #	2438-72-4
Related CAS #	2438-72-4
PubChem CID	2466
Appearance	White to off-white solid powder
Density	1.1±0.1 g/cm3
Melting Point	161 - 162ºC
Index of Refraction	1.530
LogP	1.7
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	6
Heavy Atom Count	16
Complexity	200
Defined Atom Stereocenter Count	0
InChi Key	MXJWRABVEGLYDG-UHFFFAOYSA-N
InChi Code	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)
Chemical Name	2-(4-butoxyphenyl)-N-hydroxyacetamide
Synonyms	CP 1044 J 3;Bufexamic acid; CP-1044-J-3; CP1044J3; Droxaryl; Parfenac
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Leukotriene A4 Hydrolase (LTA4H) (IC50: 0.87 ± 0.06 μM for Bufexamac (Bufexamic acid) against human recombinant LTA4H) [3]
ln Vitro	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. 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]
ln Vivo	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]
Enzyme Assay	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]
Cell Assay	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]
Animal Protocol	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]
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Method of application affects the extent of cutaneous absorption. Following rectal administration as suppositories, the systemic absorption was reported to be low. Following topical administration of 5% bufexamac, the recovery in the urine was 3.5% of the applied dose within 144 hours. Studies in healthy volunteers receiving an oral dose of 125 to 500 mg indicate that an average of 80% of the total dose is excreted in the urine within 48 hours. No data available. No data available. Metabolism / Metabolites No data available. Biological Half-Life No data available.
Toxicity/Toxicokinetics	Protein Binding No data available. 1. Mouse toxicity: In the 1-day acute lung injury study, Bufexamac (Bufexamic acid) at 10 mg/kg and 30 mg/kg (oral) had no significant effect on mouse body weight (final weight: 20.5 ± 1.2 g and 19.8 ± 1.1 g, respectively, vs. LPS group 20.1 ± 1.3 g). Serum alanine transaminase (ALT: 46 ± 5 U/L vs. control 43 ± 4 U/L) and creatinine (0.50 ± 0.04 mg/dL vs. control 0.48 ± 0.03 mg/dL) levels in the 30 mg/kg group were within normal ranges [3] 2. Equine toxicity: In the 72-hour joint injection study, bufexamac (20 mg/joint, intra-articular) caused no adverse clinical signs (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. Synovial fluid from bufexamac-treated joints showed no evidence of cytotoxicity (e.g., increased cell necrosis) [5]
References	[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.
Additional Infomation	Bufexamac is a hydroxamic acid derived from phenylacetamide in which the benzene moiety is substituted at C-4 by a butoxy group. It has anti-inflammatory, analgesic, and antipyretic properties. It has a role as a non-narcotic analgesic, a non-steroidal anti-inflammatory drug and an antipyretic. It is a hydroxamic acid and an aromatic ether. Bufexamac is a non-steroidal anti-inflammatory drug (NSAID) under the market name Droxaryl, Malipuran, Paraderm and Parfenac. It is typically administered topically for the treatment of subacute and chronic eczema of the skin, including atopic eczema and other inflammatory dermatoses, as well as sunburn and other minor burns, and itching. It has also been used in suppositories in combination with local anaesthetics indicated for haemorrhoids. The use of bufexamac has been discontinued in Canada and the United States, which may be due to undetermined clinical efficacy and a high prevalence of contact sensitization. Bufexamac was also withdrawn by the EMA in April 2010. A benzeneacetamide with anti-inflammatory, analgesic, and antipyretic action. It is administered topically, orally, or rectally. Drug Indication Indicated for the treatment of various skin conditions, such as atopic eczema and other inflammatory dermatoses. Mechanism of Action The full mechanism of action is unclear. It is proposed that bufexamac acts similarly to other non-steroidal anti-inflammatory drugs to inhibit prostaglandin biosynthesis _in vitro_, via inhibiting cyclo-oxygenase (COX) enzymes. Systematically administered bufexamac may accumulate preferentially in the adrenal cortex of rats and may play a role in adrenal stimulation; however its topical anti-inflammatory action is likely to be independent of this effect. Pharmacodynamics Bufexamac is a topically-active anti-inflammatory agent that inhibits the cyclooxygenase enzyme. In cutaneous and deep experimental inflammation, topical administration of bufexamac exerted a dose-related anti-inflammatory effect. In guinea pigs, bufexamax was shown to be more active than topical acetylsalicylic acid 5% or phenylbutazone 5% in delaying the local increase in temperature resulting from UV exposure. Bufexamac is unlikely to have any effect on wound healing. 1. Bufexamac (Bufexamic acid) is a non-steroidal anti-inflammatory drug (NSAID) with a unique mechanism of targeting LTA4H: it inhibits LTA4H-mediated LTB4 synthesis, thereby reducing LTB4-induced inflammatory cell recruitment and cytokine secretion—this differs from traditional NSAIDs that target COX enzymes [3] 2. In veterinary medicine, bufexamac shows potential for treating joint inflammation: intra-articular injection in healthy horses reduces synovial fluid inflammatory markers (white blood cells, PGE2) without causing tissue damage, supporting its use in equine osteoarthritis or post-traumatic joint inflammation [5] 3. The anti-inflammatory effect of bufexamac in acute lung injury is not associated with COX inhibition (no effect on COX-2 expression in RAW264.7 cells), indicating a COX-independent pathway mediated by LTA4H/LTB4 suppression [3]

Solubility Data

Solubility (In Vitro)

DMSO:45 mg/mL (201.5 mM) Water:<1 mg/mL Ethanol:3 mg/mL (13.4 mM)

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.4789 mL	22.3944 mL	44.7888 mL
	5 mM	0.8958 mL	4.4789 mL	8.9578 mL
	10 mM	0.4479 mL	2.2394 mL	4.4789 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.