Cyclooxygenase (COX-1 and COX-2). Phenylbutazone is a nonsteroidal anti-inflammatory drug (NSAID) that inhibits cyclooxygenase (COX) enzymes. COX-1 and COX-2 are enzymes that catalyze the conversion of arachidonic acid to prostaglandins and thromboxanes, which are mediators of inflammation, pain, and fever. By inhibiting COX enzymes, Phenylbutazone reduces prostaglandin synthesis, leading to its anti-inflammatory, antipyretic, and analgesic effects. The compound is a butyl-diphenyl-pyrazolidinedione that has been used in the treatment of ankylosing spondylitis, rheumatoid arthritis, and reactive arthritis. Phenylbutazone is particularly effective in the treatment of ankylosing spondylitis. In veterinary medicine, Phenylbutazone is used for the treatment of musculoskeletal disorders in horses and dogs.

The inhibitory effect of phenylbutazone on COX-1 and COX-2 is minimal [3].
Phenylbutazone demonstrates anti-inflammatory, antipyretic, and analgesic activity in vitro through COX inhibition. The compound inhibits both COX-1 and COX-2 enzymes, reducing prostaglandin synthesis. Its IC50 for COX-1 and COX-2 inhibition has been characterized in enzymatic assays. Phenylbutazone also exhibits other effects, including inhibition of leukocyte migration and superoxide production. The compound's anti-inflammatory activity has been demonstrated in various in vitro models. Phenylbutazone is a synthetic, pyrazolone derivative NSAID.

Phenylbutazone has been used clinically for the treatment of ankylosing spondylitis, rheumatoid arthritis, and reactive arthritis. In veterinary medicine, it is used for the treatment of musculoskeletal disorders in horses and dogs. The compound's anti-inflammatory, analgesic, and antipyretic effects have been established through clinical use. Phenylbutazone is particularly effective in the treatment of ankylosing spondylitis. However, its use in humans has declined due to safety concerns, including gastrointestinal ulceration, bone marrow suppression, and hepatotoxicity. Phenylbutazone is for research use only and is not for human therapeutic use.

COX enzyme inhibition assays are performed using recombinant COX-1 and COX-2 enzymes. The enzyme is incubated with the substrate arachidonic acid and a peroxidase co-substrate in assay buffer. The reaction product prostaglandin G2 is detected by measuring the oxidation of a chromogenic co-substrate (e.g., TMPD) at 590 nm. Phenylbutazone is serially diluted in DMSO and added to the reaction mixture to determine IC50 values. Each concentration is tested in duplicate, and IC50 values are calculated by non-linear regression analysis. Appropriate positive controls (e.g., indomethacin, aspirin) and vehicle controls are included to validate the assay.

Cellular COX inhibition is evaluated in cells stimulated to produce prostaglandins (e.g., macrophages stimulated with LPS). Cells are cultured in appropriate media at 37°C with 5% CO₂ and treated with Phenylbutazone at various concentrations. Prostaglandin E2 (PGE2) levels in the culture supernatant are measured by ELISA. Cell viability is assessed using MTT or LDH assays. Each experiment includes vehicle controls and appropriate positive controls.

In vivo efficacy of Phenylbutazone is evaluated in animal models of inflammation and pain (e.g., carrageenan-induced paw edema, adjuvant-induced arthritis). The compound is administered orally or intraperitoneally at doses determined by preclinical studies. Paw swelling is measured by plethysmometry. Pain responses are assessed. Clinical studies have been conducted for ankylosing spondylitis and other rheumatic conditions. Sample sizes typically range from 6-10 animals per group.

Absorption, Distribution and Excretion
Nonsteroidal anti-inflammatory drugs (NSAIDs) bind up to 95% to plasma proteins, especially albumin. Due to this high protein binding, their volume of distribution is small (0.10–0.17 L/kg). The pKa values of NSAIDs range from 3.5 to 5.2. Phenytozoline appears to be rapidly and completely absorbed from the gastrointestinal tract. In healthy, fasting men, a single oral dose of 300 mg phenytozoline resulted in a peak plasma concentration of 43.3 μg/mL over an average of 2.5 hours. Many medications are recommended to be taken with or after food. However, recent studies have shown that food intake can significantly alter the absorption rate and/or extent of absorption of many drugs. This alteration may lead to important changes in the clinical activity of these drugs. Enteric-coated phenylbutazone is recommended to be taken with food to minimize potential gastrointestinal side effects. The results of this study indicate that while food delays the absorption time of this formulation by 4–5 hours, it has no significant effect on peak concentration or area under the curve. Therefore, food is expected to have some impact on fluctuations in steady-state plasma concentrations, but the average concentration within the recommended dosing interval will remain constant. Thus, food will not affect therapeutic efficacy but may improve tolerability. After oral administration, phenylbutazone is almost completely absorbed. Most of the drug in plasma is bound to proteins and has a small volume of distribution. Phenybutazone is primarily eliminated through metabolism, with only 1% excreted unchanged in the urine. Approximately 10% of a single dose of phenylbutazone is excreted in bile as metabolites. Approximately 60% of urinary metabolites have been identified. In humans, a novel drug metabolite—C-glucuronide—is formed by the direct coupling of the pyrazolidine ring of phenylbutazone to glucuronic acid via a C-C bond. Phenybutazone is oxidized on the benzene ring or side chain to generate hydroxylated metabolites, which may further undergo O-glucuronidation. After a single dose, C-glucuronidation appears to be the dominant reaction, while oxidation gradually increases with repeated dosing. Due to the different pharmacokinetic properties of their metabolites, C-glucuronide has the highest concentration in urine, while the pharmacologically active compounds hydroxybutazolone and γ-hydroxybutazolone are mainly found in plasma. Phenybutazolone has a relatively long biological (elimination) half-life in humans, averaging approximately 70 hours, with significant inter-individual and intra-individual variability. Inter-individual variability is primarily attributed to genetic factors. For more complete data on the absorption, distribution, and excretion of 14 phenybutazolone compounds, please visit the HSDB record page. Metabolism/Metabolites Phenybutazolone is metabolized in the liver. It is oxidized to hydroxybutazolone, γ-hydroxybutazolone, β-hydroxybutazolone, γ-ketobutazolone, and p-γ-dihydroxybutazolone. Glucuronide conjugates of phenybutazolone and its metabolites are also formed. In a multi-dose study of patients with rheumatoid arthritis, the concentration of total oxyphenazole in plasma decreased with increasing phenazole dose, suggesting that long-term use of higher doses of phenazole may promote its clearance or inhibit its formation. The concentration of γ-hydroxyphenazole in plasma increased proportionally with the dose of phenazole, with significant inter-individual variability. Identified major metabolites include oxyphenazole (cyclic hydroxylation), γ-hydroxyphenazole (side-chain hydroxylation), γ-hydroxyphenazole (dihydroxy metabolite), and 4-hydroxyphenazole. In rats and horses, γ-hydroxyphenazole is the major metabolite (approximately 35%), existing in two interchangeable forms: a lactone form and a linear form. The formation of the lactone form of γ-hydroxyphenazole requires the cleavage of an amide bond. Studies have shown that the formation of this lactone isomer is a negligible reaction in humans. Other products of phenazole oxidation, but in smaller quantities, include β-hydroxy and γ-ketone derivatives of the parent compound.
Phenyrazolone exists in solution in three forms—diketo, enol, and mesocrystalline anionic. In solution, it is predominantly in the diketo form, and the conversion between these forms is slow. These conversions may lead to its chemical instability, allowing it to be converted to 4-hydroxyphenyrazolone metabolites via a peroxide-dependent co-oxidation reaction by the cyclooxygenase system.
In addition to the major metabolites, glucuronide/sulfate conjugates of these major metabolites were detected in varying proportions. Glucuronide metabolites were not found in horses; in rats, approximately 35%–40% of the metabolites were excreted in urine as conjugate metabolites; in humans, conjugates account for approximately 50% of urinary metabolites.
…A novel drug metabolite—C-glucuronide—is formed by the direct coupling of the pyrazolidine ring of phenyrazolone to glucuronic acid via a C-C bond. Phenyrazolone is oxidized on the benzene ring or side chain to generate hydroxylated metabolites, which may subsequently undergo O-glucuronidation. Following a single dose, C-glucuronidation appears to be the primary response, while oxidation becomes increasingly important with repeated administration. Due to the varying pharmacokinetic properties of the metabolites, C-glucuronide concentrations are highest in urine, while the pharmacologically active compounds hydroxybutazolone and γ-hydroxybutazolone are primarily found in plasma. ...

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Biological Half-Life
The biological half-life of phenbutazolone in canine plasma is approximately 6 hours, in guinea pigs approximately 5 hours, and in rabbits approximately 3 hours.
The biological half-life of phenbutazolone in plasma is 72 hours.
It has been reported that the plasma half-life of phenbutazolone and its metabolite hydroxybutazolone ranges from 50 to 100 hours, with significant inter- and intra-individual variability. It has been reported that the plasma half-life of phenbutazolone in children is shorter than in adults; one study showed that the plasma half-life in children aged 1-7 years was approximately 40 hours. Some studies suggest this may be due to increased cytochrome P450 enzyme activity in children or a higher liver-to-body weight ratio in children compared to adults. The plasma half-life of phenbuprofen in elderly patients may be slightly longer than in younger adults. Age-related biological and physiological changes (e.g., decreased liver and kidney function, decreased serum albumin concentration) may contribute to altered drug clearance in older patients. In patients with severely impaired liver function, the plasma half-life of phenbuprofen has been reported to be as long as 149 hours. Following a single dose in humans, the plasma concentration of the unmetabolized drug is characterized by an early peak of 36 μg/mL at 3 hours, followed by a slow decline between 7 and 336 hours, with a corresponding elimination half-life of 88 hours.
Molecular Weight: 308.37. Formula: C19H20N2O2. CAS No.: 50-33-9. IUPAC Name: 4-butyl-1,2-diphenylpyrazolidine-3,5-dione. Synonyms: Phenylbutazone. Appearance: White or off-white crystalline powder. Melting Point: 105.5-106.5°C. Solubility: Soluble in acetone, chloroform, and benzene; soluble in alcohol and ether; almost insoluble in water; soluble in sodium hydroxide solution. Storage: Typically at room temperature. Phenylbutazone is a synthetic, pyrazolone derivative NSAID.

Interactions
Because phenylbutazone and its metabolite hydroxyphenylbutazone are highly bound to proteins, they may be displaced from their binding sites by other protein-binding drugs (e.g., oral anticoagulants, phenytoin, salicylates, sulfonamides, and sulfonylureas), or vice versa. Patients taking phenylbutazone should be closely monitored for adverse reactions if they are taking any of the aforementioned drugs concurrently. As a microsomal enzyme inducer, phenylbutazone and its metabolite hydroxyphenylbutazone may accelerate the metabolism of drugs affected by this system. Conversely, concurrent administration of other drugs (e.g., barbiturates, promethazine, chlorpheniramine, rifampin, or corticosteroids) may also enhance phenylbutazone metabolism and shorten its plasma half-life, as these drugs can also induce hepatic microsomal enzymes. Phenytozoline may enhance digoxin metabolism (presumably through the induction of hepatic microsomal enzymes), leading to a decrease in digoxin plasma concentration and half-life. Phenytozoline may also enhance the metabolism of aminopyrine, hexobarbital, or corticosteroids. Concomitant use of phenylbutazone with warfarin or other coumarin or indanedione derivative anticoagulants can lead to increased free anticoagulant concentrations and an increased risk of serious bleeding. Almost all patients receiving warfarin and phenylbutazone treatment experience hypoprothrombinemia, usually within the first week of starting combination therapy, or even as early as day one. This effect is attributed to phenylbutazone and/or its metabolite oxyphenbutazone displacing the anticoagulant from its protein binding site; furthermore, phenylbutazone appears to inhibit the metabolism of the pharmacologically active S-isomer of warfarin. Phenylbutazone alone does not affect prothrombin time. The ulcerative effect of phenylbutazone and its influence on platelet function further increase the risk of concomitant use with any anticoagulant or thrombolytic agent (e.g., streptokinase). Phenylbutazone may enhance the hypoglycemic effects of acesulfame potassium, tolbutamide, and other sulfonylureas by competing for protein binding sites or urinary excretion pathways. Studies have shown that phenylbutazone can inhibit the metabolism of tolbutamide, possibly by stimulating a cytochrome P450-like enzyme system with low activity in the metabolism of tolbutamide hydroxylation, and reducing renal excretion of hydroxyhexylamine (the active metabolite of acesulfame potassium). Phenylebazole may also enhance the hypoglycemic effect of insulin. For more complete data on interactions of phenylebazole (out of 20), please visit the HSDB record page. Non-human toxicity values: Oral LD50 in rats: 245 mg/kg; Intraperitoneal LD50 in rats: 142 mg/kg; Subcutaneous LD50 in rats: 230 mg/kg; Intravenous LD50 in rats: 100 mg/kg. For more complete data on non-human toxicity values of phenylebazole (out of 17), please visit the HSDB record page.
Phenylbutazone has significant toxicity concerns. Common adverse effects may include gastrointestinal ulceration and bleeding, bone marrow suppression (agranulocytosis, aplastic anemia), and hepatotoxicity. Its use in humans has declined due to these safety concerns. It is contraindicated in patients with blood dyscrasias, peptic ulcer disease, and hypersensitivity. Phenylbutazone is primarily used in veterinary medicine. The compound is for research use only and is not for human therapeutic use.

[1]. Inactivation of prostaglandin H synthase and prostacyclin synthase by phenylbutazone. Requirement for peroxidative metabolism. Mol Pharmacol. 1985 Jan;27(1):109-14.

[2]. Phenylbutazone induces expression of MBNL1 and suppresses formation of MBNL1-CUG RNA foci in a mouse model of myotonic dystrophy. Sci Rep. 2016 Apr 29;6:25317.

[3]. COX-1 and COX-2 inhibition in horse blood by phenylbutazone, flunixin, carprofen and meloxicam: an in vitro analysis. Pharmacol Res. 2005 Oct;52(4):302-6.

Therapeutic Uses
Nonsteroidal anti-inflammatory drugs (NSAIDs)
Phenytocin was approved in 1949 for the treatment of rheumatoid arthritis and gout. However, it is no longer approved for any human use in the United States and is therefore no longer marketed. This is because some patients treated with phenytocin experienced severe toxic reactions, and other effective and less toxic drugs are now available for the same conditions. Phenytocin is known to induce blood disorders, including aplastic anemia, leukopenia, agranulocytosis, thrombocytopenia, and even death. Serum sickness-type hypersensitivity reactions have also been reported. Furthermore, phenylbutazone is classified as a carcinogen by the National Toxicology Program.
Veterinary Use: For the relief of inflammation associated with the musculoskeletal system in horses. /US Product Label Includes/
Veterinary Use: Used in veterinary medicine as an analgesic, antipyretic, and anti-inflammatory agent.
For more complete data on the therapeutic uses of phenylbutazone (10 in total), please visit the HSDB record page.

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Drug Warnings
If symptoms such as gastrointestinal upset, jaundice, or blood disorders occur, discontinue use immediately. Confirmed cases of agranulocytosis associated with this drug have occurred in humans. To prevent this, routine blood cell counts should be performed every two weeks thereafter. A significant decrease in total white blood cell count, relative granulocytopenia, or the appearance of melena or tarry stools should be considered a signal to immediately discontinue treatment and take appropriate action. Specific anti-infective therapy is required when treating inflammatory diseases related to infection.
Animals treated should not be slaughtered for consumption. Injection administration is only permitted via intravenous injection; never administer subcutaneously or intramuscularly. Use with caution in patients with a history of drug allergy.
Therefore, the use of phenylbutazone carries serious risks in patients receiving thrombolytic therapy or long-term anticoagulation therapy and should be avoided.
Significant changes in total white blood cell count, relative granulocytopenia, the presence of immature blood cells, or a decrease in hematocrit or platelet count all indicate that phenylbutazone should be discontinued immediately and a comprehensive hematological evaluation should be performed.
Hematologic toxicity may occur shortly after the start of treatment or after prolonged treatment, and may occur suddenly or gradually, and may appear days or weeks after discontinuation of the drug. For more complete data on drug warnings for phenbuzodone (31 in total), please visit the HSDB record page.

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Pharmacodynamics
Phenbuzodone is a synthetic pyrazolone derivative. It is a non-steroidal anti-inflammatory and antipyretic drug used to treat inflammatory diseases. Its significant analgesic effect is likely primarily related to its anti-inflammatory properties, stemming from its ability to reduce the production of prostaglandin H and prostacyclin. Prostaglandins act on various cells, such as vascular smooth muscle cells, causing vasoconstriction or vasodilation; on platelets, causing platelet aggregation or disaggregation; and on spinal cord neurons, causing pain. Prostacyclin can cause vasoconstriction and platelet disaggregation.
Phenylbutazone is also known as 4-butyl-1,2-diphenylpyrazolidine-3,5-dione. Its IUPAC name is 4-butyl-1,2-diphenylpyrazolidine-3,5-dione. Phenylbutazone is a synthetic, pyrazolone derivative NSAID with anti-inflammatory, antipyretic, and analgesic activities. It has been used in the treatment of ankylosing spondylitis, rheumatoid arthritis, and reactive arthritis. In veterinary medicine, Phenylbutazone is used for musculoskeletal disorders in horses and dogs. Its use in humans has declined due to safety concerns. Regulatory approvals for human use exist in some countries but use has declined. Phenylbutazone is for research use only.

C19H20N2O2

308.3743

184.984

50-33-9

Phenylbutazone(diphenyl-d10);1219794-69-0;Phenylbutazone-13C12;1325559-13-4;Phenylbutazone-d9;1189479-75-1

4781

White to off-white solid powder

1.5±0.1 g/cm3

240.4±23.0 °C at 760 mmHg

104-107 °C

99.2±22.6 °C

0.0±0.5 mmHg at 25°C

1.613

2.76

0

2

5

23

389

0

CCCCC1C(=O)N(N(C1=O)C2=CC=CC=C2)C3=CC=CC=C3

VYMDGNCVAMGZFE-UHFFFAOYSA-N

InChI=1S/C19H20N2O2/c1-2-3-14-17-18(22)20(15-10-6-4-7-11-15)21(19(17)23)16-12-8-5-9-13-16/h4-13,17H,2-3,14H2,1H3

4-butyl-1,2-diphenylpyrazolidine-3,5-dione

phenylbutazone; 50-33-9; 4-Butyl-1,2-diphenylpyrazolidine-3,5-dione; Butapirazol; Butazolidin;

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 : ≥ 100 mg/mL (~324.29 mM)
H2O : ~0.67 mg/mL (~2.17 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.11 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 (8.11 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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 (Please use freshly prepared in vivo formulations for optimal results.)