Histone Deacetylases (HDACs); endoplasmic reticulum (ER) stress

At a concentration of 2 mM, the HDAC inhibitor 4-phenylbutyric acid (4-PBA) stops the growth of NSCLC cell lines. Phenylbutyric acid and ciglitazone together can improve cancer cell growth inhibition [1]. 4-ASFV infection is inhibited by phenylbutyric acid (0–5 mM) in a dose-dependent manner. In addition to preventing ASFV-induced H3K9/K14 hypoacetylation, benzoenebutyric acid also suppresses late protein synthesis. Together, phenylbutyric acid and enrofloxacin prevent ASFV replication [2]. When bafilomycin A1 was added, LC3II accumulated; however, 4-phenylbutyric acid dramatically decreased this accumulation. Phenylbutyric acid counteracted the 48-hour decline in p62 levels caused by LPS stimulation. After 48 hours, the percentage of AVO cells induced by LPS rose, whereas 4-phenylbutyric acid markedly reduced this percentage. Particularly, following treatment with phenylbutyric acid, the proportion of cells exhibiting AVO dropped from 61.6% to 53.1%, indicating that 4-phenylbutyric acid suppresses autophagy induced by lipopolysaccharide (LPS). The positive control for autophagy inhibition employed in this study was bafilomycin A1. The percentage of LPS-induced AVO cells was decreased by bafilomycin A1 treatment. In ATG7 knockdown, there was no phenylbutyric acid treatment-induced decrease in OC area or fusion index. Phenylbutyric acid's inhibitory effect on LPS-induced effects is totally eliminated when NF-κB is inhibited using BAY 11-7082 and JSH23, which also lowers LC3 II levels following LPS stimulation [3].

LPS significantly decreased bone volume (BV/TV), trabecular thickness (Tb. Th), and bone mineral density (BMD) as compared to PBS alone. Trabecular space (Tb. Sp.) increased. LPS-induced bone loss is decreased by 4-phenylbutyric acid (4-PBA). 4-BMD, BV/TV, and Tb. Th were all elevated after phenylbutyric acid treatment. besides decreasing the rise in Tb in comparison to LPS alone. Sp., but when phenylbutyric acid was administered to mice alone, no alterations were seen. Phenylbutyric acid treatment of LPS-treated mice also resulted in a considerable decrease in OC.S/BS as measured by TRAP staining. However, OC.N/BS tended to decline in mice treated with LPS and phenylbutyric acid, albeit not in a statistically significant way. According to these findings, phenylbutyric acid causes OC in LPS-treated mice to shrink in size as opposed to increasing in number. In line with these results, phenylbutyric acid therapy of LPS-injected mice resulted in a decrease in blood CTX-1, a marker of bone resorption in vivo that was enhanced by LPS treatment. In contrast to LPS alone, phenylbutyric acid therapy did not substantially alter serum levels of osteocalcin and ALP, two indicators of bone formation in vivo. Moreover, phenylbutyric acid can lessen the rise in serum MCP-1 that is brought on by LPS, suggesting that it can lessen systemic inflammation brought on by LPS [3].

African swine fever virus (ASFV) causes a highly lethal disease in swine for which neither a vaccine nor treatment are available. Recently, a new class of drugs that inhibit histone deacetylases enzymes (HDACs) has received an increasing interest as antiviral agents. Considering studies by others showing that valproic acid, an HDAC inhibitor (HDACi), blocks the replication of enveloped viruses and that ASFV regulates the epigenetic status of the host cell by promoting heterochromatinization and recruitment of class I HDACs to viral cytoplasmic factories, the antiviral activity of four HDACi against ASFV was evaluated in this study. Results showed that the sodium phenylbutyrate fully abrogates the ASFV replication, whereas the valproic acid leads to a significant reduction of viral progeny at 48h post-infection (-73.9%, p=0.046), as the two pan-HDAC inhibitors tested (Trichostatin A: -82.2%, p=0.043; Vorinostat: 73.9%, p=0.043). Further evaluation showed that protective effects of NaPB are dose-dependent, interfering with the expression of late viral genes and reversing the ASFV-induced histone H3 lysine 9 and 14 (H3K9K14) hypoacetylation status, compatible to an open chromatin state and possibly enabling the expression of host genes non-beneficial to infection progression. Additionally, a synergic antiviral effect was detected when NaPB is combined with an ASFV-topoisomerase II poison (Enrofloxacin). Altogether, our results strongly suggest that cellular HDACs are involved in the establishment of ASFV infection and emphasize that further in vivo studies are needed to better understand the antiviral activity of HDAC inhibitors[2].

Nanomolar concentrations of trichostatin A induced growth arrest in five of seven NSCLC cell lines, whereas sodium phenylbutyrate (PB) was markedly less potent. In adenocarcinomas, trichostatin A up-regulated general differentiation markers (gelsolin, Mad, and p21/WAF1) and down-regulated markers of the type II pneumocyte progenitor cell lineage (MUC1 and SP-A), indicative of a more mature phenotype. PB had a similar effect. Simultaneous treatment with a PPARgamma ligand and PB enhanced the growth inhibition in adenocarcinomas but not in nonadenocarcinomas. Growth arrest was accompanied by markedly decreased cyclin D1 expression but not enhanced differentiation[1].

Female 10-week-old C57BL/6J mice were housed in the pathogen-free animal facility of IRC. All mice were handled in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Immunomodulation Research Center (IRC), University of Ulsan. All animal procedures were approved by the IACUC of IRC. The approval ID for this study is # UOU-2014-014. Animals were randomized into the following 4 groups: vehicle control (n = 5), vehicle + 4-PBA (n = 6), LPS (n = 6), and LPS + 4-PBA (n = 6). Mice were treated with LPS in 200 μL phosphate-buffered saline (PBS) (or with PBS as a vehicle) once a week (5mg/kg, i.p.) for 3 weeks as described [19]. 4-PBA solution was prepared by titrating equimolecular amounts of 4-PBA and sodium hydroxide to reach pH 7.4; mice were injected daily intraperitoneally in 200 μL PBS (or with PBS as a vehicle) at a dose of 240 mg/kg for 3 weeks. Mice were sacrificed by CO2 asphyxiation. To determine the bone mineral density (BMD) and microarchitecture of the long bone, the right femur was scanned in a high-resolution Micro CT (μCT) SkyScan 1176 System. Scans were performed with an effective detector pixel size of 6.9 μm and a threshold of 77–255 mg/cc. Trabecular bone was analyzed in a region 1.6 mm in length and located 0.1 mm below the distal femur growth plate. A total of 75–125 tomographic slices were acquired; 3 D analyses were performed with CT volume software. The structural parameters such as bone volume fraction (BV/TV), trabecular thickness (Tb. Th), and trabecular space (Tb. Sp.) were analyzed. In vivo markers of bone resorption were measured according to the manufacturer’s directions; serum collagen-type I fragments (CTX-1) were assessed using a RatLaps EIA assay. Serum osteocalcin was assessed using an osteocalcin EIA kit, and alkaline phosphatase (ALP) was quantitated using a colorimetric kinetic determination kit. Serum MCP-1 was quantitated by sandwich ELISA using the recommended Abs according to manufacturer’s instruction[3].

Absorption, Distribution and Excretion
Following oral administration of a single 5g dose of sodium phenylbutyrate, the Cmax was 195-218 µg/mL under fasting conditions and the Tmax was one hour. The effect of food on drug absorption is unknown.
Approximately 80–100% of the dose was excreted by the kidneys within 24 hours as the conjugation product, phenylacetylglutamine. For each gram of sodium phenylbutyrate administered, it is estimated that between 0.12–0.15 grams of phenylacetylglutamine nitrogen are produced.

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Metabolism / Metabolites
The major sites for metabolism of sodium phenylbutyrate are the liver and kidney. Phenylbutyric acid is rapidly metabolized to phenylacetate via beta-oxidation. Phenylacetate is conjugated with phenylacetyl-CoA, which in turn combines with glutamine via acetylation to form phenylacetylglutamine.

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Biological Half-Life
Following oral administration of a single 5g dose of sodium phenylbutyrate, the elimination half-life of phenylbutyric acid ranged from 0.76 to 0.77 hours.

Hepatotoxicity
While the urea cycle disorders are caused by deficiencies of hepatic enzymes responsible for the elimination of nitrogen, patients generally present with hyperammonemia without other features or biochemical evidence of hepatic injury. Thus, serum aminotransferase, alkaline phosphatase and bilirubin levels are generally normal or only mildly elevated. Newborns presenting with hyperammonemia may have hepatomegaly but other, non-urea cycle, liver function is normal as is hepatic histology. Phenylbutyrate can help to lower ammonia levels acutely and manage to keep them in the normal or near normal range, but generally does not affect other liver functions. In open label studies, a small proportion of patients (particularly with ornithine transcarbamylase [OTC] deficiency) have had ALT or AST elevations, but these have generally been attributed to the underlying condition or its complications. Phenylbutyrate has not been linked to instances of clinically apparent liver injury with jaundice.
Likelihood score: E (unlikely cause of clinically apparent liver injury, but experience with its use is limited).

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Protein Binding
When co-administered with tauroursodeoxycholic acid as a combination product, the _in vitro_ plasma protein binding of phenylbutyric acid is 82%.

[1]. Enhanced growth inhibition by combination differentiation therapy with ligands of peroxisome proliferator-activated receptor-gamma and inhibitors of histone deacetylase in adenocarcinoma of the lung. Clin Cancer Res. 2002 Apr;8(4):1206-12.

[2]. Sodium phenylbutyrate abrogates African swine fever virus replication by disrupting the virus-induced hypoacetylation status of histone H3K9/K14. Virus Res. 2017 Oct 15;242:24-29.

[3]. 4-Phenylbutyric acid protects against lipopolysaccharide-induced bone loss by modulating autophagy in osteoclasts. Biochem Pharmacol. 2018 May;151:9-17.

4-phenylbutyric acid is a monocarboxylic acid the structure of which is that of butyric acid substituted with a phenyl group at C-4. It is a histone deacetylase inhibitor that displays anticancer activity. It inhibits cell proliferation, invasion and migration and induces apoptosis in glioma cells. It also inhibits protein isoprenylation, depletes plasma glutamine, increases production of foetal haemoglobin through transcriptional activation of the gamma-globin gene and affects hPPARgamma activation. It has a role as an EC 3.5.1.98 (histone deacetylase) inhibitor, an antineoplastic agent, an apoptosis inducer and a prodrug. It is functionally related to a butyric acid. It is a conjugate acid of a 4-phenylbutyrate.
Phenylbutyric acid is a fatty acid and a derivative of [butyric acid] naturally produced by colonic bacteria fermentation. It demonstrates a number of cellular and biological effects, such as relieving inflammation and acting as a chemical chaperone. It is used to treat genetic metabolic syndromes, neuropathies, and urea cycle disorders.
Phenylbutyric acid is a Nitrogen Binding Agent. The mechanism of action of phenylbutyric acid is as an Ammonium Ion Binding Activity.
Phenylbutyrate and sodium benzoate are orphan drugs approved for the treatment of hyperammonemia in patients with urea cycle disorders, a series of at least 8 rare genetic enzyme deficiencies. The urea cycle is the major pathway of elimination of excess nitrogen including ammonia, and absence of one of the urea cycle enzymes often causes elevations in serum ammonia which can be severe, life-threatening and result in permanent neurologic damage and cognitive deficiencies. Both phenylbutyrate and sodium benzoate act by promoting an alternative pathway of nitrogen elimination. Neither phenylbutyrate nor sodium benzoate have been linked to cases of liver injury either in the form of serum enzyme elevations during therapy or clinically apparent acute liver injury.
4-Phenylbutyric acid has been reported in Streptomyces with data available.
See also: Sodium Phenylbutyrate (active moiety of); Glycerol Phenylbutyrate (is active moiety of).

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Drug Indication
Phenylbutyric acid is used for the treatment of various conditions, including urea cycle disorders, neonatal-onset deficiency, late-onset deficiency disease in patients with a history of hyperammonemic encephalopathy. Phenylbutyric acid must be combined with dietary protein restriction and, in some cases, essential amino acid supplementation. Phenylbutyric acid, as sodium phenylbutyrate, is used in combination with [tauroursodeoxycholic acid] to treat amyotrophic lateral sclerosis (ALS) in adults.

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Mechanism of Action
Sodium phenylbutyrate is the most commonly used salt used in drug products of phenylbutyric acid. Sodium phenylbutyrate is a pro-drug that rapidly metabolizes to phenylacetate. Phenylacetate is conjugated with phenylacetyl-CoA, which in turn combines with glutamine via acetylation to form phenylacetylglutamine. Phenylacetylglutamine is then excreted by the kidneys, thus providing an alternate mechanism of waste nitrogen excretion to the urea cycle. Phenylacetylglutamine is comparable to urea, as each molecule contains two moles of nitrogen.

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Pharmacodynamics
Phenylbutyric acid decreases elevated plasma ammonia glutamine levels in patients with urea cycle disorders. It increases waste nitrogen excretion in the form of phenylacetylglutamine. In the intestines, phenylbutyric acid was shown to reduce mucosal inflammation, regulate transepithelial fluid transport, and improve oxidative status. Some studies report antineoplastic properties of phenylbutyric acid, showing that phenylbutyric acid can promote growth arrest and apoptosis of cancer cells. It is suggested that phenylbutyric acid can act as an ammonia scavenger, chemical chaperone, and histone deacetylase inhibitor.

C10H12O2

164.2

164.083

C, 73.15; H, 7.37; O, 19.49

1821-12-1

Sodium 4-phenylbutyrate;1716-12-7;4-Phenylbutyric acid-d11;358730-86-6;4-Phenylbutyric acid-d5;64138-52-9;4-Phenylbutyric acid-d2;461391-24-2

4775

White to off-white solid powder

1.1±0.1 g/cm3

290.7±9.0 °C at 760 mmHg

49-52ºC

187.9±13.9 °C

0.0±0.6 mmHg at 25°C

1.535

2.42

1

2

4

12

137

0

O([H])C(C([H])([H])C([H])([H])C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O

OBKXEAXTFZPCHS-UHFFFAOYSA-N

nChI=1S/C10H12O2/c11-10(12)8-4-7-9-5-2-1-3-6-9/h1-3,5-6H,4,7-8H2,(H,11,12)

4-Phenylbutyric acid

4-Phenylbutyric acid; AI3 12065; 4-PHENYLBUTYRIC ACID; 4-Phenylbutanoic acid; 1821-12-1; Benzenebutanoic acid; Benzenebutyric acid; Phenylbutyrate; Phenylbutyric acid; gamma-Phenylbutyric acid; AI312065; AI3-12065

2934.99.03.00

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 (~609.01 mM)
H2O : ~2 mg/mL (~12.18 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.23 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 (15.23 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 (15.23 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: 33.33 mg/mL (202.98 mM) in 20% HP-β-CD in 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.)