ERK; Nrf2; Autophagy

In H9c2 cells, TBHQ (tert-butylhydroquinone; tBHQ; 0-100 μM; 48 hours) did not diminish cell viability on its own. H9c2 cells' vitality was increased by preincubating them with various tBHQ doses for 24 hours; in contrast, exposure to ethanol decreased viability in a dose-dependent way. H9c2 cardiomyocytes exposed to ethanol showed a considerable increase in viability upon receiving tBHQ therapy [3]. The number of apoptotic cells exposed to ethanol is dramatically reduced when H9c2 cells are treated with TBHQ (5 μM) for 15 minutes [3]. The pretreatment of H9c2 cells with TBHQ (5 μM) greatly reduced the increase in caspase-3 and Bax expression caused by ethanol and increased the expression of Bcl-2 [3].

Treatment with TBHQ (50 mg/kg; intraperitoneal injection; three injections every eight hours beginning one hour after ICH; CD-1 mice) increases Nrf2's DNA-binding activity and reduces immediate neurological impairments and oxidative brain damage following intracerebral hemorrhage. functional deficiency (ICH), which concurrently lowers the release of the pro-inflammatory cytokine interleukin-1β (IL-1β) and attenuates microglial activation. When administered after an accident, TBHQ is effective in reducing immediate neurological injury following ICH [4].

Determination of lipid peroxides (measured as MDA)[1]
These measurements were performed as previously described. The fresh heart tissue was rinsed and then homogenized in a buffer [10 mM Tris-HCl, 137 mM NaCl, 1 mM Na2EDTA, and 0.5 mM dithiotreitol (DTT)] with 250 mM sucrose at pH 7.4 using a homogenizer (T 18 basic Ultra-Turrax®). The homogenate was centrifuged at 1,000 × g for 15 min at 4°C. The supernatants were removed, and their total protein concentration was measured using a protein assay kit. The supernatants were used for the biochemical assay and western blot analysis. The malondialdehyde (MDA) content in the heart tissue was used as an index of the lipid superoxide level. The measurements were conducted using a spectrophotometer and a commercially available kit.

Cell Viability Assay[3]
Cell Types: H9c2 Cell
Tested Concentrations: 0 µM, 0.625 µM, 1.25 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, 50 µM and 100 µM
Incubation Duration: 48 hrs (hours)
Experimental Results: Enhanced Cell Viability H9c2 Cardiomyocytes exposed to ethanol.

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Apoptosis analysis[3]
Cell Types: H9c2 Cell
Tested Concentrations: 5 μM
Incubation Duration:
Experimental Results: The number of apoptotic cells diminished when exposed to ethanol.

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Western Blot Analysis [3]
Cell Types: H9c2 cells
Tested Concentrations: 5 μM
Incubation Duration:
Experimental Results: Inhibited ethanol-induced increase in caspase-3 and Bax expression and enhanced Bcl-2 expression.

Animal/Disease Models: Male CD-1 mice (8-10 weeks old) [4]
Doses: 50 mg/kg
Route of Administration: intraperitoneal (ip) injection; 3 injections starting 1 hour after ICH, with an interval of 8 hrs (hrs (hours)).
Experimental Results: Treatment enhanced the DNA-binding activity of Nrf2, alleviated brain oxidative damage, and weakened microglial activation and IL-1β expression.

Metabolism / Metabolites
TBHQ is readily metabolized. In mouse studies, metabolism primarily involved oxidation at the tert-butyl group, followed by formation of the glucuronide conjugate and excretion in the urine, or by excretion of the free acid in feces. In rats, 80-90% of the (14)C-radiolabel was excreted in urine or feces within 96 hours, mostly as the free acid in feces with smaller amounts in urine, and less than 0.3% in expired air. More than 43 metabolites were present in the urine and feces of mice and rats. In several studies with rats and dogs, TBHQ by the oral route was shown to be well absorbed and rapidly excreted, mainly in the urine. Primary urinary metabolites in both species are the 4-O-sulfate conjugate and the 4-O-glucuronide. Excretion seems to be essentially complete after 4 days.
The dominant metabolic pathway of the presumably carcinogenic food antioxidant 2(3)-tert-butyl-4-hydroxyanisole includes O-demethylation to 2-tert-butyl(1,4)hydroquinone and subsequent peroxidation to 2-tert-butyl(1,4)paraquinone. ...
The tert-butylsemiquinone anion radical is formed from tert-butylhydroquinone and from tert-butylquinone in rat liver microsomes.
After ip administration of 3-tert-butyl-4-hydroxyanisole to rats two previously undocumented metabolites 2-tert-butyl-5-methylthiohydroquinone and 2-tert-butyl-6-methylthiohydroquinone were identified in the urine. In addition to these metabolites 3-tert-butyl-4,5-dihydroxyanisole was also detected in the urine hydrolyzed by beta-glucuronidase/sulfatase. Administration of tert-butylhydroquinone, an O-demethylated metabolite of 3-tert-butyl-4-hydroxyanisole, also resulted in the formation of the S-containing metabolites, 2-tert-butyl-5-methylthiohydroquinone and 2-tert-butyl-6-methylthiohydroquinone. After incubation of tert-butylhydroquinone with rat liver microsomes in the presence of glutathione, two metabolites were isolated and purified by HPLC. The metabolites were identified as 2-tert-butyl-5-(glutathione-S-yl)hydroquinone and 2-tert-butyl-6-(glutathione-S-yl)hydroquinone.The formation of tert-butyl hydroquinone-glutathione conjugates required NADPH molecular oxygen and glutathione. Cytochrome p450 inhibitors such as SKF 525-A and metyrapone markedly inhibited the formation of tert-butylhydroquinone-glutathione conjugates in vitro. tert-Butylhydroquinone is converted by cytochrome p450-mediated monooxygenases to a reactive metabolite 2-tert-butyl-p-benzoquinone which then conjugates with glutathione to form tert-butylhydroquinone-glutathione conjugates. Glutathione S-transferase activities do not seen to play a role in glutathione conjugation reaction of 2-tert-butyl-p-benzoquinone because cytosol fraction from rat liver homogenates did not enhance the microsome-mediated production of tert-butylhydroquinone-glutathione conjugates.

Toxicity Summary
IDENTIFICATION AND USE: t-Butylhydroquinone (TBHQ) is a white to light tan crystalline substance with a faint odor. It is used as an antioxidant in fats and oils. HUMAN EXPOSURE AND TOXICITY: There have been reports of vision disturbances in individuals exposed to this chemical. TBHQ produced single strand DNA breaks in human cells. ANIMAL STUDIES: TBHQ produced no teratogenic effects in pregnant rats fed this chemical in the diet. In rats fed TBHQ in the diet, it caused liver enlargement. Acute neurotoxic effects of animals exposed to TBHQ included convulsions and medullary paralysis. In a 2-year study, mice were exposed to t-butyl hydroquinone in feed. There was no evidence of carcinogenic activity of t-butylhydroquinone in male or female animals. TBHQ was negative in an Ames assay in which Salmonella typhimurium strains TA97, TA98, TA100 and TA102 were tested with two types of metabolic activation. In a mouse lymphoma L5178Y forward mutation assay, TBHQ was positive with metabolic activation. TBHQ was negative in a CHO/HGPRT assay at doses of up to 6 ug/mL in the absence of metabolic activation, and 250 ug/mL in the presence of metabolic activation. ECOTOXICITY STUDIES: The transcription of various Glutathione S-Transferases (GSTs), including GSTA, GSTR2 and GSTT, were significantly induced by TBHQ in Nile tilapia.

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Toxicity Data
LCLo (rat) = 2,900 mg/m3/4h

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Interactions
... The objective of this study was to test whether tert-butylhydroquinone (tBHQ), a well-known synthetic Nrf2 inducer, could protect human hepatocytes against arsenic-induced cytotoxicity and oxidative injuries. Our results showed that 5 and 25 umol/L tBHQ pretreatment suppressed the arsenic-induced hepatocellular cytotoxicity, reactive oxygen species generation, and hepatic lipid peroxidation, while relieved the arsenic-induced disturbances of intracellular glutathione balance. In addition, we also observed that tBHQ treatment promoted the arsenic biomethylation process and upregulated Nrf2-regulated downstream heme oxygenase-1 and NADPH: quinine oxidoreductase 1 mRNA expressions. Collectively, we suspected that Nrf2 signaling pathway may be involved in the protective effects of tBHQ against arsenic invasion in hepatocytes. These data suggest that phenolic Nrf2 inducers, such as tBHQ, represent novel therapeutic or dietary candidates for the population at high risk of arsenic poisoning.
... Pretreatment of MIN6 beta-cells with NRF2 activators, including CDDO-Im, dimethyl fumarate (DMF), and tert-butylhydroquinone (tBHQ), protected the cells from high levels of H2O2-induced cell damage. ...
... Copper has been shown to be capable of mediating the activation of several xenobiotics producing reactive oxygen and other radicals. Since copper exists in the nucleus and is closely associated with chromosomes and DNA bases, in this study we have investigated whether the activation of 1,4-hydroquinone (1,4-HQ) and a variety of other phenolic compounds by copper can induce strand breaks in double-stranded phi X-174 RF I DNA (phi X-174 relaxed form I DNA). In the presence of micromolar concentrations of Cu(II), DNA strand breaks were induced by 1,4-HQ and other phenolic compounds including 4,4'-biphenol, catechol, 1,2,4-benzenetriol, 2-methoxyestradiol, 2-hydroxyestradiol, diethylstilbestrol, butylated hydroxytoluene, butylated hydroxyanisole, tert-butylhydroquinone, ferulic acid, caffeic acid, chlorogenic acid, eugenol, 2-acetamidophenol, and acetaminophen. Structure-activity analysis shows that in the presence of Cu(II), the DNA cleaving activity for phenolic compounds with a 1,4-hydroquinone structure, such as 1,2,4-benzenetriol and tert-butylhydroquinone is greater than those with a catechol group (catechol, 2-hydroxyestradiol and caffeic acid). Those compounds having one phenol group, such as eugenol, 2-acetamidophenol, and acetaminophen, are the least reactive. In addition, the induced DNA strand breaks could be inhibited by bathocuproinedisulfonic acid, a Cu(I)-specific chelator, or catalase indicating that a Cu(II)/Cu(I) redox cycle and H2O2 generation are two major determinants involved in the observed DNA damage. Using reactive oxygen scavengers, it was observed that the DNA strand breaks induced by the 1,4-HQ/Cu(II) system could not be efficiently inhibited by hydroxyl radical scavengers, but could be protected by singlet oxygen scavengers, suggesting that either singlet oxygen or a singlet oxygen-like entity, possibly a copper-peroxide complex, but not free hydroxyl radical probably plays a role in the DNA damage. The above results would suggest that macromolecule-associated copper and reactive oxygen generation may be important factors in the mechanism of 1,4-HQ and other phenolic compound-induced DNA damage in target cells.
The effects of diethyl maleate on the cytotoxicity of phenyl-hydroquinone and other hydroquinones were studied in freshly isolated rat hepatocytes. ... Among other hydroquinones (0.5 mM), tert-butyl-hydroquinone-induced cytotoxicity was decreased by diethyl maleate (1.25 mM) ... .
For more Interactions (Complete) data for T-BUTYLHYDROQUINONE (13 total), please visit the HSDB record page.

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Non-Human Toxicity Values
LD50 Rat (male) oral 951 mg/kg bw
LD50 Rat (female) oral 1131 mg/kg bw
LD50 Rat oral 615 mg/kg bw
LD50 Rat oral 750-950 mg/kg, depending on the presence of food in the intestinal tract
For more Non-Human Toxicity Values (Complete) data for T-BUTYLHYDROQUINONE (14 total), please visit the HSDB record page.

[1]. Tert-butylhydroquinone ameliorates doxorubicin-induced cardiotoxicity by activating Nrf2 and inducing the expression of its target genes. Am J Transl Res. 2015; 7(10): 1724–1735.

[2]. Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes.Int J Mol Med. 2016 Jul; 38(1): 123–130.

[3]. Dehydrocorydaline inhibits cell proliferation, migration and invasion via suppressing MEK1/2-ERK1/2 cascade in melanoma.Onco Targets Ther. 2019 Jul 2;12:5163-5175.

[4]. Post-Injury Administration of Tert-butylhydroquinone Attenuates Acute Neurological Injury AfterIntracerebral Hemorrhage in Mice.J Mol Neurosci. 2016 Apr;58(4):525-31.

Tert-butylhydroquinone appears as white to light tan crystalline powder or a fine beige powder. Very slight aromatic odor. (NTP, 1992)
2-tert-butylhydroquinone is a member of the class of hydroquinones in which one of the ring hydrogens of hydroquinone is replaced by a tert-butyl group. It has a role as a food antioxidant.
tert-Butylhydroquinone has been reported in Paeonia suffruticosa and Glycyrrhiza glabra with data available.
2-tert-Butyl-1,4-benzenediol is found in fats and oils. 2-tert-Butyl-1,4-benzenediol is an antioxidant used in food, e.g. oils and fats. Polym. inhibitor
2-tert-Butyl-1,4-benzenediol belongs to the family of Cumenes. These are aromatic compounds containing a prop-2-ylbenzene moiety.
See also: Anoxomer (monomer of).

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Mechanism of Action
... In this study, tert-butylhydroquinone (tBHQ), a widely used Nrf2 activator, was initially employed to investigate the potential protective role of Nrf2 activation in saturated fatty acid (SFA)-induced hepatoxicity. As expected, SFA-induced hepatocyte cell death was prevented by tBHQ in both AML-12 mouse hepatocytes and HepG2 human hepatoma cells. However, the protective effect of tBHQ is Nrf2-independent, because the siRNA-mediated Nrf2 silencing did not abrogate tBHQ-conferred protection. Alternatively, our results revealed that autophagy activation was critically involved in the protective effect of tBHQ on lipotoxicity. tBHQ induced autophagy activation and autophagy inhibitors abolished tBHQ's protection. The induction of autophagy by tBHQ exposure was demonstrated by the increased accumulation of LC3 puncta, LC3-II conversion, and autophagic flux (LC3-II conversion in the presence of proteolysis inhibitors). Subsequent mechanistic investigation discovered that tBHQ exposure activated AMP-activated protein kinase (AMPK) and siRNA-mediated AMPK gene silencing abolished tBHQ-induced autophagy activation, indicating that AMPK is critically involved in tBHQ-triggered autophagy induction. Furthermore, our study provided evidence that tBHQ-induced autophagy activation is required for its Nrf2-activating property. Collectively, our data uncover a novel mechanism for tBHQ in protecting hepatocytes against SFA-induced lipotoxicity. tBHQ-triggered autophagy induction contributes not only to its hepatoprotective effect, but also to its Nrf2-activating property.
/An/ outbreak of H7N9 influenza in China /was/ of high concern to public health. H7 hemagglutinin (HA) plays a critical role in influenza entry and thus HA presents an attractive target for antivirals. Previous studies have suggested that the small molecule tert-butyl hydroquinone (TBHQ) inhibits the entry of influenza H3 HA by binding to the stem loop of HA and stabilizing the neutral pH conformation of HA, thereby disrupting the membrane fusion step. Based on amino acid sequence, structure and immunogenicity, H7 is a related Group 2 HA. In this work we show, using a pseudovirus entry assay, that TBHQ inhibits H7 HA-mediated entry, as well as H3 HA-mediated entry, with an IC50 ~ 6 uM. Using NMR, we show that TBHQ binds to the H7 stem loop region. STD NMR experiments indicate that the aromatic ring of TBHQ makes extensive contact with the H7 HA surface. Limited proteolysis experiments indicate that TBHQ inhibits influenza entry by stabilizing the H7 HA neutral pH conformation. Together, this work suggests that the stem loop region of H7 HA is an attractive target for therapeutic intervention and that TBHQ, which is a widely used food preservative, is a promising lead compound.

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Therapeutic Uses
/EXPL THER/ /The objective was/ to investigate the protective effect of tert-butylhydroquinone on bone marrow cells in rats from cytotoxicity induced by benzene in vitro. The bone marrow cells in rats were divided into two groups randomizedly. Cells of the control group were stimulated by 0, 5, 10, 15, 20 mmol/L benzene for 2, 4, 6 hours respectively. Cells of the tBHQ-pretreated group were treated by 100 umol/L tBHQ for 12 hours followed by the same conditions as the control group. The DNA damage was detected by single cell gel electrophoresis assay (SCGE) and cell apoptosis was examined by flow cytometry. The activities of NAD (P) H: quinone oxidoreductase (NQO1) in bone marrow cells of rats were also measured before benzene treatment in two groups. In the control group, the DNA damage and the apoptosis of bone marrow cells was increased with the growing concentration and time of benzene treatment. The DNA migration and the lengths of DNA migration of the bone marrow cells in the rats under 5, 10, 15, 20 mmol/L benzene treatment in the tBHQ-pretreated group were significantly lower than those in control group at the same time point (p<0.05). The apoptosis of the bone marrow cells in the rats stimulated by 15, 20 mmol/L benzene for 2 hours and 10, 15, 20 mmol/L benzene for 4 hours as well as 5, 10, 15, 20 mmol/L benzene for 6 hours were also significantly lower than those in control group (p<0.05). The activities of NQO1 in the bone marrow cells in the rats were increased after tBHQ treatment (p<0.01) (1.62 +/- 0.16 min(-1).mg(-1) vs. the control group: 0.95 +/- 0.08 min(-1).mg(-1)). Benzene can induce the DNA damage and the apoptosis of bone marrow cells in rats in a time dependent and dose dependent manner to some extent. tBHQ can protect the bone marrow cells in rats from the cytotoxicity induced by benzene, which can be partly explained by the increase of the NQO1 activity induced by tBHQ.
/EXPL THER/ ... In this study, we evaluated whether tBHQ pretreatment prevented renal damage induced by ischemia and reperfusion (I/R). Four groups of rats were studied: (a) control-sham (CT), (b) tBHQ-sham (tBHQ), (c) I/R and (d) tBHQ + I/R. Intraperitoneal (i.p.) injections of tBHQ (50 mg/kg) were given to the tBHQ and tBHQ + I/R groups and 3% ethanol/isotonic saline solution to the CT and I/R groups. Animals were killed 24 hours after I/R. tBHQ attenuated I/R-induced renal dysfunction, structural damage, oxidative/nitrosative stress, glutathione depletion and the decrease in several antioxidant enzymes. The renoprotective effect of tBHQ on I/R injury was associated with the attenuation in oxidative/nitrosative stress and the preservation of antioxidant enzymes.
/EXPL THER/ Cis-diamminedichloroplatinum II (CDDP)-induced nephrotoxicity is associated with the overproduction of reactive oxygen species. ... The purpose of this study was to investigate the ability of tBHQ to prevent the nephrotoxic effect of CDDP in rats as well as the mechanisms involved. Thirty-six Wistar rats divided in the following groups were used: control, tBHQ (12.5 mg/kg), CDDP (7.5 mg/kg) and tBHQ+CDDP. Twenty-four hr urine was collected at the beginning and at the end of the experiment and the rats were sacrificed 72 hr after CDDP-administration. Histological studies were performed and markers of renal function and oxidative/nitrosative stress were measured. In addition, the activity of the following antioxidant enzymes was measured: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione reductase (GR) and glutathione-S-transferase (GST). CDDP-induced renal dysfunction, structural damage and oxidative/nitrosative were prevented by tBHQ. In addition, tBHQ completely prevented the CDDP-induced fall in GPx and GST activities. In conclusion, the present study indicates that the antioxidant activity of tBHQ is associated with its nephroprotective effect against CDDP-induced acute kidney injury in rats.
/EXPL THER/ The present study was aimed at determining the role of paraquat (PQ) in the activation of the NF-E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway and the possible neuroprotective effects of tert-butylhydroquinone (tBHQ) pretreatment on PQ-induced neurodegeneration in vivo and in vitro. 7 mg/kg PQ treatment of male C57BL/6 mice caused decreased spontaneous locomotor activity, decreased tyrosine hydroxylase (TH)-positive neurons, increased terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL)-positive cells in the substantia nigra, as well as increased protein levels of both nuclear Nrf2 and HO-1. In PQ-treated mice, pretreatment with 1% tBHQ (w/w) significantly attenuated impairments in behavioral performance, decreased TH-positive neurons, and increased TUNEL-positive cells in the substantia nigra, as well as increased protein expression of both nuclear Nrf2 and HO-1. Pretreatment with 40 uM tBHQ protected PC12 cells against 100 and 300 uM PQ-mediated cytotoxicity. The dual-luciferase reporter gene also revealed that the transcriptional activation of HO-1 gene expression of the antioxidant responsive element via Nrf2 occurred as a consequence of 100 and 300 uM PQ exposure. Collectively, these results clearly indicated for the first time that the Nrf2/HO-1 pathway in the substantia nigra was activated by PQ, and pretreatment with tBHQ conferred neuroprotection against PQ-induced Parkinsonism presumably by increasing Nrf2 and HO-1 expression.
For more Therapeutic Uses (Complete) data for T-BUTYLHYDROQUINONE (7 total), please visit the HSDB record page.

166.099

C, 72.26; H, 8.49; O, 19.25

1948-33-0

16043

White to off-white solid powder

1.1±0.1 g/cm3

291.4±20.0 °C at 760 mmHg

127-129 °C(lit.)

138.7±16.4 °C

0.0±0.6 mmHg at 25°C

1.545

2.33

2

2

1

12

148

0

BGNXCDMCOKJUMV-UHFFFAOYSA-N

InChI=1S/C10H14O2/c1-10(2,3)8-6-7(11)4-5-9(8)12/h4-6,11-12H,1-3H3

2-(tert-butyl)benzene-1,4-diol

tert-Butylhydroquinone TBHQ

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 : ≥ 56.66 mg/mL (~340.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (15.04 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 (15.04 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 (15.04 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: 20 mg/mL (120.32 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear 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.)