Absorption, Distribution and Excretion
Tributyrate is absorbed by the intestinal mucosa of rats but not by the skin of guinea pigs. In humans, once-daily oral administration of 50-400 mg/kg tributyrate for 3 weeks resulted in peak plasma concentrations 0.25-3 hours post-administration. Peak plasma concentrations ranged from 0.1-0.45 mM in the 200 mg/kg dose group. No higher plasma concentrations were observed in higher dose groups. In mice and rats, peak plasma butyrate concentrations after oral administration of 10.3 g/kg tributyrate were 1.75 mM and 3.07 mM, respectively. Plasma concentrations ≥1 mM were observed 10-60 minutes post-administration in mice and 30-90 minutes in rats. …Female CD2F1 mice were administered tributyrate orally, or sodium butyrate via intravenous bolus or oral gavage. Mice were administered tributylate orally at doses of 3.1, 5.2, 7.8, and 10.3 g/kg. Sodium butyrate was administered intravenously at doses of 0.31, 0.62, 0.94, and 1.25 g/kg. Sodium butyrate was also administered orally at a dose of 5 g/kg. Similar studies were subsequently conducted in female Sprague-Dawley rats. Rats were administered tributylate orally at doses of 3.6, 5.2, or 10.3 g/kg, or sodium butyrate intravenously at a dose of 500 mg/kg. Plasma butyrate concentrations were determined by gas chromatography. Results: Plasma butyrate concentrations were detectable as early as 5 minutes after oral administration of tributylate to mice, reaching peak concentrations between 15 and 60 minutes post-administration. Peak plasma butyrate concentrations increased proportionally with increasing tributylate dose, but the increase in the area under the plasma butyrate concentration-time curve (AUC) was greater than the dose-proportional increase with increasing oral tributylate dose. When tributylate was administered at a dose of 10.3 g/kg, the peak plasma butyrate concentration was approximately 1.75 mM, and remained above 1 mM for 10 to 60 minutes post-administration. However, approximately 10% of mice treated at this dose experienced acute death. Following administration of tributylate at a dose of 7.8 g/kg, the plasma butyrate concentration reached approximately 1 mM at 15 minutes post-administration and remained between 0.8 and 1 mM until 60 minutes post-administration. No acute death occurred in mice treated at this dose. Mice administered tributylate at doses of 5.2 g/kg and 3.1 g/kg reached peak plasma butyrate concentrations of approximately 0.9 mM and 0.5 mM, respectively, at 45 minutes post-administration. The plasma butyrate concentrations in these mice remained above 0.1 mM for 120 minutes and 90 minutes post-administration, respectively. Results from four intravenous injections of sodium butyrate showed that the plasma concentration-time curve also exhibited nonlinear pharmacokinetic characteristics and could be well described using a one-compartment model with saturation elimination. The recorded Michaelis constant (Km) and maximum response rate (Vmax) ranged from 1.02 to 5.65 mM and 0.60 to 1.82 mmol/min, respectively. The central compartment volume (Vc) ranged from 0.48 to 0.72 L/kg. Following intravenous administration of 1.25 g/kg sodium butyrate, peak plasma butyrate concentrations ranged from 10.5 to 17.7 mM, and remained above 1 mM for 20 to 30 minutes. Following oral administration of 5 g/kg sodium butyrate, peak plasma butyrate concentrations in mice were approximately 9 mM at 15 minutes post-administration, and remained above 1 mM for 90 minutes. In rats, following oral administration of 10.3 g/kg glyceryl butyrate, peak plasma butyrate concentrations were approximately 3 mM at 75 minutes, and exceeded 1 mM between 30 and 90 minutes. Plasma butyrate concentrations in rats at doses of 5.2 g/kg and 3.6 g/kg were lower than those in the 10.3 g/kg dose group, and no nonlinear relationship was observed. Following intravenous injection of 500 mg/kg sodium butyrate, the peak plasma butyrate concentration in rats was approximately 11 mM, and the concentration decreased over time, consistent with saturation clearance. The peak plasma butyrate concentration occurred between 0.25 and 3 hours after administration and increased with increasing dose, ranging from 0 to 0.45 mM. In three patients receiving dose escalation therapy, peak plasma concentrations did not increase. There was no difference in butyrate pharmacokinetics between day 1 and day 15. Because peak plasma concentrations were reached close to the in vitro effective concentration (0.5–1 mM), but butyrate disappeared from plasma within 5 hours after administration, we are currently conducting dose escalation therapy, administering the drug three times daily, starting at a dose of 450 mg/kg/day. Metabolites… Tributyratease… is an enzyme that specifically hydrolyzes tributyrate. This enzyme can be inhibited by certain organic compounds, particularly certain fluorophosphates. …A fluoride-sensitive tributylate enzyme has been isolated from rat adipose tissue, which releases butyric acid from 1-monobutyric acid, 1,2-dibutyric acid, or tributyric acid.
The liver and kidney microsomes of pigs exhibit the highest hydrolytic activity for tributyric acid. Liver microsomes of frogs, pigs, rats, cats, rabbits, guinea pigs, sheep, pigeons, and dogs (but not fish) also possess esterase activity against tributylic acid glycerides.
Studies on the hydrolysis of glycerol fatty acid esters (including tributylic acid glycerides…) have shown that they are completely hydrolyzed to glycerol and the corresponding fatty acids, namely butyric acid, 5-hydroxydecanoic acid, and 5-hydroxydodecanoic acid.

Interactions
Effect of tributylate administration on diisopropyl fluorophosphate (DFP) toxicity in mice. 36 hours after administration of tributylate, DFP toxicity was 1.7 times the normal value; after 14 days of administration, the toxicity was only 1.15 times the normal value. Non-human toxicity values Rats oral LD50: 3.2 g/kg Mice oral LD50: 12.8 mg/kg

[1]. Tributyrin, a stable and rapidly absorbed prodrug of butyric acid, enhances antiproliferative effects of dihydroxycholecalciferol in human colon cancer cells. J Nutr. 2001 Jun;131(6):1839-43.

[2]. Tributyrin-induced differentiation promotes apoptosis of LS 174T colon cancer cells in vitro. Int J Oncol. 2002 Jan;20(1):195-200.

Tributyrate is a triglyceride derived from the three hydroxyl groups of butyrate-acylated glycerol. It possesses multiple functions, including as an EC 3.5.1.98 (histone deacetylase) inhibitor, protectant, apoptosis inducer, prodrug, and antitumor agent. It is a triglyceride and butyrate ester, functionally related to butyrate. Tributyrate has been used in trials for the treatment of prostate cancer and certain adult solid tumors (protocol-specific). It has been reported to exist in Euglena and Caenorhabditis elegans, with relevant data. Tributyrate is a butyrate prodrug with potential antitumor activity. Butyrate is the active metabolite of tributyrate, which inhibits histone deacetylase, leading to increased differentiation, decreased proliferation, cell cycle arrest, and apoptosis in certain tumor cell lines. (NCI04)

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Mechanism of Action
……Human gastric cancer SGC-7901 cells were exposed to tributylate at concentrations of 0.5, 1, 2, 5, 10, and 50 mmol/L for 24–72 hours. Cell proliferation was assessed using the MTT/2H-tetrazole, 2-(4,5-dimethyl-2-thiazol-3) assay. [(3)H]-TdR uptake was measured to determine DNA synthesis. Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. tributylate-induced apoptosis was detected by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Expression of PARP/polyADP-ribose polymerase, Bcl-2, and Bax was detected by Western blot. Tributylate inhibited the growth of SGC-7901 cells in a dose- and time-dependent manner. Compared with the control group, after treatment with 2 mmol/L tributyrate for 48 h, the uptake of [(3)H]-TdR in SGC-7901 cells decreased to 33.6% (P<0.05). TUNEL assay showed apoptotic morphology. Flow cytometry results indicated that tributyrate induced apoptosis in SGC-7901 cells in a dose-dependent manner. After incubation with 2 mmol/L(-1) tributyrate for 48 hours, Bcl-2 protein levels decreased and Bax protein levels increased in SGC-7901 cells, accompanied by PARP cleavage. ...HT-29 colon cancer cells exposed to PB (phenylbutyrate) and TB (tributyrate) showed growth inhibition, accompanied by caspase-3 activation-mediated apoptosis induction. G1/S phase cell cycle arrest was also observed after PB and TB exposure, accompanied by decreased CDK2 (cyclin-dependent kinase) protein levels and hypophosphorylation of retinoblastoma proteins. We investigated the role of tributyrate in inducing growth arrest and apoptosis in MCF-7 human breast cancer cells. Four hours after treatment with tributyrate, a transient increase in the mitochondrial-associated protein Bax, dissipation of mitochondrial membrane potential (Δψm), and cleavage of caspase-3-independent poly(ADP-ribose) polymerase were observed. Subsequently, transient accumulation of mitochondrial cytochrome c in the cytosol led to the generation and accumulation of sub-diploid DNA-rich cells. During periods of elevated mitochondrial Bax levels, δψm channel disruption, and detection of cytochrome c in the cytoplasm, we found that p21WAF1/Cip1 was induced without increased p53 expression, and cells arrested in the G2-M phase.

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Therapeutic Use
…This study included 20 patients with advanced solid tumors who had no other treatment options, a life expectancy of more than 12 weeks, and normal organ function. They received tributyrate treatment at doses of 150 to 200 mg/kg three times daily. Blood samples were collected before administration and at 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, and 4 hours after administration for pharmacokinetic analysis. The enrolled patients included 15 men and 5 women, with a median age of 61 years (range 30–74 years). Previous treatment regimens included chemotherapy (median 2 cycles, range 0–5 cycles), radiotherapy (1 patient), and no treatment (1 patient). No dose-limiting toxicities were observed. Due to the large number of capsules required, the dose was escalated to 200 mg/kg three times daily and then discontinued. The median butyrate concentration was 52 μM, but varied considerably among patients. No objective response was observed. Four patients experienced prolonged disease stability, lasting from 3 to 23 months; the median progression-free survival was 55 days. As of the time of this report, two patients with chemotherapy-refractory non-small cell lung cancer had survived for more than one year without disease progression.

C15H26O6

302.36334

302.172

C, 59.58; H, 8.67; O, 31.75

60-01-5

60-01-5

6050

COLORLESS
Oily liquid

1.1±0.1 g/cm3

307.5±0.0 °C at 760 mmHg

-75 °C

173.9±0.0 °C

0.0±0.6 mmHg at 25°C

1.448

2.95

0

6

14

21

304

0

CCCC(=O)OCC(COC(=O)CCC)OC(=O)CCC

UYXTWWCETRIEDR-UHFFFAOYSA-N

InChI=1S/C15H26O6/c1-4-7-13(16)19-10-12(21-15(18)9-6-3)11-20-14(17)8-5-2/h12H,4-11H2,1-3H3

2,3-di(butanoyloxy)propyl butanoate

Tributyrin; NSC 661583; NSC-661583; NSC661583

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 (~330.7 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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