Apoptosis

BTR-1 (10, 50, 100, and 250 µM, 5 days) causes cytotoxicity in human leukemic cells, with IC50s of 8 and 6 µM at 48 and 72 h, respectively[1].

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BTR-1 potently inhibited the proliferation of human colon cancer Caco-2 cells. After 96 hours of treatment, its IC50 value was 0.85 ± 0.9 mmol/L, compared to 2.2 ± 0.5 mmol/L for butyrate, indicating greater potency. Treatment for 120 hours with 1 mmol/L BTR-1 reduced cell growth by 22–53%, whereas 1 μmol/L dihydroxycholecalciferol alone reduced growth by 8–15% and 2 mmol/L butyrate by 11–23%. A combination of 1 mmol/L BTR-1 and 1 μmol/L dihydroxycholecalciferol showed a synergistic effect, reducing growth to 55 ± 4% after 120 hours, which was significantly more effective than butyrate or BTR-1 alone.[1]
BTR-1 effectively induced differentiation of Caco-2 cells, with potency greater than butyrate or dihydroxycholecalciferol. Assessed by alkaline phosphatase activity, treatment with BTR-1 for 9 days increased activity to 393 ± 28 mU/mg protein, compared to 301 ± 15 mU/mg protein for butyrate. The combination of BTR-1 and dihydroxycholecalciferol further enhanced differentiation (535 ± 31 mU/mg protein).[1]
BTR-1 upregulated vitamin D receptor expression in Caco-2 cells. RT-PCR analysis showed that treating cells with 5 × 10⁻⁵ mol/L and 5 × 10⁻⁴ mol/L BTR-1 for 12 hours increased vitamin D receptor mRNA levels by 70% and 150%, respectively.[1]
Pretreatment with BTR-1 (0.5 mmol/L for 48 hours) significantly enhanced the binding of dihydroxycholecalciferol to its receptor. Maximum specific binding increased by over 100% (from 14,321 molecules/cell in controls to 31,657 molecules/cell), with no significant change in receptor affinity (KD values approximately 3-4.8 × 10⁻¹⁰ mol/L).[1]

Cell Proliferation Assay: Caco-2 cells were seeded at a density of 28,500 cells/cm² in plastic culture plates. After allowing attachment for 24 hours, cells were treated daily with medium supplemented with varying concentrations (0–5 mmol/L) of butyrate or BTR-1. Following 96 hours of treatment, cells were stained with crystal violet and counted to assess proliferation. Additionally, changes in DNA synthesis rate were evaluated by measuring the incorporation of 5-bromo-2′-deoxyuridine (BrdU) during the initial 48 hours of culture.[1]
Cell Differentiation Assay: Caco-2 cells were treated daily with medium containing specified concentrations of dihydroxycholecalciferol, butyrate, BTR-1, or their combination. At various time points (e.g., days 0, 3, 6, 9), cells were harvested. Cells were washed with cold PBS, scraped, sonicated, and centrifuged. Alkaline phosphatase activity in the supernatant was measured by the hydrolysis rate of p-nitrophenyl phosphate at pH 9.8 and 25°C. Total protein content was determined using a Coomassie blue assay. Enzyme activity was expressed as milliunits per milligram of protein.[1]
Vitamin D Receptor Binding Assay: Synchronized subconfluent Caco-2 cells were treated with 0.5 mmol/L BTR-1 for 48 hours (in medium with 1% fetal calf serum). After treatment, cells were washed twice with culture medium and then incubated at 16°C for 8 hours with increasing concentrations (0.2–2 nmol/L) of [³H]-labeled dihydroxycholecalciferol, in the presence or absence of unlabeled dihydroxycholecalciferol (1 μmol/L). Incubation was terminated by removing the medium and washing cells with cold Hanks' buffered saline. Cells were solubilized with 0.5 mL of 1 mol/L NaOH for 30 minutes at room temperature, and radioactivity was measured by liquid scintillation counting. Saturation binding kinetics were analyzed by Scatchard plot.[1]
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR): Caco-2 cells were cultured in serum-free or 1% fetal calf serum-containing medium and then treated with 5–500 μmol/L BTR-1 for 12 hours. Total RNA was extracted and quantified. After DNase digestion, 2 μg of total RNA was reverse-transcribed into cDNA using an oligo d(T) primer. PCR amplification was performed using this cDNA template and specific primers for the vitamin D receptor. The amplification profile consisted of an initial 5-minute denaturation at 94°C, followed by 50 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds. Amplified products were separated by electrophoresis on a 2% agarose gel, visualized by ethidium bromide staining under UV light. β-actin mRNA served as an internal control. The optical density of PCR products was analyzed using computer software and normalized to the density of β-actin.[1]

Reference [1] points out that BTR-1, as a prodrug, has superior pharmacokinetic properties compared to butyrate. It is chemically stable in plasma, rapidly absorbed, and can be hydrolyzed by intracellular lipases to directly release therapeutically effective butyrate into the cells at millimolecular concentrations. Other studies cited in the article show that liquid BTR-1 in gelatin capsules can achieve millimolecular concentrations of butyrate in both plasma and cells. However, this study itself does not provide specific ADME/PK parameters (e.g., half-life, oral bioavailability). [1]

[1]. Novel rhodanine derivatives induce growth inhibition followed by apoptosis. Bioorg Med Chem Lett. 2010 Nov 1;20(21):6297-301.

BTR-1 (tributyrate) is a prodrug of natural butyrate. Butyrate is a normal component of the contents of the colon, produced by bacterial fermentation of unabsorbed complex carbohydrates. In normal mucosa, it is the main energy source; however, it exhibits antiproliferative and differentiation-inducing effects in a variety of cancer cells. Natural butyrate has pharmacokinetic defects as a drug because it is rapidly metabolized and difficult to reach pharmacological concentrations in tumor cells. [1] The mechanism of action of BTR-1 involves at least partly the upregulation of vitamin D receptor expression, thereby enhancing the antiproliferative and differentiation-inducing effects of dihydroxycholecalciferol. This synergistic effect suggests a potential nutritional therapy strategy for the chemoprevention and treatment of colorectal cancer. [1] This study suggests that the combination of BTR-1 with dihydroxycholecalciferol (or its fluorinated analogue), or delivery methods such as liposome encapsulation, may be promising for the treatment of future cancer patients. [1]

C12H11NOS2

249.35184

249.028

C, 57.80; H, 4.45; N, 5.62; O, 6.42; S, 25.72

18331-34-5

18331-34-5

2244563

Solid

1.34g/cm3

372.8ºC at 760mmHg

179.3ºC

9.34E-06mmHg at 25°C

1.692

2.845

0

3

2

16

332

0

C(N1C(=O)/C(=C\C2C=CC=CC=2)/SC1=S)C

ZQDPYAPUFMILTB-UHFFFAOYSA-N

InChI=1S/C12H11NOS2/c1-2-13-11(14)10(16-12(13)15)8-9-6-4-3-5-7-9/h3-8H,2H2,1H3

5-benzylidene-3-ethyl-2-sulfanylidene-1,3-thiazolidin-4-one

BTR-1

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: 13~25 mg/mL (52.1~100.26 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.34 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 20.8 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: ≥ 2.08 mg/mL (8.34 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 20.8 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.)