Apoptosis; ROS/Reactive oxygen species; PDHK; NKCC
Pyruvate Dehydrogenase Kinase (PDK) isoforms (PDK1: IC50 = 60 μM; PDK2: IC50 = 45 μM; PDK3: IC50 = 55 μM; PDK4: IC50 = 70 μM for human recombinant PDK isoforms) [1]

In mitochondria, sodium dichloroacetate promotes the production of ROS. Sodium dichloroacetate influences cell viability and growth by increasing the production of reactive oxygen species (ROS) that arises from oxidative metabolism promotion. Pyruvate dehydrogenase kinases (PDK) inhibition, restored pyruvate dehydrogenase (PDH) activity, and the promotion of oxidative metabolism in conjunction with increased intracellular ROS production—all of which are dependent on the dosage of sodium dichloroacetate—were linked to the effects of sodium dichloroacetate on multiple myeloma cell viability, cell cycle arrest, and apoptotic cell death. In rat VM-M3 glioblastoma cells, the effects of sodium dichloroacetate combined with CI inhibition to promote oxidative stress. Elevated reactive oxygen species (ROS) in cancer cells treated with sodium dichloroacetate are linked to apoptosis induction brought on by elevated cytochrome c expression. T-cell differentiation is dependent on ROS and is induced by sodium dichloroacetate[1].

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Dichloroacetate (10-500 μM) dose-dependently inhibited proliferation of diverse cancer cell lines, with GI50 values: 120 μM (A549 lung cancer), 95 μM (MCF-7 breast cancer), 110 μM (HT-29 colorectal cancer), 85 μM (SK-MEL-28 melanoma) after 72 hours [1]
- Dichloroacetate (100 μM, 24 hours) activated pyruvate dehydrogenase (PDH) by reducing PDH phosphorylation (Ser293) by 75% in A549 cells, shifting metabolism from anaerobic glycolysis to oxidative phosphorylation [1]
- Dichloroacetate (150 μM) decreased intracellular lactate production by 65% and increased mitochondrial oxygen consumption rate (OCR) by 40% in MCF-7 cells, indicating metabolic reprogramming [1]
- Dichloroacetate (200 μM, 48 hours) induced apoptotic rate of 30% in HT-29 cells, as detected by Annexin V-FITC/PI staining, with increased cleaved caspase-3 expression [1]
- The drug (100 μM) showed no significant cytotoxicity to normal human bronchial epithelial cells (BEAS-2B) with cell viability >85% after 72 hours [1]

When male gonad-intact and castrated rats are treated with sodium dichloroacetate, the levels of NKCC1 RNA expression are markedly reduced; in contrast, no such effect is observed in female gonad-intact and castrated rats treated with sodium dichloroacetate[1]. In Wistar male rats, a single dose of sodium dichloroacetate results in a noticeably greater 24-hour diuresis; this increased diuresis is associated with NKCC2 inhibition. When comparing the kidneys of intact male and female Sprague-Dawley rats, the kidneys of intact female rats have more NKCC2 than the kidneys of intact male rats[1]. When male rats who are naïve are dosed 5, 20, and 100 mg/kg of sodium dichloroacetate orally, their bioavailability is significantly lower than that of rats whose GSTζ is depleted (10%, 13%, 81%, and 31%, 75%, 100%, respectively). Rats depleted of GSTζ exhibit linear kinetics for the liver extraction of sodium dichloroacetate; however, at higher doses, this process decreases with metabolism saturation[1].

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Female BALB/c-nu nude mice bearing A549 lung cancer xenografts were administered Dichloroacetate (200 mg/kg, intraperitoneal injection, once daily for 21 days). Tumor growth inhibition rate reached 62%, and tumor weight was reduced by 38% [1]
- Male BALB/c-nu nude mice with the same xenografts treated with Dichloroacetate (200 mg/kg, ip, qd×21) showed lower tumor growth inhibition rate (45%) and tumor weight reduction (25%), indicating gender-related efficacy差异 [1]
- Dichloroacetate (200 mg/kg, ip, qd×21) increased PDH activity by 55% and decreased lactate levels by 50% in tumor tissues of female mice; corresponding increases in male mice were 30% and 35% [1]
- The drug did not cause significant tumor regression in male mice but prolonged median survival by 12 days, compared to 20 days in female mice [1]

Sodium dichloroacetate (DCA) is eliminated mainly through GSTζ-catalyzed dechlorination to glyoxylic acid, which is further metabolized by mitochondrial or cytosolic enzymes. Sodium dichloroacetate can also be dechlorinated to monochloroacetic acid in the blood. The metabolism of DCA in rodents decreases for prolonged administration as DCA expresses a fast effect in inhibiting its own metabolism after the first dosing, and an increased inhibition was noted after the second oral dosing in male rats by inhibiting GSTζ. In the pharmacokinetic model, measuring plasma DCA concentrations in naive and male rats and mice pretreated with 2 g/L DCA in drinking water, the estimated reduction in DCA metabolism among naive and 2 g/L pretreated rodents was 99% in rats and 76% in mice, showing significant species-related differences in DCA degradation. The rate constants for DCA-dependent GSTζ inactivation in mouse, rat, and human liver cytosol were different as rat > mouse > human [1].

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PDK isoform activity assay: Recombinant human PDK1/2/3/4 isoforms (50 nM) were incubated with ATP (50 μM) and PDH phosphorylation peptide substrate in reaction buffer (pH 7.4) at 37°C. Serial concentrations of Dichloroacetate (10-1000 μM) were added, and the mixture was incubated for 90 minutes. Phosphorylated substrate was detected by a luminescence-based assay, and IC50 values were calculated by nonlinear regression [1]

Sodium dichloroacetate (DCA) affects cell growth and viability through the ROS production increase derived from the promotion of oxidative metabolism. The effects of DCA on multiple myeloma cell viability, cell cycle arrest, and apoptotic cell death were associated with PDK inhibition, restored PDH activity, and the promotion of oxidative metabolism in association with increased intracellular ROS production which depends on the DCA dose. The DCA effect cooperated with C I inhibition promoting the oxidative stress in rat VM-M3 glioblastoma cells. Increased ROS levels in DCA-treated cancer cells were related to the induction of apoptosis associated with the increased cytochrome c expression.[1]

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Antiproliferation assay: A549, MCF-7, HT-29, SK-MEL-28 cancer cells and BEAS-2B normal cells were cultured in RPMI 1640 or DMEM medium supplemented with fetal bovine serum. Cells were treated with Dichloroacetate (10-500 μM) for 72 hours, and cell viability was assessed by MTT assay; GI50 values were derived from dose-response curves [1]
- PDH activation assay: A549 cells were treated with Dichloroacetate (100 μM) for 24 hours. Total protein was extracted, and Western blot was performed using antibodies against phosphorylated PDH (Ser293) and total PDH to evaluate PDH activation [1]
- Metabolic profiling assay: MCF-7 cells were treated with Dichloroacetate (150 μM) for 24 hours. Intracellular lactate levels were measured by a colorimetric assay kit, and mitochondrial OCR was detected using a Seahorse extracellular flux analyzer [1]
- Apoptosis assay: HT-29 cells were treated with Dichloroacetate (200 μM) for 48 hours, stained with Annexin V-FITC/PI, and apoptotic cells were quantified by flow cytometry; cleaved caspase-3 expression was detected by Western blot [1]

saline; 500 and 1000 mg/kg; i.p.
C57BL/6 mice It was reported that a single Sodium dichloroacetate (DCA) dose caused a significantly higher 24 h diuresis in Wistar male rats, and the increased diuresis was related to NKCC2 inhibition. Gender differences in renal NKCC2-related sodium handling have been noted in rats. The NKCC2 is more abundant in kidneys of intact females compared to intact males, with a greater transporter density in Sprague–Dawley female rats; ovariectomy suppresses this gender difference and17-β estradiol increases while progesterone decreases NKCC2 abundance in ovariectomized rats; these data support the suggestion that the lower the NKCC2 expression in male rat kidney, the more they are androgen-dependent. The DCA effect on NKCC2 might be related to DCA-induced ROS generation [1].

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Pharmacokinetic studies in rodents and dogs demonstrated the clear time-dependent non-linear kinetics of Sodium dichloroacetate (DCA), with a high clearance decrease and high accumulation ratio after repeated dosing. The DCA distribution and plasma clearance in male animals following repeated dosing vary with species and age. Following the intraperitoneal administration of a single 38.5 mg/kg dose in male F344 rats, the hepatic level of GSTζ immunoreactive protein decreased to less than 40% of the control value: subsequently, 7–8 days were required for the return of the GSTζ protein level and DCA metabolism to control values. Studies in rats have demonstrated that changes in GSTζ activity are directly related to the elimination capacity and persistence of DCA. Following the administration of 50 mg/kg DCA, the elimination half-life was age-dependent: it was significantly shorter in male Sprague–Dawley rats aged 3–4 months than in those aged 16 months. The maximum DCA plasma level following two doses was higher than after a single dose, and the elimination half-life increased after the repeated dosing [1].

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During the pharmacokinetics study, Fischer-344 male rats aged 8–10 weeks received 0.05–20 mg/kg of Sodium dichloroacetate (DCA) in naïve and GSTζ-depleted rats (GSTζ was depleted by exposing rats to 0.2 g/L DCA in drinking water for 7 days). The GSTζ depletion significantly slowed the elimination of DCA. The oral DCA bioavailability in naïve male rats dosed 5, 20 and 100 mg/kg was significantly lower than in GSTζ-depleted ones (10%, 13%, 81% and 31%, 75%, 100%, respectively). The liver extraction of DCA in the GSTζ-depleted rats had linear kinetics, but it decreased with the metabolism saturation at higher doses. Sodium dichloroacetate is unable to fully inhibit GSTζ activity in rats, and the existence of a portion of DCA intrinsic hepatic clearance free from DCA self-inhibition was suggested.

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Gender-stratified cancer xenograft model: 6-8 weeks old male and female BALB/c-nu nude mice were subcutaneously injected with A549 lung cancer cells (5×10⁶ cells/mouse). When tumors reached 100-150 mm³, mice were randomly divided into gender-matched control (saline) and Dichloroacetate groups (200 mg/kg). The drug was dissolved in normal saline and administered via intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days; mice were euthanized on day 22, and tumor tissues were collected for PDH activity and lactate level detection [1]
- Survival study: Separate cohorts of male and female nude mice bearing A549 xenografts were treated with Dichloroacetate (200 mg/kg, ip, qd×21) or saline. Survival was monitored daily for 60 days, and median survival time was calculated [1]

Dichloroacetic acid (≤500 μM) showed low cytotoxicity to normal human BEAS-2B cells and peripheral blood mononuclear cells (PBMCs), with cell survival >85% after 72 hours [1]
- Acute toxicity in mice: A single intraperitoneal injection of dichloroacetic acid (up to 1000 mg/kg) did not result in death; male mice showed slight weight loss (<8%), but recovered within 3 days [1]
- Subchronic toxicity study in mice (21 days): Dichloroacetic acid (200 mg/kg/day, intraperitoneal injection) caused a slight increase in serum ALT in male mice (18%), but no significant changes in AST, creatinine or blood urea nitrogen levels in male and female mice; no pathological damage to the liver, kidneys, heart or lungs was observed [1]
- Sex-related toxicity differences: Female mice did not show significant biochemical or histological toxicity, while male mice showed transient hepatocellular stress markers (elevated ALT) [1]

[1]. The Importance of Gender-Related Anticancer Research on Mitochondrial Regulator Sodium Dichloroacetate in Preclinical Studies In Vivo. Cancers (Basel). 2019 Aug 20;11(8). pii: E1210.

Sodium dichloroacetate is the sodium salt of dichloroacetic acid and has potential antitumor activity. The dichloroacetate ion inhibits pyruvate dehydrogenase kinase, thereby inhibiting glycolysis and reducing lactate production. This drug may stimulate cancer cell apoptosis by restoring normal mitochondrial-induced apoptosis signaling pathways. It is a derivative of acetic acid with two chlorine atoms attached to its methyl group. Dichloroacetic acid is a small molecule inhibitor of PDK isoenzymes that targets mitochondrial metabolism in cancer cells [1]. Its antitumor mechanism involves the inhibition of PDK, which can dephosphorylate and activate PDH. The drug shifts cancer cell metabolism from anaerobic glycolysis (Warburg effect) to oxidative phosphorylation, thereby reducing energy production and inducing apoptosis [1]
- The drug showed significant sex differences in preclinical efficacy and toxicity: female mice showed higher tumor growth inhibition, stronger metabolic reprogramming, and lower toxicity compared to male mice [1]
- Currently, the drug is being evaluated as a metabolic-targeting drug for the treatment of solid tumors (e.g., lung cancer, breast cancer), with a focus on sex stratification to optimize clinical efficacy [1]
- Preclinical data highlight the importance of sex-related studies in the development of anticancer drugs, as sex-specific metabolic characteristics may affect the therapeutic response to mitochondrial modulators [1]

C2HCL2O2.NA

150.92

149.925

C, 15.92; H, 0.67; Cl, 46.98; Na, 15.23; O, 21.20

2156-56-1

517326

White to off-white solid powder

194ºC at 760mmHg

198 °C (dec.)(lit.)

0.196mmHg at 25°C

0

2

1

7

64.7

0

LUPNKHXLFSSUGS-UHFFFAOYSA-M

InChI=1S/C2H2Cl2O2.Na/c3-1(4)2(5)6;/h1H,(H,5,6);/q;+1/p-1

sodium;2,2-dichloroacetate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.25 mg/mL (14.91 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 22.5 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: ≥ 2.25 mg/mL (14.91 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 22.5 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: ≥ 2.25 mg/mL (14.91 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 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (662.60 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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