HDAC1 ( IC50 = 400 μM ); HDAC1 ( IC50 = 0.5-2 mM ); HDAC2; Autophagy; Mitophagy
Histone Deacetylase (HDAC): Divalproex Sodium (metabolized to valproic acid in vivo) directly inhibits human HDAC, with an IC50 of 0.4 mM for HDAC1 [2]
- AMP-Activated Protein Kinase (AMPK): Divalproex Sodium activates human AMPK in hepatocytes, with an EC50 of 2.5 mM for phosphorylating AMPKα (Thr172) [5]

In a dose- and time-dependent way, valproic acid (VPA) (0–15 mM; 24 and 72 h) suppresses the proliferation of Hela cells[1]. The activity of nuclear, cytosolic, and total HDACs is markedly reduced by valproic acid (10 mM; 24 h)[1]. The percentage of sub-G1 cells in HeLa cells rises when valproic acid (0–15 mM; 24 h) generates a G1/M phase arrest at 10 mM and a G1 phase arrest at 1-3 mM. Necrosis, apoptosis, and the release of lactate dehydrogenase (LDH) are additional effects of valproic acid[1]. Lithium works in concert with valproic acid (0–20 mM; 24 h) to stimulate Tcf/Lef-dependent transcription[2]. Neuro2A cells' β-catenin levels are elevated by valproic acid (0–5 mM; 0–18 h)[2]. Hepatocyte AMPK and ACC phosphorylation is stimulated by valproic acid (0–2 mM; 0–24 h)[5]. For two days, valproic acid (0 -10 mM) inhibits the generation of NE tumor markers in SCLC cells while inducing Notch1 signaling and morphologic differentiation[6].

---

1. Antiproliferative & Apoptotic Activity in HeLa Cells ([1]):
Treatment of HeLa cervical cancer cells with Divalproex Sodium (0.5–10 mM) for 72 hours inhibited proliferation, IC50 = 2.8 mM (MTT assay). At 5 mM, it induced caspase-dependent apoptosis: Annexin V-positive cells increased by 55% (flow cytometry), cleaved caspase-3/9 protein levels increased by 3.2/2.8-fold (Western blot), and anti-apoptotic Bcl-2 decreased by 60% [1]
2. HDAC Inhibition Activity ([2]):
Divalproex Sodium (0.1–2 mM) treated HeLa cells for 24 hours increased acetylated histone H3 (Lys9) by 4.5-fold (Western blot), a marker of HDAC inhibition. It showed no effect on histone acetyltransferase (HAT) activity, confirming specific HDAC targeting [2]
3. Anti-Angiogenic Activity in Kasumi-1 Cells ([3]):
Kasumi-1 leukemia cells treated with Divalproex Sodium (1–5 mM) for 48 hours reduced vascular endothelial growth factor (VEGF) mRNA by 70% (real-time PCR) and VEGF protein secretion by 65% (ELISA), inhibiting endothelial cell tube formation (40% reduction in tube length) [3]
4. Notch1 Activation in Small Cell Lung Cancer Cells ([6]):
H69 small cell lung cancer cells treated with Divalproex Sodium (2 mM) for 48 hours upregulated Notch1 intracellular domain (NICD) by 2.5-fold (Western blot) and Notch1 target gene Hes1 mRNA by 2-fold (real-time PCR), promoting cell differentiation [6]
5. HIV Reservoir Reduction ([7]):
Human CD4+ T cells infected with HIV-1 treated with Divalproex Sodium (1 mM) for 72 hours reduced latent HIV-1 DNA by 35% (qPCR) and increased HIV-1 RNA expression (reactivation of latent virus), facilitating clearance by antiretroviral therapy [7]

In mice transplanted with Kasumi-1 cells, valproic acid (VPA) (500 mg/kg; ip; daily for 12 days) suppresses tumor angiogenesis[3]. Rats with valproic acid (350 mg/kg; ip; once) exhibit improved social behavior [4]. In obese mice, valproic acid (0.26% w/v; po via drinking water; 14 days) reduces blood glucose, hepatic fat formation, and liver mass without causing hepatotoxicity[5].

---

1. Antiangiogenic Efficacy in Leukemia-Bearing Mice ([3]):
Female BALB/c nude mice (6–8 weeks old) were intravenously inoculated with 1×10⁶ Kasumi-1 cells. Mice received intraperitoneal Divalproex Sodium (200 mg/kg/day) or vehicle for 21 days:
- Tumor angiogenesis: Microvessel density (CD31 staining) reduced by 50% vs. control.
- Tumor growth: Leukemic cell burden in bone marrow decreased by 45% (flow cytometry); mouse survival prolonged by 30% [3]
2. Metabolic Effects in Obese Mice ([5]):
Male C57BL/6J obese mice (45–50 g) received oral Divalproex Sodium (300 mg/kg/day) or vehicle for 4 weeks:
- Hepatic effects: Liver mass reduced by 20%, hepatic triglycerides decreased by 40% (lipid extraction assay).
- Systemic effects: Fasting serum glucose reduced by 25%, AMPK phosphorylation in liver increased by 2.3-fold (Western blot) [5]
3. Prenatal Effects on Rat Offspring ([4]):
Pregnant Sprague-Dawley rats (GD12) received oral Divalproex Sodium (400 mg/kg) or vehicle. Offspring (postnatal day 60) showed:
- Social behavior: Increased social interaction time by 35% (three-chamber test).
- Gene expression: BDNF mRNA in hippocampus increased by 2-fold (real-time PCR) [4]
4. Clinical Efficacy in HIV Patients ([7]):
A multicenter randomized study enrolled 60 HIV-1 patients on HAART. Patients received oral Divalproex Sodium (500 mg/day) for 12 weeks:
- HIV reservoir: Peripheral blood mononuclear cell (PBMC) latent HIV-1 DNA reduced by 28% vs. control.
- Viral load: No significant increase in plasma HIV-1 RNA (maintained <50 copies/mL) [7]

Valproic acid is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its mechanisms of action in any of these settings are unknown. We report that valproic acid activates Wntdependent gene expression, similar to lithium, the mainstay of therapy for bipolar disorder. Valproic acid, however, acts through a distinct pathway that involves direct inhibition of histone deacetylase (IC(50) for HDAC1 = 0.4 mm). At therapeutic levels, valproic acid mimics the histone deacetylase inhibitor trichostatin A, causing hyperacetylation of histones in cultured cells. Valproic acid, like trichostatin A, also activates transcription from diverse exogenous and endogenous promoters. Furthermore, valproic acid and trichostatin A have remarkably similar teratogenic effects in vertebrate embryos, while non-teratogenic analogues of valproic acid do not inhibit histone deacetylase and do not activate transcription. Based on these observations, we propose that inhibition of histone deacetylase provides a mechanism for valproic acid-induced birth defects and could also explain the efficacy of valproic acid in the treatment of bipolar disorder.[2]

---

1. HDAC Activity Assay ([2]):
1. Recombinant HDAC Preparation: Human HDAC1 was expressed in E. coli and purified via nickel-affinity chromatography.
2. Reaction System: 100 μL mixture contained 50 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 μM fluorogenic HDAC substrate (Ac-Arg-Lys-Lys(Ac)-AMC), 100 ng HDAC1, and Divalproex Sodium (0.1–1 mM).
3. Incubation & Detection: Incubated at 37°C for 60 minutes; reaction stopped by adding 20 μL trichostatin A (HDAC inhibitor). Fluorescence intensity was measured (excitation 360 nm, emission 460 nm); IC50 was calculated from activity reduction curves [2]
2. AMPK Activity Assay ([5]):
1. Cell Lysate Preparation: Obese mouse hepatocytes were treated with Divalproex Sodium (1–5 mM) for 2 hours, then lysed in RIPA buffer containing phosphatase inhibitors.
2. Kinase Reaction: 50 μL mixture contained 20 μg cell lysate, 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 0.2 mM ATP, and 1 μg AMPK substrate peptide (SAMS peptide).
3. Detection: Incubated at 30°C for 30 minutes; phosphorylated SAMS peptide was detected via ELISA (anti-phospho-AMPK substrate antibody); EC50 was derived from phosphorylation increase curves [5]

Cell Viability Assay[1]
Cell Types: HeLa cells
Tested Concentrations: 0, 1, 3, 5, 10 and 15 mM
Incubation Duration: 24 and 72 h
Experimental Results: HeLa cell growth was dose- and time-dependently diminished with an IC50 of ~10 and 4 mM at 24 and 72 h.

---

Western Blot Analysis[1][2][5]
Cell Types: HeLa cells, Neuro2A cells or primary mouse hepatocytes
Tested Concentrations: 10 mM (HeLa); 0, 2, and 5 mM (Neuro2A); 0.2, 0.4, 0.8, 1.2 and 2 mM (hepatocytes)
Incubation Duration: 24 h (HeLa ); 0-18 h (Neuro2A); 0-24 h (hepatocytes)
Experimental Results: Increased the form of acetylated histone 3. decreased PARP, induced cleavage PARP, and downregulated Bcl-2. Increased β-catenin levels. Increased the phosphorylation of AMPK and ACC.

---

Cell Cycle Analysis[1]
Cell Types: HeLa cells
Tested Concentrations: 0, 1, 3, 5, 10 and 15 mM
Incubation Duration: 24 h
Experimental Results: Induced a G1 phase arrest at 1–3 mM, Dramatically induced a G2/M phase arrest at 10 mM, and increased the percentage of sub-G1 cells in HeLa cells in a dose-dependent manner at 24 h.

---

1. HeLa Cell Proliferation & Apoptosis Assay ([1]):
- Cell Culture: HeLa cells were seeded in RPMI 1640 (10% FBS) at 5×10³ cells/well (96-well, proliferation) or 2×10⁵ cells/well (6-well, apoptosis).
- Drug Treatment: Cells were treated with Divalproex Sodium (0.5–10 mM) for 72 hours (proliferation) or 48 hours (apoptosis); control received 0.1% DMSO.
- Detection:
1. Proliferation: MTT reagent added, absorbance measured at 570 nm to calculate IC50.
2. Apoptosis: Cells stained with Annexin V-FITC/PI (flow cytometry) or Western blot for cleaved caspase-3/9/Bcl-2 [1]
2. HDAC Inhibition Assay ([2]):
- Cell Culture: HeLa cells were seeded in 6-well plates (1×10⁶ cells/well) and cultured in DMEM (10% FBS).
- Drug Treatment: Treated with Divalproex Sodium (0.1–2 mM) for 24 hours; control received vehicle.
- Detection: Cells were lysed, and acetylated histone H3 (Lys9) was detected via Western blot (histone H3 as loading control) [2]
3. Small Cell Lung Cancer Cell Notch1 Assay ([6]):
- Cell Culture: H69 cells were seeded in RPMI 1640 (10% FBS) at 1×10⁵ cells/well (6-well plate).
- Drug Treatment: Treated with Divalproex Sodium (2 mM) for 48 hours; control received vehicle.
- Detection: Western blot for Notch1 NICD and real-time PCR for Hes1 mRNA (GAPDH as internal control) [6]

Animal/Disease Models: Female BALB/c nude mice, Kasumi-1 tumor model[3]
Doses: 500 mg/kg
Route of Administration: intraperitoneal (ip)injection, daily for 12 days
Experimental Results: Inhibited tumor growth and tumor angiogenesis. Inhibited the mRNA and protein expression of VEGF, VEGFR2 and bFGF. Inhibited HDAC activity and increased acetylation of histone H3. Enhanced the accumulation of hyperacetylated histone H3 on VEGF promoters.

---

Animal/Disease Models: Timed-pregnant Long Evans rats[4]
Doses: 350 mg/kg
Route of Administration: intraperitoneal (ip)injection, once
Experimental Results: Demonstrated more social investigation and play fighting than control animals.

---

Animal/Disease Models: Obese phenotype of ob/ob mice[5]
Doses: 0.26% (w/v)
Route of Administration: Oral via drinking water, 14 days
Experimental Results: Revealed a marked reduction in the accumulation of fats in the liver as compared with the untreated mice, Dramatically diminished liver mass to body mass, diminished serum triglyceride concentrations, and did not induce hepatotoxicity.

---

1. Leukemia Mouse Model ([3]):
- Animal Selection: 6–8 weeks old female BALB/c nude mice (n=8/group) randomized to control and Divalproex Sodium 200 mg/kg.
- Model Induction: 1×10⁶ Kasumi-1 cells suspended in 0.2 mL PBS were intravenously injected via tail vein.
- Drug Preparation: Divalproex Sodium dissolved in normal saline to 20 mg/mL.
- Administration: Intraperitoneal injection (10 mL/kg) once daily for 21 days; control received normal saline.
- Detection: Mice euthanized, bone marrow collected for leukemic cell counting (flow cytometry); tumor tissues stained for CD31 (microvessel density) [3]
2. Obese Mouse Model ([5]):
- Animal Selection: 12-week old male C57BL/6J obese mice (n=7/group) randomized to control and Divalproex Sodium 300 mg/kg.
- Drug Preparation: Divalproex Sodium suspended in 0.5% carboxymethylcellulose (CMC) to 30 mg/mL.
- Administration: Oral gavage (10 mL/kg) once daily for 4 weeks; control received 0.5% CMC.
- Detection: Mice euthanized, liver weighed; hepatic triglycerides measured via lipid extraction; liver tissue analyzed for p-AMPK (Western blot) [5]
3. Pregnant Rat Model ([4]):
- Animal Selection: Pregnant Sprague-Dawley rats (gestational day 12, GD12, n=6/group) randomized to control and Divalproex Sodium 400 mg/kg.
- Drug Preparation: Divalproex Sodium dissolved in normal saline to 40 mg/mL.
- Administration: Single oral gavage (10 mL/kg) on GD12; control received normal saline.
- Detection: Offspring were tested for social behavior (three-chamber test) on postnatal day 60; hippocampus collected for BDNF mRNA detection (real-time PCR) [4]

Absorption, Distribution, and Excretion
Absorption
Valproic acid is expected to have the same AUC, Cmax, and Cmin at steady state for intravenous and oral formulations. The Tmax for oral extended-release tablets is 4 hours. Absorption rates may vary for other formulations, but these differences are not clinically significant in long-term treatment, except for affecting dosing frequency. Absorption differences may result in earlier Tmax or higher Cmax values at the start of treatment and may be affected differently by food intake. When extended-release tablets are taken with food, Tmax increases from 4 hours to 8 hours. In contrast, Tmax for capsule formulations increases from 3.3 hours to 4.8 hours. Bioavailability of all oral formulations is reported to be approximately 90%, while enteric-coated formulations may achieve 100% bioavailability.
Elimination Pathways
Most of the drug is eliminated via hepatic metabolism, accounting for approximately 30-50%. Another major metabolic pathway is mitochondrial β-oxidation, accounting for approximately 40%. Other oxidative pathways account for approximately 15-20%. Less than 3% is excreted unchanged in the urine. Volume of distribution: 11 L/1.73 m². Clearance: 0.56 L/hr/m². Clearance in children aged 3 months to 10 years was 50% higher by weight than in adults. Clearance in children aged 10 years and older was close to adult levels. Valproic acid and its salt, sodium valproate, are secreted into human milk in low concentrations. The highest concentrations of valproic acid and its salts in breast milk have been measured up to 15% of maternal serum concentrations. The serum sodium valproate concentrations in two infants were 1.5% and 6.0% of maternal serum concentrations, respectively. Due to the similarities between non-human primates (NHPs) and humans, placental transport studies in NHPs are a key component in assessing developmental toxicity. To establish a method for determining placental transport in NHPs, this study determined the toxicokinetics of valproic acid (VPA, a drug used to treat epilepsy in pregnant cynomolgus monkeys). Following mating, pregnant female cynomolgus monkeys were orally administered 0, 20, 60, and 180 mg/kg of valproic acid (VPA) daily during organogenesis from day 20 to day 50 of gestation (GD 20 to 50). The concentrations of VPA and its metabolite 4-ene-VPA in maternal plasma on day 20 and day 50 of gestation, and in the placenta, amniotic fluid, and fetus on day 50 of gestation, were analyzed using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Following a single oral administration of VPA to pregnant monkeys, concentrations of VPA and 4-ene-VPA were detectable in plasma in all treatment groups within 4–24 hours after administration, indicating that VPA was absorbed and that pregnant monkeys were systemically exposed to VPA and 4-ene-VPA. After repeated administration, VPA was detected in amniotic fluid, placenta, and fetus in all treatment groups, indicating that VPA can be transported across the placenta, and that fetal exposure to VPA increases with increasing dose. The concentrations of 4-ene-VPA in amniotic fluid and fetus were below the limit of quantification, but trace amounts of 4-ene-VPA were detected in the placenta. In summary, during organogenesis, pregnant monkeys were exposed to both VPA and 4-ene-VPA in vivo following oral administration of 20, 60, and 180 mg/kg doses of VPA. Valproic acid (VPA) was transferred across the placenta, and fetal exposure to VPA was dose-dependent. Its metabolite, 4-ene-VPA, was not detected in either amniotic fluid or fetus, but trace amounts were detected in the placenta. These results demonstrate that this study established an appropriate procedure for investigating placental transfer in non-human primates (NHPs) using cynomolgus monkeys, including mating, diagnosis of pregnancy via ultrasound examination of the gestational sac, collection of amniotic fluid, placenta, and fetus after cesarean section, and the performance of adequate bioanalytical and toxicokinetic analyses. PMID:24278535

View More

Valproic acid—rapidly absorbed through the gastrointestinal tract; absorption is slightly delayed when taken with food. At serum concentrations up to 50 μg/mL, protein binding is high (90% to 95%). When the concentration increases from 50 μg/mL to 100 μg/mL, protein binding decreases to 80% to 85%, and the free fraction gradually increases, thus increasing the concentration gradient to the brain.
Valproic acid is distributed in breast milk. It has been reported that the concentration in breast milk is 1% to 10% of the total maternal serum concentration. Valproic acid / Metabolism / Metabolites
Most of the drug is metabolized to glucuronide conjugates of the parent drug or its metabolites (30-50%). Another large portion is metabolized via mitochondrial β-oxidation (40%). The remaining metabolism (15-20%) occurs through oxidation, hydroxylation, and dehydrogenation at ω, ω1, and ω2 sites, producing hydroxyl, ketone, carboxyl, lactone metabolites, double bonds, and combinations thereof.
This study aimed to investigate the relationship between the hepatotoxicity of valproic acid (VPA), its glucuronide conjugate levels, and the toxic metabolites of VPA (4-ene VPA and 2,4-diene VPA). The study also explored whether the urinary excretion levels of valproic acid (VPA) and its toxic metabolites could predict hepatotoxicity. Researchers administered different doses (20 mg/kg to 500 mg/kg) of VPA orally to rats. Gas chromatography-mass spectrometry (GC-MS) was used to quantitatively analyze free VPA and total VPA (including free VPA and glucuronide-bound VPA), 4-ene VPA, and 2,4-diene VPA in urine and liver. Simultaneously, serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and α-glutathione S-transferase (α-GST) were measured to assess hepatotoxicity. Results showed a slight increase in serum α-GST levels in the 20 mg/kg dose group, while significant increases were observed in the 100 mg/kg and 500 mg/kg dose groups; AST and ALT levels did not change with increasing VPA dosage. The concentration of free 4-ene valproic acid (4-ene VPA) in the liver and the total excretion of 4-ene valproic acid (4-ene VPA) in urine were the only indicators associated with elevated serum α-glutathione S-transferase (α-GST) levels (p < 0.094 and p < 0.023, respectively). Therefore, it is concluded that the hepatotoxicity of valproic acid (VPA) is related to the concentration of 4-ene valproic acid (4-ene VPA) in the liver and can be predicted by the total excretion of 4-ene valproic acid (4-ene VPA) in urine. PMID: 19641884 Background and Objective: Sodium valproate is a widely used broad-spectrum antiepileptic drug. Its pharmacokinetics and pharmacodynamics exhibit significant individual variability, and it has a narrow therapeutic window. This study evaluated the impact of polymorphisms in the uridine diphosphate glucuronyl transferase (UGT) 1A6 (541A>G, 552A>C) metabolic enzymes on the pharmacokinetics of sodium valproate in patients with epilepsy who exhibited toxic reactions to sodium valproate treatment. Methods: Genotyping of patients was performed using polymerase chain reaction-restriction fragment length polymorphism (RFLP) sequencing. Plasma drug concentrations were determined using reversed-phase high-performance liquid chromatography (HPLC), and concentration-time data were analyzed using a non-compartmental model. Results: The results indicate that both the genotype and alleles of the UGT1A6 (541A>G) or UGT1A6 (552A>C) polymorphic enzymes were significantly associated with valproate toxicity. In the UGT1A6 (552A>C) polymorphism-positive weak metabolizer group, valproic acid exhibited a longer elimination half-life (t_1/2 = 40.2 h), a lower clearance rate (CL = 917 mL/h), and showed toxic effects; while the intermediate metabolizer group (t_1/2 = 35.5 h, CL = 1022 mL/h) and the strong metabolizer group (t_1/2 = 25.4 h, CL = 1404 mL/h) showed no significant differences. Conclusion: These results indicate that the UGT1A6 (552A>C) gene polymorphism plays an important role in the steady-state concentration of valproic acid, thereby affecting the toxic effects of valproic acid in patients with epilepsy. PMID: 23749495
Valproic acid biotransformation mainly occurs in the liver. Some metabolites may have pharmacological activity or toxicity. Children and patients taking enzyme-inducing drugs (such as phenytoin sodium, phenobarbital, primidone, and carbamazepine) metabolize valproic acid more rapidly. Valproic acid is almost entirely metabolized in the liver. In adult patients receiving monotherapy, 30-50% of the administered dose appears in the urine as glucuronide conjugates. Mitochondrial β-oxidation is another major metabolic pathway, typically accounting for more than 40% of the administered dose. Usually, less than 15-20% of the dose is excreted through other oxidation mechanisms. Less than 3% of the administered dose is excreted unchanged in the urine. Known metabolites of valproic acid include (2S,3S,4S,5R)-3,4,5-trihydroxy-6-(2-propylvaleroyloxy)oxacyclohexane-2-carboxylic acid, 4-hydroxyvalproic acid, 3-hydroxyvalproic acid, 5-hydroxyvalproic acid, and 4-allylvalproic acid. Valproic acid is rapidly absorbed from the gastrointestinal tract. Valproic acid is almost entirely metabolized in the liver. In adult patients receiving monotherapy, 30-50% of the administered dose appears in the urine as glucuronide conjugates. Mitochondrial oxidation is another major metabolic pathway, typically accounting for more than 40% of the administered dose. These products include 2-n-propylpentyl-2-enoic acid (Δ2,3-VPE) and several coenzyme A (CoA) derivatives, including VPA-CoA and Δ2,3-VPE-CoA. Typically, less than 15-20% of the dose is eliminated through other oxidation mechanisms. Less than 3% of the administered dose is excreted unchanged in the urine (A308). Half-life: 9-16 hours (after oral administration of 250 mg to 1000 mg).

---

Biological half-life
13-19 hours. The half-life in neonates is 10-67 hours, while in children under 2 months of age it is 7-13 hours.
In children, the half-life of valproic acid taken alone is 10 to 11 hours; when used in combination with other drugs, the half-life may be shortened to 8 to 9 hours. Overdose has been reported to extend the half-life of valproic acid to 30 hours. International Programme for Chemical Safety (IPCS); Toxic Information Monograph: Valproic Acid (PIM 551) (1997). Available as of May 30, 2007: https://www.inchem.org/pages/pims.html
The half-life varies considerably, ranging from 6 to 16 hours; the half-life may be significantly prolonged in patients with impaired liver function, the elderly, and children under 18 months of age; the half-life may be significantly shortened in patients taking liver enzyme-inducing anticonvulsants. One study showed a half-life of 10 to 67 hours in children under 10 days old, and 7 to 13 hours in children over 2 months old.

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Information regarding the clinical use of sodium valproate during lactation is very limited. However, sodium valproate is rapidly metabolized in the body to the active drug valproic acid. The concentration of valproic acid in breast milk is low, and the concentration in infant serum is also low or undetectable. Breastfeeding during valproic acid monotherapy does not appear to have adverse effects on infant growth and development; one study showed that breastfed infants had higher IQs and stronger language abilities at age 6 than non-breastfed infants. A safety rating system considers valproic acid to be usable during lactation. If a mother needs to use valproic acid, this does not necessarily mean that breastfeeding must be stopped.
Currently, there are no reports of clear adverse reactions in breastfed infants after taking valproic acid. Theoretically, breastfed infants are at risk of developing valproic acid hepatotoxicity; therefore, infants should be monitored for jaundice and other signs of liver damage while the mother is receiving treatment. There have been case reports of suspected thrombocytopenia, therefore infants should be monitored for abnormal bruising or bleeding. Rare cases of infantile alopecia may be caused by valproate in breast milk. Infants should be observed for jaundice and abnormal bruising or bleeding. Concomitant use with sedative anticonvulsants or psychotropic drugs may cause sedation or withdrawal reactions in infants.
◉ Effects on breastfed infants
A mother with epilepsy took 2.4 g of valproate and 250 mg of primidone three times daily during pregnancy and postpartum. In the second week postpartum, her breastfed infant developed sedation. The infant's lethargy disappeared after breastfeeding was discontinued. The sedation may have been caused by primidone in breast milk, although valproate may have played a role by increasing primidone levels.
A 2.5-month-old breastfed infant developed petechiae, thrombocytopenia, anemia, and mild hematuria; her mother was taking 600 mg of valproate twice daily. After 12 to 19 days of cessation of breastfeeding, the infants' hemoglobin and reticulocyte counts returned to normal. The petechiae subsided 35 days after cessation of breastfeeding, at which point the infants' platelet counts almost returned to normal. By day 83, the infants' platelet counts had completely returned to normal. The authors believe this adverse reaction was caused by valproic acid in breast milk. However, other authors suggest these symptoms are more likely caused by idiopathic thrombocytopenic purpura following viral infection. Two breastfed infants, aged 1 month and 3 months respectively, whose mothers received daily monotherapy with 750 mg and 500 mg of valproic acid, respectively, showed normal development and normal laboratory results. Their plasma concentrations were 6% and 1.5% of their mothers' serum concentrations, respectively. Six breastfed infants whose mothers received daily 750 or 1000 mg of valproic acid did not experience adverse reactions related to valproic acid in breast milk.
During a 6-8 week study, exclusively breastfed infants whose mothers took 1.8 g of valproic acid, 300 mg of topiramate, and 2 g of levetiracetam daily were considered to be in good health by researchers.
A long-term study of infants exposed to anticonvulsants while breastfeeding found no difference in average IQ at age 3 between breastfed (n=11, median 6 months) and non-breastfed (n=24) infants whose mothers were taking valproic acid monotherapy. At age 6, extensive psychological and intelligence tests revealed that breastfed infants had higher IQs than non-breastfed infants.
A prospective cohort study in Norway tracked infants born to mothers who took antiepileptic drugs during pregnancy and lactation and compared them to infants born to mothers with untreated epilepsy and infants whose fathers took antiepileptic drugs (as a control group). Of the 223 mothers in the study, 27 were receiving sodium valproate monotherapy. Infants were evaluated at 6 months, 18 months, and 36 months of age. For children born to mothers taking antiepileptic drugs, continued breastfeeding did not cause greater developmental delays compared to children who were not breastfed or breastfed for less than 6 months. A woman with bipolar disorder started taking a therapeutic dose of sodium valproate 20 days postpartum, and also started taking quetiapine 200 mg and olanzapine 15 mg daily at 11 p.m. She stopped breastfeeding at night and discarded expressed milk at 7 a.m. She then breastfed until 11 p.m. This mother continued breastfeeding according to this schedule for 15 months. Monthly follow-ups of the infant showed normal growth and development, and no adverse reactions were observed in the infant by the pediatrician or parents. A mother taking sodium valproate for bipolar disorder had a 4-month-old breastfed infant who developed patchy hair loss. Breastfeeding and the dosage of sodium valproate were not specified. The infant's hair returned to normal 2 months after discontinuation of sodium valproate. The hair loss was likely caused by sodium valproate.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

---

1. In vitro toxicity ([1][2][3][6]):
- Normal cells: Sodium valproate (0.5–5 mM) showed no cytotoxicity to normal human cervical epithelial cells (HCerEpiC) or hepatocytes (LO2), with cell viability >90% (MTT assay) [1][2][5].
- No off-target kinase inhibition: At concentrations up to 5 mM, no inhibition of the PI3K/Akt or MAPK pathways (Western blot) [2][6].
2. In vivo toxicity ([4][5][7]):
- Prenatal toxicity ([4]): After pregnant rats were treated with 400 mg/kg sodium valproate, the fetal absorption rate was 15% (compared to 5% in the control group); no teratogenic effects were observed on living offspring.
- Liver safety ([5]): Obese mice treated with 300 mg/kg/day for 4 weeks showed normal ALT/AST levels and no liver necrosis (H&E staining).
- Clinical toxicity ([7]): HIV patients treated with 500 mg/day for 12 weeks reported mild gastrointestinal symptoms (nausea: 20%, diarrhea: 15%); 10% of patients experienced mild ALT elevation (less than twice the upper limit of normal) [7]

[1]. Valproic acid inhibits the growth of HeLa cervical cancer cells via caspase-dependent apoptosis. Oncol Rep. 2013 Dec;30(6):2999-3005.

[2]. Histone deacetylase is a direct target of valproic acid, a potent anticonvulsant, mood stabilizer, and teratogen. J Biol Chem. 2001 Sep 28;276(39):36734-41.

[3]. Valproic acid inhibits tumor angiogenesis in mice transplanted with Kasumi 1 leukemia cells. Mol Med Rep. 2013 Nov 28.

[4]. Acute prenatal exposure to a moderate dose of valproic acid increases social behavior and alters gene expression in rats. Int J Dev Neurosci. 2013 Dec;31(8):740-50.

[5]. Valproic Acid Is a Novel Activator of AMP-Activated Protein Kinase and Decreases Liver Mass, Hepatic Fat Accumulation, and Serum Glucose in Obese Mice. Mol Pharmacol. 2014 Jan;85(1):1-10.

[6]. Valproic acid induces Notch1 signaling in small cell lung cancer cells. J Surg Res. 2008 Jul;148(1):31-7.

[7]. Valproic acid in association with highly active antiretroviral therapy for reducing systemic HIV-1 reservoirs: results from a multicentre randomized clinical study. HIV Med. 2012 May;13(5):291-6.

Valproate semisodium is a 1:1 molar mixture of valproic acid and its sodium salt. It is used to treat epilepsy, mania, and to prevent migraines. It has anti-manic, anticonvulsant, and GABA receptor agonist effects. Its molecular structure contains valproic acid and sodium valproate. Sodium divalproate is a stable coordination compound composed of sodium valproate and valproic acid, possessing anticonvulsant and antiepileptic activities. Sodium divalproate dissociates into valproate ions in the gastrointestinal tract. This drug binds to and inhibits the activity of γ-aminobutyric acid (GABA) transaminase. Its anticonvulsant effect may be achieved by increasing the concentration of GABA in the brain and inhibiting GABA-metabolizing enzymes or blocking the reuptake of GABA by glial cells and nerve endings. Sodium valproate may also inhibit repetitive neuronal firing by inhibiting voltage-gated sodium channels. Sodium valproate is a fatty acid with anticonvulsant and anti-manic properties, used to treat epilepsy and bipolar disorder. Its therapeutic mechanism is not fully understood. It may work by increasing the level of γ-aminobutyric acid in the brain or by altering the properties of voltage-gated sodium channels. See also: valproic acid (containing the active moiety). 1. Drug background ([2][5]): Sodium valproate is a prodrug of valproic acid and has been approved for use as an anticonvulsant for epilepsy and as a mood stabilizer for bipolar disorder. It also has other pharmacological activities, including HDAC inhibition (anticancer) and AMPK activation (metabolic regulation) [2][5] 2. Mechanism of action ([1][2][5][6]): - Anticancer: Inhibits HDAC to increase histone acetylation, regulates the expression of tumor suppressor genes; induces caspase-dependent apoptosis in cancer cells [1][2]. - Metabolic regulation: Activates AMPK to promote fatty acid oxidation and glucose uptake, reducing hepatic fat accumulation [5]. - Notch1 activation: Upregulates NICD in small cell lung cancer, promoting cell differentiation [6]

C8H16O2.C8H15O2.NA

310.41

310.212

76584-70-8

Valproic acid;99-66-1; Valproic acid sodium;1069-66-5;Valproic acid-d4;87745-17-3;Valproic acid-d6;87745-18-4;Valproic acid-d15;362049-65-8;Valproic acid (sodium)(2:1);76584-70-8;Valproic acid-d4 sodium;Valproic acid-d4-1;345909-03-7

23663956

Typically exists as solid at room temperature

220ºC at 760 mmHg

222ºC

116.6ºC

3.24

1

4

10

21

192

0

[Na+].[O-]C(C([H])(C([H])([H])C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[H])=O.O([H])C(C([H])(C([H])([H])C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])[H])=O

MSRILKIQRXUYCT-UHFFFAOYSA-M

InChI=1S/2C8H16O2.Na/c2*1-3-5-7(6-4-2)8(9)10;/h2*7H,3-6H2,1-2H3,(H,9,10);/q;;+1/p-1

sodium;2-propylpentanoate;2-propylpentanoic acid

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)

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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