HDAC1 ( IC50 = 400 μM ); HDAC1 ( IC50 = 0.5-2 mM ); HDAC2; Autophagy; Mitophagy
Valproic acid targets histone deacetylase 1 (HDAC1) (IC50 = 0.4 mM) [2]
Valproic acid targets AMP-activated protein kinase (AMPK) [5]
Valproic acid indirectly targets Notch1 signaling pathway, VEGF/VEGFR2/bFGF (angiogenic factors), caspase-3/-8/-9 [1,3,6]

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].

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1. In HeLa cervical cancer cells, Valproic acid (10 mM, 24 h) induced G2/M cell cycle arrest (DNA flow cytometric analysis) and caspase-dependent apoptosis, accompanied by PARP cleavage, activation of caspase-3/-8/-9, and loss of mitochondrial membrane potential (∆Ψm); all tested caspase inhibitors blocked VPA-induced apoptosis, while TNF-α enhanced it; VPA increased ROS levels and depleted GSH, but NAC/BSO did not affect VPA-induced cell death (IC50 for growth inhibition = 10 mM at 24 h) [1]
2. Valproic acid (0.4 mM) directly inhibited HDAC1 activity, mimicking trichostatin A (TSA) to induce histone hyperacetylation in cultured cells and activate transcription from exogenous/endogenous promoters; non-teratogenic VPA analogs did not inhibit HDAC or activate transcription [2]
3. In Kasumi-1 acute myeloid leukemia cells, Valproic acid downregulated mRNA/protein expression of VEGF, VEGFR2, and bFGF (semi-quantitative RT-PCR, Western blot, immunohistochemistry); it inhibited HDAC1 expression, increased histone H3 acetylation, and enhanced hyperacetylated H3 accumulation on VEGF promoters (ChIP assay) [3]
4. In primary mouse/human hepatocytes, Valproic acid (200 μM–2 mM, 2 h) increased phosphorylation of AMPKα and acetyl-CoA carboxylase (ACC) in a dose-dependent manner; pretreatment with Compound C (AMPK inhibitor) abrogated ACC phosphorylation; VPA metabolites (2-ene-VPA, 4-ene-VPA, 3-OH-VPA, 3-keto-VPA, 20 μM) also induced AMPK/ACC phosphorylation, and 1-aminobenzotriazole (CYP inhibitor) blocked VPA-mediated AMPK phosphorylation [5]
5. In DMS53 small cell lung cancer (SCLC) cells, Valproic acid (1–10 mM, 2 d) induced morphological changes (flatter, rounder, spindle-shaped with neuronal projections), upregulated full-length Notch1 and active Notch intracellular domain (NICD), increased Hes-1 (Notch1 downstream target) expression, and downregulated neuroendocrine markers chromogranin A and ASCL-1 (Western blot); it inhibited cell proliferation in a dose-dependent manner (MTT assay, 8 d) [6]
6. In in vitro HIV reservoir models, Valproic acid (500 mg bid, therapeutic range) did not reduce replication-competent HIV in CD4+ T cells (quantitative culture assay) [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].

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1. In mice transplanted with Kasumi-1 leukemia cells, intraperitoneal injection of Valproic acid (500 mg/kg body weight, 0.2 ml daily for 2 weeks) inhibited tumor growth (tumor volume measured every 3 days) and angiogenesis (CD34 immunostaining for microvessel density); VPA downregulated VEGF/VEGFR2/bFGF expression in tumor tissues (RT-PCR, Western blot, immunohistochemistry) and increased histone H3 acetylation on VEGF promoters [3]
2. In pregnant Long Evans rats (gestational day 13), intraperitoneal injection of Valproic acid (350 mg/kg) increased social investigation and play fighting in offspring (modified social interaction test) during adolescence/adulthood; no effect on social preference/locomotor activity was observed; in adult offspring, VPA altered gene expression in anterior amygdala, cerebellar vermis, and orbitofrontal cortex, with dysregulated genes enriched in acetylation-modulated proteins and epigenetic regulation-related isoforms [4]
3. In ob/ob obese mice, Valproic acid (0.26% w/v in drinking water, 14 d) decreased liver mass (liver/body weight ratio), hepatic fat accumulation (H&E staining), and serum glucose (colorimetric assay); effects were comparable to metformin (0.5% w/v); targeted metabolomics showed 9 serum metabolites with differential abundance in VPA-treated mice; no increase in serum ALT (hepatotoxicity marker) was observed [5]
4. In a multicenter randomized clinical study (56 HIV-infected patients on suppressive HAART), Valproic acid (500 mg bid, adjusted to therapeutic range) added to HAART for 16/32 weeks did not reduce latent HIV reservoirs in CD4+ T cells (quantitative culture assay: median IUPB at baseline/week 16/week 48: arm 1 = 2.55/1.80/2.70, P=0.87; arm 2 = 2.55/1.64/2.51, P=0.50) [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]

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1. HDAC1 activity assay: Crude nuclear extracts from mouse hepatocytes or recombinant human HDAC1 were incubated with Valproic acid (0.4 mM), TSA (1 μM), or VPA metabolites (20 μM–2 mM); deacetylation activity was measured using a fluorimetric assay with acetylated histone substrates; VPA inhibited HDAC1 activity in a dose-dependent manner, while non-teratogenic analogs showed no inhibition [2,5]
2. AMPK activity assay: Primary mouse/human hepatocytes were treated with Valproic acid (200 μM–2 mM) or its metabolites (20 μM) for 2 h; cell lysates were immunoblotted for phosphorylated AMPKα (p-AMPKα) and total AMPKα; AMPK was immunoprecipitated and blotted for acetylated lysine to assess acetylation status; Compound C (10 μM) was used to confirm AMPK dependence of ACC phosphorylation [5]
3. Caspase activity assay: HeLa cells treated with Valproic acid (10 mM) were lysed; caspase-3/-8/-9 activity was measured using fluorometric substrates; the effect of caspase inhibitors/TNF-α on caspase activation was evaluated by Western blot and cell viability assays [1]

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.

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Western Blot Analysis[1][2][5]
Cell Types: HeLa cells, Neuro2A cells or primary mouse hepatocytes
Tested Concentrations: 10mM (HeLa); 0, 2, and 5mM (Neuro2A); 0.2, 0.4, 0.8, 1.2 and 2mM (hepatocytes)
Incubation Duration: 10mM (HeLa ); 0, 2, and 5 mM (Neuro2A); 0.2, 0.4, 0.8, 1.2 and 2 Mm (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.

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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

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1. HeLa cell proliferatiopoptosis assay: HeLa cells were seeded in 96-well plates and treated with Valproic acid (0–10 mM) for 24–72 h; cell viability was measured by MTS assay (IC50 calculation); cell cycle distribution was analyzed by flow cytometry (propidium iodide staining); apoptosis was assessed by PARP cleavage (Western blot), caspase activation (fluorometric assay), and mitochondrial membrane potential (JC-1 staining); ROS/GSH levels were measured by fluorescent probes [1]
2. Kasumi-1 cell angiogenic factor assay: Kasumi-1 cells were treated with Valproic acid (gradient concentrations); total RNA/protein was extracted for semi-quantitative RT-PCR (VEGF/VEGFR2/bFGF primers) and Western blot; ChIP assay was performed with anti-acetyl-H3 antibody to detect H3 enrichment on VEGF promoters [3]
3. Hepatocyte AMPK activation assay: Primary mouse/human hepatocytes were isolated and seeded in 6-well plates; treated with Valproic acid (200 μM–2 mM) or metabolites (20 μM) for 0–24 h; cell lysates were immunoblotted for p-AMPKα, AMPKα, p-ACC, ACC, and β-actin; densitometry analysis was performed to quantify phosphorylation levels [5]
4. DMS53 SCLC cell assay: DMS53 cells were treated with Valproic acid (1–10 mM) for 2–8 d; cell morphology was observed by light microscopy (20× magnification); Western blot was used to detect Notch1/NICD/Hes-1 and neuroendocrine markers (chromogranin A, ASCL-1); cell proliferation was measured by MTT assay every 2 d for 8 d [6]
5. CD4+ T cell HIV reservoir assay: CD4+ T cells from HIV-infected patients were isolated; treated with Valproic acid (therapeutic concentration); replication-competent HIV was quantified by quantitative culture assay (IU per log10 billion cells) [7]

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.

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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.

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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.

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1. Kasumi-1 leukemia xenograft mice: Mice were intraperitoneally injected with 0.2 ml Valproic acid (500 mg/kg) or saline daily for 2 weeks; tumor volume was measured every 3 days (volume = length × width²/2); mice were euthanized, tumors were collected for RT-PCR, Western blot, immunohistochemistry (VEGF/VEGFR2/bFGF), CD34 staining (microvessel density), and ChIP assay (VEGF promoter H3 acetylation) [3]
2. Prenatal VPA exposure in rats: Pregnant Long Evans rats were intraperitoneally injected with 350 mg/kg Valproic acid or saline on gestational day 13; offspring were tested for social behavior (modified social interaction test) at P28, P42, and P95; adult offspring were euthanized, brain tissues (anterior amygdala, cerebellar vermis, orbitofrontal cortex) were collected for microarray analysis and exon expression profiling [4]
3. Ob/ob obese mice: Ob/ob mice were given 0.26% (w/v) Valproic acid, 0.5% (w/v) metformin, or untreated water via drinking water for 14 days; liver tissues were collected for H&E staining (hepatic fat accumulation) and liver mass measurement (liver/body weight ratio); serum was collected for glucose/triglyceride/ALT assays (colorimetric) and targeted metabolomics (uHPLC-MS/MS) [5]
4. HIV clinical trial: 56 virologically suppressed HIV patients were randomized into two arms: arm 1 (VPA + HAART for 16 weeks, then HAART alone for 32 weeks) and arm 2 (HAART alone for 16 weeks, then VPA + HAART for 32 weeks); Valproic acid was administered at 500 mg twice daily, adjusted to therapeutic range; CD4+ T cells were collected at baseline, week 16, and week 48 for HIV reservoir quantification (quantitative culture assay) [7]

Absorption, Distribution and Excretion
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 the effect of food may differ. When extended-release tablets are taken with food, Tmax increases from 4 hours to 8 hours. In contrast, Tmax for powder and capsule formulations increases from 3.3 hours to 4.8 hours. The bioavailability of all oral formulations is reported to be approximately 90%, while enteric-coated formulations may achieve 100% bioavailability. Most of the drug is eliminated via hepatic metabolism, 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.
11 L/1.73 m²
.
0.56 L/hr/m²
The clearance rate (by body weight) in children aged 3 months to 10 years was 50% higher than that in adults. The clearance rate in children aged 10 years and older was close to that in adults.
Valproic acid and its salt sodium valproate are secreted into human milk in low concentrations. The highest concentrations of valproic acid and its salts measured in breast milk have reached up to 15% of the corresponding concentrations in maternal serum. The serum valproic acid levels in two infants were 1.5% and 6.0% of the maternal levels, respectively.
Due to the similarity between non-human primates (NHPs) and humans, placental transport studies are a key component in assessing developmental toxicity. To establish a method for measuring placental transport in non-human primates, we determined the toxicokinetics of valproic acid (VPA), a drug used to treat epilepsy in pregnant women, in pregnant cynomolgus monkeys. Following mating, pregnant female monkeys were orally administered valproic acid (VPA) daily at doses of 0, 20, 60, and 180 mg/kg 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, both VPA and 4-ene-VPA were detectable in plasma within 4–24 hours after administration in all treatment groups, indicating VPA absorption and systemic exposure in the monkeys. After repeated administration of VPA to the monkeys, VPA was detected in the amniotic fluid, placenta, and fetus in all treatment groups, indicating that VPA can be transported across the placenta, exposing the fetus to VPA, with exposure increasing with dose. The concentrations of 4-ene-VPA in amniotic fluid and fetus were below the limit of quantification, but trace amounts were detected in the placenta. In summary, during organogenesis, pregnant monkeys were exposed to VPA and its metabolite 4-ene-VPA after oral administration of 20, 60, and 180 mg/kg doses. VPA is transplacentally transported, and fetuses are exposed to VPA in a dose-dependent manner. The metabolite 4-ene-VPA was not detected in either amniotic fluid or fetus, but trace amounts were detected in the placenta. This study established an appropriate procedure for investigating placental transport in non-human primates 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 comprehensive bioanalysis and toxicokinetic analysis. Valproic acid—rapidly absorbed via the gastrointestinal tract; absorption is slightly delayed when taken with food. High protein binding (90% to 95%) is observed at serum concentrations up to 50 μg/mL. When the concentration increases from 50 μg/mL to 100 μg/mL, the protein binding rate decreases to 80% to 85%, while the free fraction gradually increases, thus increasing the concentration gradient in the brain. Valproic acid can be distributed into breast milk. It has been reported that the concentration of valproic acid in breast milk is 1% to 10% of the total maternal serum concentration. /Valproic acid/ For more complete data on the absorption, distribution, and excretion of valproic acids (8 in total), please visit the HSDB record page.

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Metabolism/Metabolites
Most drugs are metabolized to glucuronide conjugates of the parent drug or metabolites (30-50%). Another large portion is metabolized via mitochondrial β-oxidation (40%). The remaining metabolism (15-20%) occurs through oxidation, hydroxylation, and dehydrogenation reactions 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 valproic acid (VPA) glucuronide conjugate levels and hepatotoxicity, as well as the relationship between the levels of VPA's toxic metabolites (4-enyl VPA and 2,4-dienyl VPA). This study also examined whether urinary excretion levels of VPA and its toxic metabolites could predict hepatotoxicity. Rats were administered VPA orally at doses ranging from 20 mg/kg to 500 mg/kg. Gas chromatography-mass spectrometry (GC-MS) was used to quantitatively analyze free valproic acid (VPA), total valproic acid (including free valproic acid and glucuronide conjugate valproic acid), 4-allyl valproic acid, and 2,4-dienyl valproic acid in urine and liver. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and α-glutathione S-transferase (α-GST) were simultaneously measured to assess hepatotoxicity. Serum α-GST levels were slightly elevated in the 20 mg/kg dose group, and significantly elevated in the 100 mg/kg and 500 mg/kg dose groups; AST and ALT levels did not change with increasing VPA dose. The concentration of free 4-allyl valerate in the liver and the total excretion of 4-allyl valerate in urine were the only indicators associated with elevated serum α-GST levels (p < 0.094 and p < 0.023, respectively). Based on these results, it can be concluded that the hepatotoxicity of valproic acid is related to the concentration of 4-allyl valerate in the liver and can be predicted by the total excretion of 4-allyl valerate in urine.
Background and Objective: Sodium valproate is a widely used broad-spectrum antiepileptic drug. Its pharmacokinetics and pharmacodynamics exhibit high inter-individual variability, and its therapeutic window is narrow. This study aimed to evaluate the effects of polymorphic uridine diphosphate glucuronosyltransferase (UGT) 1A6 (541A>G, 552A>C) metabolic enzymes on the pharmacokinetics of sodium valproate in patients with epilepsy exhibiting therapeutic toxicity. Methods: Genotyping of patients was performed using polymerase chain reaction-restriction fragment length polymorphism (RFLP) sequencing. Plasma drug concentrations were determined by reversed-phase high-performance liquid chromatography (HPLC), and concentration-time data were analyzed using a non-compartmental model. Results: The results indicate a significant genotypic and allele association between the UGT1A6 (541A>G) or UGT1A6 (552A>C) polymorphic enzymes and valproate toxicity. In individuals with slower metabolism due to the UGT1A6 (552A>C) polymorphism, valproic acid exhibited a longer elimination half-life (t_1/2 = 40.2 h), lower clearance (CL = 917 mL/h), and showed toxic effects. Conversely, in individuals with slower metabolism, valproic acid had a shorter elimination half-life (t_1/2 = 35.5 h), shorter clearance (CL = 1022 mL/h), and shorter overall clearance (CL = 1404 mL/h). Conclusion: These findings suggest that the UGT1A6 (552A>C) gene polymorphism plays a significant role in the steady-state concentration of valproic acid, thereby influencing the toxicity of valproic acid use in patients with epilepsy. Valproic acid biotransformation primarily occurs in the liver. Some metabolites may possess 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 by 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 cleared by other oxidative mechanisms. Less than 3% of the administered dose is excreted unchanged in the urine. Known metabolites of valproic acid include 4-hydroxyvalproic acid, 5-hydroxyvalproic acid, (2S,3S,4S,5R)-3,4,5-trihydroxy-6-(2-propylvaleroyloxy)oxacyclohexane-2-carboxylic acid, 3-hydroxyvalproic acid, and 4-allylvallic acid. Valproic acid is rapidly absorbed from the gastrointestinal tract. Valproic acid is almost entirely metabolized by 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).

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Biological half-life
13-19 hours. The half-life in neonates is 10-67 hours, while in pediatric patients 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. In cases of overdose, the half-life can be as long as 30 hours. 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 that the half-life in children under 10 days old was 10 to 67 hours, while the half-life in children over 2 months of age was 7 to 13 hours.

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1. Valproic acid is biotransformed in hepatocytes by cytochrome P450 (CYP) enzymes; its metabolites (2-en-valproic acid, 4-en-valproic acid, 3-hydroxy-valproic acid, 3-keto-valproic acid) can activate AMPK; 1-aminobenzotriazole (a CYP inhibitor) can block VPA-mediated AMPK phosphorylation [5]

Toxicity Summary
Identification and Uses: Valproic acid is a colorless to pale yellow viscous liquid. It is an antiepileptic drug that can be used alone or in combination with other anticonvulsants to treat simple (petit mal) and complex absence seizures. Valproic acid may be effective for myoclonic and atonic seizures in young children. Human Exposure and Toxicity: After oral administration, the drug is rapidly absorbed from the gastrointestinal tract and metabolized in the liver. Fatal liver failure has been reported in patients receiving valproic acid, especially those on long-term use. Pancreatitis has also been reported in patients receiving normal therapeutic doses. Acute toxicity is reported to be rare and usually has a good prognosis. The most common adverse reactions are anorexia, nausea, and vomiting. Central nervous system side effects include drowsiness, possibly accompanied by apathy and withdrawal, confusion, restlessness, and hyperactivity. Less common side effects are seizures and coma. Sedation is more pronounced when used in combination with other antiepileptic drugs. Hematopoietic side effects include thrombocytopenia, abnormal bleeding time and partial thromboplastin time, accompanied by decreased fibrinogen levels and prolonged prothrombin time, leading to bruising, petechiae, hematomas, and epistaxis. The drug can cause pruritic rashes and transient hair loss. Thyroid dysfunction has been reported. Death is rare, but if it occurs, it is usually due to cardiopulmonary arrest secondary to liver failure. The safety of valproic acid use during pregnancy has not been established. Although some reports suggest an association between valproic acid use in pregnant women with epilepsy and an increased incidence of birth defects, particularly neural tube defects, in their offspring, the causal relationship remains to be determined. The drug can cross the placental barrier and is found in breast milk. The mechanism of action of valproic acid is unclear. Its effects may be at least partially related to increased concentrations of the inhibitory neurotransmitter GABA in the brain. Animal studies: Animal studies have shown that valproic acid inhibits GABA transferase and succinate dehydrogenase, both of which are essential for the catabolism of GABA. One study indicated that the drug inhibits neuronal activity by increasing potassium conductance. In animal studies, valproic acid prevented seizures induced by electrical stimulation and those induced by pentylenetetrazol. In 2-year rat and chronic mouse studies, the incidence of subcutaneous fibrosarcoma increased in male rats at high doses, and the incidence of benign lung adenomas in male mice also showed a dose-related increase. The implications of these findings for humans are unclear. Adverse fetal effects were observed in reproductive studies in rats and mice. No evidence of mutagenicity has been found. Valproic acid binds to and inhibits the activity of GABA transaminase. This leads to elevated brain concentrations of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Acute valproic acid poisoning can cause severe central nervous system depression, including coma, confusion, somnolence, dizziness, or hallucinations. Hypotension, respiratory depression, and hypothermia/hyperthermia are also common. Valproic acid also exhibits hepatotoxicity, likely due to its mitochondrial toxicity. Valproic acid (VPA) appears to exert its mitochondrial toxicity by impairing mitochondrial function, leading to oxidative stress and cytochrome c excretion, ultimately resulting in apoptosis (A15078). Due to its teratogenicity, VPA is contraindicated in pregnant women. VPA is a known folic acid antagonist that can cause neural tube defects in developing fetuses. Therefore, folic acid supplementation in pregnant women may help mitigate teratogenic problems associated with VPA use. VPA and its metabolites inhibit carnitine biosynthesis by reducing the concentration of α-ketoglutarate (directly inhibiting α-ketoglutarate dehydrogenase) and may lead to carnitine deficiency. It is speculated that carnitine supplementation may increase VPA β-oxidation, thereby limiting cytoplasmic ω-oxidation and the production of toxic metabolites involved in hepatotoxicity and ammonia accumulation. VPA-induced hepatotoxicity and hyperammonemia-induced encephalopathy may be due to pre-existing carnitine deficiency or carnitine deficiency caused by VPA itself. Valproic acid (VPA) has been shown to downregulate levels of superoxide dismutase (SOD), glutathione (GSH), histone deacetylase (HDAC), and folic acid. It has also been shown to upregulate hydrogen peroxide (H2O2) and homocysteine levels. Elevated hydrogen peroxide levels negatively affect the NADPH reduction system of dihydrofolate reductase (DHFR) and methylenetetrahydrofolate reductase (MTHFR) (A15079).
Toxicity Data

Oral LD50 in mice: 1098 mg/kg; Oral LD50 in rats: 670 mg/kg.
Typically, during controlled treatment, serum or plasma valproic acid concentrations range from 20–100 mg/L, but can reach 150–1500 mg/L after acute poisoning.
Interactions
In 15 healthy volunteers (10 men and 5 women), a single oral dose of 50 mg amitriptyline, combined with sodium valproate (500 mg twice daily), resulted in a 21% decrease in plasma clearance of amitriptyline and a 34% decrease in net clearance of nortriptyline. Post-marketing reports indicate that concomitant use of sodium valproate and amitriptyline can lead to elevated amitriptyline levels, a rare occurrence. Concomitant use of sodium valproate and amitriptyline rarely causes toxicity. Monitoring of amitriptyline plasma concentrations should be considered in patients taking both sodium valproate and amitriptyline. A dose reduction of amitriptyline/nortriptyline should be considered when taking sodium valproate.
Significantly reported decreases in serum valproic acid concentrations have been observed in patients receiving carbapenem antibiotics (e.g., ertapenem, imipenem, meropenem; this list is not exhaustive), which may lead to poor seizure control. The mechanism of this interaction is not fully understood. Serum valproic acid concentrations should be monitored frequently after initiating carbapenem antibiotic therapy. If serum valproic acid concentrations decrease significantly or seizure control worsens, alternative antibacterial or anticonvulsant treatment regimens should be considered…
In patients with epilepsy, concomitant administration of valproic acid and carbamazepine (CBZ) resulted in a 17% decrease in serum carbamazepine levels and a 45% increase in carbamazepine-10,11-epoxide (CBZ-E) levels.
A study of 10 patients with epilepsy showed that concomitant administration of 1200 mg/day of non-amino esters and valproic acid increased the mean peak concentration of valproic acid by 35% (from 86 μg/mL to 115 μg/mL) compared to valproic acid alone. Increasing the non-amino ester dose to 2400 mg/day further increased the mean peak concentration of valproic acid to 133 μg/mL (another 16% increase). When starting non-amino ester therapy, a reduction in the valproic acid dose may be necessary. For more complete data on interactions of valproic acid (61 items in total), please visit the HSDB record page.

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Non-human toxicity values
Guinea pig oral LD50 824 mg/kg
Mouse subcutaneous LD50 860 mg/kg
Mouse intraperitoneal LD50 470 mg/kg
Mouse oral LD50 1098 mg/kg
For more complete data on non-human toxicity values of valproic acid (7 items in total), please visit the HSDB record page. 1. Valproic acid is a teratogenic agent in vertebrate embryos (with similar teratogenic effects to TSA); non-teratogenic valproic acid analogs do not inhibit HDAC [2]. 2. In ob/ob mice, the addition of valproic acid (0.26% w/v, 14 days) to drinking water did not increase serum ALT levels (a marker of hepatotoxicity) [5]. 3. In HeLa cells, valproic acid (10 mM) is cytotoxic (24-hour growth inhibition IC50 = 10 mM), but has no off-target effects on ROS/GSH-mediated cell death [1].

[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. 2014 Feb;9(2):443-9.

[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.

Therapeutic Uses

/Experimental Treatment/ Emerging evidence suggests that adenomyosis, like endometriosis, may be an epigenetic disease. This study evaluated the effects of valproic acid (VPA) on adenomyosis in neonatal ICR mice induced by tamoxifen. We assessed the body weight and thermal response of all mice at 4, 8, and 12 weeks post-administration, as well as by hot plate and tail-flick tests, and then treated the mice with low and high doses of VPA, progesterone (P4), P4 + VPA, or the carrier alone. Three weeks after treatment, body weight and thermal response were reassessed before sacrifice, and the depth of myometrial invasion was evaluated. We found that: (i) adenomyosis induces progressive generalized hyperalgesia (measured by hot plate and tail-flick tests) and weight loss; (ii) VPA, P4, or a combination of both effectively improves generalized hyperalgesia; and (iii) drug treatment appears to reduce myometrial infiltration, but the difference was not statistically significant. Therefore, as reported in some recent human case series studies, VPA appears to be a promising treatment for adenomyosis.
/Experimental Treatment/ Objective: 5-AZA is a DNA hypomethylating agent. Valproic acid is a histone deacetylase inhibitor. The combination of a hypomethylating agent and a histone deacetylase inhibitor produces synergistic anticancer activity in vitro and in vivo. Based on this, we conducted a phase I clinical trial to evaluate the efficacy of 5-AZA and valproic acid in combination for the treatment of patients with advanced cancer. Experimental Design: Subcutaneous injection of 5-AZA daily for 10 days. Valproic acid was administered orally daily, with the goal of titrating plasma concentrations to 75–100 μg/mL (the concentration for treating seizures). The treatment cycle was 28 days. The starting dose of 5-azacytidine (5-AZA) was 20 mg/m², with dose escalation performed using an adaptive algorithm based on toxicity profiles from previous cohorts (6+6 design). On days 1 and 10 of each treatment cycle, with patient consent, global DNA methylation and histone H3 acetylation levels in peripheral blood mononuclear cells were measured using long discrete nucleotide element pyrosequencing and Western blotting, respectively. Results: A total of 55 patients were included. The median age was 60 years (range: 12–77 years). The maximum tolerated dose of 5-AZA in combination with valproic acid was 75 mg/m². Dose-limiting toxicities were neutropenic fever and thrombocytopenia, occurring at a 5-azacytidine (5-AZA) dose of 94 mg/m². Fourteen patients (25%) had stable disease for 4 to 12 months (median 6 months). Significantly reduced overall DNA methylation and induced histone acetylation were observed. Conclusion: For patients with advanced malignant tumors, the combined use of 5-AZA and valproic acid, with a maximum 5-AZA dose of 75 mg/m², is safe.
/Experimental Treatment/ Amphetamine (AMPH)-induced hyperkinesis has been well-established in animal models of mental illnesses such as drug addiction and bipolar disorder. /This Study/ Investigated the effect of long-term microinjection of valproic acid into the nucleus accumbens (NAcc) on amphetamine-induced motor activity. Rats with bilaterally implanted guiding catheters were divided into three groups and received daily microinjections of saline or valproic acid (100 or 300 μg/0.5 μL/side) into the NAcc for 7 consecutive days. On day 8, half of the rats in each group received either saline or amphetamine (AMPH, 1 mg/kg, intraperitoneal injection), and their motor activity was measured over 2 hours. Compared with the saline group, the increase in horizontal movement and orthostatic behavior in rats pre-injected with valproic acid was attenuated in a dose-dependent manner. These results suggest that chronic valproic acid-induced changes in nucleus accumbens neurons can modulate amphetamine-induced motor activity.
/Experimental Treatment/ Objective: Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancers and is the most common cause of cancer death in industrialized countries. Epigenetic modifications are prevalent in the tumorigenesis of lung cancer. Developing epigenetic modulatory drugs using the synergistic effect of hypomethylating agents and histone deacetylase (HDAC) inhibitors provides a novel treatment approach for NSCLC. Methods: This study conducted a phase I clinical trial to evaluate the efficacy of 5-aza-2'-deoxycytidine (decitabine) combined with valproic acid (VPA) in the treatment of patients with advanced NSCLC. Patients received decitabine (5-15 mg/m²) intravenously for 10 consecutive days, in 28-day cycles, with oral VPA (10-20 mg/kg/day) on days 5-21. Pharmacokinetic and pharmacodynamic analyses included the pharmacokinetics of decitabine and fetal hemoglobin expression. Results: This phase I clinical trial enrolled 8 patients. All patients had advanced NSCLC and had previously received chemotherapy. Eastern Cooperative Oncology Group (ECOG) performance status scores ranged from 0 to 2. Major toxicities included myelosuppression and neurotoxicity. Dose-limiting toxicities occurred in two patients who experienced grade 3 neurotoxicity during the first treatment cycle, including disorientation, somnolence, memory impairment, and ataxia (dose level 1). One patient developed grade 3 neutropenia after dose tapering. No objective response was observed; one patient's condition remained stable. Fetal hemoglobin levels increased in all seven evaluable patients after the first treatment cycle. Conclusion: This study observed that the combination of decitabine and valproic acid effectively activated hypermethylated genes, manifested as re-expression of fetal hemoglobin. However, in patients with advanced stage IV non-small cell lung cancer (NSCLC), this combination therapy caused unacceptable neurotoxicity at relatively low doses, thus limiting its use. Further exploration of the combination of hypomethylating agents with other HDAC inhibitors without valproic acid toxicity is warranted. For more complete data on the therapeutic uses of valproic acid (8 in total), please visit the HSDB record page.

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Drug Warnings
/Black Box Warning/ Warning: Life-threatening adverse reactions. Hepatotoxicity: General population: Liver failure leading to death has occurred in patients treated with valproic acid. These events typically occur within the first six months of treatment. Severe or fatal hepatotoxicity may initially present with nonspecific symptoms such as malaise, weakness, somnolence, facial edema, anorexia, and vomiting. Poor seizure control may also occur in patients with epilepsy. Patients should be closely monitored for these symptoms. Serum liver function tests should be performed before treatment, and regular follow-up examinations are necessary after treatment, especially during the first six months. Children under two years of age have a significantly increased risk of fatal hepatotoxicity, particularly those taking multiple antiepileptic drugs, those with congenital metabolic disorders, those with severe seizures accompanied by intellectual disability, and those with organic brain disease. When Depakine is used in such patients, extreme caution should be exercised, and it should only be used as a monotherapy. The benefits and risks of treatment should be weighed. The incidence of fatal hepatotoxicity decreases significantly with increasing patient age. Patients with mitochondrial diseases: Patients with inherited neurometabolic syndromes caused by mutations in the mitochondrial DNA polymerase γ (POLG) gene (e.g., Alpert-Hutenloch syndrome) have an increased risk of valproate-induced acute liver failure and death. Depakine is contraindicated in patients with known mitochondrial diseases caused by POLG gene mutations and in children under two years of age clinically suspected of having mitochondrial diseases. For patients over two years of age clinically suspected of having inherited mitochondrial diseases, valproate should only be used after other antiepileptic drug treatments have failed. For older patients, close monitoring for acute liver injury is necessary during sodium valproate treatment, with regular clinical evaluations and serum liver function tests. POLG gene mutation screening should be performed according to current clinical practice. Fetal risks: Sodium valproate can cause serious congenital malformations, especially neural tube defects (e.g., spina bifida). Furthermore, fetal IQ may be lowered after intrauterine exposure to sodium valproate. Sodium valproate should only be considered for treating epilepsy during pregnancy when other medications fail to control symptoms or when the patient cannot tolerate other medications. Sodium valproate should not be given to women of childbearing age unless it is essential for treating their condition. This is especially important when considering sodium valproate for treating conditions that generally do not cause permanent damage or death (e.g., migraines). Women should use effective contraception while using sodium valproate. Pancreatitis: Life-threatening cases of pancreatitis have been reported in children and adults taking sodium valproate. Some cases presented as hemorrhagic pancreatitis, with rapid progression from initial symptoms to death. Cases of pancreatitis have been reported both shortly after initial administration and several years after administration. Patients and their caregivers should be informed that abdominal pain, nausea, vomiting, and/or anorexia may be symptoms of pancreatitis and require immediate medical attention. If pancreatitis is diagnosed, sodium valproate should generally be discontinued. Replacement therapy for the primary disease should be initiated based on clinical indications. Due to these variations in valproate clearance, increased monitoring of valproate and its concomitant medication concentrations is recommended when introducing or discontinuing enzyme-inducing drugs. Because valproate may interact with concomitant enzyme-inducing drugs, it is recommended to periodically measure plasma concentrations of valproate and its concomitant medications at the beginning of treatment. Valproate is partially excreted in the urine as a ketone metabolite, which may lead to misinterpretation of urine ketone test results. For more complete data on drug warnings for valproate (58 total), please visit the HSDB record page. Pharmacodynamics: Valproate has been shown to reduce the incidence of complex partial seizures and migraines. It can also improve symptom control in bipolar disorder mania. Although the exact mechanism is unclear, valproate is generally believed to enhance cortical inhibition, thereby helping to control neural synchronization. Furthermore, valproate is thought to have neuroprotective effects, preventing damage and neurodegeneration in epilepsy, migraines, and bipolar disorder. Valproate has hepatotoxicity and teratogenicity. The reasons for these are unclear, but are thought to be related to the drug's genomic effects. A small proof-of-concept study found that valproate, when used in combination with highly active antiretroviral therapy (HAART), could promote viral clearance by activating the virus, thereby increasing the clearance rate of human immunodeficiency virus (HIV). However, a larger, multicenter trial failed to show a significant effect of valproate combined with HAART on the HIV viral reservoir. The U.S. Food and Drug Administration (FDA) drug label includes a warning about HIV viral activation during valproate use. Valproic acid is a clinically approved anticonvulsant and mood stabilizer with anticancer, antiangiogenic and metabolic regulatory effects; it exerts its anticancer effects by inhibiting HDAC, caspase-dependent apoptosis, activating Notch1 and antiangiogenic [1-3,6]
2. Valproic acid can activate AMPK in hepatocytes, reduce liver mass, liver fat accumulation and serum glucose levels in obese mice, similar to metformin [5]
3. Prenatal exposure to moderate doses of valproic acid (350 mg/kg) in rats increased social behavior in offspring and altered gene expression in brain regions associated with social behavior, with dysregulated genes enriched in acetylated proteins [4]
4. Adding valproic acid to highly active antiretroviral therapy (HAART) does not reduce the latent HIV reservoir in patients with viral suppression, indicating no clinical benefit in reducing the HIV reservoir [7]
5. The anti-angiogenic effect induced by valproic acid in leukemia is related to the inhibition of VEGF/VEGFR2/bFGF and the enhancement of histone acetylation on the VEGF promoter [3]

C8H16O2

144.21144

144.115

C, 66.63; H, 11.18; O, 22.19

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

3121

Colorless to light yellow liquid

0.9±0.1 g/cm3

220.0±0.0 °C at 760 mmHg

120 - 130ºC

111.1±0.0 °C

0.0±0.9 mmHg at 25°C

1.435

2.8

1

2

5

10

93.4

0

NIJJYAXOARWZEE-UHFFFAOYSA-N

InChI=1S/C8H16O2/c1-3-5-7(6-4-2)8(9)10/h7H,3-6H2,1-2H3,(H,9,10)

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (17.34 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 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 (17.34 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 (17.34 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 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: 2 mg/mL (13.87 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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Solubility in Formulation 5: 20 mg/mL (138.69 mM) in 0.5% CMC/saline water (add these co-solvents sequentially from left to right, and one by one), suspension 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.)