Liquid chromatography–tandem mass spectrometry method for simultaneous determination of valproic acid and its ene-metabolites in epilepsy patient plasma

A simple and high throughput method was developed and validated for simultaneous determination of valproic acid and its two toxicant ene-metabolites, 2-enevalproic acid and 4-enevalproic acid in epilepsy patient plasma using liquid chromatography-tandem mass spectrometry. Probenecid was used as in- ternal standard and solid-phase extraction was selected for sample preparation. A chromatographic separation was performed on an Agilent Poroshell SB-C18 column （50 mm × 4.6 mm i.d., 2.7μm） by an optimized gradient elution at a flow rate of 0.9 mL/min. The total run time was 7 rain. Electrospray ionization was used in negative ion mode by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 143.0→143.0 for valproic acid, m/z 140.9 →140.9 for 2-enevalproic acid and 4-enevalproic acid for their poor fragments, and m/z 283.9→239,9 for probenecid. The results showed good linearity ofvalproic acid, 2-enevalproic acid and 4-enevalproic acid in their respective linear ranges. The correlation coefficients were more than 0.998, The intra- and inter-day precision of the assay was less than 11.0% and the accuracy ranged from 2% to 12%. This analytical method was successfully applied to assay plasma concentrations of valproic acid and its two ene-metabolites in epilepsy patient plasma and used for therapeutic drug monitoring.

Valproic acid protects neurons and promotes neuronal regeneration after brachial plexus avulsion

Valproic acid has been shown to exert neuroprotective effects and promote neurite outgrowth in several peripheral nerve injury models. However, whether valproic acid can exert its beneficial effect on neurons after brachial plexus avulsion injury is currently unknown. In this study, brachial plexus root avulsion models, established in Wistar rats, were administered daily with valproic acid dis-solved in drinking water （300 mg/kg） or normal water. On days 1, 2, 3, 7, 14 and 28 after avulsion injury, tissues of the C 5-T 1 spinal cord segments of the avulsion injured side were harvested to in-vestigate the expression of Bcl-2, c-Jun and growth associated protein 43 by real-time PCR and western blot assay. Results showed that valproic acid significantly increased the expression of Bcl-2 and growth associated protein 43, and reduced the c-Jun expression after brachial plexus avulsion. Our findings indicate that valproic acid can protect neurons in the spinal cord and enhance neuronal regeneration fol owing brachial plexus root avulsion.

Scorpion ethanol extract and valproic acid effects on hippocampal glial fibrillary acidic protein expression in a rat model of chronic-kindling epilepsy induced by lithium chloride-pilocarpine

The present study analyzed the effects of ethanol extracts of scorpion on epilepsy prevention and hippocampal expression of glial fibrillary acidic protein in a lithium chloride-pilocarpine epileptic rat model. Results were subsequently compared with valproic acid. Results showed gradually- increased hippocampal glial fibrillary acidic protein expression following model establishment; glial fibrillary acidic protein mRNA expression was significantly increased at 3 days, reached a peak at 7 days, and then gradually decreased thereafter. Ethanol extracts of scorpion doses of 580 and 1 160 mg/kg, as well as 120 mg/kg valproic acid, led to a decreased number of glial fibrillary acidic protein-positive cells and glial fibrillary acidic protein mRNA expression, as well as decreased seizure grades and frequency of spontaneously recurrent seizures. The effects of 1 160 mg/kg ethanol extracts of scorpion were equal to those of 120 mg/kg valproic acid. These results suggested that the anti-epileptic effect of ethanol extracts of scorpion were associated with decreased hippocampal glial fibrillary acidic protein expression in a rat model of lithium chlofide-pilocarpine induced epilepsy.

Effect of valproic acid on endogenous neural stem cell proliferation in a rat model of spinal cord injury

BACKGROUND： Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells. OBJECTIVE： To investigate the effects of valproic acid on proliferation of endogenous neural stem cells in a rat model of spinal cord injury. DESIGN, TIME AND SETTING： A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007. MATERIALS： A total of 45 adult, Wistar rats were randomly divided into sham surgery （n = 5）, injury （n = 20）, and valproic acid （n = 20） groups. Valproic acid was provided by Sigma, USA. METHODS： Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours （twice in total）.MAIN OUTCOME MEASURES： Nestin expression （5 mm from injured center） was detected using immunohistochemistry at 1,3 days, 1, 4, and 8 weeks post-injury. RESULTS： Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week （P 〈 0.05）. Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was no significant difference in nestin expression between the valproic acid and injury groups. At 1 week, there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group （P 〈 0.05）. Expression reached a peak by 4 weeks, and it was still present at 8 weeks. CONCLUSION： Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.

Valproic acid as a micro RNA modulator to promote neurite outgrowth

Valproic acid （VPA） has been a first-choice drug for clinical treatment of epilepsy and manic disorder. For decades, its phar- macological action was believed to act on inhibition of gam- ma-aminobutyric acid （GABA） transaminase, in turn, increas- ing GABA in inhibitory synapses. However, in recent years, VPA has been investigated on other therapeutic actions. Those investigations demonstrate that VPA shows neuroprotective ef- fects by promoting neurogenesis, neuronal differentiation, and neuroregeneration （Foti et al., 2013）.

Valproic Acid Enhances the Anti-tumor Effect of(-)-gossypol to Burkitt Lymphoma Namalwa Cells

Burkitt lymphoma is a highly aggressive B-cell neoplasm. New therapeutic methods are needed to overcome the adverse effect of intensive chemotherapy regimens. Valproic acid and （-）-gossypol are two kinds of chemical compounds used as new anti-tumor drugs in recent years.

Simultaneous determination of asenapine and valproic acid in human plasma using LC-MS/MS:Application of the method to support pharmacokinetic study

Combination of asenapine with valproic acid received regulatory approval for acute treatment of schizophrenia and maniac episodes of bipolar disorders. A simple LC-MS/MS method was developed and validated for simultaneous quantification of asenapine and valproic acid in human plasma. Internal standards were added to 300 ~L of plasma sample prior to liquid-liquid extraction using methyl tertiary butyl ether （MTBE）. Chromatographic separation was achieved on Phenomenex C18 column （50 mm ~ 4.6 mm, 5 pm） in isocratic mode at 40 ~C. The mobile phase used was 10 mM ammonium formate-acetonitrile （5：95, v/v） at a constant flow rate of 0.8 mL/min monitored on triple quadrupole mass spectrometer, operating in the multiple reaction monitoring （MRM） mode. The injection volume used for LC-MS/MS analysis was 15 taL and the run time was 2.5 min. These low run time and small injection volume suggest the high efficiency of the proposed method. The method was validated over the concentration range of 0.1-10.02ng/mL and 10-20,000ng/mL for asenapine and valproic acid respectively. The method recoveries of asenapine （81.33%）, valproic acid （81.70%）, gliclazide （78.45%） and benzoic acid （79.73） from spiked plasma samples were consistent and reproducible. The application of this method was demonstrated by a pharmacokinetic study in 8 healthy male volunteers with 5 mg asenapine and 250 mg valproic acid administration.

Valproic acid therapy decreases serum 25-hydroxyvitamin D level in female infants and toddlers with epilepsy-a pilot longitudinal study

To evaluate if valproic acid(VPA)therapy is associated with vitamin D deficiency among infants and toddlers with epilepsy,a cross-sectional clinical study was conducted in 25 children with epilepsy taking VPA.Blood levels of calcium,phosphorus,alkaline phosphatase,and 25-hydroxy vitamin D[25(OH)D]and plasma VPA level were measured at 1-to 3-month intervals.At the initial and final measurements,vitamin D deficiency or insufficiency was recognized in 8(32%)and 12(42%),respectively.In girls,a decreasing trend in serum25(OH)D levels(P<0.05)was observed.Polytherapy had a significant negative effect on the longitudinal change of 25(OH)D(P<0.05)in girls.In conclusion,our study indicates that a high proportion of girls after VPA therapy had hypovitaminosis D.

Valproic acid enhances neurosphere formation in cultured rat embryonic cortical cells through TGFβ1 signaling

This study aimed to investigate the effect and mechanism of valproic acid(VPA)on the neurosphere formation in rat embryonic cortical cells.We used free-floating neurosphere formation as a model system to evaluate the VPA on the proliferation of neural stem cells(NSCs).We found a time-and dose-dependent increase in neurosphere formation and NSC proliferation after VPA treatment.Further RNA-seq analysis demonstrated that the upregulated TGFβ1 signaling might attribute to the effect of VPA on the neurosphere formation and NSC proliferation.Consistently,the neurosphere formation and NSC proliferation were blocked by the treatment with SB431542,an inhibitor of TGFβ1 receptor.Moreover,in a coculture system,NSCs treated with VPA significantly reduced the oxygen-glucose deprivation-induced neuronal apoptosis.Taken together,our results showed that VPA could enhance neurosphere formation and NSC proliferation by activating TGFβ1,which might be a novel therapeutic strategy for neurological disorders.

Valproic acid alters differential protein expression in SH-SY5Y cells

This study sought to identify differentially expressed proteins in SH-SY5Y cells treated with valproic acid, using two-dimensional difference gel electrophoresis analysis. Three proteins were unambi-guously identified： the eukaryotic translation initiation factor 4A isoform 1 and ATP6V1B2 protein were downregulated, while the heterogeneous nuclear ribonucleoprotein K was upregulated. Moreover, all three proteins are associated with altered expression due to oxidative stress. Ma-trix-assisted laser desorption/ionization-time of flight mass spectrometry and protein immunoblotting assay confirmed the differential expression of eukaryotic translation initiation factor 4A isoform 1. The results indicate that valproic acid exerts an antioxidation effect by regulating the expression of eukaryotic translation initiation factor 4A isoform 1.

Differential Effects of Valproic Acid on Immobility Responses and Locomotor Activity in Female and Male Rats

Valproic acid (VPA) is used in the treatment of epilepsy and behavioral disorders. However, the exposure to VPA during pregnancy increases the risk of having offspring with autism spectrum disorder (ASD). Reports indicate that men are more likely to suffer ASD than women who were exposed to VPA prenatally. Few studies have related the sex differences and behavioral changes in the ASD rat model. Our aim was to determinate whether male and female Wistar rats whose mothers were exposed to either VPA (600 mg/kg;animal model for ASD) or saline (0.9%) i.p. at 12.5 day of gestation, have different effects on immobility induce by clamping (IC), dorsal immobility (DI), catalepsy, locomotor activity, stereotypes, and analgesia (tail flick). For this purpose, we made four groups (n = 8). Group: A) saline male rats, B) saline female rats, C) VPA male rats and D) VPA female rats. At 35 (prepubertal age), 56 (postpubertal age) and 180 days, we tested the behaviors previously mentioned. Finding that VPA has the same effect on IC, catalepsy, and analgesia in male and female rats, the time of these tests was increased. However, VPA only has an effect on DI in males but not in female rats. On the contrary, there is hyperactivity and an increase of stereotypes in female but not in male rats. Thereby, VPA has an effect on the three immobility responses tested (IC, DI and catalepsy), locomotor activity and analgesia but in a differential way on DI, stereotypes and locomotor activity between male and female rats.

Valproic Acid,a Drug with Multiple Molecular Targets Related to Its Potential Neuroprotective Action

Valproic acid (VA) is used worldwide as an antiepileptic drug and a mood stabilizer. Recently, VA was shown to act on cell growth, differentiation and apoptosis, by regulating gene expression at the molecular level, through epigenetic mechanisms. Thus, VA was demonstrated to act on the chromatin remodeling what is a consequence of the drug inhibition of histone deacetylases (HDACs) activity. Other studies uncovered the potential of VA to interfere with multiple regulatory mechanisms besides HDACs, as the GSK3 alpha and beta, Akt, ERK and phosphoinositol pathways, tricarboxylic acid cycle, GABA and OXPHOS system. The review focuses on the mechanisms of action of VA, showing that HDAC inhibitors, as VA, can be successfully used in the treatment of neurodegenerative disorders. This molecule, whose biological activities range from interactions with receptors and ion channels to the regulation of many catalytic reactions, has a central role in cellular cascades that regulate gene expression. Thus, inhibitors of HDACs, by positively affecting both neuronal degeneration and cognitive deficits, appear as promising drugs against various pathological conditions and neurodegenerative diseases. VA is known to present anti-inflammatory and antioxidative properties. And, since inflammation and oxidative stress are common links in neurodegeneration, VA is a drug that, from a clinical point of view, shows a great potential as a candidate for the treatment of neurodegenerative diseases related to excitotoxic events.

Valproic acid and nonalcoholic fatty liver disease: A possible association?

Valproic acid(VPA) is one of the most prescribed drugs in children with newly diagnosed epilepsy. Weight gain and obesity have been observed as side effects of VPA. These are often linked with other metabolic disturbances such as development of insulin resistance, dyslipidemia, metabolic syndrome(Met S) and nonalcoholic fatty liver disease or nonalcoholic fatty liver disease(NAFLD). NAFLD refers to a group of liver disorders with marked hepatic steatosis. It is associated with an increased incidence of cardiovascular diseases and overall reduced life expectancy. NAFLD occurs in 20%-25% of the general population and it is known to be the most common cause of chronic liver disease. NAFLD therefore represents a major public health issue worldwide. This study reviews and summarizes relevant literature that supports the existence of an association between VPA therapy and the development of NAFLD in children. Long-term VPA-therapy appears to be associated with an increased risk of developing NAFLD. Further studies are needed to clarify the pathogenic mechanisms that lie behind this association and to standardize the options for the use of this drug in overweight patients and in those with risks for developing Met S and NAFLD.

Valproic acid's effects on visual acuity in retinitis pigmentosa: a systemic review and Meta-analysis

AIM: To gain a better understanding of the overall efficacy of valproic acid(VPA) treatment for retinitis pigmentosa(RP). METHODS: Publications in PubMed, EMBASE, Cochrane Library, Web of Science and Clinicaltrials.gov were searched for clinical trials of patients with RP assigned to treatment with VPA. Patients' pre-and post-treatment visual field(VF) and best-corrected visual acuity(BCVA) scores were extracted and compared to assess changes. RESULTS: A total of 78 reports were retrieved and 6 studies involving 116 patients were included in the Meta-analysis.The combined results showed a significant decrease in logarithm of minimal angle of resolution(logMAR) scores,calculated using baseline and post-treatment BCVA(P<0.00001, mean difference=-0.05, 95%CI:-0.05,-0.04,I2=36%) scores, which means there was considerable improvement in visual acuity. Meanwhile, more BCVA changes were observed in short-term(≤6 mo) treatment studies(P<0.00001, mean difference=-0.05, 95%CI:-0.05,-0.04, I2=38%), studies conducted in Asia(P<0.00001,mean difference=-0.05, 95%CI:-0.05,-0.04, I2=4%), studies with a sample size of 30 or fewer patients(P<0.00001,mean difference=-0.05, 95%CI:-0.05,-0.04, I2=38%) and prospective studies(P<0.00001, mean difference=-0.05,95%CI:-0.05,-0.04, I2=0%). However, VPA's effect on VF was inconsistent across studies(P=0.75, mean difference=-22.76, 95%CI:-160.56, 115.05, I2=68%). CONCLUSION: This Meta-analysis reveals that most RP patients who were treated with VPA showed improvement in BCVA. However, its effect on VF remains inconsistent.VPA may be a promising treatment for RP.

Rikkunshi-to Partially Reverses Cancer Chemotherapy-Induced Decrease in Plasma Valproic Acid Concentration in a Patient with Malignant Lymphoma

A fifty-five-year-old male patient with malignant lymphoma who took oral valproic acid (VPA) tablets and itraconazole (ITZ) capsles received 3 courses of cancer chemotherapy, including 2 courses of a combination of rituximab/methotrexate/ifosphamide/etoposide/ carboplatin/ methylpredonisolon (R-IMVP16/CBDCA regimen) and subsequent one course of a combination of rituximab/ranimustine/citara bine/etoposide/merphalan (R-MEAM regimen). Plasma concentration of VPA dramatically decreased below the therapeutic concentration after the first and second chemotherapy and seizures appeared in both cases. Plasma concentration of ITZ was also lowered after the second chemotherapy course. At the third chemotherapy, Rikkunshi-to, a Japanese herbal medicine, was prescribed for 14 days. Plasma VPA concentration decreased, though to a lesser extent, after chemotherapy, in which the level was near the border of therapeutic concentration. No convulsion was observed. Therefore, care should be taken to monitor plasma drug concentration during cancer chemotherapy. Rikkunshi-to may be useful to alleviate the chemotherapy-induced decrease in plasma concentrations of orally administered drugs.

In vitro and in vivo study of formulated valproic acid loaded nanoemulsion in rats for epilepsy treatment

OBJECTIVE To investigate the in vitro and in vivo potential of formulated valproic acid-encapsulated nanoemulsion in terms of drug penetrability across the blood-brain barrier. METHODS Human cerebral microvascular endothelial cells(hCMEC/D3)and human cortical astrocytes(SC-1800)were cultured on the membrane of the transwell inserts as to build the co-culture model of in vitro BBB model.Drug penetration analysis was then conducted at various time intervals for 24 h.The in vivostudy of this formulated nanoemulsion was also investigated using Sprague Dawley rats.They were treated with VANE 60mg·kg-1 or valproic acid 60mg·kg-1 via intraperitoneal route before being anesthetized and sacrificed by cardiac puncture at different time points after administration.The blood and brain were collected and processed.The samples were analyzed with HPLC to determine the pharmacokinetic profile and biodistribution of the drug in the brain.RESULTS The formulated VANE had apparent permeability coefficient of 0.205cm·s-1 in vitro studies.It was 1.06-fold better than the drug solution.It showed that the VANE can penetrate across the BBB faster than the drug solution.The cumulative amount was reported to be 1.12-fold higher that the drug solution.This briefly suggests the VANE can be good candidate to promote the uptake of drug into the brain.In in vivo study,the formulated nanoemulsion showed remarkably improved pharmacokinetic parameters with 3.85-fold higher of total area under the curve-time curve(AUC)and 2.81-fold higher in maximum concentration of drug in blood plasma(cmax).More interestingly,the half-life(T1/2)is prolonged by 1.80-fold and its clearance(Cl)was reduced by 3.85-fold.CONCLUSION The formulated nanoemulsions were able to improve most of the pharmacokinetic parameter thus this formulation technique improves the bioavailability of the drug in the brain compared to the drug solution alone.

Anticancer Effects of Valproic Acid on Esophageal Stem-like Carcinoma Cells

Valproic acid(VPA) is a histone deacetylase inhibitor that has been an object of interest to clinicians for its promising potency in cancer therapy, as it induces apoptosis and differentiation, and enhances of chemotherapy sensitivity. Esophageal squamous cell carcinoma(ESCC) is a malignant disease with growing incidence and low survival rate. Due to limited information on VPA activity in ESCC cells, we aimed to determine effects of VPA on chemotherapy responsiveness and expression of malignant markers in ESCC stem-like cells. Upon coadministration of non-toxic VPA + cisplatin(DDP), paclitaxel and 5-fluorouracil, viability of KYSE30 cells was assessed, and induced apoptosis was evaluated by DAPI staining, DNA laddering and flow cytometry. In addition, real time RT-PCR was performed to study changes in the expression of P21, CD44 and BMI-1 upon treatments. MTT test demonstrated that VPA significantly(P < 0.05) increased toxicity of DDP, which was confirmed by DNA laddering, flow cytometry analysis and significant(P < 0.05) overexpression of P21. Moreover, real time RT-PCR results indicated significant(P < 0.05) down regulation of CD44 and BMI-1 after VPA administration. Present attempt provided evidence, for the first time, that VPA not only improved responsiveness of esophageal stem-like cancer cells to DDP, also negatively regulated cancer stem cells markers in these cells.

Valproic Acid Decreases Cell Proliferation and Color Preference in the Zebrafish Larvae

Valproic acid (VPA) is widely used as an antiepileptic drug or mood stabilizer. In this study, we evaluated the effects of treatment with 2 mM VPA for 3 h on cell proliferation in the telencephalic area of zebrafish larvae using bromodeoxyuridine (BRDU) to label dividing cells. It was demonstrated that 2 mM VPA exposure for 3 h at 2 and 3 days post-fertilization (dpf) larvae decreased cell proliferation in the telencephalic area of 5 dpf larvae. The reduced cell proliferation was not restored at 10 dpf larvae. The quantitative real-time PCR (qRT-PCR) data indicated that mRNA expression levels of WNT signaling pathway-related factors such as β-catenin, LEF1, and gsk3β were altered in the zebrafish larvae treated with 2 mM VPA at 2 and 3 pdf. It was also demonstrated that 2 mM VPA exposure affected color preference of the zebrafish larvae, reducing blue color preference at 5 dpf larvae. The altered color preference was restored at 10 dpf larvae. These results suggest that VPA exposure may cause molecular, cellular, and behavioral alterations in early developmental stage of the zebrafish.

Effects of Lithium and Valproic Acid on the Production of Brain-Derived Neurotrophic Factor in Astrocytoma

No systematic investigation considering BDNF and its receptor TrkB has been conducted on the effects of mood-stabilizing drugs. We therefore decided to quantify BDNF production and the expression of TrkB-T1 receptor and PLCγ in astrocytoma. 1321N human astrocytoma cells were grown to a sufficient quantity in 5% fetal calf serum (FCS). The mood-stabilizing drugs Li and VPA were added to the therapeutic concentrations of 1 mM and 600 μM, respectively. The production of BDNF was determined by an enzyme-linked immunosorbent assay. The expressions of TrkB-T1 and PLCγ were determined by Western blot. The production of BDNF was significantly higher on Day 7 in the VPA samples (P < 0.05) and was significantly suppressed beginning on Day 1 in the Li samples (P < 0.05). TrkB-T1 expression, in contrast to BDNF production, was significantly higher in the VPA samples (P γ expression did not change. Li and VPA seem differently affect BDNF production and TrkB-T1 (BDNF receptor) expression.

Altered Mental Status and Hyperammonemia after Overdose of Valproic Acid with Therapeutic Valproic Acid Concentrations

Valproic acid is used in the treatment of multiple disorders. Adverse effects from valproic acid include hepatotoxicity, hypotension, metabolic acidosis, and decreased mental status. Valproic acid also causes hyperammonemia. Many physicians assume that this is due to a supratherapeutic valproic acid concentration;when in fact, it can occur with therapeutic valproic acid concentrations. This is because the hyperammonemia may be related to carnitine deficiency and disruption of the urea cycle, which can both occur with therapeutic valproic acid concentrations. We report a patient presented to the emergency department with alteration of mental status after ingesting valproic acid for recreational purposes, who developed hyperammonemia with a therapeutic valproic acid concentration.