Valproic Acid Glucuronide Research Articles

Serum metabolomic profiling reveals potential biomarkers in systemic sclerosis

BackgroundSystemic sclerosis (SSc) is a chronic and systemic autoimmune disease marked by the skin and visceral fibrosis. Metabolic alterations have been found in SSc patients; however, serum metabolomic profiling has not been thoroughly conducted. Our study aimed to identify alterations in the metabolic profile in both SSc patients before and during treatment, as well as in mouse models of fibrosis. Furthermore, the associations between metabolites and clinical parameters and disease progression were explored. MethodsHigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS)/MS was performed in the serum of 326 human samples and 33 mouse samples. Human samples were collected from 142 healthy controls (HC), 127 newly diagnosed SSc patients without treatment (SSc baseline), and 57 treated SSc patients (SSc treatment). Mouse serum samples were collected from 11 control mice (NaCl), 11 mice with bleomycin (BLM)-induced fibrosis and 11 mice with hypochlorous acid (HOCl)-induced fibrosis. Both univariate analysis and multivariate analysis (orthogonal partial least-squares discriminate analysis (OPLS-DA)) were conducted to unravel differently expressed metabolites. KEGG pathway enrichment analysis was performed to characterize the dysregulated metabolic pathways in SSc. Associations between metabolites and clinical parameters of SSc patients were identified by Pearson's or Spearman's correlation analysis. Machine learning (ML) algorithms were applied to identify the important metabolites that have the potential to predict the progression of skin fibrosis. ResultsThe newly diagnosed SSc patients without treatment showed a unique serum metabolic profile compared to HC. Treatment partially corrected the metabolic changes in SSc. Some metabolites (phloretin 2’-O-glucuronide, retinoyl b-glucuronide, all-trans-retinoic acid, and betaine) and metabolic pathways (starch and sucrose metabolism, proline metabolism, androgen and estrogen metabolism, and tryptophan metabolism) were dysregulated in new-onset SSc, but restored upon treatment. Some metabolic changes were associated with treatment response in SSc patients. Metabolic changes observed in SSc patients were mimicked in murine models of SSc, indicating that they may reflect general metabolic changes associated with fibrotic tissue remodeling. Several metabolic changes were associated with SSc clinical parameters. The levels of allysine and all-trans-retinoic acid were negatively correlated, while D-glucuronic acid and hexanoyl carnitine were positively correlated with modified Rodnan skin score (mRSS). In addition, a panel of metabolites including proline betaine, phloretin 2’-O-glucuronide, gamma-linolenic acid and L-cystathionine were associated with the presence of interstitial lung disease (ILD) in SSc. Specific metabolites identified by ML algorithms, such as medicagenic acid 3-O-b-D-glucuronide, 4’-O-methyl-(−)-epicatechin-3’-O-beta-glucuronide, valproic acid glucuronide, have the potential to predict the progression of skin fibrosis. ConclusionsSerum of SSc patients demonstrates profound metabolic changes. Treatment partially restored the metabolic changes in SSc. Moreover, certain metabolic changes were associated with clinical manifestations such as skin fibrosis and ILD, and could predict the progression of skin fibrosis.

Computational study of the competitive binding of valproic acid glucuronide and carbapenem antibiotics to acylpeptide hydrolase

The efficacy of the antiepileptic drug VPA is decreased by co-administered carbapenems (CBPMs). The mechanism of CBPM selective inhibition of acylpeptide hydrolase (APEH) hydrolysis of VPA-glucuronide (VPA-G) to VPA is unclear due to the lack of APEH structural information. Here we performed homology modeling of the three-dimensional structure of APEH and subsequent docking simulations with a modeled structure to understand this mechanism. Docking simulations indicated that four groups of binding structures were involved in the binding of VPA-G, panipenem, and meropenem to APEH, but only one or two binding structures were involved in the binding of meropenem with an open β-lactam ring structure and other antibiotics involving ampicillin. One of the four VPA-G binding structures was close enough to the APEH catalytic triad to facilitate VPA-G hydrolysis. This binding structure was also the most stable binding structure for panipenem, suggesting potential inhibition of VPA-G hydrolysis by panipenem. Fragment molecular orbital calculations of interaction energies of amino acid residues of APEH with VPA-G, panipenem, and meropenem indicated that the binding structure for panipenem closest to the catalytic triad is stabilized upon APEH interaction. These data suggest that APEH binding characteristics with CBPMs may help explain the selective inhibition of APEH by CBPMs.

In vivo inhibition of acylpeptide hydrolase by carbapenem antibiotics causes the decrease of plasma concentration of valproic acid in dogs

1. Our previous in vitro studies suggest that inhibition of the acylpeptide hydrolase (APEH) activity as valproic acid glucuronide (VPA-G) hydrolase by carbapenems in human liver cytosol is a key process for clinical drug–drug interaction (DDI) of valproic acid (VPA) with carbapenems. Here, we investigated whether in vivo DDI of VPA with meropenem (MEPM) was caused via inhibition of APEH in dogs.2. More rapid decrease of plasma VPA levels and increased urinary excretion of VPA-G were observed after co-administration with MEPM compared with those after without co-administration, whereas the plasma level and bile excretion of VPA-G showed no change.3. Dog VPA-G hydrolase activity, inhibited by carbapenems, was mainly located in cytosol from both the liver and kidney. APEH-immunodepleted cytosols lacked VPA-G hydrolase activity. Hepatic and renal APEH activity was negligible even at 24 h after dosing of MEPM to a dog.4. In conclusion, DDI of VPA with carbapenems in dogs is caused by long-lasting inhibition of APEH-mediated VPA-G hydrolysis by carbapenems, which could explain the delayed recovery of plasma VPA levels to the therapeutic window even after discontinuation of carbapenems in humans.

Evaluation of in situ generated valproyl 1-O-β-acyl glucuronide in valproic acid toxicity in sandwich-cultured rat hepatocytes.

Acyl glucuronides are reactive electrophilic metabolites implicated in the toxicity of carboxylic acid drugs. Valproyl 1-O-β-acyl glucuronide (VPA-G), which is a major metabolite of valproic acid (VPA), has been linked to the development of oxidative stress in VPA-treated rats. However, relatively little is known about the toxicity of in situ generated VPA-G and its contribution to VPA hepatotoxicity. Therefore, we investigated the effects of modulating the in situ formation of VPA-G on lactate dehydrogenase (LDH) release (a marker of necrosis), BODIPY 558/568 C12 accumulation (a marker of steatosis), and cellular glutathione (GSH) content in VPA-treated sandwich-cultured rat hepatocytes. VPA increased LDH release and BODIPY 558/568 C12 accumulation, whereas it had little or no effect on total GSH content. Among the various uridine 5'-diphospho-glucuronosyltransferase inducers evaluated, β-naphthoflavone produced the greatest increase in VPA-G formation. This was accompanied by an attenuation of the increase in BODIPY 558/568 C12 accumulation, but did not affect the change in LDH release or total GSH content in VPA-treated hepatocytes. Inhibition of in situ formation of VPA-G by borneol was not accompanied by substantive changes in the effects of VPA on any of the toxicity markers. In a comparative study, in situ generated diclofenac glucuronide was not toxic to rat hepatocytes, as assessed using the same chemical modulators, thereby demonstrating the utility of the sandwich-cultured rat hepatocyte model. Overall, in situ generated VPA-G was not toxic to sandwich-cultured rat hepatocytes, suggesting that VPA glucuronidation per se is not expected to be a contributing mechanism for VPA hepatotoxicity.

Soybean greatly reduces valproic acid plasma concentrations: A food–drug interaction study

The aim of this study was to investigate the effects of soy on the pharmacokinetics and pharmacodynamics of valproic acid (VPA). In a preclinical study, rats were pretreated with two different amounts of soy extract for five days (150 mg/kg and 500 mg/kg), which resulted in decreases of 57% and 65% in the Cmax of VPA, respectively. AUC of VPA decreased to 83% and 70% in the soy pretreatment groups. Interestingly, the excretion rate of VPA glucuronide (VPAG) was higher in the soy-fed groups. Levels of UDP-glucuronosyltransferase (UGT) UGT1A3, UGT1A6, UGT2B7 and UGT2B15 were elevated in the soy-treated group, and GABA concentrations were elevated in the brain after VPA administration. However, this was less pronounced in soy extract pretreated group than for the untreated group. This is the first study to report the effects of soy pretreatment on the pharmacokinetics and pharmacodynamics of VPA in rodents.

Dysregulations of UDP-glucuronosyltransferases in rats with valproic acid and high fat diet induced fatty liver

Both high fat diet (HFD) and valproic acid (VPA) interfere with mitochondrial β-oxidation of fatty acids, which subsequently triggers microvesicular fatty liver and hepatic dysfunction. UDP-glucuronosyltransferases, the major phase II drug metabolism enzymes, play a pivotal role in detoxifying various exogenous and endogenous compounds. This study aimed to investigate the dysregulation patterns of major UDP-glucuronosyltransferases (UGTs) induced by VPA and/or HFD. Biochemical and histopathological results showed that chronic treatments of VPA and HFD induced fatty liver and liver dysfunction in a synergistic manner. VPA upregulated the mRNA levels of UGT1A1, 1A6, 1A7, and UGT2B1. Notably, the protein expression and enzymatic activity of UGT1A6 were significantly increased in rats treated with HFD or VPA alone, and were further enhanced by HFD and VPA co-treatment. This dysregulation pattern was largely recapitulated in the in vitro HepG2 cells assay by using VPA and oleic acid treatment. Moreover, the induction of UGTs was accompanied by the increased expression of constitutive androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα). In line with the up-regulation of UGT1A1 and UGT1A6, urine recovery of VPA glucuronide (VPA-G) was sharply increased by VPA treatment, and the co-treatment of HFD further aggravated this change. Since VPA is necessarily prescribed for long-term and the prevalence of HFD life style nowadays, the combined effect of HFD and VPA on disturbing UGTs should take concerns in the clinics.

A rapid and sensitive assay to quantify valproyl 1-O-acyl glucuronide in supernatants of sandwich-cultured rat hepatocytes using ultra-high performance liquid chromatography–tandem mass spectrometry

A rapid and sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of valproyl-1-O-acyl glucuronide (VPA-G) levels in hepatocyte culture medium. Chromatographic separation was achieved using a Waters Acquity UPLC(®) BEH C18 column (1.7μm, 2.1mm×50mm) with gradient elution and a total run time of 4min. [(2)H6]-VPA-G was used as internal standard (IS). Quantification was performed in the multiple reaction monitoring (MRM) mode using the total ion current of the MRM transition pairs m/z 319.1→142.7 and m/z 319.1→175.2 for VPA-G, and m/z 325.1→149.3 and m/z 325.1→174.9 for the IS under negative electrospray ionization mode. The assay was linear over the VPA-G concentrations of 0.5-500ng/mL, with a r(2) value of 0.995±0.002 (mean±SD). The intra- and inter-day accuracy (% deviation) ranged from -10.2% to 11.1%, whereas the intra- and inter-day precision (% RSD) were ≤7.43%. The method was applied successfully to the quantification of VPA-G levels in culture supernatants of sandwich-cultured rat hepatocytes treated with valproic acid (VPA). No significant difference in the levels of VPA-G over a culture period of 6 days was observed in an experiment that investigated the effect of the age of hepatocyte culture on the extent of VPA glucuronidation. The method presented here for the direct quantification of VPA-G is an improvement of existing methods in the literature and offers a shorter run time and greater sensitivity that enables the use of small volumes of sample. To the best of our knowledge, this is the first validated UHPLC-MS/MS method applied to the quantification of VPA-G in cell culture supernatants.

Valproic acid pathway

Valproic acid (VPA) is a branched short-chain fatty acid derived from naturally occurring valeric acid. VPA is used primarily in the treatment of epilepsy and seizures, but is also used in migraine, bipolar, mood, anxiety, and psychiatric disorders [1]. Recent work has explored its use as an adjuvant agent in cancer, HIV therapy, and neurodegenerative disease because of its action as histone deacetylase (HDAC) inhibitor [2]. VPA is widely used in pediatric epilepsy because of its multiple mechanisms of action and acceptable safety profile [3]. The dose requirements for VPA are highly variable (10-fold differences in mean dose in adults) [4] and interactions with other drugs are common (discussed below). Therapeutic drug monitoring is commonly used, although both the clinical and toxic effects of the drug are considered to be poorly correlated with total serum concentrations [3]. The drug label carries a black box warning for life-threatening adverse drug reactions (ADR) including hepatoxocity, teratogenicity, and pancreatitis [1]. Compared with adults, children appear to be at an increased risk for severe hepatotoxic reactions to VPA, in particular those younger than 2 years of age undergoing polytherapy, with existing developmental delays and coincident metabolic disorders [1]. Hyperammonemia is also a documented ADR of VPA treatment, although this is usually successfully resolved by cessation of VPA and treatment with carnitine [5]. The Food and Drug Administration has issued recommendations for patient testing and advises that VPA is contraindicated in patients with known urea cycle disorders (see Clinical PGx tab at http://www.pharmgkb.org/drug/PA451846). However, specific genetic tests for the determination of at-risk patients are not mentioned. The aim of this study was to introduce candidate genes in the pharmacokinetics (PK) (Fig. 1), pharmacodynamics (PD) of VPA (Fig. 2), and discuss results from pharmacogenomic studies so far. Fig. 1 Graphic representation of the candidate genes involved in valproic acid (VPA) pharmacokinetics. A fully interactive version of this pathway is available online at PharmGKB at http://www.pharmgkb.org/pathway/PA165964265. CYP, cytochrome P450. Fig. 2 Graphic representation of the candidate genes involved in valproic acid (VPA) pharmacodynamics. A fully interactive version of this pathway is available online at PharmGKB at http://www.pharmgkb.org/pathway/PA165959313. Pharmacokinetics VPA is highly protein bound (87–95%) resulting in low clearance (6–20 ml/h/kg) [6]. There are at least three routes of VPA metabolism in humans: glucuronidation, β oxidation in the mitochondria (both considered major routes accounting for 50 and 40% of dose, respectively), and cytochrome P450 (CYP)-mediated oxidation (considered a minor route, ~10%) [7-9]. Valproate glucuronide is the major urinary metabolite of VPA (~30–50%) [8]. In-vitro studies of human liver microsomes and purified recombinant proteins have reported glucuronidation of VPA by UGT1A3, UGT1A4, UGT1A6, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15 [8,10,11]. Other studies have disputed the role of UGT2B15, suggesting that VPA inhibits UGT2B15, but is not glucuronidated by it [12]. UGT1A1 does not have activity against VPA in vitro [8,12]. VPA is a fatty acid and can be metabolized through endogenous pathways in the mitochondria (Fig. 1). It has been observed that some of the mitochondrial metabolites of VPA generated by this pathway are hepatotoxic. The current understanding of VPA bioactivation involves the entry of 4-ene-VPA into the mitochondria, formation of a 4-ene-VPA-CoA ester with the help of ACADSB, and subsequent β-oxidation to form the reactive 2,4-diene-VPA-CoA ester [13,14]. Studies have demonstrated that the β-oxidation is blocked in fluorinated derivatives of 4-ene-VPA [15], and that the fluoro derivative of 4-ene-VPA cannot form a CoA ester [16], indicating a specific role of β oxidation of 4-ene-VPA in the formation of the 2,4-diene metabolite. This putative cytotoxic metabolite (2,4-diene-VPA-S-CoA) further gets conjugated with glutathione to form thiol conjugates. These chemically reactive metabolites generated from 4-ene-VPA have the potential to deplete mitochondrial glutathione pools [13] and form conjugates with CoA [17], in turn inhibiting enzymes in the β-oxidation pathway [18,19]. Identification of N-acetylcysteine conjugates of (E)-2,4-diene-VPA in human urine demonstrated that the reactive thiol conjugates of VPA arise primarily from the biotransformation of (E)-2,4-diene VPA in humans [20]. The key CYP-mediated branch of the VPA pathway is the generation of the metabolite 4-ene-VPA by CYP2C9, CYP2A6, and to a lesser extent by CYP2B6 [21,22]. In addition, these metabolizing enzymes also mediate the metabolism of VPA to the inactive 4-OH-VPA and 5-OH-VPA [23]. CYP2A6 also contributes partially to the formation of 3-OH-VPA [22].

In Silico Modeling of UDP-Glucuronosyltransferase 1A10 Substrates Using the Volsurf Approach

UDP-glucuronosyltransferase 1A10 (UGT1A10) catalyzes glucuronidation of a wide range of chemicals including many drugs. Here, we report the first in silico model quantifying the substrate selectivity and binding affinity (as K(m)) for UGT1A10. The training set for model construction comprises 32 structurally diverse compounds, which are known substrates for UGT1A10. The model was derived by applying the standard VolSurf method involving calculation of VolSurf descriptors and partial least square (PLS) analyses. The yielded PLS model with two components shows statistical significance in both fitting and internal predicting (r(2) = 0.827, q(2) = 0.774). The model predictability was further validated against a test set of 11 external compounds. The activity values for all test substrates were predicted within 1 log unit. Moreover, the model reveals an overlay of chemical features influencing the enzyme-substrate binding. Those include the size and shape, capacity factors, hydrophilic regions, hydrophobic regions, and polarizability. In conclusion, the VolSurf approach is successfully utilized to establish a predictive model for UGT1A10. The derived model should be an efficient tool for high-throughput prediction of UGT1A10 metabolism.

Functional Impact and Prevalence of Polymorphisms Involved in the Hepatic Glucuronidation of Valproic Acid

Metabolism of valproic acid, a widely used drug, is only partially understood. It is mainly metabolized through glucuronidation and acts as a substrate for various UDP-glucuronosyltransferases (UGTs). UGTs metabolizing valproic acid in the liver are UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, with UGT1A6 and UGT2B7 being the most prominent. Polymorphisms in genes expressing these enzymes may have clinical consequences, regarding dosing, blood levels of the drug and adverse reactions. Not all genes are well studied and studies, where they exist, report conflicting results. Prevalence of polymorphisms and various haplotypes is also of great importance, as it may suggest different therapeutic approaches in various populations. Presented here is a review of currently known polymorphisms, their functional impact, when known, and their prevalence in different populations, highlighting the current state of understanding and areas where there is a lack of data and suggesting new perspectives for further research.

Atypical Kinetics of Valproic Acid Glucuronidation In vitro and In vivo in Humans

Atypical kinetics has been observed in vitro for both cytochrome P450 and UDP-glucuronosyltransferase catalyzed enzymatic reactions. The in vitro and in vivo kinetics of valproic acid- glucuronide (VPA-glucuronide) formation was investigated in humans. VPA-glucuronide formation kinetics was investigated in vitro using human cryoperserved hepatocytes (pool of 10). Estimates of Vmax app (39.5 ± 3.3 pmol/min/106 cells), S50 app (224 ± 34 µM) and n (2.34 ± 0.28) was obtained by fitting VPA-glucuronide formation rate vs VPA incubation concentration data to the Hill equation. In vitro EadieHofstee plots were “hooked” and characteristic of atypical sigmoidal/autoactivation kinetics. In vivo Eadie-Hofstee plots of VPA-glucuronide formation rate (calculated from urinary excretion rate data for VPA-glucuronide) were constructed using data from four human subjects given a 1000 mg VPA oral dose [1]. The positive slope of linear regression lines for these plots was consistent with in vivo atypical sigmoidal/autoactivation kinetics. In summary, these data build upon our previous observations in sheep and provide the first demonstration that VPA-glucuronide formation exhibits atypical kinetics in vitro in human hepatocytes. Available in vivo data is consistent with in vitro results suggesting that VPA-glucuronidation exhibits atypical sigmoidal/autoactivation kinetics in vivo in humans.

Inhibition mechanism of carbapenem antibiotics on acylpeptide hydrolase, a key enzyme in the interaction with valproic acid

We have reported that inhibition of acylpeptide hydrolase (APEH), identified as valproic acid glucuronide hydrolase in human liver cytosol, by carbapenem antibiotics could lead to a decrease of plasma levels of valproic acid. In this study, we examined the inhibition mechanism using human liver cytosol and purified porcine APEH with a similar property to human counterpart.After preincubation of human liver cytosol with panipenem or meropenem for 30 min, the inhibition of APEH activity was 20-fold stronger than that without preincubation. Porcine APEH activity inhibited by meropenem did not recover after dialysis. Meropenem bound to porcine APEH and the binding was blocked by a serine hydrolase inhibitor, diisopropyl fluorophosphate.Open β-lactam ring form of meropenem did not affect APEH activity in human liver cytosol. Likewise, other antibiotics, which have a different heterocycle adjacent to the β-lactam ring with an opposite configuration of the side chain from carbapenems, did not inhibit APEH activity.In conclusion, carbapenems inhibit APEH in both reversible and true irreversible manner and the irreversible inhibition is partially explained by binding to the active serine of APEH. The closed β-lactam ring is essential for inhibition and the heterocycle and/or the configuration of side chain would be important.

Identification of Valproic Acid Glucuronide Hydrolase As a Key Enzyme for the Interaction of Valproic Acid with Carbapenem Antibiotics

Plasma levels of valproic acid (VPA) are decreased by concomitant use with carbapenem antibiotics, such as panipenem (PAPM). One of the plausible mechanisms of this interaction is the inhibition of VPA glucuronide (VPA-G) hydrolysis by carbapenems in the liver. To elucidate this interaction mechanism, we purified VPA-G hydrolase from human liver cytosol, in which the hydrolytic activity was mainly located. After chromatographic purification, the VPA-G hydrolase was identified as acylpeptide hydrolase (APEH). APEH-depleted cytosol, prepared by an immunodepletion method, completely lacked the hydrolytic activity. These results demonstrate that APEH is a single enzyme involved in PAPM-sensitive VPA-G hydrolysis in cytosol. In addition, the hydrolytic activity of recombinant human APEH was inhibited by PAPM and the inhibition profile by typical esterase inhibitors (diisopropyl fluorophosphate, 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuribenzoic acid, and d-saccharic acid 1,4-lactone) was similar to that of human liver cytosol. Cytosolic VPA-G hydrolase activity was slightly inhibited by cholinesterase and carboxylesterase inhibitors. beta-Glucuronidase activity remained in APEH-depleted cytosol, whereas VPA-G hydrolase activity was completely abolished. Thus, either cholinesterase, carboxylesterase, or beta-glucuronidase in cytosol would not be involved in VPA-G hydrolysis. Taken together, APEH plays a major role in the PAPM-sensitive VPA-G hydrolysis in the liver. These findings suggest that APEH could be a key enzyme for the drug interaction of VPA with carbapenems via VPA-G hydrolysis.

Concentration-dependent disposition of glucuronide metabolite of valproate.

The glucuronide conjugation metabolism of valproate (VPA) has been assessed to be non-linear within the therapeutic concentration range. However, disposition of its metabolite, valproic acid glucuronide (VPAG), in relation to VPA doses is unclear. The purpose of this study was to elucidate the characteristics of dose-related disposition of VPAG. Guinea-pigs were treated with an intravenous bolus dose of sodium valproate at 20, 100, 500 or 600 mg kg(-1). Plasma was sampled on a pre-selected time schedule, and bile and urine were collected. Concentrations of VPA and VPAG in plasma, bile and urine were determined by gas chromatography. The pharmacokinetics of VPA and VPAG both were dose-dependent. However, the plasma concentration-time profiles of VPAG and VPA were not parallel. At a usual dose of VPA (20 mg kg(-1)), plasma VPAG declined with plasma VPA, whereas at a high dose of VPA (>500mg kg(-1)), plasma VPAG was elevated against the decline of plasma VPA, which suggested accumulation of plasma VPAG possibly owing to saturated elimination. The biliary and urinary clearances of VPA (vCLb and vCLu) were independent of dose. However, the clearances of plasma VPA (vCLp), plasma VPAG (gCLp), biliary and urinary VPAG (gCLb and gCLu) all were decreased against the increase in VPA doses. The dose-dependent decrease of gCLu (from 3.19 to 1.12 mL min(-1)) was less pronounced than that of gCLp (from 6.72 to 0.86 mL min(-1)) and the gCLu turned to exceed the gCLp at high doses of VPA (> 500 mg kg(-1)). These results suggest that the excess urinary VPAG might be produced in kidney. In conclusion, at a high dose of VPA, plasma VPAG is accumulated. The concentration-dependent biliary and urinary recovery of VPAG might be governed by a saturable elimination process rather than by saturable hepatic biotransformation rate. Glucuronide conjugation metabolism of VPA in kidney is speculated, which might be minor at low levels of plasma VPA, but more obvious after saturation of hepatic glucuronidation.

The Effect of Carbapenem Antibiotics on Plasma Concentrations of Valproic Acid

To critically evaluate the mechanisms of the interaction between valproic acid and carbapenem antibiotics. A PubMed search (January 1971-June 2009) was performed to identify literature on the interaction between valproic acid and carbapenem antibiotics. Additional references were identified through review of bibliographies of identified articles. Data on the mechanisms of the interaction between valproic acid and carbapenem antibiotics were extracted from identified references that were published in English. Valproic acid plasma concentrations fall markedly during concomitant administration with carbapenem antibiotics due to a combination of absorption, distribution, and metabolism mechanisms. Carbapenems appear to inhibit the intestinal transporter of valproic acid, thereby reducing absorption of orally administered valproic acid. In vivo experiments in rats demonstrate a 57% reduction in absorption of orally administered valproic acid in the presence of imipenem. Follow-up studies in Caco-2 cells suggest that the inhibition probably occurs at the basolateral membrane. In addition, enterohepatic recycling of valproic acid may be diminished due to carbapenem activity against gut flora producing beta-glucuronidase. When rabbits and rats were given intravenous valproic acid-glucuronide, the glucuronide metabolite of valproic acid, 50-90% of the conversion back into valproic acid was inhibited in the presence of a carbapenem. An increase in erythrocyte distribution of valproic acid has also been observed in the presence of carbapenems. After intravenous administration of a carbapenem and valproic acid, valproic acid plasma concentrations fell in the presence of a carbapenem, yet whole blood concentrations of valproic acid did not change significantly. Follow-up studies suggest that the mechanism of this distribution shift is that multidrug resistance proteins on adenosine triphosphate-binding cassette transporters on erythrocyte membranes are inhibited by carbapenems. Thus, valproic acid is not effluxed out of the erythrocytes. Finally, carbapenems may enhance glucuronidation of valproic acid by increasing UDP-glucuronic acid levels. In rats, UDP-glucuronic acid levels increased by 1.7-fold in the presence of panipenem, which was proportionate to the increase in valproic acid-glucuronide formation. Published data demonstrate a serious and complex interaction between valproic acid and carbapenem antibiotics. Coadministration should be avoided, but if no other antibiotic therapies exist, it is imperative to monitor valproic acid concentrations more frequently. Clinicians should anticipate higher doses of valproic acid to maintain therapeutic serum concentrations during coadministration and subsequent dose reductions upon discontinuation of the carbapenem antibiotic.

Effect of Aging on Glucuronidation of Valproic Acid in Human Liver Microsomes and the Role of UDP-Glucuronosyltransferase UGT1A4, UGT1A8, and UGT1A10

Valproic acid (VPA) is a widely used anticonvulsant that is also approved for mood disorders, bipolar depression, and migraine. In vivo, valproate is metabolized oxidatively by cytochromes P450 and beta-oxidation, as well as conjugatively via glucuronidation. The acyl glucuronide conjugate (valproate-glucuronide or VPAG) is the major urinary metabolite (30-50% of the dose). It has been hypothesized that glucuronidation of antiepileptic drugs is spared over age, despite a known decrease in liver mass. The formation rates of VPAG in a bank of elderly (65 years onward) human liver microsomes (HLMs) were measured by liquid chromatography/tandem mass spectrometry and compared with those in a younger (2-56 years) HLM bank. In vitro kinetic studies with recombinant UDP-glucuronosyltransferases (UGTs) were completed. A 5- to 8-fold variation for the formation of VPAG was observed within the microsomal bank obtained from elderly and younger donors. VPAG formation ranged from 6.0 to 53.4 nmol/min/mg protein at 1 mM substrate concentration (n=36). The average velocities at 0.25, 0.5, and 1 mM VPA were 7.0, 13.4, and 25.4 nmol/min/mg protein, respectively, in the elderly HLM bank. Rates of VPAG formation were not significantly different in the HLM bank obtained from younger subjects. Intrinsic clearances (V(max)/K(m)) for several cloned, expressed UGTs were determined. UGT1A4, UGT1A8, and UGT1A10 also were found to catalyze the formation of VPAG in vitro. This is the first reported activity of these UGTs toward VPA glucuronidation. UGT2B7 had the highest intrinsic clearance, whereas UGT1A1 demonstrated no activity. In conclusion, our investigation revealed no differences in VPAG formation in younger versus elderly HMLs and revealed three other UGTs that form VPAG in vitro.

Influence of chronic hepatic failure on disposition kinetics of valproate excretion through a phase II reaction in rats treated with carbon tetrachloride

The influence of chronic hepatic failure on the disposition kinetics of valproate (VPA) excretion via a phase II reaction was examined in rats treated with carbon tetrachloride (1.0 mg/kg, s.c., 3 times a week) for 2 or 3 months. There was no significant difference in the plasma concentration-time courses of VPA among the control and two treated groups up to 120 min after i.v. administration of VPA (75 mg/kg), but subsequently the plasma concentrations of the treated groups declined significantly below the control levels. Expression of Mrp2 mRNA in the liver of the treated groups was significantly lower than in the control group; conversely that in the kidney was significantly higher. The enzyme activity of UGTs in the liver of the treated groups decreased significantly, but UGT1A8 mRNA expression in the duodenum was increased about 3-fold. Cumulative excretion of VPA glucuronide (VPA-G) in bile of the treated groups was reduced significantly, while that in urine was markedly increased. In conclusion, the area under the VPA plasma concentration-time curve was decreased significantly in rats with chronic hepatic failure owing to increased excretion of VPA-G via the kidney as a result of induction of Mrp2, and inhibition of enterohepatic circulation of VPA-G.

Kinetics of Valproic Acid Glucuronidation: Evidence for in Vivo Autoactivation

Sigmoidal or autoactivation kinetics has been observed in vitro for both cytochrome P450- and UDP-glucuronosyltransferase-catalyzed enzymatic reactions. However, the in vivo relevance of sigmoidal kinetics has never been clearly demonstrated. In the current study we investigate the kinetics of valproic acid glucuronide (VPAG) formation both in vivo in adult sheep and in vitro in sheep liver microsomes (pool of 10). After a 100 mg/kg i.v. bolus dose of valproic acid (VPA) to adult sheep (n = 5), the majority of the dose was recovered in urine as VPAG (approximately 79%). Eadie-Hofstee plots of the VPAG formation rate (calculated from urinary excretion rate data for VPAG) were characteristic of autoactivation kinetics and provided estimates of the apparent maximum velocity of an enzymatic reaction (V(max)(app)), the substrate concentration resulting in 50% of V(max)(app) (S(50)(app)), and Hill coefficient (n) of 2.10 +/- 0.75 micromol/min/kg, 117 +/- 56 microM, and 1.34 +/- 0.14, respectively. Comparable estimates of V(max)(app) (2.63 +/- 0.33 micromol/min/kg), S(50)(app) (118 +/- 53 microM), and n (2.06 +/- 0.47) describing overall VPA elimination from plasma were obtained by fitting VPA unbound plasma concentration-time data to a two-compartment model with elimination described by the Hill equation. Consistent with our in vivo observations, Eadie-Hofstee plots of VPAG formation in sheep liver microsomes were characteristic of autoactivation kinetics. To our knowledge, these data provide the first clear demonstration that autoactivation kinetics observed in vitro in liver preparations can translate to the in vivo situation at least under certain experimental conditions and confirm its relevance.

Interaction Between Valproic Acid and Carbapenem Antibiotics

The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. This review summarized several case reports of this interaction between VPA (1–4 g dose) and carbapenem antibiotics to elucidate the possible mechanisms decreasing VPA concentration by carbapenem antibiotics. Studies to explain the decrease were carried out using rats by the following sites: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. In the intestinal absorption site, there are two possible mechanisms: inhibition of the intestinal transporter for VPA absorption by carbapenem antibiotics, and the decrease of β-glucuronidase supplied from enteric bacteria, which were killed by antibiotics. This is consistent with a view that the decrease of VPA originated from VPA-Glu, relating to entero-hepatic circulation. The second key site is in the liver, because of no decreased in VPA level by carbapenem antibiotics in hepatectomized rats. There are three possible mechanisms in the liver to explain the decreased phenomenon: first, decrease of the UDPGA level by carbapenem antibiotics. UDPGA is a co-factor for UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of VPA. Second, the direct activation of UGT by carbapenem antibiotics. This activation was observed after pre-incubation of human liver microsomes with carbapenem antibiotics. Third, the inhibition of β-glucuronidase in liver by carbapenem antibiotics and the decreased VPA amount liberated from VPA-Glu. The third site is the distribution of VPA in blood (erythrocytes and plasma). Plasma VPA distributed to erythrocytes by the inhibition of transporters (Mrp4), which efflux VPA from erythrocytes to plasma, by carbapenem antibiotics. The increase of renal excretion of VPA as VPA-Glu depends on the increase of VPA-Glu level by UGT. One or a combination of some factors in these mechanisms might relate to the carbapenem-mediated decrease of the plasma VPA level.

In Vitro Activation of Valproate Glucuronidation by Carbapenem Antibiotics

The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. Studies to explain the decrease were carried out using almost rats by the following steps: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. It is difficult to consider the inhibition of intestinal absorption, because carbapenem antibiotics are intravenously administered and do not reach the intestine at an effective concentration. The liver is the key organ for the decrease of VPA concentration by carbapenem antibiotics, because it has been reported that no decrease of the VPA level by carbapenem was found in hepatectomized rats. The most likely mechanism in liver is the activation of UDP-glucuronosyltransferase by carbapenem antibiotics. We found a 35% increase of VPA-glucuronidation activity by the pre-incubation of human liver microsomes with meropenem. We estimated that this increase fully compensates for the decrease of serum VPA level by carbapenem antibiotics in patients. Meanwhile, we could not find the in vitro activation of CYP2A6, CYP3A4, P-glycoprotein (P-gp, MDR1) and Multidrug resistance-associated protein 2 by pre-incubation with carbapenem antibiotics. Thus, we considered that the activation of VPA glucuronidation by carbapenem antibiotics is a key point in the decrease of plasma VPA level.