Transcriptome analysis of Burkitt lymphoma cells treated with anti-convulsant drugs that are inhibitors of Epstein-Barr virus lytic reactivation.

Herpesviruses have two distinct life cycle stages, latency and lytic replication. Epstein-Barr virus (EBV), a gamma-herpesvirus, establishes latency in vivo and in cultured cells. Cell lines harboring latent EBV can be induced into the lytic cycle by treatment with chemical inducing agents. In the Burkitt lymphoma cell line HH514-16 the viral lytic cycle is triggered by butyrate, a histone deacetylase (HDAC) inhibitor. Butyrate also alters expression of thousands of cellular genes. However, valproic acid (VPA), another HDAC inhibitor with global effects on cellular gene expression blocks EBV lytic gene expression in Burkitt lymphoma cell lines. Valpromide (VPM), an amide derivative of VPA, is not an HDAC inhibitor, but like VPA blocks induction of the EBV lytic cycle. VPA and VPM are the first examples of inhibitors of initial stages of lytic reactivation. We compared the effects of VPA and VPM, alone and in combination with butyrate, on host cellular gene expression using whole transcriptome analysis (RNA-seq). Gene expression was analyzed 6 h after addition of the compounds, a time before the first EBV lytic transcripts are detected. The results address two alternative, yet possibly complementary, mechanisms for regulation of EBV lytic reactivation. First, cellular genes that were up- or down-regulated by butyrate, but no longer altered in the presence of VPA or VPM, represent genes that correlated with EBV lytic reactivation. Second, genes regulated similarly by VPA and VPM in the absence and presence of butyrate are candidates for suppressors of EBV reactivation. Two genes upregulated by the lytic cycle inhibitors, CHAC1 and SLC7A11, are related to redox status and the iron-dependent cell death pathway ferroptosis. This study generates new hypotheses for control of the latency to lytic cycle switch of EBV and provides the first description of effects of the anti-convulsant drug VPM on global human cellular gene expression.

Modulation of the powder properties of lamotrigine by crystal forms.

The mechanical properties of powders determine the ease of manufacture and ultimately the quality of the oral solid dosage forms. Although poor mechanical properties of an active pharmaceutical ingredient (API) can be mitigated by using suitable excipients in a formulation, the effectiveness of that approach is limited for high dose drugs or multidrug tablets. In this context, improving the mechanical properties of the APIs through solid form optimisation is a good strategy to address such a challenge. This work explores the powder and tableting properties of various lamotrigine (LAM) solid forms with the aim to facilitate direct compression by overcoming the poor tabletability of LAM. The two drug-drug crystals of LAM with nicotinamide and valproic acid demonstrate superior flowability and tabletability over LAM. The improved powder properties are rationalised by structure analysis using energy framework, scanning electron microscopy, and Heckel analysis.

Teratogenicity of valproic acid and its constitutional isomer, amide derivative valnoctamide in mice.

OBJECTIVES: The anticonvulsant valproic acid (VPA) has a known teratogenic effect capable of inducing major congenital malformations and developmental disorders. A comparative teratogenicity study of VPA and its analog valnoctamide (VCD), which is a new generation candidate antiepileptic drug, was carried out using Swiss Vancouver (SWV) mice. METHODS: Pregnant SWV dams were treated with either a single intraperitoneal injection of VPA (1.8 and 2.7 mmol/kg), VCD (1.8 and 2.7 mmol/kg), or vehicle on E8:12 (gestational day:hour). The numbers of implantation and resorption, viable and dead fetuses, and the presence of gross fetal visceral and skeletal abnormalities were determined (E18). Real-time Polymerase chain reaction (RT-PCR) arrays were used to analyze the expression of 84 genes related to the processes of neurogenesis and neural stem cell differentiation. RESULTS: Significant decreases in pregnancy weight gain and the number of live fetuses were observed when VPA was administered at the high dose, whereas the percentage of exencephalic fetuses was significantly increased in VPA treated compared with an equivalent VCD dosage group. There was a dose-related increase in visceral defects in the VPA-exposed fetuses. Missing skull bones and fused vertebrae in fetuses occurred at the high dose of VPA. Three genes (Mtap2, Bmp8b, and Stat3) were significantly upregulated and one (Heyl) was downregulated in samples from VPA-treated dams. CONCLUSIONS: The study demonstrates that the teratogenicity of VPA was significantly greater than that of an equimolar dose of VCD. Four genes (Mtap2, Bmp8b, Stat3, and Heyl) represent candidate target genes for the underlying teratogenic mechanism responsible for VPA-induced malformations.

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.

[Observation on therapeutic effect of acupoint catgut-embedding combined western medicine for epilepsy of generalized seizures type].

OBJECTIVE: To compare the efficacy differences between acupoint catgut-embedding combined western medicine (ACCWM) and simple western medicine (WM) for treatment of epilepsy of generalized seizures type. METHODS: Fifty-two patients with epilepsy of generalized seizures type were randomly divided into an ACCWM group (n=30) and a WM group (n=22). Dazhui (GV 14), Yaoshu (GV 2), Jiuwei (CV 15), Qihai (CV 6) were selected in the ACCWM group as the main acupoints combined with routine antiepileptic drugs, Valpromide was taken orally, 2.0 g/d for adult, 50 mg/kg x d for child. The WM group was treated with routine antiepileptic drugs. The epilepsy score and the attack frequency one year before the treatment and one year during treatment were observed and the therapeutic effects in the two groups were compared. RESULTS: The epilepsy score and the attack frequency were both decreased in the two groups (P<0.01, P<0.05), and the epilepsy score in the ACCWM group decreased significantly (6.57 +/- 3.29 vs 10.73 +/- 2.54, P<0.05). The total effective rate of 90.0% (27/30) in the ACCWM group was superior to that of 68.2% (15/22) in the WM group (P<0.05). CONCLUSION: The therapeutic effect of ACCWM is superior to that of simple western medicine in treating epilepsy of generalized seizures type. It can improve significantly the attack symptoms so as to enhance life quality of the patients with epilepsy.

Dose-dependent pharmacokinetics of toxic metabolites is not related to increased toxicity following high-dose valproic acid in rats.

It has been reported that urinary excretion of two metabolites of valproic acid (VPA), 4-ene-valproic acid (4-VPA)and 2,4-diene-valproic acid (2,4-VPA), increased exponentially with the administration of high doses of VPA, and this increased formation of toxic metabolites could be related to VPA hepatotoxicity in humans. The aim of this study was to investigate whether the plasma level of 4-VPA and 2,4-VPA in rats corresponds to the urinary data for the same metabolites in humans.After the oral administration of VPA at doses of 20, 100 and 500 mg kg-1 in rats, the AUC0­24 h, 4-VPA/AUC0­24 h, VPA ratios (0.0399,0.0120 and 0.0100 for 20, 100 and 500 mg kg-1, respectively) and AUC0­24 h, 2,4-VPA/AUC0­24 h, VPA ratios (0.00104, 0.00201 and 0.00141, respectively) did not increase with increasing doses of VPA in rats. Thus, the plasma exposure of toxic metabolites normalized by dose remained unchanged (for 2,4-VPA) or even decreased (for 4-VPA) following high-dose VPA administration;this contradicts the findings of previous studies. Our results suggest that toxicity induced by high doses of VPA cannot be explained by a nonlinear increase of toxic metabolites in rats.

Carnitine in the treatment of valproic acid-induced toxicity.

INTRODUCTION: Valproic acid (VPA) is a broad-spectrum antiepileptic drug that is now used commonly for several other neurological and psychiatric indications. VPA is usually well tolerated, but serious complications, including hepatotoxicity and hyperammonemic encephalopathy, may occur. These complications may also arise following acute VPA overdose, the incidence of which is increasing. Intoxication usually only results in mild central nervous system depression, but serious toxicity and death have been reported. VALPROIC ACID AND CARNITINE: As a branched chain carboxylic acid, VPA is extensively metabolized by the liver via glucuronic acid conjugation, mitochondrial beta- and cytosolic omega-oxidation to produce multiple metabolites, some of which may be involved in its toxicity. Carnitine is an amino acid derivative that is an essential cofactor in the beta-oxidation of fatty acids. It is synthesized endogenously from the essential amino acids, methionine and lysine. VPA inhibits the biosynthesis of carnitine by decreasing the concentration of alpha-ketoglutarate and may contribute to carnitine deficiency. It is postulated that carnitine supplementation may increase the beta-oxidation of VPA, thereby limiting cytosolic omega-oxidation and the production of toxic metabolites that are involved in liver toxicity and ammonia accumulation. VPA-induced hepatotoxicity and hyperammonemic encephalopathy may be promoted either by a pre-existing carnitine deficiency or by deficiency induced by VPA per se. CARNITINE SUPPLEMENTATION: Some experimental and clinical data suggest that early intravenous supplementation with l-carnitine could improve survival in severe VPA-induced hepatotoxicity. Carnitine administration has been shown to speed the decrease of ammonemia in patients with VPA-induced encephalopathy although a correlation between ammonia concentrations and the clinical condition was not always observed. As it does not appear to be harmful, l-carnitine is commonly recommended in severe VPA poisoning, especially in children, although the clinical benefit in terms of liver protection or hastening of recovery from unconsciousness has not been established clearly. Prophylactic carnitine supplementation is also advocated during VPA therapy in high-risk pediatric patients. CONCLUSION: Further controlled, randomized, and probably multicenter trials are required to better delineate the therapeutic and prophylactic roles of l-carnitine and the optimal regimen of administration in the management of VPA toxicity.

Tetramethylcyclopropyl analogue of the leading antiepileptic drug, valproic acid: evaluation of the teratogenic effects of its amide derivatives in NMRI mice.

BACKGROUND: Although valproic acid (VPA) is used extensively for treating various kinds of epilepsy, it causes hepatotoxicity and teratogenicity. In an attempt to develop a more potent and safer second generation to VPA drug, the amide derivatives of the tetramethylcyclopropyl VPA analogue, 2,2,3,3-tetramethylcyclopropanecarboxamide (TMCD), N-methyl-TMCD (MTMCD), 4-(2,2,3,3-tetramethylcyclopropanecarboxamide)-benzenesulfonamide (TMCD-benzenesulfonamide), and 5-(TMCD)-1,3,4-thiadiazole-2-sulfonamide (TMCD-thiadiazolesulfonamide) were synthesized and shown to have more potent anticonvulsant activity than VPA. Teratogenic effects of these CNS-active compounds were evaluated in Naval Medical Research Institute (NMRI) mice susceptible to VPA-induced teratogenicity by comparing them to those of VPA. METHODS: Pregnant NMRI mice were given a single sc injection of either VPA or TMC-amide derivatives on gestation day 8.5, and then the live fetuses were examined to detect any external malformations on gestation day 18. After double-staining for bone and cartilage, their skeletons were examined. RESULTS: In contrast to VPA, which induced NTDs in a high number of fetuses at 2.4-4.8 mmol/kg, TMCD, TMCD-benzenesulfonamide, and TMCD-thiadiazolesulfonamide at 4.8 mmol/kg and MTMCD at 3.6 mmol/kg did not induce a significant number of NTDs. TMCD-thiadiazolesulfonamide exhibited a potential to induce limb defects in fetuses. Skeletal examination also revealed that fetuses exposed to all four of the tetramethylcyclopropanecarboxamide derivatives developed vertebral and rib abnormalities less frequently than those exposed to VPA. Our results established that TMCD, MTMCD, and TMCD-benzenesulfonamide are distinctly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS-active amides containing a tetramethylcyclopropanecarbonyl moiety demonstrated better anticonvulsant potency compared to VPA and a lack of teratogenicity, which makes these compounds good second-generation VPA antiepileptic drug candidates.

Hydroxamic acid and fluorinated derivatives of valproic acid: anticonvulsant activity, neurotoxicity and teratogenicity.

PURPOSE: Fluorinated and non-fluorinated valproic acid (VPA) analogues with hydroxamic acid moieties were tested for their teratogenic, anticonvulsant and neurotoxic potencies in mice. METHODS: Compounds were synthesized from their corresponding acids. The induction of neural tube defects (exencephaly) of the resulting hydroxamates (applied on day 8.25 of gestation) was tested in the offspring of pregnant animals (Han:NMRI mice). The anticonvulsant activity was evaluated in the subcutaneous pentylenetetrazole (PTZ) seizure threshold test and neurotoxicity in the rotorod neurotoxicity test. RESULTS: All tested hydroxamates showed no or greatly reduced teratogenic potency in mice compared to the free acids. Furthermore all compounds exhibited anticonvulsant activity with ED(50) doses ranging from 0.16 mmol/kg to 0.59 mmol/kg (VPA 0.57 mmol/kg). Neurotoxicity of the hydroxamates was increased compared to VPA. TD(50) doses range from 0.70 mmol/kg to 1.42 mmol/kg (VPA 1.83 mmol/kg). CONCLUSION: Hydroxamic acid derivatives of VPA with improved protective index and little or undetectable teratogenic potency compared to the free acids are described. alpha-fluorination of VPA also resulted in loss of teratogenic activity. Such fluorination of the hydroxamic acids also led to compounds with an improved anticonvulsant profile compared to non-fluorinated hydroxamates. The non-chiral 2-Fluoro-VPA-hydroxamic acid was the most promising compound with a protective index (ratio of TD(50) to ED(50)) of 4.4 compared to 3.2 for VPA. This compound combines an improved ratio of anticonvulsant potency/neurotoxicity with the advantage of not being teratogenic in the mouse neural tube defect model used.

The oral absorption of phospholipid prodrugs: in vivo and in vitro mechanistic investigation of trafficking of a lecithin-valproic acid conjugate following oral administration.

The purpose of this study was to evaluate the oral absorption characteristics of a phospholipid-drug conjugate, comprising direct conjugation between the lecithin and the drug moiety through the sn-2 position. We investigated the mechanisms involved with the trafficking of this conjugate following oral administration in the gastrointestinal (GI) lumen, within the enterocyte and further. A phospholipid-valproic acid conjugate (DP-VPA) was utilized as a model molecule. The oral absorption of this conjugate in rats was investigated following administration in long (LCT) vs. medium (MCT) chain triglyceride formulations, and in the postprandial vs. fasted state. Oral administration within the LCT solution caused more than a 3-fold increase in DP-VPA bioavailability in comparison to the MCT solution. Moreover, a significant food effect was evident for DP-VPA. Hence, we evaluated the lymphatic transport of DP-VPA in mesenteric lymph duct cannulated freely moving rats. Sixty percent of the absorbed DP-VPA was associated with lymphatic transport. Similar DP-VPA absorption was obtained in secretory type II PLA(2) knockout mice (C57BL/6) and in control mice (BALB/c). Moreover, nil DP-VPA degradation in serum and very low (4.8%) degradation by bee venom PLA(2)in vitro were obtained. In conclusion, direct conjugation between the drug and the phospholipid produces a complex having unique absorption properties that include: (1) a stable complex that does not undergo degradation in the GI tract; (2) permeation through the gut wall and entering intact to the enterocyte; and (3) association with chylomicron in the enterocyte and reaching the systemic circulation via the lymphatic route. These unique properties may be of interest in drug delivery.

New antiepileptic drugs that are second generation to existing antiepileptic drugs.

In the last decade, 10 new antiepileptic drugs (AEDs) have been introduced that offer appreciable advantages in terms of their favourable pharmacokinetics, improved tolerability and lower potential for drug interactions. However, despite the large therapeutic range of old and new AEDs, approximately 30% of the patients with epilepsy are still not seizure free and, consequently, there is a substantial need to develop new AEDs. The new AEDs currently in development can be divided into two categories: drugs with completely new chemical structures such as lacosamide (formally harkoseride), retigabine, rufinamide and talampanel; and drugs that are derivatives or analogues of existing AEDs that can be regarded as second-generation or follow-up compounds of established AEDs. This article focuses on the second category and thus critically reviews the following second-generation compounds: eslicarbazepine acetate or BIA-2-093 and 10-hydroxy carbazepine (carbamazepine derivatives); valrocemide and NPS 1776 (isovaleramide; valproic acid derivatives); pregabalin and XP13512 (gabapentin derivatives); brivaracetam (ucb 34714) and seletracetam (ucb 44212; levetiracetam derivatives); and fluorofelbamate (a felbamate derivative). In addition, a series of valproic acid derivatives that are currently in preclinical stage has also been evaluated because some lead compounds of this series have a promising potential to become new antiepileptics and CNS drugs. For any of these follow-up compounds to become a successful second generation to an existing AED, it has to be more potent, safer and possess favourable pharmacokinetics, including low potential for pharmacokinetic and pharmacodynamic drug interactions.

Preclinical profiling and safety studies of ABT-769: a compound with potential for broad-spectrum antiepileptic activity.

PURPOSE: The objective of this study was to characterize the antiseizure and safety profiles of ABT-769 [(R)-N-(2 amino-2-oxoethyl)spiro[2,5]octane-1-carboxamide]. METHODS: ABT-769 was tested for protection against maximal electroshock and pentylenetetrazol-induced seizures in the mouse and for suppression of electrically kindled amygdala seizures and spontaneous absence-like seizures in the rat. The central nervous system safety profile was evaluated by using tests of motor coordination and inhibitory avoidance. The potential for liver toxicity was assessed in vitro by using a mitochondrial fatty acid beta-oxidation assay. Teratogenic potential was assessed in the mouse. RESULTS: ABT-769 blocked maximal electroshock, subcutaneous pentylenetetrazol and intravenous pentylenetetrazol-induced seizures with median effective dose (ED50) values of 0.25, 0.38, and 0.11 mmol/kg, p.o., respectively. No tolerance was evident in the intravenous pentylenetetrazol test after twice-daily dosing of ABT-769 (0.3 mmol/kg, p.o.) for 4 days. ABT-769 blocked absence-like spike-wave discharge (ED50, 0.15 mmol/kg, p.o.) and shortened the cortical and amygdala afterdischarge duration of kindled seizures (1 and 3 mmol/kg, p.o.). The protective indices (ED50 rotorod impairment/ED50 seizure protection) were 4.8, 3.2, and 10.9 in the maximal electroshock, subcutaneous pentylenetetrazol and intravenous pentylenetetrazol seizure tests, respectively. ABT-769 did not affect inhibitory avoidance performance (0.1-1 mmol/kg, p.o.). ABT-769 did not affect mitochondrial fatty acid beta-oxidation or induce neural tube defects. CONCLUSIONS: ABT-769 is an efficacious antiseizure agent in animal models of convulsive and nonconvulsive epilepsy and has a favorable safety profile. ABT-769 has a broad-spectrum profile like that of valproic acid. Its profile is clearly different from those of carbamazepine, phenytoin, lamotrigine, topiramate, vigabatrin, and tiagabine.

Effects of valproic acid derivatives on inositol trisphosphate depletion, teratogenicity, glycogen synthase kinase-3beta inhibition, and viral replication: a screening approach for new bipolar disorder drugs derived from the valproic acid core structure.

Inositol-1,4,5-trisphosphate (InsP3) depletion has been implicated in the therapeutic action of bipolar disorder drugs, including valproic acid (VPA). It is not currently known whether the effect of VPA on InsP3 depletion is related to the deleterious effects of teratogenicity or elevated viral replication, or if it occurs via putative inhibitory effects on glycogen synthase kinase-3beta (GSK-3beta). In addition, the structural requirements of VPA-related compounds to cause InsP3 depletion are unknown. In the current study, we selected a set of 10 VPA congeners to examine their effects on InsP3 depletion, in vivo teratogenic potency, HIV replication, and GSK-3beta activity in vitro. We found four compounds that function to deplete InsP3 in the model eukaryote Dictyostelium discoideum, and these drugs all cause growth-cone enlargement in mammalian primary neurons, consistent with the effect of InsP3 depletion. No relationship was found between InsP3 depletion and teratogenic or elevated viral replication effects, and none of the VPA congeners were found to affect GSK-3beta activity. Structural requirements of VPA congers to maintain InsP3 depletion efficacy greater than that of lithium are a carboxylic-acid function without dependence on side-chain length, branching, or saturation. Noteworthy is the enantiomeric differentiation if a chiral center exists, suggesting that InsP3 depletion is mediated by a stereoselective mode of action. Thus, the effect of InsP3 depletion can be separated from that of teratogenic potency and elevated viral replication effect. We have used this to identify two VPA derivatives that share the common InsP3-depleting action of VPA, lithium and carbamazepine, but do not show the side effects of VPA, thus providing promising novel candidates for bipolar disorder treatment.

[Valpromide, Valproic acid and removal of small intestine in the treatment of a chronic depression: a case report]. / Traitement d'une dépression résistante par substitution du Dépamide par la Dépakine chez une patiente avec résection du grêle.

Valpromide (VPD) is an antiepileptic drug, derivative of Valproic acid (VPA), used as a mood-stabilizer in bipolar disorder for 25 years in several European countries. VPD is also used as an augmentation strategy in refractory depression. Despite chemical similarity between VPA and VPD, the pharmacokinetics of the 2 drugs in humans are quite distinct. We report a case of a patient, suffering from a bipolar treatment resistant depression, who dramatically improved after substituting VPD to VPA in association with fluoxetine. Mme X, 68 years old, has been hospitalized in March 2001 for the treatment of a resistant depression (TRD). She was suffering from removal of small intestine with chronic diarrhoea after a suicidal attempt two years ago. She had a bipolar disorder treated with VPD (1,200 mg/d) since 1 year. She presented a major depressive episode according to DSM IV with various symptoms like depressed mood, hypersomnia and difficulty initiating sleep, diminished ability to concentrate and to think, markedly diminished pleasure in all activities and major anxiety. Mme X fulfilled TRD diagnosis after resistance to two adequate antidepressants trials from different classes (clomipramine 175 mg/day and venlafaxine 300 mg/day). The antidepressant treatment (venlafaxine) was interrupted and she has been receiving a SSRI (fluoxetine 20 mg/day) for 4 weeks. After four weeks, she had a partial remission with persistent sleep problems, mood lability and anxiety. The VPA blood concentration was very low: 27 mg/L (normal range: 50 to 100 mg/L) in spite of a high dosage: 1,200 mg/day. Pharmacokinetic analysis of VPD shown that VPD transformation to VPA usually done in the intestine, was reduced because of the removal of hail intestine. We substituted VPD by VPA. Valproate blood concentration returned to normal range, induced dramatic improvement of depression within three days. VPD is an amide derivative of valproic acid (valproate), biotransformed by hydrolysis to its corresponding valproic acid. VPD is a prodrug of VPA. VPD is absorbed after transformation in gastro-intestinal mucous membrane. The adequate dosage of VPD (Depamide, 300 mg) is 4 to 6 tablets in acute manic phases, 2 to 4 tablets in long term treatment, 1 to 3 tablets in depressive episode. The biodisponibility of VPD is around 100% 75 and 90% of VPD is linked with protein albumin. The daily dosage determined the blood concentration of the active form (VPA), but this relation isn't linear. The optimal blood concentration of VPA (Depakine) ranges between 50 and 100 mg/L. the free form of VPA is influenced by protein disorders such as of hypoalbuminemia and by presence of fat acids in food. This case report demonstrates at a clinical level that VPD and VPA are not equivalent for treating bipolar depression. This case also suggests that a deep investigation of the pharmacokinetic of psychotropic drugs can help clinicians to resolve clinical problems of treatment of depression.

Characterization of the anticonvulsant profile of valpromide derivatives.

The antiepileptic activity of nine derivatives of valpromide is discussed. They comply with a pharmacophore model that establishes the essential structural and electronic features responsible for the protection against the MES test. The model results from the comparison of 17 structures, using density functional methodologies combined with an active analog approach. The derivatives of valpromide have been tested for anticonvulsant activity in mice. These compounds displayed a phenytoin-like profile, being active in the MES test and inactive in the PTZ test. 4-(Valproylamido)benzenesulfonamide is the most active compound, with an ED(50) of 53 micromol/kg and no neurotoxicity at doses up to 1000 micromol/kg. The pharmacological behavior of the drugs points to a sodium channel blocking effect as one of the associated mechanisms. This mechanism was tested positive for N-ethylvalpromide through its competition with the binding of [(3)H]batrachotoxin-A-20 alpha-benzoate to the voltage-dependent sodium channels from rat brain synaptosomes.

DP-VPA D-Pharm.

Shire is developing DP-VPA, a prodrug of valproic acid (VPA) licensed from D-Pharm, for the potential treatment of severe forms of epilepsy, including status epilepticus, acute repetitive seizures in children and possibly manic depression and migraine [242649], [385958]; the drug is also being developed for bipolar disorder and migraine prophylaxis [385862]. By March 2000, phase I trials had been completed [359581], [373232] and in October 2000, the compound entered phase II trials for the treatment of epilepsy, bipolar disorder and migraine [385862]; these trials were ongoing in November 2001 [429470]. In October 2001, Shire initiated multicenter, multinational phase II trials of SPD-421 as add-on therapy in the treatment of complex partial seizures [425660]. In February 2002, Shire reported that it planned to make a decision regarding the future of the compound by the second half of 2002 [441819]. DP-VPA is based on D-Pharm's regulation activation of prodrugs (RAP) technology, which designs drugs to be internalized within cells and to be activated only when the tissue becomes diseased. In the case of DP-VPA, the pathological epilepsy process activates it on demand [342433]. RAP-prodrugs are composed of the active drug moiety attached via a chemical linkage to a hydrophobic molecule which allows the agent to penetrate into the cell [182806]. In October 2000, D-Pharrm received patent US-06077837 from the USPTO covering novel 'Prodrugs with enhanced penetration into cells'. This patent claims prodrugs, comprising a pharmacologically active compound covalently linked to an intracellular transporter by a bond which is preferentially cleaved by disease-associated supranormal enzyme activity [385507]. In April 2000, Lehman Brothers predicted the launch of DP-VPA during 2003 [365103]. In December 2001, Lehman Brothers suggested that the product would be approved in 2004. The analysts estimated a 25% probability of the drug reaching the market, and envisaged peak sales of $200 million [434768]. Analysts at Schroder Salomon Smith Barney predicted in March 2002, that sales for Shire of DP-VPA would reach US $9.5 million in 2005, rising to US $31 million in 2006 [449087].

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.

Stereoselective pharmacokinetic analysis and antiepileptic activity of N-2-hydroxypropyl valpromide, a central nervous system--active chiral valproylamide.

The purpose of this study was to evaluate the anticonvulsant activity and pharmacokinetics (PK) of a novel chiral CNS-active 2-hydroxypropyl valpromide (HP-VPD), a derivative of valproic acid (VPA). The individual enantiomers, R, S, and racemic (R,S)-HP-VPD were synthesized and evaluated for their pharmacokinetics and pharmacodynamics in a stereoselective manner. A stereoselective gas chromatography (GC) assay for simultaneous quantification of HP-VPD enantiomers in plasma and urine was developed and used to investigate the pharmacokinetics of HP-VPD in dogs. Pharmacodynamic analysis in rats showed that (S)-HP-VPD was 2.5 times more potent as an anticonvulsant in the maximal electroshock seizure (MES) test than its enantiomer and approximately 10 times more potent than VPA. No significant differences were observed in major PK parameters (clearance, volume of distribution, and half-life) between S and (R)-HP-VPD, and this suggested that pharmacodynamic differences could be attributed to the intrinsic pharmacodynamics of each enantiomer rather than to a preferable pharmacokinetic profile. The pharmacokinetic (metabolic) analysis showed that the fraction metabolized to HP-VPD-glucuronide ranged from 5% to 7% and no biotransformation of HP-VPD to VPA and 2-ketopropyl valpromide was observed. This is the first report of significant stereoselectivity in the anticonvulsant activity of a valproylamide with a chiral carbon situated on the alkyl chain of the amine moiety.

Prediction of embryotoxic effects of valproic acid-derivatives with molecular in vitro methods.

Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause similar teratogenic effects (neural tube defects) in human and mice. In this study a new molecular bioassay is presented using following endpoints: differentiation of F9 teratocarcinoma cells, altered cell morphology, induction of possible targeted genes, and the induction of viral RSV-promoter. The induction of a transiently transfected viral (RSV) promoter driven luciferase gene by VPA was used to screen a set of VPA-derivatives. Structure-activity investigations showed: the longer the aliphatic side chain the more the induction of the RSV-reporter gene. The specific induction was stereoseletive. The teratogenic enantiomer S-4-yn-VPA (2-propyl-4-pentynoic acid) induced the RSV-driven reporter gene while the non teratogenic R-4-yn-VPA does not. Heptyl-4-yn-VPA was the most potent teratogen in vitro and in vivo. Non teratogenic VPA-derivatives like R-4-yn-VPA and 2-en-VPA (2-propyl-2-pentenoic acid) were ineffective in this system. Thus, the teratogenic effect of VPA and VPA-derivatives in the mouse correlated with the specific induction of the viral RSV-promoter controlled reporter in F9-cells. Acid compounds such as fatty acids are known to interact with peroxisome proliferator-activated receptors (PPARs). To test structure-activity relationships by VPA or its derivatives we used CHO cells stably expressing hybrid proteins of the ligand-binding domain of either of the PPARs. The teratogen VPA and the teratogenic derivatives of VPA activated the PPAR-delta construct in a very specific structure- and stereoselective way which correlated well with the activities in the reporter gene assay (bioassay) and those in vivo. No such correlation was found with respect to activation of PPAR-alpha or PPAR-gamma. These structure-activity relationships indicate that PPAR-delta may be a potential mediator of VPA-induced differentiation of F9 cells and may possibly be involved in the mechanism of teratogenicity of VPA in vivo. Furthermore two bioassays were designed with clearly defined endoints, amenable to automation and screening of great number of compounds. The test system allows to replace animal experiments in the preclinical development of new antiepileptics drugs with reduced teratogenic risk. Supported by BgVV-ZEBET (Berlin).

P450-catalyzed in-chain desaturation of valproic acid: isoform selectivity and mechanism of formation of Delta 3-valproic acid generated by baculovirus-expressed CYP3A1.

The mechanism of formation of the in-chain, unsaturated fatty acid metabolite, Delta3-valproic acid (Delta3-VPA) by rat liver microsomes was examined. Microsomal rates of formation of Delta3-VPA were below quantifiable limits in reactions catalyzed by control female rat liver microsomes, but were induced more than 20-fold following pretreatment with triacetyloleandomycin and pregnenolone-16alpha-carbonitrile. Microsomal incubations conducted with 3-hydroxy-VPA or [2-2H1]VPA demonstrated that Delta3-VPA did not arise by dehydration of preformed alcohol nor was it reversibly isomerized to Delta2-VPA. CYP3A1 expression was optimized in the baculovirus expression vector system, and infected insect cell membranes which were supplemented with P450 reductase catalyzed formation of 3-OH-, 4-OH-, 5-OH-, Delta3-, and Delta4-VPA in ratios of 160:35:6:3:1. Intramolecular deuterium isotope effects on metabolite formation, determined with cDNA-expressed CYP3A1 and either [3,3-2H2]VPA or [4,4-2H2]VPA, yielded kH/kD values for Delta3-VPA of 2.00 +/- 0.06 and 2.36 +/- 0.08, respectively. These values were significantly lower than the isotope effects observed in the same incubations for 3-OH-VPA formation from 3,3-D2-VPA (kH/kD = 6.04 +/- 0.08), or for 4-OH- and Delta4-VPA formation from 4, 4-D2-VPA (kH/kD > 5). Collectively, these data demonstrate the existence of a microsomal P450-dependent in-chain fatty acid desaturase system distinct from the well-documented cytochrome b5-linked CoA desaturases and suggest further that CYP3A1-dependent formation of Delta3-VPA arises via nonselective, initial hydrogen atom abstraction from either the C-3 or the C-4 position.