Flavio Moroni

Clinical Pharmacokinetics of Valproic Acid - 1988

SummarySodium valproate (valproic acid) has been widely used in the last decade and is now considered a relatively safe and effective anticonvulsant agent. Recently, several investigators have proposed its use in the treatment of anxiety, alcoholism and mood disorders, although these indications require further clinical studies.Valproic acid is available in different oral formulations such as solutions, tablets, enteric-coated capsules and slow-release preparations. For most of these formulations bioavailability approaches 100%, while the absorption half-life varies from less than 30 minutes to 3 or 4 hours depending on the type of preparation used. Once absorbed, valproic acid is largely bound to plasma proteins and has a relatively small volume of distribution (0.1 to 0.4 L/kg). Its concentration in CSF is approximately one-tenth that in plasma and is directly correlated with the concentration found in tears. At therapeutic doses, valproic acid half-life varies from 10 to 20 hours in adults, while it is significantly shorter (6 to 9 hours) in children.Valproic acid undergoes extensive liver metabolism. Numerous metabolites have been positively identified and there is reasonable evidence that several of them contribute to its pharmacological and toxic actions. In fact, several valproic acid metabolites have anti-convulsant properties, while many of the side effects it may cause (e.g. those related to hyperammonaemia or liver damage) are most often observed in patients previously treated with phenobarbitone. This could indicate that induction of liver enzymes is responsible for the formation of toxic valproic acid metabolites.

Adenosine decreases aspartate and glutamate release from rat hippocampal slices

The effect of adenosine and related compounds on the release of endogenous aspartate and glutamate from isolated, superfused rat hippocampal slices was studied at rest and during electrical stimulation of the stratum radiatum in the CA3/CA2 region, using a sensitive mass-spectrometric technique. Evoked extracellular potentials were recorded from the CA1 region. Adenosine, at 3 X 10(-4) M concentration, inhibited the stimulation-evoked potentials and prevented the stimulation-induced release of aspartate and glutamate. Similarly, 1-phenylisopropyladenosine (10(-6) M) and cyclohexyladenosine (10(-6) M) depressed both electrical and neurochemical responses to stimulation of the stratum radiatum. 8-Phenyltheophylline (5 X 10(-6) M) increased the release of aspartate and glutamate and antagonized the cyclohexyladenosine-induced inhibition of amino acid release. Our results support the hypothesis that adenosine modulates the electrophysiological responses to stimulation of stratum radiatum through a reduction of the release of the excitatory amino acids aspartate and glutamate.

Presence of kynurenic acid in the mammalian brain.

Kynurenic acid, a tryptophan metabolite able to antagonize the actions of the excitatory amino acids, has been identified and measured for the first time in the brain of mice, rats, guinea pigs, and humans by using an HPLC method. Its content was 5.8 ± 0.9 in mouse brain, 17.8 ± 2.0 in rat brain, 16.2 ± 1.5 in guinea pig brain, 26.8 ± 2.9 in rabbit brain, and 150 ± 30 in human cortex (pmol/g wet wt, mean ± SE). The regional distribution of this molecule was uneven. In rats, guinea pigs, and rabbits, the brainstem was the area richest in this compound. Tryptophan administration (100–300 mg/kg, i.p.) to rats resulted in a significant increase of the brain content of kynurenic acid. Similarly, 1 h after probenecid administration (200 mg/kg, i.p.), the brain content of kynurenate increased by fourfold, thus suggesting that its turnover rate is relatively fast.

GM1 ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system

The effect of GM1 ganglioside on the recovery of nigro-strital dopaminergic neurons was studied in rats after unilateral hemitransection. We find that repeated administration of GM1 significantly increased the HVA content, the tyrosine hydroxylase activity and the tyrosine hydroxylase-related immunofluorescence in the striatum ipsilateral to the lesion. Futhermore, GM1 reduced the sensitivity of lesioned rats to apomorphine. The data comparable with the view that a functional dopaminergic reinnervation of the striatum is facilitated by GM1 treatments after hemitransection.

Excitatory Amino Acid Release from Rat Hippocampal Slices as a Consequence of Free-Radical Formation

Abstract: The release of D‐[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hy‐droxyl‐radical scavenger D‐mannitol, or the superoxide‐radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.

Pharmacological Inhibition of Histone Deacetylases by Suberoylanilide Hydroxamic Acid Specifically Alters Gene Expression and Reduces Ischemic Injury in the Mouse Brain

Pharmacological manipulation of gene expression is considered a promising avenue to reduce postischemic brain damage. Histone deacetylases (HDACs) play a central role in epigenetic regulation of transcription, and inhibitors of HDACs are emerging as neuroprotective agents. In this study, we investigated the effect of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on histone acetylation in control and ischemic mouse brain. We report that brain histone H3 acetylation was constitutively present at specific lysine residues in neurons and astrocytes. It is noteworthy that in the ischemic brain tissue subjected to6hof middle cerebral artery occlusion, histone H3 acetylation levels drastically decreased, without evidence for a concomitant change of histone acetyl-transferase or deacetylase activities. Treatment with SAHA (50 mg/kg i.p.) increased histone H3 acetylation within the normal brain (of approximately 8-fold after 6 h) and prevented histone deacetylation in the ischemic brain. These effects were accompanied by increased expression of the neuroprotective proteins Hsp70 and Bcl-2 in both control and ischemic brain tissue 24 h after the insult. It is noteworthy that at the same time point, mice injected with SAHA at 25 and 50 mg/kg had smaller infarct volumes compared with vehicle-receiving animals (28.5% and 29.8% reduction, p < 0.05 versus vehicle, Students t test). At higher doses, SAHA was less efficient in increasing Bcl-2 and Hsp70 expression and did not afford significant ischemic neuroprotection (13.9% infarct reduction). Data demonstrate that pharmacological inhibition of HDACs promotes expression of neuroprotective proteins within the ischemic brain and underscores the therapeutic potential of molecules inhibiting HDACs for stroke therapy.

High mobility group box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo

High mobility group proteins are chromatin binding factors with key roles in maintenance of nuclear homeostasis. The evidence indicates that extracellularly released high mobility group box 1 (HMGB1) protein behaves as a cytokine, promoting inflammation and participating to the pathogenesis of several disorders in peripheral organs. In this study, we have investigated the expression levels and relocation dynamics of HMGB1 in neural cells, as well as its neuropathological potential. We report that HMGB1 is released in the culture media of neurons and astrocytes challenged with necrotic but not apoptotic stimuli. Recombinant HMGB1 prompts induction of pro‐inflammatory mediators such as inducible nitric oxide synthase (iNOS), cyclooxygenase‐2, interleukin‐1β, and tumor necrosis factor α, and increases excitotoxic as well as ischemic neuronal death in vitro. Dexamethasone reduces HMGB1 dependent immune glia activation, having no effect on the protein’s neurotoxic effects. HMGB1 is expressed in the nucleus of neurons and astrocytes of the mouse brain, and promptly (1 h) translocates into the cytoplasm of neurons within the ischemic brain. Brain microinjection of HMGB1 increases the transcript levels of pro‐inflammatory mediators and sensitizes the tissue to the ischemic injury. Together, data underscore the neuropathological role of nuclear HMGB1, and point to the protein as a mediator of post‐ischemic brain damage.

Presynaptic kynurenate-sensitive receptors inhibit glutamate release

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and α7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30–100 nm) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3–10 nm), a selective α7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nm), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mm)‐induced release of transmitter or previously taken‐up [3H]‐D‐aspartate. This inhibition was not modified by glycine, N‐methyl‐d‐aspartate or subtype‐selective kainate receptor agents, while CNQX or DNQX (10 µm), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]‐kainate (high and low affinity) or [3H]‐AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1–100 nm) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate‐induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.

Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats.

1 The effects of the Ca2+‐channel blockers verapamil and nimodipine, on the behavioural signs of naloxone (1 mg kg−1)‐induced abstinence syndrome in morphine‐dependent rats, were evaluated. The content of noradrenaline (NA) and of its metabolite 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) was measured, using high performance liquid chromatography and electrochemical detection or gas chromatography‐mass spectrometry, in various brain regions of these animals. Possible interactions of nimodipine and verapamil with opioid receptors were evaluated by examining their ability to displace [3H]‐naloxone binding to brain membranes. 2 Verapamil (5, 10 and 50 mg kg−1) and nimodipine (1,5 and 10 mg kg−1) dose‐dependently reduced most of the signs of morphine abstinence. 3 Naloxone‐precipitated abstinence decreased the NA content in the cortex, hippocampus, brainstem and cerebellum. In the same brain regions the content of MHPG increased, suggesting an increased release of the amine during morphine abstinence. 4 Nimodipine (10 mg kg−1 i.v.) did not change the content of NA or MHPG in the cortex, hippocampus and brainstem. However, nimodipine pre‐treatment markedly reduced the changes in NA and MHPG content induced by the abstinence syndrome. 5 Neither verapamil nor nimodipine displaced [3H]‐naloxone from its binding sites. 6 These results suggest that Ca2+‐channel blockers suppress the behavioural and neurochemical expressions of morphine abstinence by a mechanism that differs from those of opioids or α2‐adrenoceptor agonists.

Inhibitors of kynurenine hydroxylase and kynureninase increase cerebral formation of kynurenate and have sedative and anticonvulsant activities

Kynurenate is an endogenous antagonist of the ionotropic glutamate receptors. It is synthesized from kynurenine, a tryptophan metabolite, and a significant increase in its brain concentration could be useful in pathological situations. We attempted to increase its neosynthesis by modifying kynurenine catabolism. Several kynurenine analogues were synthesized and tested as inhibitors of kynurenine hydroxylase (E.C.1.14.13.9) and of kynureninase (E.C.3.7.1.3), the two enzymes which catalyse the conversion of kynurenine to excitotoxin quinolinate. Among these analogues we observed that nicotinylalanine, a compound whose pharmacological properties have previously been reported, had an IC50 of 900 +/- 180 microM as inhibitor of kynurenine hydroxylase and of 800 +/- 120 microM as inhibitor of kynureninase. In the search for more potent molecules we noticed that meta-nitrobenzoylalanine had an IC50 of 0.9 +/- 0.1 microM as inhibitor of kynurenine hydroxylase and of 100 +/- 12 microM as inhibitor of kynureninase. When administered to rats meta-nitrobenzoylalanine (400 mg/kg) significantly increased the concentration of kynurenine (up to 10 times) and kynurenate (up to five times) in the brain. Similar results were obtained in the blood and in the liver. Furthermore meta-nitrobenzoylalanine increased in a dose dependent, long lasting (up to 13 times and up to 4 h) manner the concentration of kynurenate in the hippocampal extracellular fluid, as evaluated with a microdialysis technique. This increase was associated with a decrease in the locomotor activity and with protection from maximal electroshock-induced seizures in rats or from audiogenic seizures in DBA/2 mice. The conclusions drawn from the present study are: (i) meta-nitrobenzoylalanine is a potent inhibitor of kynurenine hydroxylase also affecting kynureninase; (ii) the inhibition of these enzymes causes a significant increase in the brain extracellular concentration of kynurenate; (iii) this increase is associated with sedative and anticonvulsant actions, suggesting a functional antagonism of the excitatory amino acid receptors.