Giovanna Guiso

Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder involving the selective degeneration of motor neurons. In a small proportion of patients, ALS is caused by mutations in copper/zinc superoxide dismutase (SOD1), and mice overexpressing SOD1G93A mutant develop a syndrome that closely resembles the human disease. Excitotoxicity mediated by glutamate AMPA receptors has been suggested to be implicated in the selective susceptibility of motor neurons occurring in ALS. In SOD1G93A mice, we found that levels of GluR2 AMPA subunit, which plays a pivotal role in the maintenance of calcium impermeability of AMPA receptors, are decreased in spinal motor neurons before symptom onset in concomitance with a modest increase of GluR3 expression, a calcium‐permeable AMPA subunit. This effect can result in a higher number of calcium‐permeable AMPA receptors on motor neurons of SOD1G93A mice, predisposing these cells to be injured by AMPA‐mediated glutamate firing. In support of this, we showed that treatment with a new noncompetitive AMPA antagonist, ZK 187638, partially protected motor neurons, improved motor function, and prolonged the survival of SOD1G93A mice.

Comparative studies on the anorectic activity of d-fenfluramine in mice, rats, and guinea pigs

SummaryThe present study compares the anorectic activity of d-fenfluramine and its metabolite d-norfenfluramine in three animal species. d-Fenfluramine and d-norfenfluramine show anorectic activity at increasing doses (ED50) in rats, guinea pigs, and mice, d-norfenfluramine being more active than d-fenfluramine in all three species. Equiactive anorectic activities are reached with different brain levels of d-fenfluramine and d-norfenfluramine, guinea pigs being the most sensitive species, followed by rats then mice. The metabolite most probably plays a major role in the anorectic effect of d-fenfluramine in guinea pigs, contributes to the anorectic activity in rats, but adds little to the action of the parent drug in mice. The different sensitivity to d-fenfluramine and d-norfenfluramine in these three species does not appear to be explained by a number of biochemical parameters, including serotonin uptake or release, receptor subtypes, or 3H-d-fenfluramine binding and uptake.

A Pilot Study on Brain-to-Plasma Partition of 10,11-Dyhydro-10-hydroxy-5H-dibenzo(b,f)azepine-5-carboxamide and MDR1 Brain Expression in Epilepsy Patients Not Responding to Oxcarbazepine

Summary:  Purpose: We measured the brain‐to‐plasma partition of 10,11‐dihydro‐10‐hydroxy‐5H‐dibenzo(b,f)azepine‐5‐carboxamide (10‐OHCBZ) in epilepsy patients undergoing surgery to alleviate drug‐resistant seizures and administered with different oral doses of oxcarbazepine (OXC). We addressed the possible contribution of the multidrug transporter P‐glycoprotein (P‐gp or MDR1) in determining 10‐OHCBZ brain levels by measuring whether this active metabolite is a substrate of P‐gp and the relation between the level of expression of MDR1 and the drug concentration in the same brain tissue specimens.

Combining 4-aminoquinoline- and clotrimazole-based pharmacophores toward innovative and potent hybrid antimalarials.

Antimalarial agents structurally based on novel pharmacophores, synthesized by low-cost synthetic procedures and characterized by low potential for developing resistance are urgently needed. Recently, we developed an innovative class of antimalarials based on a polyaromatic pharmacophore. Hybridizing the 4-aminoquinoline or the 9-aminoacridine system of known antimalarials with the clotrimazole-like pharmacophore, characterized by a polyarylmethyl group, we describe herein the development of a unique class (4a-l and 5a-c) of antimalarials selectively interacting with free heme and interfering with Plasmodium falciparum (Pf) heme metabolism. Combination of the polyarylmethyl system, able to form and stabilize radical intermediates, with the iron-complexing and conjugation-mediated electron transfer properties of the 4(9)-aminoquinoline(acridine) system led to potent antimalarials in vitro against chloroquine sensitive and resistant Pf strains. Among the compounds synthesized, 4g was active in vivo against P. chabaudi and P. berghei after oral administration and, possessing promising pharmacokinetic properties, it is a candidate for further preclinical development.

In vitro responsiveness of human-drug-resistant tissue to antiepileptic drugs: Insights into the mechanisms of pharmacoresistance

Pharmacoresistance in epileptic patients may be ascribed to at least two, not mutually exclusive, mechanisms: a pharmacokinetic mechanism and a decreased sensitivity or availability of targets to antiepileptic drugs (AEDs; i.e., carbamazepine and phenytoin (CBZ, PHT)). Brain:plasma drug concentration ratios were determined intraoperatively during lobectomies performed to alleviate drug-resistant seizures. The brain:plasma ratio of CBZ was 1.48 when therapeutic serum levels (15-34 microM) were achieved. When concentrations of CBZ found in multiple-drug-resistant brain were directly applied to human cortical slices from drug-resistant patients made hyperexcitable and hypersynchronous by Mg(2+)-free media, bursting frequency was not significantly affected and overall excitability was reduced by 40%. Similar results were obtained for PHT. At higher AED concentrations (60-200 microM), a dose-dependent decrease of bursting frequency and amplitude was observed. Slices from drug-resistant epileptic patients made hypersynchronous/hyperexcitable by elevated potassium or inhibition of GABA-A receptors behaved similarly. Of note is the response of slices from human multiple-drug-resistant brain, which was greater than in rodent cortex from naive animals. Taken together, our results support the hypothesis that multiple drug resistance to AEDs involves cerebrovascular changes that impede the achievement of appropriate drug levels in the central nervous system.

Species differences in clobazam metabolism and antileptazol effect

The antileptazol effect of clobazam lasts longer in the mouse than in the rat. After intraperitoneal injection of clobazam (10 mg kg−1) plasma and brain concentrations of the drug and its rate of disappearance were similar in both species, whereas the metabolite N demethylclobazam was present at higher concentrations and for longer in the mouse than in the rat. Although the exact contribution of the N‐desmethylclobazam to the anticonvulsant effect of clobazam cannot be assessed, the longer duration in mice than in rats seems to be associated with different brain accumulations of the metabolite.

Determination of piribedil and its basic metabolites in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of piribedil and its p-hydroxylated, catechol and N-oxide metabolites in plasma is described. After addition of an internal standard (buspirone), the plasma samples were subjected to a three-step extraction procedure. The final extracts were evaporated to dryness under nitrogen, and the residues were reconstituted in 100 microliters of mobile phase (0.01 M phosphate buffer-acetonitrile, 50:50, v/v) and chromatographed by acetonitrile gradient elution on a C18 reversed-phase column coupled to an ultraviolet detector set at 240 nm. The method was selective for piribedil and its metabolites, and sufficiently sensitive and precise for studies aimed at elucidating the role of the metabolites in the parent drugs pharmacological effects.

Optimized synthesis of AMPA receptor antagonist ZK 187638 and neurobehavioral activity in a mouse model of neuronal ceroid lipofuscinosis.

Previous structure–activity relationship studies in the search for a potent, noncompetitive α‐amino‐3‐(3‐hydroxy‐5‐methyl‐4‐isoxazolyl)propionic acid (AMPA) receptor antagonist led to 2,3‐dimethyl‐6‐phenyl‐12H‐[1,3]dioxolo[4,5‐h]imidazo[1,2‐c][2,3]benzodiazepine (ZK 187638). However, the first synthesis had some drawbacks regarding reagents, processes, and overall yield, which furthermore decreased when the synthesis was scaled up. Therefore, we now report a new synthetic route for this compound which requires fewer steps and is suited for large‐scale production. This compound significantly relieved the symptoms of neuromuscular deficit in mnd mice, a model of neuronal ceroid lipofuscinosis with motor neuron dysfunction. After oral administration, the concentrations of the compound in the brain and spinal cord were about threefold higher than those in the plasma. In summary, this novel AMPA antagonist is accessible through an optimized synthetic route, has good neurobehavioral activity, oral bioavailability, and favorable brain penetration. This opens new possibilities for the treatment of devastating neurological diseases that are mediated by the AMPA receptor.

Determination of orally administered all-trans-retinoic acid in human plasma by high-performance liquid chromatography.

All-trans-retinoic acid (RA) is used successfully in the treatment of acute promyelocytic leukaemia (APL), although unexplained relapses occur in many of the patients. Pharmacokinetic studies may help in understanding the mechanism of resistance to RA and a simple and rapid procedure for its determination in biological samples may be advantageous. A high-performance liquid chromatographic procedure is described, involving one-step extraction of RA from plasma, isocratic elution from a reversed-phase column (LiChrosorb RP-18, 5-microns particle size) and UV detection at 340 nm. The calibration graph is linear over a wide range and the limit of detection is approximately 10 ng/ml, using 0.5 ml of human plasma. The method is selective for RA, accurate and robust and thus suitable for the routine analysis of plasma samples from patients undergoing RA therapy. Analysis of plasma in a patient on RA therapy (45 mg/m2 per day) confirmed that during continuous treatment with RA the drug plasma concentrations are markedly lower at the time of relapse than on the first day of treatment.

Studies on the Susceptibility to Convulsions in Animals Receiving Abuse Doses of Aspartame

Aspartame and its major metabolite phenylalanine were studied for their capacity to potentiate metrazol, electroshock, and quinolinic-acid-induced seizures in rats. Pretreatment with 0.75 and 1 g/kg aspartame or 0.250 and 0.500 g/kg phenylalanine administered by gavage significantly increased the number of animals undergoing convulsions after a dose of 65.9 mg/kg of metrazol. This effect was no longer significant when 1 g/kg aspartame was given as three divided doses over two hours, after a meal or overnight with food and drinking water. However, a dose of 1 g/kg aspartame or 0.500 g/kg phenylalanine did not influence the threshold electrical current necessary to elicit convulsions in 50% of animals or the limbic seizures induced by convulsant doses of quinolic acid. After doses of 1 g/kg aspartame or 0.500 g/kg phenylalanine, striatal and hippocampal levels of 5-HT, NA, DA and their metabolites were not significantly modified. Also, in vivo release of striatal DA measured by brain microdialysis was not changed by treatment with these compounds. Under different treatment schedules, significant increases in brain phenylalanine and tyrosine were observed in rats treated acutely with doses of 250 mg/kg or more.