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NEUROPHARMACOLOGY

Neuroprotective Effects of the Novel Glutamate Transporter Inhibitor (–)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]-isoxazole-4-carboxylic Acid, Which Preferentially Inhibits Reverse Transport (Glutamate Release) Compared with Glutamate Reuptake

Istituto di Ricerche Farmacologiche "Mario Negri," Milano, Italy (S.C., E.F., T.M., M.G.); Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A.J.); Dipartimento di Farmacologia Preclinica e Clinica, Università di Firenze, Firenze, Italy (E.L., D.E.P.-G.); and Istituto di Chimica Farmaceutica e Tossicologica "Pietro Pratesi," Università di Milano, Milan, Italy (P.C., A.P., M.D.A., C.D.M.)

(±)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo [3,4 -d]-isoxazole-4-carboxylic acid (HIP-A) and (±)-3-hydroxy-4,5,6, 6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B) are selective inhibitors of excitatory amino acid transporters (EAATs), as potent as DL-threo-β-benzyloxyaspartic acid (TBOA). We report here that the active isomers are (–)-HIP-A and (+)-HIP-B, being approximately 150- and 10-fold more potent than the corresponding enantiomers as inhibitors of [³H]aspartate uptake in rat brain synaptosomes and hEAAT1–3-expressing cells. Comparable IC₅₀ values were found on the three hEAAT subtypes. (–)-HIP-A maintained the remarkable property, previously reported with the racemates, of inhibiting synaptosomal glutamate-induced [³H]D-aspartate release (reverse transport) at concentrations significantly lower than those inhibiting [³H]L-glutamate uptake. New data suggest that the noncompetitive-like interaction described previously is probably the consequence of an insurmountable, long-lasting impairment of EAAT's function. Some minutes of preincubation are required to induce this impairment, the duration of preincubation having more effect on inhibition of glutamate-induced release than of glutamate uptake. In organotypic rat hippocampal slices and mixed mouse brain cortical cultures, TBOA, but not (–)-HIP-A, had toxic effects. Under ischemic conditions, a neuroprotective effect was found with 10 to 30 µM (–)-HIP-A, but not with 10 to 30 µM TBOA or 100 µM (–)-HIP-A. The effect of (–)-HIP-A suggests that, under ischemia, EAATs mediate both release (reverse transport) and uptake of glutamate. The neuroprotection with the lower (–)-HIP-A concentrations may indicate a selective inhibition of the reverse transport confirming the data obtained in synaptosomes. The selective interference with glutamate-induced glutamate release might offer a new strategy for neuroprotective action.

Address correspondence to: Dr. Marco Gobbi, Istituto di Ricerche Farmacologiche "Mario Negri," Via La Masa 19, 20156 Milan, Italy. E-mail: Gobbi{at}marionegri.it