Bioavailability, multiple-dose pharmacokinetics, and biotransformation of the aldose reductase inhibitor zopolrestat in dogs.

Zopolrestat (Alond) is a new drug that is being evaluated as an aldose reductase inhibitor for the treatment of diabetic complications. The bioavailability in dogs of a 2 mg/kg oral dose of zopolrestat was 97.2%. In a 1-year, multiple-dose, pharmacokinetic study, systemic exposure increased with increasing dose (50, 100, and 200 mg/kg/day), and there were no consistent changes in exposure with multiple dosing. Renal clearance at 1 year appeared to be higher in males. The magnitude of the potential gender difference in exposure was relatively small and was unlikely to have had a meaningful impact on the pharmacokinetics of zopolrestat in dogs. In studies with bile duct-cannulated dogs, radioactivity from [14C]zopolrestat was primarily eliminated as unchanged drug and acyl glucuronide in the bile and feces (77.3% of the dose) and in urine (18.3% of the dose). The concentrations of acyl glucuronide in urine and feces were approximately 50% of the zopolrestat concentrations. Minor metabolites (each accounting for <1% of the dose) included those resulting from hydroxylation of the phthalazinone ring and glutathione conjugation of the benzothiazole ring.

Effect of methyl bromide on regional brain glutathione, glutathione-S-transferases, monoamines, and amino acids in F344 rats.

Both metabolic and neurotransmitter changes have been implicated in the pathogenesis of monohalomethane neurotoxicity in rodents. This study in male and female F344 rats examined the effects of methyl bromide (MeBr) on regional brain glutathione-S-transferase (GST) activities and concentrations of glutathione (GSH), monoamines, and amino acid. Inhalation exposure to 150 ppm MeBr (6 hr/day x 5 days) yielded no histologic evidence of brain lesions but resulted in a number of biochemical changes. GSH depletion and GST inhibition were detected in the frontal cortex, caudate nucleus, hippocampus (examined for GSH only), brain stem, and cerebellum from animals of both sexes. Differences between sexes were detected for GSH depletion. Simultaneous treatment of rats with the inhibitor of monohalomethane toxicity, BW 755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline; 10 mg/kg bw ip, 1 hr pre- and 1 hr postexposure) completely protected against GST inhibition in all brain regions of both sexes. Partial protection by BW 755C against GSH depletion was observed in the cerebral cortex and in the cerebellum only. In males, MeBr exposure had no effect on the regional concentrations of the monoamines dopamine and serotonin and the amino acids glutamate, glutamine, taurine, and gamma-aminobutyric acid. Regional increases of brain aspartate and glycine levels were observed after exposure of males to MeBr but BW 755C had no effect on these changes induced by MeBr. Thus, of all the parameters studied, only GST, and in some brain areas GSH, correlated with inhibition of toxicity. It is concluded that, in contrast to the monoamines and the amino acids, GST and GSH are sensitive and potentially relevant indicators of MeBr neurotoxicity which could explain sex and regional differences in response to the monohalomethanes.

In vitro neurotoxicity of methyl iodide.

Methyl iodide (MeI) and two other commonly used monohalomethanes, methyl bromide and methyl chloride, are potent human neurotoxicants. In the present study neural cell cultures were used to investigate MeI neurotoxicity in vitro. In primary, dispersed mixed (neurons and glia) neural cultures from mouse embryos, MeI produced severe morphological alterations and leakage of lactate dehydrogenase into the medium (LC(50), 5-6 mm). These effects showed steep concentration-response curves. Both glial and neuronal cells from both the cerebral cortex and the cerebellum were affected. The dual cyclooxygenase-lipoxygenase inhibitor, 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW755C) protected against monohalomethane toxicity (EC(50) 100 mum). All of these observations reflected those previously reported to occur in vivo after exposure to other monohalomethanes. They indicated that MeI shared similar mechanisms of toxicity with other monohalomethanes and supported the validity of the in vitro model to study these mechanisms. Another lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), was also effective in protecting neural cultures against the effects of MeI (EC(50) 3 mum). The use of BW755C/NDGA and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) as specific inhibitors of MeI and glutamate neurotoxicity, respectively, demonstrated that MeI toxicity is not mediated by glutamate, a potential by-product of glutathione-mediated metabolism of MeI. When BW755C and NDGA were compared with other modifiers of arachidonic acid metabolism for their protection against MeI toxicity, their mechanisms of action appeared to be unrelated to inhibition of the oxygenases. Their mechanism of action could be related to their antioxidant effect. Results from this work show the value of primary neural cultures to demonstrate and study monohalomethane neurotoxicity.

Metabolism and toxicity of methyl iodide in primary dissociated neural cell cultures.

The metabolism and the toxicity of methyl iodide (Mel) has been studied in primary dissociated neuronal and glial murine cell cultures to further characterize the mechanisms of monohalomethane neurotoxicity. Measurement of intracellular glutathione (GSH) concentrations in cerebellar and cerebral cultures revealed GSH levels (21.6 +/- 1.9 and 29.1 +/- 1.9 nmol/mg protein, respectively) close to brain GSH levels measured in vivo. A GSH-depleting effect of Mel was demonstrated, with an ED50 for a 5 min exposure of 0.2 and 0.5 mM for glial and mixed (neurons + glia) cultures, respectively. Mel-induced GSH depletion was correlated with its neurotoxicity as the two powerful protective agents of monohalomethane toxicity, 3-amino-1-[m-(trifluoromethyl) phenyl]-2-pyrazoline (BW 755C, 1 mM) and nordihydroguaiaretic acid (NDGA, 10 microM) provided a 20-fold protection against depletion of GSH levels following Mel exposure. When glia and neurons from cerebral cultures were exposed in suspension to increasing concentrations of Mel for 30 min at 37 degrees C, a concentration-dependent increase in the production of formaldehyde resulted. Formaldehyde appeared to be an indicator of Mel metabolism as its production was decreased by sulfasalazine, a compound which was shown to be an inhibitor of the glutathione-S-transferases in this culture system. Since BW 755C and NDGA had no effect on formaldehyde production, while sulfasalazine as well as semicarbazide, a protective agent against formaldehyde-producing toxicants, failed to protect the cells against Mel toxicity, mechanism(s) of Mel neurotoxicity appeared independent of the GSH-mediated metabolism of this compound. It is concluded that GSH-mediated metabolic biotransformation is not necessary for the neurotoxicity of the monohalomethanes, that GSH depletion may act as a starting point in the chain of events leading to neural cell death, and that glia may be more sensitive than neurons to this primary effect. Moreover, these results demonstrate the value of primary dissociated neuronal cell cultures for studies of biochemical mechanisms of neurotoxicity.

Sprouting of GABAergic and mossy fiber axons in dentate gyrus following intrahippocampal kainate in the rat.

The present study examined the bilateral synaptic rearrangements of presumed gamma-aminobutyric acid (GABAergic) inhibitory axons and mossy fiber (presumed excitatory) recurrent collaterals following intrahippocampal kainic acid (KA) injection. Glutamate decarboxylase immunoreactivity (GAD-IR) was used to study inhibitory axon terminal sprouting, following 0.5 microgram KA/0.2 microliter injected unilaterally into the posterior hippocampus of rats (n = 16), with survival periods of 14, 28, and 120 days. The age-matched control animals (n = 9) received intrahippocampal 0.2 microliter saline (sham, n = 4) or no injection (normal, n = 5). To study mossy fiber synaptic rearrangements, 0.5 microgram KA/0.2 microliter volumes were injected unilaterally into the posterior hippocampus of rats (n = 10), with survival periods from 14, 28, and 120 days, and Timm sulfide-stained tissue sections were compared to age-matched sham (n = 4) or normal controls (n = 4). At 14 through 120 days after posterior KA injection, GAD-IR puncta were significantly increased in the ipsi- and contralateral inner molecular layers (IML) of the fascia dentata (FD) when compared to sham or normal controls. KA lesion also induced mossy fiber recurrent collateral sprouting into the ipsi- and contralateral FD IMLs. The loss of both the commissural and ipsilateral associational afferents to the FD apparently induced sprouting into their ipsi- and contralateral termination zones by granule cell mossy fibers and GAD-IR axons, thus establishing an abnormal circuitry near the observed pathology in the kainate model of epilepsy. Although reactive synaptogenesis of mossy fibers producing monosynaptic excitation may be one mechanism for KA epileptogenicity, the concurrent sprouting of GABAergic terminals in the same IML zone of the FD suggests that anomalous inhibitory synapses may contribute to chronic KA hippocampal hyperexcitability.

GABAergic neurons are spared after intrahippocampal kainate in the rat.

The present study used Nissl stains and glutamate decarboxylase immunoreactivity (GAD-IR) to quantify the acute and chronic toxicity of kainic acid (KA) on focal and remote hippocampal principal neurons (i.e., pyramidal and granule cells) and on putative inhibitory neurons (GAD-IR or GABAergic) following intrahippocampal KA administration. Concentrations of 0.5, 1.0, 1.25 or 1.5 micrograms KA/0.2 microliters were injected unilaterally into the posterior hippocampus of rats (n = 32), with survival periods of 1, 3, 5, 14, 21, 30 and 60 days. The age-matched control animals (n = 10) received an intrahippocampal injection of 0.2 microliter saline (sham control, n = 4) or no injection (normal, n = 6). The ipsilateral (KA+) cell counts demonstrated a selective vulnerability of CA3 and CA4 pyramidal neurons which was maximal at 14 days and unchanged to 60 days. However, in the same region, putative inhibitory (GAD-IR) neurons were resistant to the neurotoxic effects of KA. Contralateral (KA-) pyramidal cell and GAD-IR neuron densities were equivalent to controls. The present data demonstrate a selective resistance to KA by GABA neurons compared to the vulnerability of pyramidal neurons. Because GABA neurons are relatively spared in the KA focus, loss of GABAergic inhibitory neurons is probably not a mechanism for the seizure sensitivity in the KA model.

Tetrahydroaminoacridine selectively attenuates NMDA receptor-mediated neurotoxicity.

Addition of the acetylcholinesterase inhibitor 1,2,3,4-tetra-9-hydroaminoacridine (THA) at 1-3 mM markedly reduced the neuronal cell loss that otherwise followed brief exposure of murine cortical cell cultures to 500 microM N-methyl-D-aspartate (NMDA). This novel antagonism was selective for NMDA receptor-mediated toxicity, as it extended to glutamate toxicity but not to quisqualate toxicity, and was THA concentration-dependent between 100 microM and 3 mM, with IC50 approximately 500 microM. The antagonism was probably not due to enhancement of endogenous cholinergic action, as it was not mimicked by acetylcholine, carbachol, or bethanechol; rather, it likely reflected a recently described interaction of THA with the phencyclidine receptor. Exploration of structural specificity revealed some partial neuron-protection with high concentrations of other cholinesterase inhibitors--physostigmine, neostigmine, and edrophonium, but not the structurally related potassium channel blocker, 4-aminopyridine. Further examination of correlations between THA-like structure, and neuron-protective activity, may provide useful insights in the development of new antagonists of NMDA receptor-mediated neurotoxicity.