Richard P. Shank

An Overview of the Preclinical Aspects of Topiramate: Pharmacology, Pharmacokinetics, and Mechanism of Action

In this overview, we discuss the discovery and development of topiramate (TPM) as an anticonvulsant, including notable aspects of its chemical, biologic, and pharmacokinetic properties. In particular, we highlight its anticonvulsant profile in traditional seizure tests and animal models of epilepsy and the results of recent electrophysiological and biochemical studies using cultured neurons that have revealed a unique combination of pharmacologic properties of TPM. Finally, we present a hypothesis for the mechanistic basis of the anticonvulsant activity of TPM, which proposes that TPM binds to certain membrane ion channel proteins at phosphory‐lation sites and thereby allosterically modulates channel conductance and secondarily inhibits protein phosphorylation.

Amyloid Peptide Aβ1-42 Binds Selectively and with Picomolar Affinity to α7 Nicotinic Acetylcholine Receptors

Abstract: We have recently reported evidence that a very high affinity interaction between the β‐amyloid peptide Aβ1‐42 and the α7 nicotinic acetylcholine receptor (α7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimers disease. In the present study, the kinetics for the binding of Aβ1‐42 to α7nAChR and α4β2nAChR were determined using the subtype‐selective nicotinic receptor ligands [3H]methyllycaconitine and [3H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Aβ1‐42 bound to the α7nAChR with exceptionally high affinity, as indicated by Ki values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the α7nAChR, the affinity of Aβ1‐42 for the α4β2nAChR was ∼5,000‐fold lower, as indicated by corresponding Ki values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Aβ1‐42 and α7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.

Topiramate: Preclinical Evaluation of a Structurally Novel Anticonvulsant

Summary: Topiramate [TPM, 2,3:4,5‐his‐O‐(1‐methyl‐ethylidene)‐β‐D‐fructopyranose sulfamate] (RWJ‐17021‐000, formerly McN‐4853) is a structurally novel antiepileptic drug (AED). The preclinical anticonvulsant profile suggests that TPM acts primarily by blocking the spread of seizures. TPM was highly effective in the maximal electroshock (MES) seizure test in rats and mice. Activity was evident 0.5. h after oral administration and lasted at least 16 h. The ED50 values 4 h after oral dosing were 13.5 and 40.9 mg/kg in rats and mice, respectively. TPM blocked pentylenetetrazol (PTZ)‐induced clonic seizures at high doses in mice (ED50= 1,030 mg/kg orally, p.o.). With motor incoordination and loss of righting reflex used as indicators of neurologic impairment, the neuroprotective index (TD50/MES ED50) for TPM was equivalent or superior to that of several approved AEDs. In mice pretreated with SKF‐525A (a P450 enzyme inhibitor), the anticonvulsant potency was either increased or unaffected when TPM was tested 0.5, 1, or 2 h after i.p. administration, suggesting that TPM rather than a metabolite was the active agent. In mice pretreated with reserpine or tetrabenazine, the activity of TPM in the MES test was markedly reduced. TPM was inactive in a variety of receptor binding, neurotransmitter uptake, and ion channel tests. TPM weakly inhibited erythrocyte carbonic anhydrase (CA) activity. However, the anticonvulsant activity of TPM appears to differ mechanistically from that of acetazolamide.

Topiramate as an Inhibitor of Carbonic Anhydrase Isoenzymes

Purpose: This study investigated the effectiveness of topiramate (TPM) as an inhibitor of six isozymes of carbonic anhydrase (CA).

α-Ketoglutarate and Malate Uptake and Metabolism by Synaptosomes: Further Evidence for an Astrocyte-to-Neuron Metabolic Shuttle

Abstract: This study was undertaken to provide further evidence relevant to the hypothesis that astrocytes supply one or more citric acid cycle intermediates to syn‐aptic terminals, thereby serving an anaplerotic function necessitated by the synthesis and release of amino acid neurotransmitters. In our experiments, two populations of synaptosomes obtained from the brain of rats were separated from myelin and mitochondria by using Percoll to generate continuous density gradients. Both synapto‐somal populations readily accumulated 14C‐labelled a‐ke‐toglutarate and L‐malate by high‐affinity transport systems. Hofstee plots of uptake velocity as a function of substrate concentration were highly nonlinear, indicating that uptake was mediated by two or more carriers, or was subject to negative cooperativity. At least one carrier was selective for a‐ketoglutarate and another for malate, whereas a third carrier appeared to be present which transported both substrates. At low concentrations (1 μM), γ‐ketoglutarate transport was almost totally Na + ‐dependent, whereas malate uptake exhibited little Na+‐dependency. The transport of a‐ketoglutarate was associated with a net influx, and therefore was not due to a homoexchange process. γ‐Ketoglutarate and malate were metabolized rapidly to glutamate and aspartate, respectively, by both synaptosomal preparations; however, in all cases, label accumulated in γ‐aminobutyric acid rather slowly. The incorporation of label into glutamine from γ‐ketoglutarate was much greater in the high‐density synaptosomes than in low‐density synaptosomes, an indication that the former contained a higher proportion of astrogliasomes. The results of our study are consistent with the concept that certain types of synaptic terminals are dependent upon astrocytes for a supply of a‐ketoglutarate and/or malate in order to maintain their pools of citrate cycle intermediates and replenish their neuro‐transmitter pools.

Cerebral metabolic compartmentation as revealed by nuclear magnetic resonance analysis of D-[1-13C]glucose metabolism.

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ‐aminobutyrate (GABA), glutamine, and aspartate. d‐[1‐13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for 13C‐NMR analysis and amino acid analysis. The absolute amount of 13C present within each carbon‐atom pool was determined for C‐2, C‐3, and C‐4 of glutamate, glutamine, and GABA, for C‐2 and C‐3 of aspartate, and for C‐3 of lactate. The natural abundance 13C present in extracts from control rats was also determined for each of these compounds and for N‐acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl‐CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia‐specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from 13C‐13C coupling.

2,3-Dihydro-dithiin and -dithiepine-1,1,4,4-tetroxides: small molecule non-peptide antagonists of the human galanin hGAL-1 receptor

The neuropeptide galanin modulates several physiological functions such as cognition, learning, feeding behavior, and depression, probably via the galanin 1 receptor (GAL-R1). Using an HTS assay based on 125I-human galanin binding to the human galanin-1 receptor (hGAL-R1), we discovered a series of 1,4-dithiin and dithiipine-1,1,4,4-tetroxides that exhibited binding affinity IC50s to hGAL-R1 ranging from 190 to 2700 nM. Two of the dithiepin analogues, 7 and 23, behaved pharmacologically as hGAL-R1 antagonists in secondary assays involving adenylate cyclase activity and GTP binding to G-proteins. Analogues 7 and 23 were also active in functional assays involving galanin, reversing the inhibitory effect of galanin on acetylcholine (ACh) release in rat brain hippocampal slices and electrically-stimulated guinea pig ileum twitch.

Ion Dependence of Neurotransmitter Uptake: Inhibitory Effects of Ion Substitutes

Several ions commonly used as substitutes for Na+ or Cl‐ were found to inhibit directly the high‐affinity uptake of norepinephrine, dopamine, serotonin, and γ‐aminobutyric acid, but not glutamate or glutamine. When Na+ was partially replaced by any of several different cations or sucrose the uptake of all neurotransmitters studied except that of serotonin was reduced more than could be accounted for by just the inhibitory effect of the cation substitute. In contrast, when Cl was partially replaced by any of several anions only the uptake of dopamine was reduced more than could be accounted for by the inhibitory effect of the anion substitute. These results suggest that for most neurotransmitters the electrochemical potential for Na+, but not for Cl, contributes to the uptake driving force. When either Na+ or Cl was totally replaced by an ion substitute or by sucrose the high‐affinity uptake was virtually abolished, an exception being that glutamate uptake was not affected when isethionate was substituted for Cl. The lack of uptake in the absence of either Na+ or Cl may reflect a specific role for these ions in either increasing the affinity between the substrate and the carrier, or facilitating the translocation process. Alternatively, the transport carriers may undergo a nonspecific conformational change to an inactive form in the absence of Na+ or Cl. A partial substitution of Na+ with Li+ or sucrose differentially affected the kinetics of uptake in that replacement with Li+, but not sucrose, usually resulted in a marked increase in the Km values. The results of this study emphasize the importance of taking into consideration the effects of ions used as substitutes in experiments undertaken to elucidate the roles of Na+ and Cl in the high‐affinity uptake of monoamine and amino acid neurotransmitters.

Glutamine, glutamate, and other possible regulators of α-ketoglutarate and malate uptake by synaptic terminals

Abstract: The uptake of a‐ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of γ‐ketoglutarate by causing an apparent reduction in the substrate‐carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L‐Glutamate and L‐as‐partate were comparatively strong inhibitors of γ‐keto‐glutarate and malate uptake. N‐Acetylaspartate was a weak inhibitor of γ‐ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited γ‐ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8‐ to 14‐day‐old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of γ‐ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of γ‐ketoglutarate by low‐density synaptosomes. By comparison, the uptake of glutamate, glutamine, γ‐aminobutyric acid, and several other amino acids was not affected. The enhancement of γ‐ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U‐‐14C] γ‐ketoglutarate to glutamate, as‐partate, glutamine, and aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.

In vivo microdialysis and liquid chromatography/thermospray mass spectrometry of the novel anticonvulsant 2,3:4,5-bis-o-(1-methylethylidene)-β-D-fructopyranose sulfamate (topiramate) in rat brain fluid

The concentration of a novel anticonvulsant, 2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate (topiramate), was determined in the extracellular fluid of rat brain by in vivo microdialysis combined off-line with liquid chromatography/thermospray mass spectrometry. A microdialysis probe was stereotaxically implanted in the nucleus accumbens region of the rat brain. The maximum concentration of topiramate in the brain dialysate for a dose of 50 mg kg-1 i.v. was approximately 10 microM and occurred 45 min post-injection. The detection limit of topiramate in the extracellular fluid of rat brain was in the 0.1 microM range using selected ion monitoring techniques. The base peak, which was the ammonium adduct ion [M + NH4]+, was used for detection. An internal standard of d12-labeled topiramate was utilized for quantitation by isotope dilution analysis.