Topiramate promotes neurite outgrowth and recovery of function after nerve injury.

Topiramate is a structurally novel neurotherapeutic agent with a unique combination of pharmacological properties and currently is available in most world markets for treating several seizure disorders. Because its pharmacological profile was suggestive of possible activity as a neuroprotectant, topiramate was evaluated and found to be active in several animal models of stroke or neuropathic pain. This prompted an evaluation of topiramate as a possible neurotrophic agent. In this study, topiramate enhanced the recovery of facial nerve function after injury when administered orally at therapeutically relevant doses, and significantly increased neurite outgrowth in cell cultures derived from fetal rat cortical and hippocampal tissues.

Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors.

We have recently reported evidence that a very high affinity interaction between the beta-amyloid peptide Abeta(1-42) and the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) may be a precipitating event in the formation of amyloid plaques in Alzheimer's disease. In the present study, the kinetics for the binding of Abeta(1-42) to alpha7nAChR and alpha4beta2nAChR were determined using the subtype-selective nicotinic receptor ligands [(3)H]methyllycaconitine and [(3)H]cytisine. Synaptic membranes prepared from rat and guinea pig cerebral cortex and hippocampus were used as the source of receptors. Abeta(1-42) bound to the alpha7nAChR with exceptionally high affinity, as indicated by K(i) values of 4.1 and 5.0 pM for rat and guinea pig receptors, respectively. When compared with the alpha7nAChR, the affinity of Abeta(1-42) for the alpha4beta2nAChR was approximately 5,000-fold lower, as indicated by corresponding K(i) values of 30 and 23nM. The results of this study support the concept that an exceptionally high affinity interaction between Abeta(1-42) and alpha7nAChR 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.

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 IC50's 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.

Synthesis and structure-activity relationship of novel pyridyl ethers for the nicotinic acetylcholine receptor.

The preparation of novel pyridyl ethers as ligands for the nicotinic acetylcholine receptor (nAChR) is described. Variations of the ring size of the azacycle and substitution on the pyridine had dramatic effects on receptor binding affinity with IC50s at the alpha4beta2 nAChR ranging from 22 to >10,000 nM. The most potent molecule was (R)-2-chloro-3-(4-cyanophenyl)-5-((3-pyrrolidinyl)oxy)pyridine 27f with an IC50 of 22 nM.

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 phosphorylation sites and thereby allosterically modulates channel conductance and secondarily inhibits protein phosphorylation.

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). METHODS: The inhibition constants (Ki) of TPM and acetazolamide (AZM) for CA I, CA II, CA III, CA IV, CA V, and CA VI were determined for human (HCA), rat (RCA), or mouse (MCA). The activity of CA was studied by using purified isozymes, erythrocytes, subcellular fractions of kidney or brain, and saliva, and was assayed at 37 degrees C or 25 degrees C by 18O mass spectrometry and/or by measuring the pH shift at 0 degrees C. RESULTS: Topiramate Ki values for HCA I, HCA II, HCA IV, and HCA VI were approximately 100, 7, 10, and >100 microM, respectively. TPM Ki values for RCA I, RCA II, RCA III, RCA IV, and RCA V were approximately 180, 0.1 to 1, >100, 0.2 to 10 and 18 microM, respectively. For RCA II and RCA IV, the Ki values were temperature dependent. TPM Ki values for MCA II and MCA IV ranged between 1 and 20 microM. CONCLUSIONS: These results indicate that TPM is more potent as an inhibitor of CA II and CA IV than of CA I, CA III, and CA VI. In all three species, AZM was usually 10 to 100 times more potent than TPM as an inhibitor of CA isozymes.

Effects of topiramate on kainate- and domoate-activated [14C]guanidinium ion flux through GluR6 channels in transfected BHK cells using Cytostar-T scintillating microplates.

PURPOSE: This study was undertaken to test the hypothesis that topiramate (TPM) exerts a negative modulatory effect on some types of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)/kainate receptors by binding to the site at which protein kinase A (PKA) phosphorylates the receptor-channel complex. METHODS: The effect of TPM on kainate- or domoate-induced [14C]guanidinium ion flux through iGluR6 channels expressed in baby hamster kidney (BHK) cells was evaluated. Because the hypothesis predicts that TPM will bind only in the dephosphorylated state, a variety of experimental conditions were used to either promote or impede the phosphorylation of the receptor-channel complex. These included the use of dibutyryl cyclic adenosine monophosphate (cAMP) and forskolin to activate PKA, H-9 and H-89 to inhibit PKA, and okadaic acid to inhibit protein phosphatases. RESULTS: Kainate (1 microM) induced a gradual accumulation of [14C]guanidinium into the cells that plateaued approximately 30 min after initiation of the reaction, whereas domoate (0.1 microM) caused a rapid accumulation into the cells that peaked within 5 min; thereafter, the amount of [14C]guanidinium in the cells declined gradually. Topiramate, at 0.1 and 100 microM, did not significantly affect the [14C]guanidinium accumulation under any of the experimental conditions used. CONCLUSIONS: The results of this study are not consistent with the hypothesis tested. However, the results must be interpreted cautiously because iGluR6 receptors expressed in the BHK cells and the functional state of proteins that regulate AMPA/receptors (e.g., PSD-95) may not be sufficiently similar to the receptors and functional state in neurons to serve as a true test of the hypothesis.

beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology.

Alzheimer's disease pathology is characterized by the presence of neuritic plaques and the loss of cholinergic neurons in the brain. The underlying mechanisms leading to these events are unclear, but the 42-amino acid beta-amyloid peptide (Abeta(1-42)) is involved. Immunohistochemical studies on human sporadic Alzheimer's disease brains demonstrate that Abeta(1-42) and a neuronal pentameric cation channel, the alpha7 nicotinic acetylcholine receptor (alpha7nAChR), are both present in neuritic plaques and co-localize in individual cortical neurons. Using human brain tissues and cells that overexpress either alpha7nAChR or amyloid precursor protein as the starting material, Abeta(1-42) and alpha7nAChR can be co-immunoprecipitated by the respective specific antibodies, suggesting that they are tightly associated. The formation of the alpha7nAChR.Abeta(1-42) complex can be efficiently suppressed by Abeta(12-28), implying that this Abeta sequence region contains the binding epitope. Receptor binding experiments show that Abeta(1-42) and alpha7nAChR bind with high affinity, and this interaction can be inhibited by alpha7nAChR ligands. Human neuroblastoma cells overexpressing alpha7nAChR are readily killed by Abeta(1-42), whereas alpha7nAChR agonists such as nicotine and epibatidine offered protection. Because Abeta(1-42) inhibits alpha7nAChR-dependent calcium activation and acetylcholine release, two processes critically involved in memory and cognitive functions, and the distribution of alpha7nAChR correlates with neuritic plaques in Alzheimer's disease brains, we propose that interaction of the alpha7nAChR and Abeta(1-42) is a pivotal mechanism involved in the pathophysiology of Alzheimer's disease.

gamma-Aminobutyrate-A receptor modulation by 3-aryl-1-(arylsulfonyl)- 1,4,5,6-tetrahydropyridazines.

A series of 3-aryl-1-(arylsulfonyl)-1,4,5,6-tetrahydropyridazine allosteric modulators of the GABAA receptor was synthesized, and biological activity was examined in vitro and in vivo. Beginning with 1a, stepwise modification of the substituents and conservation of the scaffold yielded a chemical series in which the modulatory activity was enhanced by the presence of GABA. The SAR suggests, but does not establish, that the compounds bind to the steroid binding site on the GABAA receptor. The GABA shift for each compound indicates that all compounds in this series are either agonists or partial agonists.

Potential anxiolytic agents. 3. Novel A-ring modified pyrido[1,2-a]benzimidazoles.

A variety of pyrido[1,2-a]benzimidazoles (PBIs) modified on the A-ring were prepared and evaluated for affinity to the benzodiazepine binding site on the GABA-A receptor and in animal models predictive of anxiolytic activity in humans. A-ring benzo-fused derivative 7 exhibited potent activity, as did the 6- and 7-pyrido compounds 3 and 4.

New directions in anxiolytic drug research.

Agents to treat anxiety have gained in acceptance and importance in the fast pace of life in the second half of this century. The discovery and refinement of the benzodiazepines represented a quantum leap in therapy from early compounds which were essentially sedatives. With the advent of molecular biology, an understanding of the basic mechanism by which the benzodiazepines exert their effects was revealed through the discovery and isolation of the GABAA receptor and its benzodiazepine binding site. This, in turn, has enabled benzodiazepines to be classified into a broad spectrum of pharmacological types ranging from agonist to inverse agonist, thus allowing fine tuning with respect to side-effects. Consequently, newer, more promising agents have emerged which bind at the GABAA BZD site and have reduced side-effects. An example of this is RWJ-51204 (92), a member of a novel structural type which is superior to several marketed benzodiazepines in animals in terms of efficacy and side-effects. The cost-conscious environment of managed health care presents continuing challenges to the discovery and development of safe, highly efficacious, and cost-effective anxiolytic agents.

Galanin inhibits acetylcholine release from rat cerebral cortex via a pertussis toxin-sensitive G(i)protein.

Galanin has been implicated in various physiological functions including memory, feeding and pain perception. Using rat cerebral cortical slices and synaptosome preparations incubated with [(3)H]choline in Kreb's-Ringer solution, galanin was shown to inhibit both spontaneous and K(+)-stimulated [(3)H]ACh release in a concentration-related manner [EC(50)= 35 nM]. The galanin-mediated inhibition on spontaneous and K(+)-stimulated [(3)H]ACh release was respectively regulated by pertussis toxin-sensitive G(alphai3)and G(alphai1). These suggest that galanin is a negative modulator of cortical cholinergic function and most probably acting on presynaptic cholinergic terminals. Although galantide blocked the galanin-mediated inhibitory effect on [(3)H]ACh release, it mimicked galanin in blocking K(+)-stimulated [(3)H]ACh release, indicating that galantide may have a more complicated pharmacology than being a galanin receptor antagonist. In addition, we demonstrate that galanin and beta-amyloid peptide(1-42)synergistically attenuated K(+)-evoked [(3)H]ACh release from synaptosomes prepared from rat cerebral cortex. Since galanin is increased in Alzheimer's disease brain, our results suggest that galanin may be involved in cholinergic dysfunctions that occur in Alzheimer's disease.

Orally active benzamide antipsychotic agents with affinity for dopamine D2, serotonin 5-HT1A, and adrenergic alpha1 receptors.

New antipsychotic drugs are needed because current therapy is ineffective for many schizophrenics and because treatment is often accompanied by extrapyramidal symptoms and dyskinesias. This paper describes the design, synthesis, and evaluation of a series of related (aminomethyl)benzamides in assays predictive of antipsychotic activity in humans. These compounds had notable affinity for dopamine D2, serotonin 5-HT1A, and alpha1-adrenergic receptors. The arylpiperazine 1-[3-[[4-[2-(1-methylethoxy)phenyl]-1-piperazinyl]methyl]benzoyl]p ipe ridine (mazapertine, 6) was chosen because of its overall profile for evaluation in human clinical trials. The corresponding 4-arylpiperidine derivative 67 was also highly active indicating that the aniline nitrogen of 6 is not required for activity. Other particularly active structures include homopiperidine amide 14 and N-methylcyclohexylamide 31.

Structure-activity studies on anticonvulsant sugar sulfamates related to topiramate. Enhanced potency with cyclic sulfate derivatives.

We have explored the structure-activity relationship (SAR) surrounding the clinically efficacious antiepileptic drug topiramate (1), a unique sugar sulfamate anticonvulsant that was discovered in our laboratories. Systematic structural modification of the parent compound was directed to identifying potent anticonvulsants with a long duration of action and a favorable neurotoxicity index. In this context, we have probed the pharmacological importance of several molecular features: (1) the sulfamate group (6-8, 22-25, 27, 84), (2) the linker between the sulfamate group and the pyran ring (9, 10, 21a,b), (3) the substituents on the 2,3- (58-60, 85, 86) and 4, 5-fused (30-38, 43, 45-47, 52, 53) 1,3-dioxolane rings, (4) the constitution of the 4,5-fused 1,3-dioxolane ring (2, 54, 55, 63-68, 76, 77, 80, 83a-r, 84-87, 90a, 91a, 93a), (5) the ring oxygen atoms (95, 96, 100-102, 104, 105), and (6) the absolute stereochemistry (106 and 107). We established the C1 configuration as R for the predominant alcohol diastereomer from the highly selective addition of methylmagnesium bromide to aldehyde 15 (16:1 ratio) by single-crystal X-ray analysis of the major diastereomer of sulfamate 21a. Details for the stereoselective syntheses of the hydrindane carbocyclic analogues 95, 96, 100, and 104 are presented. We also report the synthesis of cyclic imidosulfites 90a and 93a, and imidosulfate 91a, which are rare examples in the class of such five-membered-ring sulfur species. Imidosulfite 93a required the preparation and use of the novel sulfur dichloride reagent, BocN=SCl2. Our SAR investigation led to the impressive 4,5-cyclic sulfate analogue 2 (RWJ-37947), which exhibits potent anticonvulsant activity in the maximal electroshock seizure (MES) test (ca. 8 times greater than 1 in mice at 4 h, ED50 = 6.3 mg/kg; ca. 15 times greater than 1 in rats at 8 h, ED50 = 1.0 mg/kg) with a long duration of action (>24 h in mice and rats, po) and very low neurotoxicity (TD50 value of >1000 mg/kg at 2 h, po in mice). Cyclic sulfate 2, like topiramate and phenytoin, did not interfere with seizures induced by pentylenetetrazole, bicucculine, picrotoxin, and strychnine; also, 2 was not active in diverse in vitro receptor binding and uptake assays. However, 2 turned out to be a potent inhibitor of carbonic anhydrase from different rat tissue sources (e. g., IC50 of 84 nM for the blood enzyme and 21 nM for the brain enzyme). An examination of several analogues of 2 (83a-r, 85-87, 90a, 91a, 93a) indicated that potent anticonvulsant activity is associated with relatively small alkyl substituents on nitrogen (Me/H, 83a; Me/Me, 83m; Et/H, 83b; allyl/H, 83e; c-Pr/H, 83j; c-Bu/H, 83k) and with limited changes in the cyclic sulfate group, such as 4,5-cyclic sulfite 87a/b. The potent anticonvulsants 83a and 83j had greatly diminished carbonic anhydrase inhibitory activity; thus, inhibition of this enzyme may not be a significant factor in the anticonvulsant activity. The alpha-L-sorbopyranoses 67, 68, and 80, which mainly possess a skew conformation (ref 29), were nearly twice as potent as topiramate (1). The L-fructose enantiomers of 1 (106) and 2 (107), synthesized from L-sorbose, were found to have moderate anticonvulsant activity, with eudysmic ratios (MES ED50 in mice at 4 h, po) of 1:106 = 1.5 and 2:107 = 3.5. The log P values for 1 and 2 were determined experimentally to be 0.53 and 0.42, respectively, which are less than the optimal 2.0 for CNS active agents. However, analogues with more favorable calculated log P (clogP) values, in conjunction with just minor steric perturbation according to the developed SAR profile, such as 47 (clogP = 2.09), 83m (1.93), and 86 (1.50), did not display improved potency: 47 is less potent than 1, 83m is equipotent with 2, and 86 is less potent than 2. Although the measured log P value for diethyl analogue 31 is 1.52, this did not translate into enhanced potency relative to 1. (ABSTRACT TRUNCATED)

In vivo microdialysis and liquid chromatography/thermospray mass spectrometry of the novel anticonvulsant 2,3:4,5-bis-O-(1-methylethylidene)-beta-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.

Azepinoindole derivatives with high affinity for brain dopamine and serotonin receptors.

We synthesized 20 and 21 as conformationally constrained analogues of the dopamine receptor antagonist SKF-83742, as well as analogues 6-9, 16, and 18-22. Although 20 and 21 were inactive, 7, 9, and 19 showed strong binding to D-1, D-2, S-2, and alpha-1 receptors, as well as antipsychotic activity in vivo.

Anticonvulsant activity of topiramate and phenytoin in a rat model of ischemia-induced epilepsy.

Topiramate, a structurally novel anticonvulsant, and phenytoin were evaluated in a rat model of ischemia-induced epilepsy. In this model a transient global cerebral ischemia is induced by cardiac compression. By precisely controlling the experimental conditions the procedure causes reproducible neurological deficits that include audiogenic epileptic seizures. The seizures can be broadly separated into three types reflecting the degree of severity: wild running, clonic seizures, and tonic extension seizures of the forelimbs and hindlimbs. Topiramate and phenytoin blocked all three types of seizures. Calculated ED50 values for topiramate 1 hr after oral administration were 8.2, 13.0 and 36.1 mg/kg for blockade of tonic extension seizures, clonic seizures and wild running, respectively. Corresponding ED50 values for phenytoin were 5.0, 10.8 and 20.7 mg/kg. These results support the concept that the anticonvulsant activity of these drugs is due primarily to an ability to block the spread of seizures.

Characterization of a benzodiazepine receptor site with exceptionally high affinity for Ro 15-4513 in the rat CNS.

The binding of [3H]Ro 15-4513, [3H]flunitrazepam and [3H]flumazenil to rat CNS membranes was studied at 2 degrees C, 22 degrees C and 37 degrees C using ligand concentrations ranging from approximately 0.06 nM to 10 microM. Analysis of the binding saturation data suggested the existence of high-affinity sites (Kd < 10 nM) and low-affinity sites (Kd > 100 nM) for each ligand. When binding was performed using very low ligand concentrations a benzodiazepine site with an exceptionally high affinity for Ro 15-4513 (Kd approximately 0.1 nM) was evident in all major regions of the CNS except the cerebellum. This site was most prevalent in the hippocampus, medulla and spinal cord where it accounted for approximately 70% of the specific binding when [3H]Ro 15-4513 was approximately 0.06 nM. The selectivity of Ro 15-4513 for this site as compared to other high-affinity sites was 20- to 60-fold depending on the incubation temperature and CNS region. The affinity for the very high-affinity site was decreased approximately 3-fold as temperature was increased from 2 degrees C to 37 degrees C (Kd approximately 0.1 nM and approximately 0.3, respectively), which was similar to the effect of temperature on other high-affinity sites (Kd approximately 2.6 nM at 2 degrees C and approximately 8 nM at 37 degrees C). Flumazenil, flunitrazepam, and diazepam did not differentiate the very high-affinity [3H]Ro 15-4513 site from other BZ sites, but alpidem exhibited a low affinity for it (IC50 approximately 5 microM). GABA at 100 microM had little effect on the Kd value for the very high-affinity site (GABA shift: approximately 0.8 to 1.0), suggesting that Ro 15-4513 is a partial inverse agonist or an antagonist at this site. These findings provide further evidence for the pharmacologic diversity of BZ sites on different subtypes of GABAA receptors.

Serotonin and norepinephrine uptake inhibiting activity of centrally acting analgesics: structural determinants and role in antinociception.

Although it is well established that the analgesic effects of morphine are mediated by opioid receptors, previous studies have shown that some opioids additionally inhibit the uptake of serotonin and norepinephrine. The present investigation of a diverse group of opioids revealed that structurally identifiable subgroups inhibited the neuronal reuptake of these monoamines. Phenanthrene opioids with an oxygen bridge between C4 and C5, such as morphine and naloxone (group I), did not block norepinephrine or serotonin uptake, whereas phenanthrene opioids without the oxygen bridge and the C6-OH moiety, such as levorphanol and levomethorphan (group II), did inhibit uptake, as did nonphenanthrene opioids, such as d-propoxyphene and methadone (group III). Affinity at the mu opioid receptor correlated with antinociceptive potency (r = 0.87, P < .05). Although the antinociceptive activity of the "active enantiomers" of group II and III compounds also correlated with their affinity at the mu opioid receptor (r = 0.85, P = .007), additional consideration of serotonin uptake inhibiting activity (but not of norepinephrine uptake inhibiting activity) significantly improved the correlation between antinociceptive potency and the in vitro activity of these compounds (r = 0.915, P = .0017). Additionally, for group II and III (but not group I) compounds, smaller differences between enantiomers in antinociceptive potency than in mu receptor affinity were noted, presumably because of the contribution of uptake inhibition to the antinociceptive activity of group II and III compounds. Evidence also is provided suggesting a broader role for the combination of mu opioid affinity and 5-hydroxytryptamine uptake inhibition in the activity of other antinociceptive agents.