Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Epilepsia ; 61(6): 1301-1311, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32420627

RESUMO

OBJECTIVE: Approximately 30% of patients with epilepsy do not experience full seizure control on their antiseizure drug (ASD) regimen. Historically, screening for novel ASDs has relied on evaluating efficacy following a single administration of a test compound in either acute electrical or chemical seizure induction. However, the use of animal models of spontaneous seizures and repeated administration of test compounds may better differentiate novel compounds. Therefore, this approach has been instituted as part of the National Institute of Neurological Disorders and Stroke Epilepsy Therapy Screening Program screening paradigm for pharmacoresistant epilepsy. METHODS: Rats were treated with intraperitoneal kainic acid to induce status epilepticus and subsequent spontaneous recurrent seizures. After 12 weeks, rats were enrolled in drug screening studies. Using a 2-week crossover design, selected ASDs were evaluated for their ability to protect against spontaneous seizures, using a video-electroencephalographic monitoring system and automated seizure detection. Sixteen clinically available compounds were administered at maximally tolerated doses in this model. Dose intervals (1-3 treatments/d) were selected based on known half-lives for each compound. RESULTS: Carbamazepine (90 mg/kg/d), phenobarbital (30 mg/kg/d), and ezogabine (15 mg/kg/d) significantly reduced seizure burden at the doses evaluated. In addition, a dose-response study of topiramate (20-600 mg/kg/d) demonstrated that this compound reduced seizure burden at both therapeutic and supratherapeutic doses. However, none of the 16 ASDs conferred complete seizure freedom during the testing period at the doses tested. SIGNIFICANCE: Despite reductions in seizure burden, the lack of full seizure freedom for any ASD tested suggests that this screening paradigm may be useful for testing novel compounds with potential utility in pharmacoresistant epilepsy.


Assuntos
Anticonvulsivantes/administração & dosagem , Ácido Caínico/toxicidade , Convulsões/induzido quimicamente , Convulsões/tratamento farmacológico , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/tratamento farmacológico , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Masculino , Ratos , Ratos Sprague-Dawley , Convulsões/fisiopatologia , Estado Epiléptico/fisiopatologia
2.
Epilepsia ; 58(6): 1073-1084, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28449218

RESUMO

OBJECTIVE: The mouse 6 Hz model of psychomotor seizures is a well-established and commonly used preclinical model for antiseizure drug (ASD) discovery. Despite its widespread use both in the identification and differentiation of novel ASDs in mice, a corresponding assay in rats has not been developed. We established a method for 6 Hz seizure induction in rats, with seizure behaviors similar to those observed in mice including head nod, jaw clonus, and forelimb clonus. METHODS: A convulsive current that elicits these seizure behaviors in 97% of rats (CC97 ) was determined using a Probit analysis. Numerous prototype ASDs were evaluated in this model using stimulus intensities of 1.5× and 2× the CC97 , which is comparable to the approach used in the mouse 6 Hz seizure model (e.g., 32 and 44 mA stimulus intensities). The ASDs evaluated include carbamazepine, clobazam, clonazepam, eslicarbazepine, ethosuximide, ezogabine, gabapentin, lacosamide, lamotrigine, levetiracetam, phenobarbital, phenytoin, rufinamide, tiagabine, topiramate, and sodium valproate. Median effective dose (ED50 ) and median toxic (motor impairment) dose (TD50 ) values were obtained for each compound. RESULTS: Compounds that were effective at the 1.5 × CC97 stimulus intensity at protective index (PI) values >1 included clobazam, ethosuximide, ezogabine, levetiracetam, phenobarbital, and sodium valproate. Compounds that were effective at the 2 × CC97 stimulus intensity at PI values >1 included ezogabine, phenobarbital, and sodium valproate. SIGNIFICANCE: In a manner similar to the use of the mouse 6 Hz model, development of a rat 6 Hz test will aid in the differentiation of ASDs, as well as in study design and dose selection for chronic rat models of pharmacoresistant epilepsy. The limited number of established ASDs with demonstrable efficacy at the higher stimulus intensity suggests that, like the mouse 6 Hz 44 mA model, the rat 6 Hz seizure model may be a useful screening tool for pharmacoresistant seizures.


Assuntos
Anticonvulsivantes/uso terapêutico , Modelos Animais de Doenças , Descoberta de Drogas , Eletroencefalografia/efeitos dos fármacos , Epilepsias Parciais/tratamento farmacológico , Epilepsias Parciais/fisiopatologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Avaliação Pré-Clínica de Medicamentos , Estimulação Elétrica , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Resultado do Tratamento
3.
PLoS Genet ; 6(6): e1000984, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20548952

RESUMO

We have identified a large expansion of an ATTCT repeat within intron 9 of ATXN10 on chromosome 22q13.31 as the genetic mutation of spinocerebellar ataxia type 10 (SCA10). Our subsequent studies indicated that neither a gain nor a loss of function of ataxin 10 is likely the major pathogenic mechanism of SCA10. Here, using SCA10 cells, and transfected cells and transgenic mouse brain expressing expanded intronic AUUCU repeats as disease models, we show evidence for a key pathogenic molecular mechanism of SCA10. First, we studied the fate of the mutant repeat RNA by in situ hybridization. A Cy3-(AGAAU)(10) riboprobe detected expanded AUUCU repeats aggregated in foci in SCA10 cells. Pull-down and co-immunoprecipitation data suggested that expanded AUUCU repeats within the spliced intronic sequence strongly bind to hnRNP K. Co-localization of hnRNP K and the AUUCU repeat aggregates in the transgenic mouse brain and transfected cells confirmed this interaction. To examine the impact of this interaction on hnRNP K function, we performed RT-PCR analysis of a splicing-regulatory target of hnRNP K, and found diminished hnRNP K activity in SCA10 cells. Cells expressing expanded AUUCU repeats underwent apoptosis, which accompanied massive translocation of PKCdelta to mitochondria and activation of caspase 3. Importantly, siRNA-mediated hnRNP K deficiency also caused the same apoptotic event in otherwise normal cells, and over-expression of hnRNP K rescued cells expressing expanded AUUCU repeats from apoptosis, suggesting that the loss of function of hnRNP K plays a key role in cell death of SCA10. These results suggest that the expanded AUUCU-repeat in the intronic RNA undergoes normal transcription and splicing, but causes apoptosis via an activation cascade involving a loss of hnRNP K activities, massive translocation of PKCdelta to mitochondria, and caspase 3 activation.


Assuntos
Apoptose , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína Quinase C-delta/metabolismo , Sequências Repetitivas de Ácido Nucleico , Ataxina-10 , Linhagem Celular , Humanos , Íntrons , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Transporte Proteico , Transcrição Gênica
4.
Proc Natl Acad Sci U S A ; 106(9): 3270-5, 2009 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-19218442

RESUMO

All DNA repeats known to undergo expansion leading to human neurodegenerative disease can form one, or several, alternative conformations, including hairpin, slipped strand, triplex, quadruplex, or unwound DNA structures. These alternative structures may interfere with the normal cellular processes of transcription, DNA repair, replication initiation, or polymerase elongation and thereby contribute to the genetic instability of these repeat tracts. We show that (CCTG) x (CAGG) repeats, in the first intron of the ZNF9 gene associated with myotonic dystrophy type 2, form slipped-strand DNA structures in a length-dependent fashion upon reduplexing. The threshold for structure formation on reduplexing is between 36 and 42 repeats in length. Alternative DNA structures also form in (CCTG)(58) x (CAGG)(58) and larger repeat tracts in plasmids at physiological superhelical densities. This represents an example of a sequence that forms slipped-strand DNA from the energy of DNA supercoiling. Moreover, Z-DNA forms in a (TG) x (CA) tract within the complex repeat sequence 5' of the (CCTG)(n) x (CAGG)(n) repeat in the ZNF9 gene. Upon reduplexing, the presence of the flanking sequence containing the Z-DNA-forming tract reduced the extent of slipped-strand DNA formation by 62% for (CCTG)(57) x (CAGG)(57) compared with 58 pure repeats without the flanking sequence. This finding suggests that the Z-DNA-forming sequence in the DM2 gene locus may have a protective effect of reducing the potential for slipped-strand DNA formation in (CCTG)(n) x (CAGG)(n) repeats.


Assuntos
DNA Forma Z/genética , Distrofia Miotônica/genética , Região 5'-Flanqueadora , Sequência de Bases , Humanos , Dados de Sequência Molecular , Distrofia Miotônica/classificação , Alinhamento de Sequência
5.
J Med Chem ; 63(11): 5865-5878, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32390424

RESUMO

Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.


Assuntos
Anticonvulsivantes/uso terapêutico , Convulsões/tratamento farmacológico , Vitamina K/análogos & derivados , Administração Oral , Animais , Anticonvulsivantes/química , Anticonvulsivantes/farmacocinética , Anticonvulsivantes/farmacologia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Convulsões/patologia , Relação Estrutura-Atividade , Vitamina K/farmacocinética , Vitamina K/farmacologia , Vitamina K/uso terapêutico , Peixe-Zebra
6.
Mol Carcinog ; 48(4): 336-49, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19306311

RESUMO

The DNA repeats (CTG).(CAG), (CGG).(CCG), (GAA).(TTC), (ATTCT).(AGAAT), and (CCTG).(CAGG), undergo expansion in humans leading to neurodegenerative disease. A genetic assay for repeat instability has revealed that the activities of RecA and RecB during replication restart are involved in a high rate of deletion of (CTG).(CAG) repeats in E. coli. This assay has been applied to (CCTG).(CAGG) repeats associated with myotonic dystrophy type 2 (DM2) that expand to 11 000 copies and to spinocerebellar ataxia type 10 (SCA10) (ATTCT).(AGAAT) repeats that expand to 4500 copies in affected individuals. DM2 (CCTG).(CAGG) repeats show a moderate rate of instability, less than that observed for the myotonic dystrophy type 1 (CTG).(CAG) repeats, while the SCA10 (ATTCT).(AGAAT) repeats were remarkably stable in E. coli. In contrast to (CTG).(CAG) repeats, deletions of the DM2 and SCA10 repeats were not dependent on RecA and RecB, suggesting that replication restart may not be a predominant mechanism by which these repeats undergo deletion. These results suggest that different molecular mechanisms, or pathways, are responsible for the instability of different disease-associated DNA repeats in E. coli. These pathways involve simple replication slippage and various sister strand exchange events leading to deletions or expansions, often associated with plasmid dimerization. The differences in the mechanisms of repeat deletion may result from the differential propensity of these repeats to form various DNA secondary structures and their differential proclivity for primer-template misalignment during replication.


Assuntos
Instabilidade Genômica , Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Deleção de Sequência/genética , Ataxias Espinocerebelares/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Humanos , Mutação/genética , Plasmídeos/genética , Transdução de Sinais
7.
Epilepsia Open ; 4(3): 452-463, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31440726

RESUMO

OBJECTIVE: The lamotrigine-resistant amygdala kindling model uses repeated administration of a low dose of lamotrigine during the kindling process to produce resistance to lamotrigine, which also extends to some other antiseizure drugs (ASDs). This model of pharmacoresistant epilepsy has been incorporated into the testing scheme utilized by the Epilepsy Therapy Screening Program (ETSP). Although some ASDs have been evaluated in this model, a comprehensive evaluation of ASD prototypes has not been reported. METHODS: Following depth electrode implantation and recovery, rats were exposed to lamotrigine (5 mg/kg, i.p.) prior to each stimulation during the kindling development process (~3 weeks). A test dose of lamotrigine was used to confirm that fully kindled rats were lamotrigine-resistant. Efficacy (unambiguous protection against electrically elicited convulsive seizures) was defined as a Racine score < 3 in the absence of overt compound-induced side effects. Various ASDs, comprising several mechanistic classes, were administered to fully kindled, lamotrigine-resistant rats. Where possible, multiple doses of each drug were administered in order to obtain median effective dose (ED50) values. RESULTS: Five sodium channel blockers tested (eslicarbazepine, lacosamide, lamotrigine, phenytoin, and rufinamide) were either not efficacious or effective only at doses that were not well-tolerated in this model. In contrast, compounds targeting either GABA receptors (clobazam, clonazepam, phenobarbital) or GABA-uptake proteins (tiagabine) produced dose-dependent efficacy against convulsive seizures. Compounds acting to modulate Ca2+ channels show differential activity: Ethosuximide was not effective, whereas gabapentin was moderately efficacious. Ezogabine and valproate were also highly effective, whereas topiramate and levetiracetam were not effective at the doses tested. SIGNIFICANCE: These results strengthen the conclusion that the lamotrigine-resistant amygdala kindling model demonstrates pharmacoresistance to certain ASDs, including, but not limited to, sodium channel blockers, and supports the utility of the model for helping to identify compounds with potential efficacy against pharmacoresistant seizures.

8.
Biotechniques ; 38(2): 247-53, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15727131

RESUMO

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.


Assuntos
Clonagem Molecular/métodos , Doenças Neurodegenerativas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sequência de Bases , Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA