RESUMO
Biotherapeutics are highly efficacious, but the pain and inconvenience of chronic injections lead to poor patient compliance and compromise effective disease management. Despite innumerable attempts, oral delivery of biotherapeutics remains unsuccessful due to their degradation in the gastrointestinal (GI) environment and poor intestinal absorption. We have developed an orally ingestible robotic pill (RP) for drug delivery, which protects the biotherapeutic drug payload from digestion in the GI tract and auto-injects it into the wall of the small intestine as a safe, pain-free injection since the intestines are insensate to sharp stimuli. The payload is delivered upon inflation of a balloon folded within the RP, which deflates immediately after drug delivery. Here we present results from two clinical studies demonstrating the safety, tolerability and performance of the RP in healthy humans. In the first study, three versions of the RP (A, B and C) were evaluated, which were identical in all respects except for the diameter of the balloon. The RP successfully delivered a biotherapeutic (octreotide) in 3 out of 12 subjects in group A, 10 out of 20 subjects in group B and 16 out of 20 subjects in group C, with a mean bioavailability of 65 ± 9% (based on successful drug deliveries in groups A and B). Thus, reliability of drug delivery with the RP ranged from 25 to 80%, with success rate directly related to balloon size. In a separate study, the deployment of the RP was unaffected by fed or fasting conditions suggesting that the RP may be taken with or without food. These promising clinical data suggest that biotherapeutics currently administered parenterally may be safely and reliably delivered via this versatile, orally ingestible drug delivery platform.
Assuntos
Procedimentos Cirúrgicos Robóticos , Administração Oral , Disponibilidade Biológica , Sistemas de Liberação de Medicamentos , Voluntários Saudáveis , Humanos , Reprodutibilidade dos TestesRESUMO
Biotherapeutic agents must be administered parenterally to obtain therapeutic blood concentrations, lowering patient compliance and complicating care. An oral delivery platform (ODP) was developed to deliver drugs into the small intestinal wall. This proof-of-concept study was performed in 17 anesthetized, laparotomized swine. In 8 swine weighing 17.4 ± 1.2 kg (mean ± SEM), 20 IU of recombinant human insulin (RHI) were auto-injected into the jejunal wall by placing the ODP inside the jejunum via an enterotomy. In 9 control swine weighing 17.0 ± 0.4 kg, 20 IU of RHI were injected subcutaneously. In both groups, under a 60-80 mg/dL euglycemic glucose clamp, blood glucose was measured with a handheld glucometer and serum insulin was measured using ELISA, at 10-minute intervals between -20 and +420 minutes after RHI delivery. The peak serum concentration of RHI was 517 ± 109 pmol/L in the ODP and 342 ± 50 pmol/L in the subcutaneous group (ns). The areas under the insulin concentration curves (83 ± 18 and 81 ± 10 nmol/L·min) were also similar in both groups. The mean time to peak serum concentration of insulin was 139 ± 42 minutes in the ODP and 227 ± 24 minutes in the subcutaneous group (ns). In conclusion, (a) The bioactivity of RHI was preserved after its delivery into the jejunal wall, (b) the intrajejunal route delivered insulin as rapidly and physiologically as the subcutaneous route, and (c) these pharmacokinetic and pharmacodynamic characteristics of RHI after intrajejunal delivery suggest that drugs currently administered parenterally, such as basal insulin, could be successfully delivered into the proximal intestinal wall via the ingestible capsule.
Assuntos
Insulina/administração & dosagem , Insulina/farmacocinética , Jejuno/química , Administração Oral , Animais , Glicemia/análise , Cápsulas , Feminino , Injeções Subcutâneas , Estudo de Prova de Conceito , SuínosRESUMO
With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1), gave rise to similarly potent in vivo neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11ß-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Fármacos Neuroprotetores/farmacologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Análise de Variância , Carbenoxolona/farmacologia , Descoberta de Drogas/métodos , Glucocorticoides/metabolismo , Hipocampo/citologia , Humanos , Neurônios/efeitos dos fármacos , Propídio , Estatísticas não ParamétricasRESUMO
Npas4 is a transcription factor, which is highly expressed in the brain and regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity. A deregulation of the inhibitory-excitatory balance has been associated with a variety of human developmental disorders such as schizophrenia and autism. However, not much is known about the role played by inhibitory synapses and inhibitory pathways in the development of nervous system disorders. We hypothesized that alterations in the inhibitory pathways induced by the absence of Npas4 play a major role in the expression of the symptoms observed in psychiatric disorders. To test this hypothesis we tested mice lacking the transcription factor (Npas4 knock-out mice (Npas4-KO)) in a battery of behavioral assays focusing on general activity, social behaviors, and cognitive functions. Npas4-KO mice are hyperactive in a novel environment, spend less time exploring an unfamiliar ovariectomized female, spend more time avoiding an unfamiliar male during a first encounter, show higher social dominance than their WT littermates, and display pre-pulse inhibition, working memory, long-term memory, and cognitive flexibility deficits. These behavioral deficits may replicate schizophrenia-related symptomatology such as social anxiety, hyperactivity, and cognitive and sensorimotor gating deficits. Immunohistochemistry analyses revealed that Npas4 expression is induced in the hippocampus after a social encounter and that Npas4 regulates the expression of c-Fos in the CA1 and CA3 regions of the hippocampus after a cognitive task. Our results suggest that Npas4 may play a major role in the regulation of cognitive and social functions in the brain with possible implications for developmental disorders such as schizophrenia and autism.
Assuntos
Agressão , Transtorno Autístico/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Cognição , Deficiências do Desenvolvimento/metabolismo , Tonsila do Cerebelo/metabolismo , Análise de Variância , Animais , Ansiedade/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Deficiências do Desenvolvimento/fisiopatologia , Deficiências do Desenvolvimento/psicologia , Feminino , Regulação da Expressão Gênica , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Masculino , Aprendizagem em Labirinto , Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Proteínas Proto-Oncogênicas c-fos/metabolismo , Reflexo de Sobressalto , Esquizofrenia/metabolismo , Filtro Sensorial , Predomínio SocialRESUMO
Alzheimer's disease (AD), the most common form of dementia, is an age-dependent progressive neurodegenerative disorder. ß-amyloid, a metabolic product of the amyloid precursor protein (APP), plays an important role in the pathogenesis of AD. The Thy1-hAPP(Lond/Swe+) (line 41) transgenic mouse overexpresses human APP751 and contains the London (V717I) and Swedish (K670M/N671L) mutations. Here, we used a battery of behavioral tests to evaluate general activity, cognition, and social behavior in six-month-old male Thy1-hAPP(Lond/Swe+) mice. We found hyperactivity in a novel environment as well as significant deficits in spontaneous alternation behavior. In fear conditioning (FC), Thy1-hAPP(Lond/Swe+) mice did not display deficits in acquisition or in memory retrieval in novel context of tone-cued FC, but they showed significant memory retrieval impairment during contextual testing in an identical environment. Surprisingly, in a standard hidden platform water maze, no significant deficit was detected in mutant mice. However, a delayed-matching-to-place paradigm revealed a significant deficit in Thy1-hAPP(Lond/Swe+) mice. Lastly, in the social novelty session of a three-chamber test, Thy1-hAPP(Lond/Swe+) mice exhibited a significantly decreased interest in a novel versus a familiar stranger compared to control mice. This could possibly be explained by decreased social memory or discrimination and may parallel disturbances in social functioning in human AD patients. In conclusion, the Thy1-hAPP(Lond/Swe+) mouse model of AD displayed a behavioral phenotype that resembles, in part, the cognitive and psychiatric symptoms experienced in AD patients.
RESUMO
Epidemiological studies suggest that early life infections may contribute to the development of psychiatric disorders characterized by cognitive deficits. Here, we studied the effects of a neonatal influenza A/WSN/33 virus infection on locomotor activity, working memory and emotional behavior in adult mice. In addition to wild type mice, immunodeficient (Tap1(-/-)) mice lacking functional CD8(+) T cells, were included in the study to model the potential influence of a genetic deficit relating to virus clearance. Three to four months after the infection, infected Tap1(-/-) mice, but not wild type mice, exhibited deficits in working memory as well as increased rearing activity and anxiety. In the medial prefrontal cortices of these infected Tap1(-/-) mice reduced levels of type III Nrg1 transcripts were observed supporting a role for neuregulin 1 signaling in neuronal circuits involved in working memory. Virus replication, distribution or clearance did not differ between the two genotypes. The lack of CD8(+) T cells, however, appeared to contribute to a more pronounced glia response in Tap1(-/-) than in wild type mice. Thus, the present study suggest that the risk of developing deficits in cognitive and emotional behavior following a CNS infection during brain development is influenced by genetic variation in genes involved in the immune response.
Assuntos
Animais Recém-Nascidos , Vírus da Influenza A , Memória de Curto Prazo/fisiologia , Proteínas do Tecido Nervoso/genética , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/psicologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Ansiedade/genética , Ansiedade/psicologia , Encéfalo/patologia , Encéfalo/virologia , Sinais (Psicologia) , Imuno-Histoquímica , Inflamação/genética , Inflamação/patologia , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Neuregulina-1 , Infecções por Orthomyxoviridae/metabolismo , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A number of studies indicate that glutamatergic N-methyl-D-aspartate (NMDA) neurotransmission is disturbed in schizophrenia partly based on the findings that NMDA receptor antagonists such as phencyclidine (PCP) can reproduce a schizophrenia-like syndrome in both humans and rodents. This study investigated whether repeated administration of low doses of PCP can induce cognitive dysfunctions in mice at doses which produce no sensorimotor disturbances. In addition, the effects on cognition were related to the expression of two genes, Arc and spinophilin, which have been related to neuronal plasticity and learning. Adult male C57Bl/6J mice received daily s.c. doses of PCP (0.5-2.0 mg/kg) or saline for 7 d. Testing was performed 24 h after the last day of treatment. Only the 2.0 mg/kg PCP dose produced a consistent impairment in spatial learning and working memory performed in the water-maze task without any apparent sensorimotor deficits. Importantly, the 2.0 mg/kg PCP dose produced no impairment in a non-spatial learning paradigm in the water-maze task. PCP treatment altered Arc mRNA levels in the hippocampus and retrosplenial agranular cortex while leaving the striatum and prefrontal cortex unaffected. The mRNA expression of spinophilin was down-regulated in striatum by repeated PCP treatment. These results demonstrate that repeated treatment with low doses of PCP in mice can produce specific cognitive deficits which are associated with alterations in gene expression in brain regions that appear to play a role in the pathophysiology of schizophrenia. These results suggest that the low-dose PCP model may have significant potential in characterizing the behavioural and molecular mechanisms underlying cognitive deficits seen in schizophrenia patients.
Assuntos
Cognição/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Alucinógenos/farmacologia , Fenciclidina/farmacologia , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Memória de Curto Prazo/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Atividade Motora/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Teste de Desempenho do Rota-Rod/métodos , Comportamento Espacial/efeitos dos fármacosRESUMO
Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.
Assuntos
Éxons/genética , Duplicação Gênica , Neurônios/fisiologia , Proteína 25 Associada a Sinaptossoma/genética , Processamento Alternativo , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas do Domínio Duplacortina , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
The effect of phencyclidine (PCP), a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, was examined in the water maze, a spatial learning and memory task dependent on hippocampal functions. Male adult C57Bl/6J mice received daily (s.c.) injections of either saline or PCP (0.25-4.0 mg/kg) for 12 days. During the last 5 days, the injections were followed by water maze training. Repeated PCP treatments disrupted spatial learning and memory in the 0.5-4.0 mg/kg dose range. Severe sensorimotor disturbances, observed at the 2.0 and 4.0 mg/kg doses of PCP, precluded further swim maze testing. The 0.5 mg/kg but not the 1.0 mg/kg dose of PCP impaired spatial learning and memory without any apparent sensorimotor deficits. PCP, at 1.0 mg/kg, produced impairment in non-spatial learning in the swim maze task and motor disturbances in the rotarod test. Repeated daily treatment with either the "atypical" antipsychotic drug clozapine (0.5 mg/kg i.p.) or the "typical" antipsychotic drug haloperidol (0.05 mg/kg i.p.) failed to influence spatial performances. The spatial impairment caused by the 0.5 mg/kg dose of PCP was blocked by concomitant treatment with clozapine (0.5 mg/kg), but not with haloperidol (0.05 mg/kg). The results suggest that it is possible, at low doses of PCP, to dissociate the spatial learning impairment in the water maze from the adverse behavioral effects of NMDA receptor blockade. This model may provide a basis for the analysis of the mechanisms underlying declarative memory disturbances in schizophrenia and the differences in mechanisms between typical and atypical antipsychotic drugs.
Assuntos
Antipsicóticos/uso terapêutico , Clozapina/uso terapêutico , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Deficiências da Aprendizagem , Fenciclidina/administração & dosagem , Comportamento Espacial/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Esquema de Medicação , Haloperidol/uso terapêutico , Deficiências da Aprendizagem/induzido quimicamente , Deficiências da Aprendizagem/tratamento farmacológico , Deficiências da Aprendizagem/fisiopatologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Retenção Psicológica/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Fatores de TempoRESUMO
In this study, we tested the hypothesis that exposure to a maternal infection during fetal life can lead to the appearance of alterations in the brain later in life. C57BL/6 mice were infected intranasally with influenza A/WSN/33 virus on day 14 of gestation. The levels of transcripts encoding neuroleukin and fibroblast growth factor 5 were significantly elevated in the brains of the virus-exposed offspring at 90 and 280 days of age, but not at earlier time-points. For neuroleukin, this difference could also be observed at the protein level. Thus, a maternal influenza A virus infection can give rise to alterations in gene expression in the brain that become apparent only after a prepubertal latency period.
Assuntos
Encéfalo/metabolismo , Viroses do Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Exposição Materna , Efeitos Tardios da Exposição Pré-Natal , Animais , Encéfalo/virologia , Viroses do Sistema Nervoso Central/virologia , Feminino , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Influenza Humana/genética , Influenza Humana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , GravidezRESUMO
We investigated sex differences in the motor responses to the full and selective dopamine D1-like receptor agonist, (+/-)-6-chloro-7,8-dihydroxyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297; 0.3, 3, and 10 mg/kg, s.c.), in non-habituated adult rats. In general, SKF-81297 produced a biphasic effect on motor activity (including locomotion, rearing and exploratory activity) which consisted of an initial short inhibition followed by a long-lasting stimulation. These effects were dose- and sex-dependent. The inhibitory phase was more pronounced in males than females while the opposite was true for the stimulatory phase. Importantly, the motor inhibitory effects of SKF-81297 were not due to an increase in stereotypy (e.g., grooming activity). These biphasic effects on several motor parameters suggest the presence of two distinct dopamine D1 receptor populations which have opposite effects on motor activity and which are, in part, sexually dimorphic.