RESUMEN
BACKGROUND: Alzheimer's disease is a large and growing unmet medical need. Clinical trial designs need to assess disease-related outcomes earlier to accelerate the development of better treatments for Alzheimer's disease. ACU193 is a monoclonal antibody that selectively targets amyloid ß oligomers, thought to be the most toxic species of Aß that accumulates early in AD and contributes to downstream pathological effects. Nonclinical data indicate that ACU193 can reduce the toxic effects of amyloid ß oligomers. ACU193 is currently being investigated in a phase 1 clinical trial designed with the properties described in this report. This phase 1 trial is designed to provide data to enable a go/no-go decision regarding the initiation of a subsequent phase 2/3 study. OBJECTIVES: To design a phase 1 study that assesses target engagement and incorporates novel measures to support more rapid development of a potential disease-modifying treatment for Alzheimer's disease. DESIGN: The INTERCEPT-AD trial for ACU193 is an ongoing randomized, placebo-controlled phase 1a/b study that assesses safety, tolerability, pharmacokinetics, target engagement, clinical measures, and several Alzheimer's disease biomarkers, including novel digital and imaging biomarkers. SETTING: For INTERCEPT-AD, brief inpatient stays for patients in the single ascending dose portion of the study, with the remainder of the evaluations being performed as outpatients at multiple clinical trial sites in the U.S. PARTICIPANTS: Patients with early Alzheimer's disease (mild cognitive impairment or mild dementia with a positive florbetapir positron emission tomography scan). INTERVENTION: ACU193 administered intravenously at doses of 2- 60 mg/kg. MEASUREMENTS: Safety assessments including magnetic resonance imaging for the presence of amyloid-related imaging abnormalities, clinical assessments for Alzheimer's disease including the Alzheimer's Disease Rating Scale-cognition and Clinical Dementia Rating scale, pharmacokinetics, a measure of target engagement, and digital and imaging biomarkers, including a computerized cognitive test battery and a measure of cerebral blood flow using arterial spin labelling magnetic resonance imaging. RESULTS: A phase 1 study design was developed for ACU193 that allows collection of data that will enable a go/no-go decision for initiation of a subsequent adaptive phase 2/3 study. CONCLUSIONS: A phase 1a/b trial and an overall clinical development plan for an Alzheimer's disease treatment can be designed that maintains patient safety, allows informed decision-making, and achieves an accelerated timeline by using novel biomarkers and adaptive study designs.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/psicología , Péptidos beta-Amiloides , Anticuerpos Monoclonales/efectos adversos , Biomarcadores , Planificación Social , Ensayos Clínicos Fase I como AsuntoRESUMEN
A novel, local inhibitory circuit in layer 2/3 of rat somatosensory cortex is described that connects pyramidal cells reciprocally with GABAergic vasoactive intestinal polypeptide-immunoreactive bipolar interneurons. In paired whole-cell recordings, the glutamatergic unitary responses (EPSPs or EPSCs) in bipolar cells evoked by repetitive (10 Hz) stimulation of a pyramidal cell show strong frequency-dependent depression. Unitary IPSPs evoked in pyramidal cells by repetitive stimulation of bipolar cells, on average, maintained their amplitude. This suggests that the excitatory synapses on bipolar cells act as a low-pass filter in the reciprocal pyramid-to-bipolar circuit. The EPSCs in bipolar cells are mediated predominantly by AMPA receptor (AMPAR) channels. AMPARs desensitize rapidly and recover slowly from desensitization evoked by a brief pulse of glutamate. In slices, reduction of AMPAR desensitization by cyclothiazide (50-100 microm) or conditioning steady-state desensitization induced by application of extracellular AMPA (50 nm) or glutamate (50 microm) strongly reduced synaptic depression. It is concluded that in the local circuits between pyramidal and bipolar cells the desensitization of AMPARs in bipolar cells contributes to low-pass feedback inhibition of layer 2/3 pyramidal neurons by bipolar cells.
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Interneuronas/metabolismo , Canales Iónicos/metabolismo , Neocórtex/metabolismo , Receptores AMPA/metabolismo , Transmisión Sináptica/fisiología , Animales , Axones/ultraestructura , Benzotiadiazinas/farmacología , Dendritas/ultraestructura , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/fisiología , Retroalimentación/fisiología , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Interneuronas/ultraestructura , Lisina/análogos & derivados , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neocórtex/efectos de los fármacos , Neocórtex/ultraestructura , Técnicas de Placa-Clamp , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Células Piramidales/ultraestructura , Ratas , Ratas Wistar , Receptores de Glutamato/metabolismo , Corteza Somatosensorial/efectos de los fármacos , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/ultraestructura , Sinapsis/ultraestructura , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
High Ca(2+) permeability and its control by voltage-dependent Mg(2+) block are defining features of NMDA receptors. These features are lost if the principal NR1 subunit carries an asparagine (N) to arginine (R) substitution in a critical channel site at NR1 position 598. NR1(R) expression from a single allele in gene-targeted NR1(+/R) mice is lethal soon after birth, precluding analysis of altered synaptic functions later in life. We therefore employed the forebrain specific alphaCaMKII promoter to drive tTA-mediated tetracycline sensitive transcription of transgenes for NR1(R) and for lacZ as reporter. Transgene expression was observed in cortex, striatum, hippocampus, amygdala and olfactory bulb and was mosaic in all these forebrain regions. It was highest in olfactory bulb granule cells, in most of which Ca(2+) permeability and voltage-dependent Mg(2+) block of NMDA receptors were reduced to different extents. This indicates significant impairment of NMDA receptor function by NR1(R) in presence of the wild-type NR1 complement. Indeed, even though NR1(R) mRNA constituted only 18% of the entire NR1 mRNA population in forebrain, the transgenic mice died during adolescence unless transgene expression was suppressed by doxycycline. Thus, glutamate receptor function can be altered in the mouse by regulated NR1(R) transgene expression.
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Antibacterianos/farmacología , Doxiciclina/farmacología , Neuronas/fisiología , Bulbo Olfatorio/citología , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Expresión Génica/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Operón Lac , Magnesio/farmacología , Ratones , Ratones Transgénicos , Mosaicismo , Técnicas de Cultivo de Órganos , Tasa de Supervivencia , Transgenes/genética , beta-Galactosidasa/genéticaRESUMEN
To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC). Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice. During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system. In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles. KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.
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Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Integrasas/metabolismo , Receptores de Ácido Kaínico/metabolismo , Proteínas Virales , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Genes Reporteros/fisiología , Inmunohistoquímica , Integrasas/genética , Operón Lac/fisiología , Ratones , Ratones Transgénicos/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de Ácido Kaínico/genética , Distribución TisularRESUMEN
In excitable cells, small-conductance Ca2+-activated potassium channels (SK channels) are responsible for the slow after-hyperpolarization that often follows an action potential. Three SK channel subunits have been molecularly characterized. The SK3 gene was targeted by homologous recombination for the insertion of a gene switch that permitted experimental regulation of SK3 expression while retaining normal SK3 promoter function. An absence of SK3 did not present overt phenotypic consequences. However, SK3 overexpression induced abnormal respiratory responses to hypoxia and compromised parturition. Both conditions were corrected by silencing the gene. The results implicate SK3 channels as potential therapeutic targets for disorders such as sleep apnea or sudden infant death syndrome and for regulating uterine contractions during labor.
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Trabajo de Parto/fisiología , Canales de Potasio Calcio-Activados , Canales de Potasio/fisiología , Fenómenos Fisiológicos Respiratorios , Regiones no Traducidas 5' , Potenciales de Acción , Animales , Encéfalo/metabolismo , Cruzamientos Genéticos , Técnicas de Cultivo , Doxiciclina/farmacología , Femenino , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Marcación de Gen , Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Canales de Potasio/genética , Embarazo , Canales de Potasio de Pequeña Conductancia Activados por el CalcioRESUMEN
This manuscript summarizes our recent attempts to regulate in vitro and in vivo the expression of genes encoding components and regulators of the postsynaptic machinery along with marker genes such as lacZ and GFP. In particular, we studied tTA-dependent regulation and utilized Cre in combination with reversible silencing by intron engineering of dominant negative alleles. We further present a "knockin" approach for on-site artificial regulation of chromosomal genes.
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Encéfalo/metabolismo , Regulación de la Expresión Génica/genética , Proteínas Virales , Animales , Encéfalo/citología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Genes Reporteros/genética , Integrasas/genética , Ratones , Ratones Transgénicos , Recombinación Genética , Transmisión Sináptica/genética , Tetraciclina/farmacología , Transactivadores/genéticaRESUMEN
The tet regulatory system in which doxycycline (dox) acts as an inducer of specifically engineered RNA polymerase II promoters was transferred into transgenic mice. Tight control and a broad range of regulation spanning up to five orders of magnitude were monitored dependent on the dox concentration in the water supply of the animals. Administration of dox rapidly induces the synthesis of the indicator enzyme luciferase whose activity rises over several orders of magnitude within the first 4 h in some organs. Induction is complete after 24 h in most organs analyzed. A comparable regulatory potential was revealed with the tet regulatory system where dox prevents transcription activation. Directing the synthesis of the tetracycline-controlled transactivator (tTA) to the liver led to highly specific regulation in hepatocytes where, in presence of dox, less than one molecule of luciferase was detected per cell. By contrast, a more than 10(5)-fold activation of the luciferase gene was observed in the absence of the antibiotic. This regulation was homogeneous throughout but stringently restricted to hepatocytes. These results demonstrate that both tetracycline-controlled transcriptional activation systems provide genetic switches that permit the quantitative control of gene activities in transgenic mice in a tissue-specific manner and, thus, suggest possibilities for the generation of a novel type of conditional mutants.
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Doxiciclina/farmacología , Regulación de la Expresión Génica , Ingeniería Genética/métodos , Ratones Transgénicos , Animales , Citomegalovirus/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hibridación in Situ , Hígado/fisiología , Ratones , Regiones Promotoras Genéticas , ARN Mensajero/genética , Distribución Tisular , Activación TranscripcionalRESUMEN
To use single-pass cDNA sequencing to characterize low-frequency cDNA clones from a region of the brain that includes the primary site of neurodegeneration in human Parkinson disease, we have developed a prescreening procedure using single brain region first-strand cDNA probes. Selection of cDNA clones giving low hybridization signals allowed the elimination of clones resulting from abundant messages and enrichment for clones corresponding to low-copy messages. Comparative sequencing of standard and prescreened cDNA libraries (191 and 124 clones, respectively) showed that this procedure raised the frequency of novel sequences encountered from 54 to 81%. The increased proportion of novel ESTs justifies the labor of prescreening. Automation of this procedure will accelerate the molecular description of genes expressed in any brain region, or any tissue, and represents a way to maximize access to cDNA sequences for human and mouse genome characterization. In total, the comparative sequencing experiments generated 207 new mouse and 11 new rat brain ESTs.