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1.
J Neuroinflammation ; 21(1): 37, 2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38297405

RESUMO

Neuroinflammation is a key driver of neurodegenerative disease, however the tools available to model this disease biology at the systems level are lacking. We describe a translational drug discovery platform based on organotypic culture of murine cortical brain slices that recapitulate disease-relevant neuroinflammatory biology. After an acute injury response, the brain slices assume a chronic neuroinflammatory state marked by transcriptomic profiles indicative of activation of microglia and astrocytes and loss of neuronal function. Microglia are necessary for manifestation of this neuroinflammation, as depletion of microglia prior to isolation of the brain slices prevents both activation of astrocytes and robust loss of synaptic function genes. The transcriptomic pattern of neuroinflammation in the mouse platform is present in published datasets derived from patients with amyotrophic lateral sclerosis, Huntington's disease, and frontotemporal dementia. Pharmacological utility of the platform was validated by demonstrating reversal of microglial activation and the overall transcriptomic signature with transforming growth factor-ß. Additional anti-inflammatory targets were screened and inhibitors of glucocorticoid receptors, COX-2, dihydrofolate reductase, and NLRP3 inflammasome all failed to reverse the neuroinflammatory signature. Bioinformatics analysis of the neuroinflammatory signature identified protein tyrosine phosphatase non-receptor type 11 (PTPN11/SHP2) as a potential target. Three structurally distinct inhibitors of PTPN11 (RMC-4550, TN0155, IACS-13909) reversed the neuroinflammatory disease signature. Collectively, these results highlight the utility of this novel neuroinflammatory platform for facilitating identification and validation of targets for neuroinflammatory neurodegenerative disease drug discovery.


Assuntos
Doenças Neurodegenerativas , Humanos , Camundongos , Animais , Doenças Neurodegenerativas/metabolismo , Doenças Neuroinflamatórias , Microglia/metabolismo , Inflamassomos/metabolismo , Biologia
2.
Neurosci Biobehav Rev ; 108: 453-458, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31783058

RESUMO

Increased healthcare and pharmaceutical understanding has led to the eradication of many childhood, infectious and preventable diseases; however, we are now experiencing the impact of aging disorders as the lifespan increases. These disorders have already become a major burden on society and threaten to become a defining challenge of our generation. Indications such as Alzheimer's disease gain headlines and have focused the thinking of many towards dementia and cognitive decline in aging. Indications related to neurological function and related behaviors are thus an extremely important starting point in the consideration of therapeutics.However, the reality is that pathological aging covers a spectrum of significant neurological and peripheral indications. Development of therapeutics to treat aging and age-related disorders is therefore a huge need, but represents a largely unexplored path. Fundamental scientific questions need to be considered as we embark towards a goal of improving health in old age, including how we 1) define aging as a therapeutic target, 2) model aging preclinically and 3) effectively translate from preclinical models to man. Furthermore, the challenges associated with identifying novel therapeutics in a financial, regulatory and clinical sense need to be contemplated carefully to ensure we address the unmet need in our increasingly elderly population. The complexity of the challenge requires different perspectives, cross-functional partnerships and diverse concepts. We seek to raise issues to guide the field, considering the current state of thinking to aid in identifying roadblocks and important challenges early. The need for therapeutics that address aging and age-related disorders is acute, but the promise of effective treatments provides huge opportunities that, as a community, we all seek to enable effectively as soon as possible.


Assuntos
Envelhecimento , Doença Crônica , Desenvolvimento de Medicamentos , Longevidade , Envelhecimento/efeitos dos fármacos , Animais , Doença Crônica/terapia , Desenvolvimento de Medicamentos/economia , Desenvolvimento de Medicamentos/legislação & jurisprudência , Humanos , Longevidade/efeitos dos fármacos
3.
Front Pharmacol ; 6: 125, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26150790

RESUMO

The recent approval of a therapeutic for a circadian disorder has increased interest in developing additional medicines for disorders characterized by circadian disruption. However, previous experience demonstrates that drug development for central nervous system (CNS) disorders has a high failure rate. Personalized medicine, or the approach to identifying the right treatment for the right patient, has recently become the standard for drug development in the oncology field. In addition to utilizing Companion Diagnostics (CDx) that identify specific genetic biomarkers to prescribe certain targeted therapies, patient profiling is regularly used to enrich for a responsive patient population during clinical trials, resulting in fewer patients required for statistical significance and a higher rate of success for demonstrating efficacy and hence receiving approval for the drug. This personalized medicine approach may be one mechanism that could reduce the high clinical trial failure rate in the development of CNS drugs. This review will discuss current circadian trials, the history of personalized medicine in oncology, lessons learned from a recently approved circadian therapeutic, and how personalized medicine can be tailored for use in future clinical trials for circadian disorders to ultimately lead to the approval of more therapeutics for patients suffering from circadian abnormalities.

4.
Bioorg Med Chem Lett ; 25(7): 1621-6, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25708617

RESUMO

Early lead compounds in this gamma secretase modulator series were found to potently inhibit CYP3A4 and other human CYP isoforms increasing their risk of causing drug-drug-interactions (DDIs). Using structure-activity relationships and CYP3A4 structural information, analogs were developed that minimized this DDI potential. Three of these new analogs were further characterized by rat PK, rat PK/PD and rat exploratory toxicity studies resulting in selection of SPI-1865 (14) as a preclinical development candidate.


Assuntos
Azetidinas/farmacologia , Produtos Biológicos/farmacologia , Citocromo P-450 CYP3A/metabolismo , Esteroides/farmacologia , Animais , Azetidinas/química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Conformação Molecular , Ratos , Ratos Sprague-Dawley , Esteroides/química , Relação Estrutura-Atividade
5.
Alzheimers Res Ther ; 5(2): 19, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23597079

RESUMO

INTRODUCTION: Modulation of the gamma-secretase enzyme, which reduces the production of the amyloidogenic Aß42 peptide while sparing the production of other Aß species, is a promising therapeutic approach for the treatment of Alzheimer's disease. Satori has identified a unique class of small molecule gamma-secretase modulators (GSMs) capable of decreasing Aß42 levels in cellular and rodent model systems. The compound class exhibits potency in the nM range in vitro and is selective for lowering Aß42 and Aß38 while sparing Aß40 and total Aß levels. In vivo, a compound from the series, SPI-1865, demonstrates similar pharmacology in wild-type CD1 mice, Tg2576 mice and Sprague Dawley rats. METHODS: Animals were orally administered either a single dose of SPI-1865 or dosed for multiple days. Aß levels were measured using a sensitive plate-based ELISA system (MSD) and brain and plasma exposure of drug were assessed by LC/MS/MS. RESULTS: In wild-type mice using either dosing regimen, brain Aß42 and Aß38 levels were decreased upon treatment with SPI-1865 and little to no statistically meaningful effect on Aß40 was observed, reflecting the changes observed in vitro. In rats, brain Aß levels were examined and similar to the mouse studies, brain Aß42 and Aß38 were lowered. Comparable changes were also observed in the Tg2576 mice, where Aß levels were measured in brain as well as plasma and CSF. CONCLUSIONS: Taken together, these data indicate that SPI-1865 is orally bioavailable, brain penetrant, and effective at lowering Aß42 in a dose responsive manner. With this unique profile, the class of compounds represented by SPI-1865 may be a promising new therapy for Alzheimer's disease.

6.
J Neurosci Methods ; 213(1): 14-21, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23219895

RESUMO

γ-Secretase modulators (GSM), which reduce amyloidogenic Aß(42) production while maintaining total Aß levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aß peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate. However, predicting selectivity for different substrates across cell systems may reduce the reliability of such ratios such that the in vitro data are not useful for predicting in vivo safety margins. This is especially concerning since the IC(50)'s of some GSIs vary depending upon the level of APP expression in a cell line. To circumvent this problem, we utilized the SUP-T1 cell line which expresses a truncated Notch receptor fragment that does not need sheddase cleavage to be a γ-secretase substrate. When combined with a sensitive method of measuring Aß production, this assay system allows both substrates to be measured simultaneously, reducing the potential to calculate imprecise selectivity margins. To demonstrate the value of this system, known GSIs and GSMs were examined in the SUP-T1 dual substrate assay. IC(50)'s were determined for both substrates and the in vitro selectivity margin was calculated. These data suggest using a single cell line is a more accurate prediction of the fold difference between NICD inhibition and Aß(42) lowering for therapeutically promising GSIs and GSMs.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/efeitos dos fármacos , Receptores Notch/efeitos dos fármacos , Alanina/análogos & derivados , Alanina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Azepinas/farmacologia , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Humanos , Oxidiazóis/farmacologia , Receptores Notch/metabolismo , Extração em Fase Sólida , Especificidade por Substrato , Sulfonamidas/farmacologia , Tiofenos/farmacologia
7.
J Med Chem ; 55(21): 9270-82, 2012 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-23030762

RESUMO

A series of triterpene-based γ-secretase modulators is optimized. An acetate present at the C24 position of the natural product was replaced with either carbamates or ethers to provide compounds with better metabolic stability. With one of those pharmacophores in place at C24, morpholines or carbamates were installed at the C3 position to refine the physicochemical properties of the analogues. This strategy gave compounds with low clearance and good distribution into the central nervous system (CNS) of CD-1 mice. Two of these compounds, 100 and 120, were tested for a pharmacodynamic effect in the strain and lowered brain Aß42 levels.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Produtos Biológicos/química , Triterpenos/química , Administração Oral , Peptídeos beta-Amiloides/metabolismo , Animais , Disponibilidade Biológica , Produtos Biológicos/farmacocinética , Produtos Biológicos/farmacologia , Barreira Hematoencefálica/metabolismo , Carbamatos/química , Carbamatos/farmacocinética , Carbamatos/farmacologia , Éteres/química , Éteres/farmacocinética , Éteres/farmacologia , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Fragmentos de Peptídeos/metabolismo , Permeabilidade , Ratos , Relação Estrutura-Atividade , Triterpenos/farmacocinética , Triterpenos/farmacologia
8.
Restor Neurol Neurosci ; 30(4): 283-9, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22596355

RESUMO

PURPOSE: Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery in experimental models of stroke in rats when treatment is delayed and without effect on infarct volume. PDE5A is expressed to only a very limited extent in forebrain tissues, raising the possibility that the locus of effect for the inhibitors is outside the brain. To start to address this question, we determined whether PDE5A inhibitors must have the ability to cross the blood brain barrier to improve recovery. METHOD: After permanent middle cerebral artery occlusion in rats, PF-5 and UK-489,791, PDE5A inhibitors that do or do not pass the blood brain barrier, were administered starting 24 h after occlusion and continued for 1 week. Motor function was assessed at intervals to 28 days using body swing and limb placement measures. RESULTS: Both PF-5 and UK-489,791 produced improvement in motor scores over 28 days that were significantly greater than in vehicle treated animals. There was no difference in efficacy between the two PDE5A inhibitors. CONCLUSIONS: Brain penetrability appears not to be critical to the ability of a PDE5A inhibitor to improve functional recovery after experimental stroke in rats. This finding is discussed with regard to the cellular target(s) for PDE5A inhibitors mediating this effect.


Assuntos
Infarto da Artéria Cerebral Média/tratamento farmacológico , Inibidores da Fosfodiesterase 5/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo
9.
Int J Alzheimers Dis ; 2012: 210756, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23320246

RESUMO

The Amyloid Hypothesis states that the cascade of events associated with Alzheimer's disease (AD)-formation of amyloid plaques, neurofibrillary tangles, synaptic loss, neurodegeneration, and cognitive decline-are triggered by Aß peptide dysregulation (Kakuda et al., 2006, Sato et al., 2003, Qi-Takahara et al., 2005). Since γ-secretase is critical for Aß production, many in the biopharmaceutical community focused on γ-secretase as a target for therapeutic approaches for Alzheimer's disease. However, pharmacological approaches to control γ-secretase activity are challenging because the enzyme has multiple, physiologically critical protein substrates. To lower amyloidogenic Aß peptides without affecting other γ-secretase substrates, the epsilon (ε) cleavage that is essential for the activity of many substrates must be preserved. Small molecule modulators of γ-secretase activity have been discovered that spare the ε cleavage of APP and other substrates while decreasing the production of Aß(42). Multiple chemical classes of γ-secretase modulators have been identified which differ in the pattern of Aß peptides produced. Ideally, modulators will allow the ε cleavage of all substrates while shifting APP cleavage from Aß(42) and other highly amyloidogenic Aß peptides to shorter and less neurotoxic forms of the peptides without altering the total Aß pool. Here, we compare chemically distinct modulators for effects on APP processing and in vivo activity.

10.
ACS Med Chem Lett ; 3(11): 908-13, 2012 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-24900406

RESUMO

The discovery of a new series of γ-secretase modulators is disclosed. Starting from a triterpene glycoside γ-secretase modulator that gave a very low brain-to-plasma ratio, initial SAR and optimization involved replacement of a pendant sugar with a series of morpholines. This modification led to two compounds with significantly improved central nervous system (CNS) exposure.

11.
J Neurosci Methods ; 204(1): 179-188, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22019329

RESUMO

Advances in imaging technology have enabled automated approaches for quantitative image analysis. In this study, a high content object based image analysis method was developed for quantification of ß-amyloid (Aß) plaques in postmortem brains of Alzheimer's disease (AD) subjects and in transgenic mice over overexpressing Aß. Digital images acquired from immunohistochemically stained sections of the superior frontal gyrus were analyzed for Aß plaque burden using a Definiens object-based segmentation approach. Blinded evaluation of Aß stained sections from AD and aged matched human subjects accurately identified AD cases with one exception. Brains from transgenic mice overexpressing Aß (PS1APP mice) were also evaluated by our Definiens object based image analysis approach. We observed an age-dependent increase in the amount of Aß plaque load that we quantified in both the hippocampus and cortex. From the contralateral hemisphere, we measured the amount of Aß in brain homogenates biochemically and observed a significant correlation between our biochemical measurements and those that we measured by our object based Definiens system in the hippocampus. Assessment of Aß plaque load in PS1APP mice using a manual segmentation technique (Image-Pro Plus) confirmed the results of our object-based image analysis approach. Image acquisition and analysis of 32 stained human slides and 100 mouse slides were executed in 8 h and 22 h, respectively supporting the relatively high throughput features of the Definiens platform. The data show that digital imaging combined with object based image analysis is a reliable and efficient approach to quantifying Aß plaques in human and mouse brain.


Assuntos
Algoritmos , Encéfalo/patologia , Interpretação de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia Confocal/métodos , Reconhecimento Automatizado de Padrão/métodos , Placa Amiloide/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Humanos , Aumento da Imagem/métodos , Masculino , Camundongos , Camundongos Transgênicos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
Bioorg Med Chem Lett ; 21(9): 2631-6, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21269825

RESUMO

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a ß-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aß in guinea pigs. The therapeutic index between Aß reductions and changes in B-cell populations were studied for compound 10 h.


Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Imidazóis/síntese química , Imidazóis/farmacologia , Aminação/efeitos dos fármacos , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/metabolismo , Animais , Bioensaio , Diamida/síntese química , Diamida/química , Diamida/farmacologia , Inibidores Enzimáticos/química , Cobaias , Células HeLa , Humanos , Imidazóis/química , Concentração Inibidora 50 , Estrutura Molecular , Relação Estrutura-Atividade
13.
Bioorg Med Chem Lett ; 21(9): 2637-40, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21269827

RESUMO

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aß in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Tetra-Hidronaftalenos/síntese química , Tetra-Hidronaftalenos/farmacologia , Valina/análogos & derivados , Animais , Bioensaio , Desenho de Fármacos , Inibidores Enzimáticos/química , Cobaias , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/química , Valina/síntese química , Valina/química , Valina/farmacologia
14.
Psychopharmacology (Berl) ; 210(4): 569-76, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20407760

RESUMO

INTRODUCTION: Casein kinase I epsilon/delta phosphorylates certain clock-related proteins as part of a complex arrangement of transcriptional/translational feedback loops that comprise the circadian oscillator in mammals. Pharmacologic inhibition leads to a delay of the oscillations with the magnitude of this effect dependent upon the timing of drug administration. OBJECTIVE: Earlier studies by our lab described the actions of a selective CKI epsilon/delta inhibitor, PF-670462, on circadian behavior following acute dosing; the present work extended these studies to chronic once-daily treatment. METHODS: Gross motor activity was used to estimate the circadian rhythms of rats maintained under a 12 L:12 D cycle. PF-670462, 10 or 30 mg/kg/day s.c., was administered once daily for 20 days either at ZT6 or ZT11 (i.e., 6 or 11 h after light onset). RESULTS: Chronic administration of PF-670462, performed at a fixed time of day, produced delays in the activity onsets of rats that cumulated with the duration of dosing. Dosing at ZT11 yielded more robust delays than dosing at ZT6 in keeping with earlier phase-response analyses with this agent. CONCLUSIONS: The magnitude of the shifts in activity onsets achieved with chronic dosing of PF-670462 appears to be a function of the dose and the previously established phase relationship. Its cumulative effect further suggests that the pharmacodynamic t (1/2) of the drug greatly exceeds its pharmacokinetic one. Most importantly, these changes in circadian behavior occurred in the presence of a fixed L:D cycle, confirming the drug to be a robust modulator of circadian phase in the presence of the natural zeitgeber.


Assuntos
Caseína Quinase 1 épsilon/antagonistas & inibidores , Caseína Quinase Idelta/antagonistas & inibidores , Ritmo Circadiano/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Animais , Relógios Biológicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Atividade Motora/efeitos dos fármacos , Pirimidinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
15.
J Pharmacol Exp Ther ; 334(1): 269-77, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20363853

RESUMO

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Tetra-Hidronaftalenos/farmacologia , Tetra-Hidronaftalenos/farmacocinética , Valina/análogos & derivados , Animais , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/química , Escherichia coli/genética , Feminino , Cobaias , Humanos , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos , Estrutura Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Baço/citologia , Baço/efeitos dos fármacos , Tetra-Hidronaftalenos/efeitos adversos , Tetra-Hidronaftalenos/química , Distribuição Tecidual , Transfecção , Valina/efeitos adversos , Valina/química , Valina/farmacocinética , Valina/farmacologia
16.
J Pharmacol Exp Ther ; 331(3): 842-50, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19729580

RESUMO

Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery after middle cerebral artery occlusion (MCA-o) in rats. We used the PDE5A inhibitor 3-(4-(2-hydroxyethyl)piperazin-1-yl)-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one hydrochloride (PF-5) to determine the timing, duration, and degree of inhibition that yields maximum efficacy. We also investigated the localization of PDE5A to determine the tissues and cells that would be targets for PDE5 inhibition and that may mediate efficacy. Nearly complete inhibition of PDE5A, starting 24 h after MCA-o and continued for 7 days, resulted in nearly complete recovery of sensorimotor function that was sustained for 3 months. Delaying administration until 72 h after MCA-o resulted in equivalent efficacy, whereas delaying treatment for 14 days was ineffective. Treatment for 7 days was equivalently efficacious to 28 or 84 days of treatment, whereas treatment for 1 day was less effective. In the normal forebrain, PDE5A immunoreactivity was prominent in smooth muscle of meningeal arteries and a few smaller blood vessels, with weak staining in a few widely scattered cortical neurons and glia. At 24 and 48 h after MCA-o, the number and intensity of blood vessel staining increased in the infarcted cortex and striatum. PDE5A immunoreactivity also was increased at 48 h in putative microglia in penumbra, whereas there was no change in staining of the scattered cortical neurons. Given the window for efficacy and the PDE5A distribution, we hypothesize that efficacy results from an effect on vasculature, and perhaps modulation of microglial function, both of which may facilitate recovery of neuronal function.


Assuntos
Encéfalo/efeitos dos fármacos , Inibidores da Fosfodiesterase 5 , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Masculino , Microglia/efeitos dos fármacos , Microglia/enzimologia , Atividade Motora/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo
17.
Chronobiol Int ; 24(3): 407-11, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17612940

RESUMO

The degu (Octodon degus) is a diurnal rodent, although phase inversions to nocturnal behavior have been reported under specific laboratory conditions. The reliability of this animal as a diurnal model of sleep therefore requires further characterization of intrinsic circadian pacemaker properties. A phase response curve to light has been reported in the degu, and is consistent with other diurnal animals. This study reports a phase response curve to melatonin in the degu, which is distinct in orientation from the light curve.


Assuntos
Ritmo Circadiano/fisiologia , Melatonina/farmacologia , Octodon/fisiologia , Animais , Relógios Biológicos/fisiologia , Ritmo Circadiano/efeitos dos fármacos , Escuridão , Injeções Intraperitoneais , Masculino , Melatonina/administração & dosagem , Sono/efeitos dos fármacos , Sono/fisiologia
18.
J Pharmacol Exp Ther ; 322(2): 730-8, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17502429

RESUMO

Casein kinase Iepsilon (CKIepsilon) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC(50) = 7.7 +/- 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKIepsilon in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (-1.97 +/- 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose- and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these drug-induced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t(1/2) = 0.46 +/- 0.04 h). Together, these results suggest that inhibition of CKIepsilon yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.


Assuntos
Caseína Quinase 1 épsilon/antagonistas & inibidores , Ritmo Circadiano/fisiologia , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Células COS , Caseína Quinase 1 épsilon/genética , Caseína Quinase 1 épsilon/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Escuridão , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Masculino , Camundongos , Estrutura Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção
19.
J Pharmacol Exp Ther ; 320(3): 1144-52, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17202404

RESUMO

beta-Amyloid peptides, tentatively regarded as the principal neurotoxins responsible for Alzheimer's Disease, make up a set of products that varies significantly among different biological systems. The full implications of this complexity and its variations have yet to be defined. In this work, Abeta peptide populations were extracted from animal brain tissue or cell-conditioned media, immunoprecipitated with specific antibodies, and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. (15)N-Substituted Abeta internal standards were added to gauge variations in the profile of captured peptides. Results from a range of species, including guinea pig, dog, rabbit, and wild-type and transgenic mice, showed that the Abeta peptide population in each system was mainly determined by the species of origin of the amyloid precursor protein (APP) and not by the host tissue or cell line. The same method was used to gauge the effect on the Abeta peptide profile of an inhibitor of gamma-secretase, one of the two proteinases that excises Abeta peptides from the precursor protein with different effects on specific peptides. Overall, the results demonstrate that the species of origin of the APP substrate dictates the outcome of APP processing to a greater extent than the origin of the processing enzymes, an important consideration in rationalizing the properties of different model systems.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/biossíntese , Encéfalo/enzimologia , Encéfalo/metabolismo , Fragmentos de Peptídeos/biossíntese , Alanina/análogos & derivados , Alanina/farmacologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/genética , Animais , Anticorpos Monoclonais/farmacologia , Azepinas/farmacologia , Células Cultivadas , Meios de Cultivo Condicionados , Cães , Inibidores Enzimáticos/farmacologia , Cobaias , Humanos , Imunoprecipitação , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/genética , Coelhos , Ratos , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Ressonância de Plasmônio de Superfície
20.
J Pharmacol Exp Ther ; 319(2): 924-33, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16920992

RESUMO

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.


Assuntos
Alanina/análogos & derivados , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/sangue , Azepinas/farmacologia , Inibidores Enzimáticos/farmacologia , Alanina/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Cobaias , Masculino , Camundongos , Fatores de Tempo
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