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1.
Geroscience ; 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38563864

RESUMO

Epidemiological studies report an elevated risk of Parkinson's disease (PD) in patients with type 2 diabetes mellitus (T2DM) that is mitigated in those prescribed dipeptidyl peptidase 4 (DPP-4) inhibitors. With an objective to characterize clinically translatable doses of DPP-4 inhibitors (gliptins) in a well-characterized PD rodent model, sitagliptin, PF-00734,200 or vehicle were orally administered to rats initiated either 7-days before or 7-days after unilateral medial forebrain bundle 6-hydroxydopamine (6-OHDA) lesioning. Measures of dopaminergic cell viability, dopamine content, neuroinflammation and neurogenesis were evaluated thereafter in ipsi- and contralateral brain. Plasma and brain incretin and DPP-4 activity levels were quantified. Furthermore, brain incretin receptor levels were age-dependently evaluated in rodents, in 6-OHDA challenged animals and human subjects with/without PD. Cellular studies evaluated neurotrophic/neuroprotective actions of combined incretin administration. Pre-treatment with oral sitagliptin or PF-00734,200 reduced methamphetamine (meth)-induced rotation post-lesioning and dopaminergic degeneration in lesioned substantia nigra pars compacta (SNc) and striatum. Direct intracerebroventricular gliptin administration lacked neuroprotective actions, indicating that systemic incretin-mediated mechanisms underpin gliptin-induced favorable brain effects. Post-treatment with a threefold higher oral gliptin dose, likewise, mitigated meth-induced rotation, dopaminergic neurodegeneration and neuroinflammation, and augmented neurogenesis. These gliptin-induced actions associated with 70-80% plasma and 20-30% brain DPP-4 inhibition, and elevated plasma and brain incretin levels. Brain incretin receptor protein levels were age-dependently maintained in rodents, preserved in rats challenged with 6-OHDA, and in humans with PD. Combined GLP-1 and GIP receptor activation in neuronal cultures resulted in neurotrophic/neuroprotective actions superior to single agonists alone. In conclusion, these studies support further evaluation of the repurposing of clinically approved gliptins as a treatment strategy for PD.

2.
Geroscience ; 2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38532069

RESUMO

The endogenous incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) possess neurotrophic, neuroprotective, and anti-neuroinflammatory actions. The dipeptidyl peptidase 4 (DPP-4) inhibitor sitagliptin reduces degradation of endogenous GLP-1 and GIP, and, thereby, extends the circulation of these protective peptides. The current nonhuman primate (NHP) study evaluates whether human translational sitagliptin doses can elevate systemic and central nervous system (CNS) levels of GLP-1/GIP in naive, non-lesioned NHPs, in line with our prior rodent studies that demonstrated sitagliptin efficacy in preclinical models of Parkinson's disease (PD). PD is an age-associated neurodegenerative disorder whose current treatment is inadequate. Repositioning of the well-tolerated and efficacious diabetes drug sitagliptin provides a rapid approach to add to the therapeutic armamentarium for PD. The pharmacokinetics and pharmacodynamics of 3 oral sitagliptin doses (5, 20, and 100 mg/kg), equivalent to the routine clinical dose, a tolerated higher clinical dose and a maximal dose in monkey, were evaluated. Peak plasma sitagliptin levels were aligned both with prior reports in humans administered equivalent doses and with those in rodents demonstrating reduction of PD associated neurodegeneration. Although CNS uptake of sitagliptin was low (cerebrospinal fluid (CSF)/plasma ratio 0.01), both plasma and CSF concentrations of GLP-1/GIP were elevated in line with efficacy in prior rodent PD studies. Additional cellular studies evaluating human SH-SY5Y and primary rat ventral mesencephalic cultures challenged with 6-hydroxydopamine, established cellular models of PD, demonstrated that joint treatment with GLP-1 + GIP mitigated cell death, particularly when combined with DPP-4 inhibition to maintain incretin levels. In conclusion, this study provides a supportive translational step towards the clinical evaluation of sitagliptin in PD and other neurodegenerative disorders for which aging, similarly, is the greatest risk factor.

3.
Proc Natl Acad Sci U S A ; 105(1): 33-8, 2008 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-18162540

RESUMO

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.


Assuntos
Canais de Cálcio/química , Sirolimo/química , Proteínas de Ligação a Tacrolimo/química , Animais , Cálcio/metabolismo , Eletrofisiologia/métodos , Humanos , Imunofilinas/metabolismo , Imunossupressores/farmacologia , Ligantes , Modelos Químicos , Neuritos/metabolismo , Neuroblastoma/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Ratos , Acidente Vascular Cerebral/metabolismo
4.
Proc Natl Acad Sci U S A ; 105(25): 8754-9, 2008 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-18559859

RESUMO

The amyloid hypothesis states that a variety of neurotoxic beta-amyloid (Abeta) species contribute to the pathogenesis of Alzheimer's disease. Accordingly, a key determinant of disease onset and progression is the appropriate balance between Abeta production and clearance. Enzymes responsible for the degradation of Abeta are not well understood, and, thus far, it has not been possible to enhance Abeta catabolism by pharmacological manipulation. We provide evidence that Abeta catabolism is increased after inhibition of plasminogen activator inhibitor-1 (PAI-1) and may constitute a viable therapeutic approach for lowering brain Abeta levels. PAI-1 inhibits the activity of tissue plasminogen activator (tPA), an enzyme that cleaves plasminogen to generate plasmin, a protease that degrades Abeta oligomers and monomers. Because tPA, plasminogen and PAI-1 are expressed in the brain, we tested the hypothesis that inhibitors of PAI-1 will enhance the proteolytic clearance of brain Abeta. Our data demonstrate that PAI-1 inhibitors augment the activity of tPA and plasmin in hippocampus, significantly lower plasma and brain Abeta levels, restore long-term potentiation deficits in hippocampal slices from transgenic Abeta-producing mice, and reverse cognitive deficits in these mice.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Fibrinolisina/metabolismo , Fibrinolíticos/metabolismo , Animais , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Inativadores de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tecidual/metabolismo
5.
Neurodegener Dis ; 7(1-3): 153-9, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20197696

RESUMO

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative condition characterized by an increasing loss of dopaminergic neurons resulting in motor dysfunction. However, cognitive impairments in PD patients are a common clinical feature that has gained increased attention. OBJECTIVE: The purpose of the current study was to evaluate the effects of an MPTP-induced dopaminergic lesion in mice on social odor recognition (SOR) memory. METHODS: Mice were acutely treated with MPTP and evaluated for memory impairments in the SOR assay and characterized using biochemical and immunohistochemical methods approximately 2 weeks later. RESULTS: Here we demonstrate that SOR memory is sensitive to MPTP treatment and that it correlates with multiple measures of nigrostriatal integrity. MPTP treatment of C57BL/6N mice produced a profound decrease in dopamine levels, dopamine transporter binding and tyrosine hydroxylase immunoreactivity in the striatum. These impairments in stratial dopaminergic function were blocked by pretreatment with the MAO-B inhibitor deprenyl. Changes in the dopaminergic system parallel those observed in SOR with MPTP treatment impairing recognition memory in the absence of a deficit in odor discrimination during learning. Deprenyl pretreatment blocked the MPTP-induced impairment of SOR memory. CONCLUSION: The use of the SOR memory model may provide a preclinical method for evaluating cognitive therapies for PD.


Assuntos
Intoxicação por MPTP/complicações , Intoxicação por MPTP/psicologia , Transtornos da Memória/etiologia , Reconhecimento Psicológico/fisiologia , Predomínio Social , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Análise de Variância , Animais , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Intoxicação por MPTP/induzido quimicamente , Intoxicação por MPTP/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Substância Negra/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo
6.
J Neurosci ; 28(45): 11445-53, 2008 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-18987181

RESUMO

Inheritance of the apoE4 allele (epsilon4) increases the risk of developing Alzheimer's disease; however, the mechanisms underlying this association remain elusive. Recent data suggest that inheritance of epsilon4 may lead to reduced apoE protein levels in the CNS. We therefore examined apoE protein levels in the brains, CSF and plasma of epsilon2/2, epsilon3/3, and epsilon4/4 targeted replacement mice. These apoE mice showed a genotype-dependent decrease in apoE levels; epsilon2/2 >epsilon3/3 >epsilon4/4. Next, we sought to examine the relative contributions of apoE4 and apoE3 in the epsilon3/4 mouse brains. ApoE4 represented 30-40% of the total apoE. Moreover, the absolute amount of apoE3 per allele was similar between epsilon3/3 and epsilon3/4 mice, implying that the reduced levels of total apoE in epsilon3/4 mice can be explained by the reduction in apoE4 levels. In culture medium from epsilon3/4 human astrocytoma or epsilon3/3, epsilon4/4 and epsilon3/4 primary astrocytes, apoE4 levels were consistently lower than apoE3. Secreted cholesterol levels were also lower from epsilon4/4 astrocytes. Pulse-chase experiments showed an enhanced degradation and reduced half-life of newly synthesized apoE4 compared with apoE3. Together, these data suggest that astrocytes preferentially degrade apoE4, leading to reduced apoE4 secretion and ultimately to reduced brain apoE levels. Moreover, the genotype-dependent decrease in CNS apoE levels, mirror the relative risk of developing AD, and suggest that low levels of total apoE exhibited by epsilon4 carriers may directly contribute to the disease progression, perhaps by reducing the capacity of apoE to promote synaptic repair and/or Abeta clearance.


Assuntos
Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Polimorfismo Genético , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Humanos , Hidrocarbonetos Fluorados/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , RNA Mensageiro/metabolismo , Sulfonamidas/farmacologia , Fatores de Tempo
7.
J Pharmacol Exp Ther ; 331(3): 827-35, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19741150

RESUMO

Src kinase signaling has been implicated in multiple mechanisms of ischemic injury, including vascular endothelial growth factor (VEGF)-mediated vascular permeability that leads to vasogenic edema, a major clinical complication in stroke and brain trauma. Here we report the effects of two novel Src kinase inhibitors, 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methyl-1-piperazinyl)propoxy]-3-quinolinecarbonitrile (SKI-606) and 4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[4-(4-methypiperazin-1-yl)but-1-ynyl]-3-quinolinecarbonitrile (SKS-927), on ischemia-induced brain infarction and short- and long-term neurological deficits. Two well established transient [transient middle cerebral artery occlusion (tMCAO)] and permanent [permanent middle cerebral artery occlusion (pMCAO)] focal ischemia models in the rat were used with drug treatments initiated up to 6 h after onset of stroke to mimic the clinical scenario. Brain penetration of Src inhibitors, their effect on blood-brain barrier integrity and VEGF signaling in human endothelial cells were also evaluated. Our results demonstrate that both agents potently block VEGF-mediated signaling in human endothelial cells, penetrate rat brain upon systemic administration, and inhibit postischemic Src activation and vascular leakage. Treatment with SKI-606 or SKS-927 (at the doses of 3-30 mg/kg i.v.) resulted in a dose-dependent reduction in infarct volume and robust protection from neurological impairments even when the therapy was initiated up to 4- to 6-h after tMCAO. Src blockade after pMCAO resulted in accelerated improvement in recovery from motor, sensory, and reflex deficits during a long-term (3 weeks) testing period poststroke. These data demonstrate that the novel Src kinase inhibitors provide effective treatment against ischemic conditions within a clinically relevant therapeutic window and may constitute a viable therapy for acute stroke.


Assuntos
Compostos de Anilina/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Nitrilas/uso terapêutico , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Quinases da Família src/antagonistas & inibidores , Compostos de Anilina/administração & dosagem , Compostos de Anilina/química , Compostos de Anilina/farmacocinética , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/enzimologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Isquemia Encefálica/enzimologia , Permeabilidade Capilar , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Citometria de Fluxo , Humanos , Injeções Intravenosas , Masculino , Estrutura Molecular , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Testes Neuropsicológicos , Nitrilas/administração & dosagem , Nitrilas/química , Nitrilas/farmacocinética , Piperazinas/administração & dosagem , Piperazinas/química , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Quinolinas/administração & dosagem , Quinolinas/química , Quinolinas/farmacocinética , Ratos , Ratos Wistar , Fatores de Tempo
8.
J Pharmacol Exp Ther ; 331(2): 598-608, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19671883

RESUMO

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Adolescente , Adulto , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ligação Competitiva , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Cães , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Medo/psicologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Receptores Notch/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacocinética , Sulfonamidas/toxicidade , Tiofenos/farmacocinética , Tiofenos/toxicidade , Adulto Jovem
9.
Neuropharmacology ; 56(2): 329-41, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19007799

RESUMO

Ischemic stroke is the second most common cause of death worldwide and a major cause of disability. Intravenous thrombolysis with rt-PA remains the only available acute therapy in patients who present within 3h of stroke onset other than the recently approved mechanical MERCI device, substantiating the high unmet need in available stroke therapeutics. The development of successful therapeutic strategies remains challenging, as evidenced by the continued failures of new therapies in clinical trials. However, significant lessons have been learned and this knowledge is currently being incorporated into improved pre-clinical and clinical design. Furthermore, advancements in imaging technologies and continued progress in understanding biological pathways have established a prolonged presence of salvageable penumbral brain tissue and have begun to elucidate the natural repair response initiated by ischemic insult. We review important past and current approaches to drug development with an emphasis on implementing principles of translational research to achieve a rigorous conversion of knowledge from bench to bedside. We highlight current strategies to protect and repair brain tissue with the promise to provide longer therapeutic windows, preservation of multiple tissue compartments and improved clinical success.


Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Fibrinolíticos/uso terapêutico , Acidente Vascular Cerebral/terapia , Animais , Ensaios Clínicos como Assunto , Humanos , Acidente Vascular Cerebral/complicações
10.
Brain Res ; 1711: 140-145, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30664847

RESUMO

Highly specific and sensitive biomarkers for pathologies related to dysfunctions in the basal ganglia circuit are of great value to assess therapeutic efficacy not only clinically to establish an early diagnosis, but also in terms of monitoring the efficacy of therapeutic interventions and decelerated neurodegeneration. The phosphodiesterase 10A (PDE10A) enzyme plays a central role in striatal signaling and is implicated in several neuropsychiatric disorders involving striatal pathology, such as Huntingtons disease (HD) and schizophrenia. Inhibition of PDE10A activates the neurons in the striatum and consequently leads to alteration of behavioral aspects modulated by the striatal circuit. [18F]MNI-659, (2-(2-(3-(4-(2-[18F]fluoroethoxy)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione), is a newly developed PET radioligand that shows a high binding to PDE10A in the human brain in vivo. In the present study, we examined the in vitro binding of [18F]MNI-659 in human postmortem brain to gain a better understanding of the presence, density, disease-related alterations and therapy related to changes in PDE10A expression. The results show high specific binding of [18F]MNI-659 in the caudate nucleus, putamen and the hippocampal formation. Low specific [18F]MNI-659 binding was detected in nucleus accumbens in comparison to the caudate nucleus and putamen. In vitro binding studies with [18F]MNI-659 will facilitate in elucidating better understanding of the role of PDE10A activity in health and disease that may lead to new diagnostic opportunities in HD.


Assuntos
Encéfalo/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Ftalimidas , Tomografia por Emissão de Pósitrons/métodos , Quinazolinonas , Adulto , Idoso , Gânglios da Base/enzimologia , Gânglios da Base/metabolismo , Encéfalo/diagnóstico por imagem , Cadáver , Corpo Estriado/enzimologia , Corpo Estriado/metabolismo , Feminino , Radioisótopos de Flúor , Humanos , Masculino , Pessoa de Meia-Idade , Neostriado/enzimologia , Neostriado/metabolismo , Compostos Radiofarmacêuticos
11.
J Cereb Blood Flow Metab ; 28(1): 217-9, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17579658

RESUMO

The continued failure in approving new drugs for treatment of acute stroke has been recently set back by the failure of the NXY-059 (Stroke-Acute Ischemic NXY Treatment (SAINT) II) trial. The disappointment was heightened by the latter study being viewed as a most promising compound for stroke drug development program based on the preclinical data. Since the SAINT I/II development program included many of the STAIR (Stroke Therapy Academic Industry Round table) guidelines, yet have still failed to achieve the expected efficacy, there is a clear need to continue and analyze the path forward for stroke drug discovery. To this end, this review calls for a consortium approach including academia, government (FDA/NIH), and pharmaceutical industry partnerships to define this path. It is also imperative that more attention is given to the evolving discipline of Translational Medicine. A key issue in this respect is the need to devote more attention to the characteristics of the drug candidate nature-target interaction, and its relationship to pharmacodynamic treatment end points. It is equally important that efforts are spent to prove that phenotypic outcomes are linked to the purported mechanism of action of the compound. Development of technologies that allows a better assessment of these parameters, especially in in vivo models are paramount. Finally, rational patient selection and new outcome scales tailored in an adaptive design model must be evaluated.


Assuntos
Benzenossulfonatos/farmacocinética , Isquemia Encefálica/tratamento farmacológico , Fármacos Cardiovasculares/farmacocinética , Aprovação de Drogas , Desenho de Fármacos , Seleção de Pacientes , Benzenossulfonatos/uso terapêutico , Fármacos Cardiovasculares/uso terapêutico , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Indústria Farmacêutica , Determinação de Ponto Final , Guias como Assunto , Humanos , Modelos Cardiovasculares , National Institutes of Health (U.S.) , Acidente Vascular Cerebral , Resultado do Tratamento , Estados Unidos , United States Food and Drug Administration
12.
Neurobiol Dis ; 31(3): 334-41, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18606547

RESUMO

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drug's inhibition of central acid-sensing ion channels (ASICs). Given that Parkinson's disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinson's disease.


Assuntos
Acidose Láctica/tratamento farmacológico , Amilorida/farmacologia , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/tratamento farmacológico , Substância Negra/efeitos dos fármacos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Canais Iônicos Sensíveis a Ácido , Acidose Láctica/etiologia , Acidose Láctica/fisiopatologia , Animais , Antiparkinsonianos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Modelos Animais de Doenças , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Peptídeos , Ensaio Radioligante , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Venenos de Aranha/farmacologia , Substância Negra/metabolismo , Substância Negra/fisiopatologia , Tirosina 3-Mono-Oxigenase/metabolismo
13.
Artigo em Inglês | MEDLINE | ID: mdl-18824419

RESUMO

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson's disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP+), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP+ in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP+ in brain extracts with 0.4M perchloric acid (HClO4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP+ using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP+ established the limits of detection (LOD) (S/N=3) at 0.34pg/mg tissue and 0.007pg/mg tissue (5microl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D.<6%), and sensitive over a range of 0.001-1ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP+ concentrations in vivo. Two reference agents, R-(-) Deprenyl and MK-801, known to alter the concentration of MPP+ in MPTP treated mice were evaluated.


Assuntos
1-Metil-4-fenilpiridínio/análise , Cromatografia Líquida/métodos , Corpo Estriado/metabolismo , Espectrometria de Massas em Tandem/métodos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , 1-Metil-4-fenilpiridínio/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Reprodutibilidade dos Testes
14.
J Neurosci Methods ; 163(2): 310-20, 2007 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-17490751

RESUMO

Immunophilins are protein receptors for the immunosuppressant drugs FK506, cyclosporin A (CsA), and rapamycin. Two categories of immunophilins are the FK506-binding proteins (FKBPs), which bind to FK506, rapamycin, and CCI-779 and the cyclophilins, which bind to CsA. Reports have shown that immunophilins are expressed in the brain and spinal cord, are 10-100-fold higher in CNS tissue than immune tissue, and their expression is increased following nerve injury, suggesting that their chemical ligands may have therapeutic utility in the treatment of neurodegenerative diseases. In this study, we report the development and utility of a rapid neurofilament (NF) enzyme-linked immunosorbent assay (ELISA) to quantify neuronal survival and the Cellomics ArrayScan platform to quantify neurite outgrowth following treatment with immunophilin ligands. Cultured neurons or F-11 cells were treated with various immunophilin ligands for 72 or 96h and their promotion of neuronal survival and neurite outgrowth were determined. The results showed that all immunophilin ligands, in a concentration-dependent manner, significantly increased neuronal survival and neurite outgrowth, when compared to control cultures. Taken together, these results demonstrate the potential utility of the neurofilament ELISA and Cellomics ArrayScan platform to efficiently quantify neurotrophic effects of immunophilin ligands on cultured neurons and cell lines.


Assuntos
Bioensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Imunofilinas/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Proteínas de Neurofilamentos/análise , Proteínas de Neurofilamentos/efeitos dos fármacos , Animais , Bioensaio/instrumentação , Contagem de Células/instrumentação , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Crescimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Imunofilinas/metabolismo , Imunossupressores/farmacologia , Ligantes , Neuritos/metabolismo , Neuritos/ultraestrutura , Óptica e Fotônica/instrumentação , Proteínas Quinases/efeitos dos fármacos , Proteínas Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR
15.
J Med Chem ; 60(18): 7764-7780, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28817277

RESUMO

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Piridinas/farmacologia , Piridinas/farmacocinética , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Receptor de Glutamato Metabotrópico 5/metabolismo , Animais , Feminino , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/efeitos adversos , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Masculino , Simulação de Acoplamento Molecular , Piridinas/efeitos adversos , Piridinas/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade
16.
Neuron ; 92(6): 1220-1237, 2016 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-27916455

RESUMO

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Doença de Huntington/fisiopatologia , Neostriado/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Pirazóis/farmacologia , Quinolinas/farmacologia , Animais , Gânglios da Base/diagnóstico por imagem , Gânglios da Base/efeitos dos fármacos , Gânglios da Base/metabolismo , Gânglios da Base/fisiopatologia , Córtex Cerebral/diagnóstico por imagem , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Doença de Huntington/metabolismo , Camundongos , Neostriado/diagnóstico por imagem , Neostriado/metabolismo , Neostriado/fisiopatologia , Diester Fosfórico Hidrolases , Tomografia por Emissão de Pósitrons , Núcleo Subtalâmico/diagnóstico por imagem , Núcleo Subtalâmico/efeitos dos fármacos , Núcleo Subtalâmico/metabolismo , Núcleo Subtalâmico/fisiopatologia , Trítio
17.
Cell Signal ; 16(9): 1013-21, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15212762

RESUMO

NAcht Leucine-rich-repeat Protein 1 (NALP1) contains a putative nucleotide binding site, a region of leucine-rich repeats, and death domain folds at both termini providing protein/protein association functions such as caspase recruitment. We report here that NALP1 gene expression was induced in primary cerebellar granule neurons (CGN) upon injury. Up-regulation of NALP1 was also observed in a model of transient focal ischemia induced by middle cerebral artery occlusion. We investigated the biological consequence of over-expression of NALP1 in both HeLa cells and in CGN. Expression of recombinant NALP1 stimulated cell death in both HeLa cells and CGN by an apoptotic mechanism, demonstrated by the induction of apoptotic nuclear morphology and activation of the apoptotic enzyme caspase-3. Also described here are studies on the mechanism of action studies including deletion analyses and investigations of nucleotide binding, which begin to elucidate a regulatory function for NALP1 in neuronal apoptosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Cerebelo/citologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxiadenina/farmacologia , Ativação Enzimática , Células HeLa , Humanos , Mutagênese , Proteínas NLR , Ligação Proteica , RNA Mensageiro/metabolismo , Ratos , Transfecção
18.
Curr Drug Targets CNS Neurol Disord ; 2(6): 349-56, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14683462

RESUMO

There is a significant unmet need for therapeutic agents in the treatment of neurodegenerative diseases. Given their clinical importance, prototypical molecules that clearly exhibit both neuroprotective and neuroregenerative activities have been highly sought after. The journey led to the exploitation of neurotrophins, a family of proteins that had extraordinary therapeutic properties in pre-clinical models of neurodegeneration. Although experimentally promising, clinical development of neurotrophins for various neurological indications, such as Alzheimer's Disease, Amyotrophic Lateral Sclerosis, and Parkinson's Disease was met with severe obstacles and setbacks, such as the inability to deliver these large proteins to target population of neurons, instability of the proteins, and non-specific activity. Immunophilins are proteins that act as receptors for immunosuppresant drugs, i.e. FK506 (tacrolimus), cyclosporin A, and rapamycin (sirolimus, Rapamune). Studies indicate immunophilins are expressed 10-100 fold higher in CNS and PNS tissue than in immune tissue. Subsequent studies revealed potent neuroprotective and neuroregenerative properties of immunophilin ligands in both culture and animal models. In contrast to neurotrophins, most immunophilin ligands are highly stable, small molecules that can readily cross the blood-brain barrier and are orally bioavailable. Taken together, these data prompted the development of nonimmunosuppressive immunophilin ligands with potent therapeutic activities, although the potency of select compounds has come into question in more recent studies. This review will examine the experimental evidence supporting the use of immunophilin ligands for the treatment of neurodegenerative diseases and the current progression of these molecules in clinical trials.


Assuntos
Imunofilinas/uso terapêutico , Doenças Neurodegenerativas/tratamento farmacológico , Animais , Humanos , Imunofilinas/química , Imunofilinas/metabolismo , Imunossupressores/metabolismo , Imunossupressores/uso terapêutico , Ligantes , Doenças Neurodegenerativas/metabolismo
19.
Brain Res ; 982(2): 146-55, 2003 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-12915250

RESUMO

Rodent models of focal and global ischemia were used to examine caspase activation. Several readouts were employed on identical tissue to provide correlative measurement of caspase induction, activation and enzymatic activity. In a rat focal ischemia model, caspase-3 enzymatic activity, as recorded by DEVD-AMC cleavage, peaked in penumbral cortex at 6-12 h following ischemia, correlating with increases in caspase 3-cleaved substrates of PARP and alpha-spectrin and subsequent disappearance of caspase-3 zymogen. Although induction of caspases 8 and 2 proteins was detectable as early as 6 h following ischemia, examination of the same tissues for caspase 8 or 2 enzymatic activities did not show significant modulation up to 12 h after ischemic insult. Caspase 9 induction was evident only after 24 h postischemia and did not correlate with elevated LDHD-AMC cleavage. Following global ischemia in gerbils, levels of caspase-3 enzyme activity peaked at 12 h in hippocampal tissue extracts. Cleaved caspase-3 signal was prominent in NeuN-positive layers in the CA1 region 6-12 h following ischemia. Interestingly, strong caspase-3 immunoreactivity was also detected in the subgranular zone of the dentate gyrus, a known region of ischemia-induced neurogenesis. Caspase-3 activation may be responsible for the loss of these cells, thereby hindering the endogenous recovery process.


Assuntos
Isquemia Encefálica/enzimologia , Caspases/metabolismo , Modelos Animais de Doenças , Hipocampo/enzimologia , Animais , Ativação Enzimática/fisiologia , Gerbillinae , Masculino , Ratos , Ratos Wistar
20.
J Pharm Biomed Anal ; 34(2): 359-68, 2004 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-15013150

RESUMO

A method using reverse phase liquid chromatography-tandem mass spectrometry and cassette administration was developed for in vivo brain and plasma exposure profiling to assist early CNS drug discovery programs. Three to four compounds were grouped in cassettes for dosing and analysis. Compounds in the cassettes were selected to minimize possible analytical interference from each other, as well as from their potential metabolites. In order to improve the confidence of cassette administration, an analogue of the study compounds, with well-established brain penetration data, was included in each cassette as a "biological internal standard". Compounds were administered to rats by intraperitoneal injection and extracted from plasma or brain homogenate by simple protein precipitation. Fast chromatographic separation was achieved by using a short narrow-bore column at a flow rate of 1.0ml/min with a fast gradient. The brain penetration of the compounds was evaluated by comparing their C(max) and AUC values in brain and plasma. This approach rapidly provided early brain penetration and plasma exposure information, thus making more of this data available to teams. Comparing the brain exposures to the EC(50) values (i.e. in vitro potency) of series compounds in the same discovery program provided another dimension of information to select lead compounds for future in vivo assessment. The method described here has been used for providing early brain penetration information in several CNS exploratory and discovery programs.


Assuntos
Química Encefálica , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Tecnologia Farmacêutica/métodos , Animais , Encéfalo/metabolismo , Química Encefálica/fisiologia , Cromatografia Líquida de Alta Pressão/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Masculino , Preparações Farmacêuticas/análise , Ratos , Ratos Sprague-Dawley
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