RESUMO
Accumulation of amyloid-ß peptide (Aß) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aß aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aß accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aß-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aß homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aß accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aß monomers, oligomers, and fibrils. Levels of all soluble Aß species declined thereafter, as Aß42 turned progressively insoluble and accumulated in Aß-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aß was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aß42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aß42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aß accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aß in endosomes and, in TgCRND8 mice, Aß overproduction causes rapid loss of Aß42 solubility that curtails ECE-mediated degradation.SIGNIFICANCE STATEMENT Deposition of aggregated Aß in extracellular plaques is a defining feature of AD. Aß aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aß is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aß may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aß aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aß proteostasis.
Assuntos
Doença de Alzheimer , Amiloidose , Neuroblastoma , Humanos , Camundongos , Animais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Neurônios/metabolismo , Neuroblastoma/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Endossomos/metabolismo , Placa Amiloide/metabolismoRESUMO
BACKGROUND: Demonstration of intrathecal production of Borrelia-specific antibodies (ITAb) is considered the most specific diagnostic marker of Lyme neuroborreliosis (LNB). Limitations include delayed detectability in early infection and continued presence long after successful treatment. Markers of active inflammation-increased cerebrospinal fluid (CSF) leukocytes, protein, and CXCL13-provide nonspecific markers of active infection. To assess the utility of CSF CXCL13, we measured its concentration in 132 patients with a broad spectrum of neuroinflammatory disorders, including LNB. METHODS: CSF CXCL13 was measured by immunoassay. Spearman rank correlation test was performed to explore its relationship to conventional markers of neuroinflammation and Borrelia-specific ITAb production. RESULTS: In non-LNB neuroinflammatory disorders, CSF CXCL13 elevation correlated with CSF immunoglobulin G (IgG) synthesis and leukocyte count. In LNB, CXCL13 concentration was far greater than expected from overall CSF IgG synthesis, and correlated with Borrelia-specific ITAb synthesis. Median CSF CXCL13 concentration in ITAb-positive LNB patients was > 500 times greater than in any other group. CONCLUSIONS: Intrathecal CXCL13 and IgG production are closely interrelated. CXCL13 is disproportionately increased in "definite LNB," defined as having demonstrable Borrelia-specific ITAb, but not "probable LNB," without ITAb. This disproportionate increase may help identify patients with very early infection or those with active vs treated LNB, or may help to differentiate ITAb-defined active LNB from other neuroinflammatory disorders. However, its reported specificity is closely related to the diagnostic requirement for ITAb. It may add little specificity to the demonstration of a pleocytosis or increased overall or specific IgG production in the CSF.
Assuntos
Quimiocina CXCL13/líquido cefalorraquidiano , Neuroborreliose de Lyme , Biomarcadores , Borrelia , Humanos , Imunoensaio , Testes Imunológicos , Neuroborreliose de Lyme/diagnósticoRESUMO
Photodynamic therapy (PDT) holds great promise for the treatment of head and neck (H&N) carcinomas where repeated loco-regional therapy often becomes necessary due to the highly aggressive and recurrent nature of the cancers. While interstitial light delivery technologies are being refined for PDT of H&N and other cancers, a parallel clinically relevant research area is the formulation of photosensitizers in nanovehicles that allow systemic administration yet preferential enhanced uptake in the tumor. This approach can render dual-selectivity of PDT, by harnessing both the drug and the light delivery within the tumor. To this end, we report on a cell-targeted nanomedicine approach for the photosensitizer silicon phthalocyanine-4 (Pc 4), by packaging it within polymeric micelles that are surface-decorated with GE11-peptides to promote enhanced cell-selective binding and receptor-mediated internalization in EGFR-overexpressing H&N cancer cells. Using fluorescence spectroscopy and confocal microscopy, we demonstrate in vitro that the EGFR-targeted Pc 4-nanoformulation undergoes faster and higher uptake in EGFR-overexpressing H&N SCC-15 cells. We further demonstrate that this enhanced Pc 4 uptake results in significant cell-killing and drastically reduced post-PDT clonogenicity. Building on this in vitro data, we demonstrate that the EGFR-targeted Pc 4-nanoformulation results in significant intratumoral drug uptake and subsequent enhanced PDT response, in vivo, in SCC-15 xenografts in mice. Altogether our results show significant promise toward a cell-targeted photodynamic nanomedicine for effective treatment of H&N carcinomas.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Fotoquimioterapia/métodos , Animais , Transporte Biológico Ativo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Química Farmacêutica , Sistemas de Liberação de Medicamentos , Receptores ErbB/metabolismo , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Indóis/administração & dosagem , Indóis/farmacocinética , Camundongos , Camundongos SCID , Nanomedicina/métodos , Nanopartículas/administração & dosagem , Compostos de Organossilício/administração & dosagem , Compostos de Organossilício/farmacocinética , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacocinética , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular perturbations and cerebral hypometabolism are emerging as important components of Alzheimer's disease (AD). While various in vivo imaging modalities have been designed to detect changes of cerebral perfusion and metabolism in AD patients and animal models, study results were often heterogenous with respect to imaging techniques and animal models. We therefore evaluated cerebral perfusion and glucose metabolism of two popular transgenic AD mouse strains, TgCRND8 and 5xFAD, at 7 and 12 months-of-age under identical conditions and analyzed possible molecular mechanisms underlying heterogeneous cerebrovascular phenotypes. Results revealed disparate findings in these two strains, displaying important aspects of AD progression. TgCRND8 mice showed significantly decreased cerebral blood flow and glucose metabolism with unchanged cerebral blood volume (CBV) at 12 months-of-age whereas 5xFAD mice showed unaltered glucose metabolism with significant increase in CBV at 12 months-of-age and a biphasic pattern of early hypoperfusion followed by a rebound to normal cerebral blood flow in late disease. Finally, immunoblotting assays suggested that VEGF dependent vascular tone change may restore normoperfusion and increase CBV in 5xFAD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/fisiopatologia , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Circulação Cerebrovascular , Modelos Animais de Doenças , Glucose/metabolismo , Humanos , Camundongos TransgênicosRESUMO
The c-Myc oncoprotein is overexpressed in many tumors and is essential for maintaining the proliferation of transformed cells. To function as a transcription factor, c-Myc must dimerize with Max via the basic helix-loop-helix leucine zipper protein (bHLH-ZIP) domains in each protein. The small molecule 7-nitro-N-(2-phenylphenyl)-2,1,3-benzoxadiazol-4-amine (10074-G5) binds to and distorts the bHLH-ZIP domain of c-Myc, thereby inhibiting c-Myc/Max heterodimer formation and inhibiting its transcriptional activity. We report in vitro cytotoxicity and in vivo efficacy, pharmacodynamics, pharmacokinetics, and metabolism of 10074-G5 in human xenograft-bearing mice. In vitro, 10074-G5 inhibited the growth of Daudi Burkitt's lymphoma cells and disrupted c-Myc/Max dimerization. 10074-G5 had no effect on the growth of Daudi xenografts in C.B-17 SCID mice that were treated with 20 mg/kg 10074-G5 intravenously for 5 consecutive days. Inhibition of c-Myc/Max dimerization in Daudi xenografts was not seen 2 or 24 h after treatment. Concentrations of 10074-G5 in various matrices were determined by high-performance liquid chromatography-UV, and metabolites of 10074-G5 were identified by liquid chromatography/tandem mass spectrometry. The plasma half-life of 10074-G5 in mice treated with 20 mg/kg i.v. was 37 min, and peak plasma concentration was 58 µM, which was 10-fold higher than peak tumor concentration. The lack of antitumor activity probably was caused by the rapid metabolism of 10074-G5 to inactive metabolites, resulting in tumor concentrations of 10074-G5 insufficient to inhibit c-Myc/Max dimerization. Our identification of 10074-G5 metabolites in mice will help design new, more metabolically stable small-molecule inhibitors of c-Myc.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Oxidiazóis/farmacologia , Oxidiazóis/farmacocinética , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Estruturas Animais/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/uso terapêutico , Fezes/química , Feminino , Glucuronatos/metabolismo , Glucuronidase/metabolismo , Células HL-60 , Humanos , Concentração Inibidora 50 , Fígado/metabolismo , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Neoplasias/patologia , Oxidiazóis/metabolismo , Oxidiazóis/uso terapêutico , Oxidiazóis/toxicidade , Plasma/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Espectrometria de Massas em Tandem , Tiazóis/farmacologia , Resultado do Tratamento , Urina/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Human milk oligosaccharides (HMO) have been recognized for the protective effects they may elicit among high risk infants. One HMO, disialyllacto-N-tetraose (DSLNT), has been shown to reduce the risk for developing necrotizing enterocolitis in preterm infants. RESEARCH AIMS: To measure DSLNT content in the human milk from mothers of preterm infants, and (1) assess variability; (2) establish correlations between maternal factors and/or an infant's risk for developing necrotizing enterocolitis; and (3) determine the effect of pasteurization. METHODS: DSLNT was measured in 84 samples of preterm milk, in human donor milk, and in Holder and flash pasteurized samples. Preterm infant outcomes were assessed by medical record review. RESULTS: DSLNT content of mother's own milk was highly variable and decreased significantly with increasing postnatal age. Four preterm infants (6.7%) developed necrotizing enterocolitis (Bell stage II or greater), 4 (6.7%) developed spontaneous intestinal perforation, and 1 developed both. DSLNT z-score was below the age-specific M within 8 (89%) of the 9 milk samples from mothers whose babies developed necrotizing enterocolitis (p = 0.039), but the DSLNT content did not differ between infants with necrotizing enterocolitis, spontaneous intestinal perforation, or neither condition (p > 0.1). DSLNT levels were significantly reduced in samples of donor milk compared to mothers' own milk (p = 0.0051). Pasteurization did not significantly reduce DSLNT content. CONCLUSIONS: DSLNT content of human milk is variable and may be lower in milk from mothers whose infants developed necrotizing enterocolitis. DSLNT content is unaffected by flash or Holder pasteurization.
Assuntos
Recém-Nascido Prematuro/metabolismo , Leite Humano/química , Mães/estatística & dados numéricos , Oligossacarídeos/análise , Adulto , Feminino , Humanos , Lactente , Recém-Nascido , Recém-Nascido Prematuro/fisiologia , Masculino , New Jersey , Oligossacarídeos/metabolismo , Estudos RetrospectivosRESUMO
PURPOSE: The interaction of p53 with its negative regulators Mdm2/4 has been widely studied (Khoury and Domling in Curr Pharm Des 18(30):4668-4678, 2012). In p53(+/+) cells, expression of Mdm2/4 leads to p53 turnover, inhibition of downstream transcription, decreasing cell cycle arrest, or apoptosis. We report in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of YH264, YH263, and WW751, three proposed small molecule inhibitors of the Mdm2/4-p53 interaction. METHODS: MTT cytotoxicity assays were performed, and alterations in proteins were examined using western blots. Mice were dosed 150 mg/kg YH264 or YH263 IV or PO QDx5. Mice were IV dosed 88, 57, or 39 mg/kg WW751 for 3, 5, or 5 days. YH264, YH263, and WW751 and metabolites were quantitated by LC-MS/MS. RESULTS: IC50 values for YH264, YH263, and WW751 against p53 wild-type HCT 116 cells after 72 h of incubation were 18.3 ± 2.3, 8.9 ± 0.6, and 3.1 ± 0.2 µM, respectively. Only YH264 appeared to affect p53 expression in vitro. None of the compounds affected the growth of HCT 116 xenografts in C.B-17 SCID mice. YH264 plasma half-life was 147 min; YH263 plasma half-life was 263 min; and WW751 plasma half-life was less than 120 min. CONCLUSIONS: Despite dosing the mice at the maximum soluble doses, we could not achieve tumor concentrations equivalent to the intracellular concentrations required to inhibit cell growth in vitro. YH263 and WW751 do not appear to affect p53/Mdm2, and none of the three were active in a subcutaneous HCT 116 p53(+/+) xenograft model.
Assuntos
Antineoplásicos/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/química , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos SCID , Transplante de Neoplasias , Pirazóis/farmacocinética , Pirazóis/farmacologiaRESUMO
INTRODUCTION: Benzaldehyde dimethane sulfonate (BEN, DMS612, NSC281612) is a bifunctional alkylating agent currently in clinical trials. We previously characterized the degradation products of BEN in plasma and blood. The conversion of BEN to its carboxylic acid analogue (BA) in whole blood, but not plasma, suggests that an enzyme in RBCs may be responsible for this conversion. BEN conversion to BA was observed in renal carcinoma cells and appeared to correlate with IC50. To better understand the pharmacology of BEN, we aimed to evaluate the metabolism and enzymes potentially responsible for the conversion of BEN to BA. METHODS: Human red blood cells (RBC) were used to characterize kinetics and susceptibility to enzyme-specific inhibitors. Recombinant enzymes were used to confirm metabolism of BEN to BA. Analytes were quantitated with established LC-MS/MS methods. RESULTS: Average apparent Vmax and Km were 68 ng/mL min(-1) [10% RBC](-1) and 373 ng/mL, respectively. The conversion of BEN to BA in RBC was not inhibited by carbon monoxide, nitrogen gas, or menadione, an inhibitor of aldehyde oxidase. The conversion was inhibited by disulfiram, an inhibitor of ALDH. Of available ALDH isoforms ALDH1A1, ALDH3A1, ALDH2, and ALDH5A1, only ALDH1A1 converted BEN to BA. CONCLUSION: The activating conversion of BEN to BA is mediated not by CYP450 enzymes or aldehyde oxidase, but by ALDH1A1. This enzyme, a potential stem cell marker, may be a candidate biomarker for clinical activity of BEN.
Assuntos
Antineoplásicos Alquilantes/sangue , Benzaldeídos/sangue , Carcinoma de Células Renais/sangue , Neoplasias Renais/sangue , Carcinoma de Células Renais/enzimologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Humanos , ImunoprecipitaçãoRESUMO
PURPOSE: Benzaldehyde dimethane sulfonate (DMS612, NSC281612, BEN) is an alkylator with activity against renal cell carcinoma, currently in phase I trials. In blood, BEN is rapidly metabolized into its highly reactive carboxylic acid (BA), presumably the predominant alkylating species. We hypothesized that BEN is metabolized to BA by aldehyde dehydrogenase (ALDH) and aimed to increase BEN exposure in blood and tissues by inhibiting ALDH with disulfiram, thereby shifting BA production from blood to tissues. METHODS: Female CD2F1 mice were dosed with 20 mg/kg BEN iv alone or 24 h after 300 mg/kg disulfiram ip. BEN, BA, and metabolites were quantitated in plasma and urine, and toxicities were assessed. RESULTS: BEN had a plasma t½ <5 min and produced at least 12 products. The metabolite half-lives were <136 min. Disulfiram increased BEN plasma exposure 368-fold (AUC0-inf from 0.11 to 40.5 mg/L min), while plasma levels of BA remained similar. Urinary BEN excretion increased (1.0-1.5 % of dose), while BA excretion was unchanged. Hematocrit, white blood cell counts, and percentage lymphocytes decreased after BEN administration. Coadministration of disulfiram appeared to enhance these effects. Profound liver pathology was observed in mice treated with disulfiram and BEN. CONCLUSIONS: BEN plasma concentrations increased after administration of disulfiram, suggesting that ALDH mediates the rapid metabolism of BEN in vivo, which may explain the increased toxicity seen with BEN after administration of disulfiram. Our results suggest that the coadministration of BEN with drugs that inhibit ALDH to patients that are ALDH deficient may cause liver damage.
Assuntos
Aldeído Desidrogenase/antagonistas & inibidores , Antineoplásicos/farmacocinética , Benzaldeídos/farmacocinética , Dissulfiram/farmacologia , Inibidores Enzimáticos/farmacologia , Animais , Antineoplásicos/toxicidade , Área Sob a Curva , Benzaldeídos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Interações Medicamentosas , Feminino , Meia-Vida , CamundongosRESUMO
PURPOSE: Protein kinase D (PKD) mediates diverse biological responses including cell growth and survival. Therefore, PKD inhibitors may have therapeutic potential. We evaluated the in vitro cytotoxicity of two PKD inhibitors, kb-NB142-70 and its methoxy analogue, kb-NB165-09, and examined their in vivo efficacy and pharmacokinetics. METHODS: The in vitro cytotoxicities of kb-NB142-70 and kb-NB165-09 were evaluated by MTT assay against PC-3, androgen-independent prostate cancer cells, and CFPAC-1 and PANC-1, pancreatic cancer cells. Efficacy studies were conducted in mice bearing either PC-3 or CPFAC-1 xenografts. Tumor-bearing mice were euthanized between 5 and 1,440 min after iv dosing, and plasma and tissue concentrations were measured by HPLC-UV. Metabolites were characterized by LC-MS/MS. RESULTS: kb-NB142-70 and kb-NB165-09 inhibited cellular growth in the low-mid µM range. The compounds were inactive when administered to tumor-bearing mice. In mice treated with kb-NB142-70, the plasma C (max) was 36.9 nmol/mL, and the PC-3 tumor C (max) was 11.8 nmol/g. In mice dosed with kb-NB165-09, the plasma C (max) was 61.9 nmol/mL, while the PANC-1 tumor C (max) was 8.0 nmol/g. The plasma half-lives of kb-NB142-70 and kb-NB165-09 were 6 and 14 min, respectively. Both compounds underwent oxidation and glucuronidation. CONCLUSIONS: kb-NB142-70 and kb-NB165-09 were rapidly metabolized, and concentrations in tumor were lower than those required for in vitro cytotoxicity. Replacement of the phenolic hydroxyl group with a methoxy group increased the plasma half-life of kb-NB165-09 2.3-fold over that of kb-NB142-70. Rapid metabolism in mice suggests that next-generation compounds will require further structural modifications to increase potency and/or metabolic stability.
Assuntos
Compostos Heterocíclicos com 3 Anéis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiazepinas/farmacologia , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Compostos Heterocíclicos com 3 Anéis/metabolismo , Humanos , Camundongos , Camundongos SCID , Ligação Proteica , Inibidores de Proteínas Quinases/metabolismo , Espectrometria de Massas em Tandem , Tiazepinas/metabolismo , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Cytidine drugs, such as gemcitabine, undergo rapid catabolism and inactivation by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU is only 20% orally bioavailable, which limits its preclinical evaluation and clinical use. Therefore, we characterized THU pharmacokinetics after the administration to mice of the more lipophilic pro-drug triacetyl-THU (taTHU). METHODS: Mice were dosed with 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally and compartmentally. RESULTS: taTHU did not inhibit CD. THU, after 150 mg/kg taTHU i.v., had a 235-min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. Renal excretion accounted for 40-55% of the i.v. taTHU dose, 6-12% of the p.o. taTHU dose. A two-compartment model of taTHU generating THU fitted the i.v. taTHU data best. taTHU, at 150 mg/kg p.o., produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122-min half-life. Approximately 68% of i.v. taTHU is converted to THU. Approximately 30% of p.o. taTHU reaches the systemic circulation as THU. CONCLUSIONS: The availability of THU after p.o. taTHU is 30%, when compared to the 20% achieved with p.o. THU. These data will support the clinical studies of taTHU.