RESUMEN
Neuromedin U (NMU) is a neuropeptide known to regulate food intake and energy homeostasis that is widely distributed in the gastrointestinal tract, hypothalamus, and pituitary. A short form of NMU, porcine NMU-8 has potent agonist activity for the receptors NMUR1 and NMUR2; however, its short half-life precludes its effective use in vivo. To address this limitation, we designed and synthesized NMU-8 analogs modified by polyethylene glycol (PEG) with a molecular weight of 30kDa (PEG30k) via a variety of linkers (i.e., ω-amino- and ω-imino-carboxylic acid linker). Integrated evaluation of NMUR1 and NMUR2 binding affinities in vitro and anorectic activity in mice revealed that the introduction of a linker with a rigid ring group, e.g., 2-(piperazin-1-yl)acetic acid (PipAc), yielded a highly potent anorectic peptide, PEG30k-PipAc-NMU-8 (14), possessing improved receptor binding affinity. Subsequent optimization of the molecular weight of the PEG moiety led to the discovery of a PEG20k conjugate (15), which exhibited significant anti-obesity effect upon once-daily subcutaneous administration in diet-induced obese mice with 10% and 22% body weight loss at doses of 10 and 30nmol/kg, respectively. In addition, 15 reduced the weights of the liver and adipose tissue in a dose-dependent manner and improved the plasma biochemical parameters, e.g., insulin, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and total cholesterol. Thus, our results suggest that 15 (NMU-0002), which showed potent and long-lasting biological profiles in vivo, represents a candidate peptide for investigating the central and peripheral actions of NMU and its potential for clinical use.
Asunto(s)
Fármacos Antiobesidad/farmacología , Neuropéptidos/farmacología , Polietilenglicoles/química , Animales , Fármacos Antiobesidad/farmacocinética , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/química , Neuropéptidos/farmacocinética , Porcinos , Pérdida de Peso/efectos de los fármacosRESUMEN
Pathological proteins including tau are produced in neurons and released into interstitial fluid (ISF) in a neural activity-dependent manner during wakefulness. Pathological proteins in ISF can be removed from the brain via the glymphatic pathway during nighttime. Thus, in individuals with Alzheimer's disease (AD) that have dysregulated sleep/wake rhythm, application of orexin receptor 2 (OX2R) agonists during daytime could recover the efflux of pathological proteins to ISF and indirectly promote the glymphatic pathway by improving the quality of nighttime sleep after proper daytime arousal, resulting in increased removal of these proteins from the brain. We investigated this hypothesis using OX-201, a novel OX2R-selective agonist with a 50% effective concentration of 8.0 nM. Diurnal rhythm of tau release into hippocampal ISF correlated well with neuronal activity and wakefulness in wild-type mice. In both wild-type and human P301S tau transgenic mice, OX-201 induced wakefulness and promoted tau release into hippocampal ISF. Human P301S tau transgenic mice, tested under our conditions, showed longer wakefulness time, which differs from individuals with AD. OX-201 treatment over 2 months did not alter hippocampal tau levels. Although further studies are required, at a minimum OX2R agonists may not exacerbate tau accumulation in individuals with tauopathy, including AD.
Asunto(s)
Enfermedad de Alzheimer , Hipocampo , Ratones Transgénicos , Receptores de Orexina , Proteínas tau , Animales , Proteínas tau/metabolismo , Ratones , Receptores de Orexina/metabolismo , Receptores de Orexina/agonistas , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Vigilia/efectos de los fármacos , Masculino , Líquido Extracelular/metabolismo , Líquido Extracelular/efectos de los fármacos , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacosRESUMEN
Aldehyde dehydrogenases (ALDHs), enzymes responsible for detoxification and retinoic acid biosynthesis, are considered a potent functional stem cell marker of normal and malignant cells in many tissues. To date, however, there are no available data on ALDH distributions and functions in oral mucosa. This study aims to clarify the levels and types of ALDH expression using immunohistochemistry with accompanying mRNA expression as well as an ALDEFLUOR assay, and to assess phenotypic and histological changes after manipulation of the ALDH activity of oral keratinocytes to increase the potency of a tissue-engineered oral mucosa by a specific ALDH inhibitor, diethylaminobenzaldehyde (DEAB), together with small interfering RNA of ALDH1A3 and ALDH3A1. Results showed the mRNA and cytoplasmic protein expression of ALDH1A3 and ALDH3A1 to be mostly localized in the upper suprabasal layer although no ALDH1A1 immunoreaction was detected throughout the epithelium. Oral keratinocytes with high ALDH activity exhibited a profile of differentiating cells. By pharmacological inhibition, the phenotypic analysis revealed the proliferating cell-population shifting to a more quiescent state compared with untreated cells. Furthermore, a well-structured epithelial layer showing a normal differentiation pattern and a decrease in Ki-67 immunopositive basal cells was developed by DEAB incubation, suggesting a slower turnover rate efficient to maintain undifferentiated cells. Histological findings of a regenerated oral epithelium by ALDH1A3 siRNA were similar to those when treated with DEAB while ALDH3A1 siRNA eradicated the epithelial regenerative capacity. These observations suggest the effects of phenotypic and morphological alterations by DEAB on oral keratinocytes are mainly consequent to the inhibition of ALDH1A3 activity.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Inhibidores Enzimáticos/farmacología , Silenciador del Gen , Queratinocitos/enzimología , Mucosa Bucal/enzimología , ARN Interferente Pequeño/genética , p-Aminoazobenceno/análogos & derivados , Adulto , Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Aldehído Oxidorreductasas/antagonistas & inhibidores , Aldehído Oxidorreductasas/genética , Proliferación Celular/efectos de los fármacos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica/métodos , Queratinocitos/patología , Antígeno Ki-67/metabolismo , Masculino , Mucosa Bucal/patología , ARN Mensajero/metabolismo , Regeneración/efectos de los fármacos , p-Aminoazobenceno/farmacologíaRESUMEN
Accumulation of tau protein is a key pathology of age-related neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Those diseases are collectively termed tauopathies. Tau pathology is associated with axonal degeneration because tau binds to microtubules (MTs), a component of axon and regulates their stability. The acetylation state of MTs contributes to stability and histone deacetylase 6 (HDAC6) is a major regulator of MT acetylation status, suggesting that pharmacological HDAC6 inhibition could improve axonal function and may slow the progression of tauopathy. Here we characterize N-[(1R,2R)-2-{3-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-5-oxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}cyclohexyl]-2,2,3,3,3-pentafluoropropanamide (T-518), a novel, potent, highly selective HDAC6 inhibitor with clinically favorable pharmacodynamics. T-518 shows potent inhibitory activity against HDAC6 and superior selectivity over other HDACs compared with the known HDAC6 inhibitors in the enzyme and cellular assays. T-518 showed brain penetration in an oral dose and blocked HDAC6-dependent tubulin deacetylation at Lys40 in mouse hippocampus. A 2-week treatment restored impaired axonal transport and novel object recognition in the P301S tau Tg mouse, tauopathy model, while a 3-month treatment also decreased RIPA-insoluble tau accumulation. Pharmaceutical inhibition of HDAC6 is a potential therapeutic strategy for tauopathy, and T-518 is a particularly promising drug candidate.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Histona Desacetilasa 6/antagonistas & inhibidores , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/administración & dosificación , Transducción de Señal/efectos de los fármacos , Acetilación , Administración Oral , Animales , Transporte Axonal/efectos de los fármacos , Axones/efectos de los fármacos , Axones/metabolismo , Células Cultivadas , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Histona Desacetilasa 6/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transducción de Señal/genéticaRESUMEN
The interactions between carbon nanotubes and important biomolecules, above all collagen molecules, have not been studied in detail. This situation is partly due to the fact that CNT are solid entities, while most of the biomolecules can be prepared in solution. We used turbidity as a means of evaluating the interaction between CNT and collagen molecules. To a stable suspension of CNT (10 ppm in 0.1% Triton), collagen solution was added to obtain a final concentration of 25 ppm. The degree of aggregation was evaluated by measuring the turbidity of the suspension at 660 nm. It was found that native collagen induced distinct aggregation with CNT, while denaturation of this protein at 60 degrees C for 1 hr deprived the molecules of their ability to aggregate with CNT. Also other globular molecules, albumin and lysozyme, did not induce aggregation of CNT. These results indicate that the rigid rod-like structure of the native collagen triple helix is essential for interaction with CNT to cause aggregation. The mechanisms are considered to be dependent upon geometric properties of rod-like collagen molecules. The findings in this paper will open a new avenue to clarify the detailed mechanism of the interaction between collagen molecules and CNT.
Asunto(s)
Colágeno/química , Colágeno/ultraestructura , Modelos Químicos , Modelos Moleculares , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestructura , Sitios de Unión , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Carbon nanotubes (CNTs) have excellent chemical durability, mechanical strength and electrical properties. Therefore, there is interest in CNTs for not only electrical and mechanical applications, but also biological and medical applications. We coated titanium, a common material for dental implants, with multiwalled carbon nanotubes (MWCNTs). First, titanium was aminated and covered with collagen. Then, the carboxylated MWCNTs were coated onto the collagen attached to the titanium plate. The collagen-coated titanium plate had a homogeneous MWCNT coating, which showed strong attachment to the titanium surface as a thin layer. The surface roughness was significantly increased with the MWCNT coating. MC3T3-E1 cells were cultured on the MWCNT-coated Ti plate, and showed good cell proliferation and strong cell adhesion. Therefore, the MWCNT coating for titanium could be useful for improvement of cell adhesion on titanium implants.
Asunto(s)
Adhesión Celular/fisiología , Cristalización/métodos , Nanotecnología/métodos , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestructura , Titanio/química , Animales , Células 3T3 BALB , Sustancias Macromoleculares/química , Ensayo de Materiales , Ratones , Conformación Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Carbon nanotubes (CNTs) are one of the most interesting nanomaterials because of their excellent characteristics. In this study, a transparent CNTs coating for cell culture dishes was developed and its properties for cell culture were estimated. Carboxylated multiwalled carbon nanotubes (MWCNTs) were dispersed in aqueous sodium cholate solution and applied on a collagen type I-coated cell culture dish (cover glass). The dish surface was homogeneously covered by MWCNTs without aggregation. The MWCNT-coated dish was slightly gray and had good transparency, so conventional optical microscopic observation of the cells on the MWCNT-coated dish was possible. Rat osteoblast-like cells cultured on the MWCNT-coated dish showed slightly lower viability and proliferation compared to the collagen-coated dish. The cell adhesion on the MWCNT-coated dish was much higher than that on the collagen-coated dish. Therefore, MWCNT-coating for dishes will be a useful new material for cell culture.
Asunto(s)
Técnicas de Cultivo de Célula/instrumentación , Materiales Biocompatibles Revestidos/química , Colágeno Tipo I/química , Nanotubos de Carbono/química , Animales , Adhesión Celular/fisiología , Recuento de Células , Línea Celular , Proliferación Celular , Supervivencia Celular/fisiología , Células Cultivadas , Colorimetría , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Fenómenos Ópticos , Osteoblastos/fisiología , Ratas , Colato de Sodio/química , Espectrofotometría , Propiedades de Superficie , Factores de TiempoRESUMEN
Despite considerable efforts to develop efficient carriers, the major target organ of short-interfering RNAs (siRNAs) remains limited to the liver. Expanding the application outside the liver is required to increase the value of siRNAs. Here we report on a novel platform targeted to muscular organs by conjugation of siRNAs with anti-CD71 Fab' fragment. This conjugate showed durable gene-silencing in the heart and skeletal muscle for one month after intravenous administration in normal mice. In particular, 1µg siRNA conjugate showed significant gene-silencing in the gastrocnemius when injected intramuscularly. In a mouse model of peripheral artery disease, the treatment with myostatin-targeting siRNA conjugate by intramuscular injection resulted in significant silencing of myostatin and hypertrophy of the gastrocnemius, which was translated into the recovery of running performance. These data demonstrate the utility of antibody conjugation for siRNA delivery and the therapeutic potential for muscular diseases.
Asunto(s)
Inmunoconjugados/uso terapéutico , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Miostatina/genética , Enfermedad Arterial Periférica/terapia , ARN Interferente Pequeño/uso terapéutico , Animales , Antígenos CD/inmunología , Células Cultivadas , Femenino , Inmunoconjugados/genética , Inmunoconjugados/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Enfermedad Arterial Periférica/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , Tratamiento con ARN de Interferencia , Ratas , Receptores de Transferrina/inmunologíaRESUMEN
Modifications of metastin(45-54) produced peptide analogues with higher metabolic stability than metastin(45-54). N-terminally truncated nonapeptide 4 ([D-Tyr46,D-Pya(4)47,azaGly51,Arg(Me)53]metastin(46-54)) is a representative compound with both potent agonistic activity and metabolic stability. Although 4 had more potent testosterone-suppressant activity than metastin, it possessed physicochemical instability at pH 7 and insufficient in vivo activity. Instability at pH 7 was dependent upon Asn48 and Ser49; substitution of Ser49 with Thr49 reduced this instability and maintained KISS1 receptor agonistic activity. Furthermore, [D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54) (14) showed 2-fold greater [Ca2+]i-mobilizing activity than metastin(45-54) and an apparent increase in physicochemical stability. N-terminal acetylation of 14 resulted in the most potent analogue, 22 (Ac-[D-Tyr46,D-Trp47,Thr49,azaGly51,Arg(Me)53,Trp54]metastin(46-54)). With continuous administration, 22 possessed 10-50-fold more potent testosterone-suppressive activity in rats than 4. These results suggested that a controlled release of short-length KISS1 receptor agonists can suppress the hypothalamic-pituitary-gonadal axis and reduce testosterone levels. Compound 22 was selected for further preclinical evaluation for hormone-dependent diseases.
Asunto(s)
Kisspeptinas/farmacología , Oligopéptidos/farmacología , Receptores Acoplados a Proteínas G/agonistas , Testosterona/antagonistas & inhibidores , Animales , Células CHO , Química Física , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Kisspeptinas/administración & dosificación , Kisspeptinas/química , Masculino , Conformación Molecular , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Ratas , Ratas Sprague-Dawley , Receptores de Kisspeptina-1 , Relación Estructura-Actividad , Testosterona/metabolismoRESUMEN
OBJECTIVE: This study aimed to clarify the effects of zoledronic acid (ZOL) on human primary oral mucosal keratinocytes growing in a monolayer culture and on a tissue-engineered oral mucosal construct. DESIGN: Changes in the viability and proliferation of oral keratinocytes incubated with ZOL were measured. Following treatment with 10 µM ZOL, histological examinations and immunohistochemical analyses for Ki-67, Geminin, and phospho-histone (γ)-H2A.X were performed on tissue-engineered oral mucosa. Cell cycle distribution and the degree of apoptosis were also measured by flow cytometry. Additionally, we measured the expression of cell cycle regulatory proteins as well as phospho-Chk1 and -Chk2. RESULTS: ZOL treatment suppressed cell viability and proliferation in a dose-dependent manner. Compared with untreated tissue-engineered oral mucosa, ZOL treatment resulted in a thinner epithelium in which the basal cells appeared less-organised. This is consistent with the observed significant reduction in the Ki-67 labelling index. In contrast, the Geminin labelling index was significantly higher than that in the untreated sample. In spite of the presence of a few apoptotic cells, ZOL induced an arrest in S-phase, which was confirmed by our observed alterations in the expression levels of cyclin A, B1, p27(KIP1), Rb and phospho-Rb. When the proteasome inhibitor MG132 was added to the ZOL-treated cells, we observed a partial restoration of the expression of cyclin A, cyclin B1, and p27(KIP1). Expression of phospho-Chk1 was detected, and a significant increase in the labelling index of γ-H2A.X was also seen. CONCLUSIONS: These results indicate that a 10-µM ZOL treatment induces a DNA damage response in oral keratinocytes that activates the ubiquitin-mediated proteolysis of cell cycle regulators, resulting in cell cycle arrest and repressive effects on cell viability, proliferation, and epithelial turnover.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/efectos de los fármacos , Daño del ADN , Difosfonatos/farmacología , Imidazoles/farmacología , Queratinocitos/efectos de los fármacos , Fase S/efectos de los fármacos , Análisis de Varianza , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Geminina , Humanos , Queratinocitos/metabolismo , Mucosa Bucal/efectos de los fármacos , Mucosa Bucal/metabolismo , Fase S/genética , Ácido ZoledrónicoRESUMEN
Metastin/kisspeptin, a hypothalamic peptide, plays a pivotal role in controlling GnRH neurons. Here we studied the effect of chronic sc administration of two kisspeptin analogs, KISS1-305 and TAK-448, on hypothalamic-pituitary-gonadal function in male rats in comparison with a GnRH analogue leuprolide or bilateral orchiectomy (ORX). The prototype polypeptide, KISS1-305 (1-4 nmol/h), caused substantial elevations of plasma LH and testosterone, followed by abrupt reductions of both hormone levels. Notably, testosterone levels were reduced to castrate levels within 3 d and remained depleted throughout the 4-wk dosing period, an effect that was faster and more pronounced than leuprolide (1 nmol/h) dosing. KISS1-305 also reduced genital organ weight more profoundly than leuprolide. In mechanistic studies, chronic KISS1-305 administration only transiently induced c-Fos expression in GnRH neurons, suggesting that GnRH-neural response was attenuated over time. Hypothalamic GnRH content was reduced to 10-20% of control at 3 wk without any changes in Gnrh mRNA expression. Dosing with the investigational peptide TAK-448 was also studied to extend our understanding of hypothalamic-pituitary functions. Similar to ORX, TAK-448 (0.1 nmol/h) depleted testosterone and decreased GnRH content by 4 wk. However, in contrast to ORX, TAK-448 decreased gonadotropin levels in pituitary and plasma samples, implying the suppression of GnRH pulses. These results suggest that chronic administration of kisspeptin analogs disrupts endogenous kisspeptin signals to suppress intrinsic GnRH pulses, perhaps by attenuating GnRH-neural response and inducing continuous GnRH leakage from the hypothalamus. The potential utility of kisspeptin analogs as novel agents to treat hormone-related diseases, including prostate cancer, is discussed.
Asunto(s)
Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Kisspeptinas/farmacología , Neuronas/efectos de los fármacos , Testículo/efectos de los fármacos , Testosterona/sangre , Animales , Hormona Liberadora de Gonadotropina/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Leuprolida/farmacología , Hormona Luteinizante/sangre , Masculino , Neuronas/metabolismo , Orquiectomía , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Ratas , Ratas Sprague-Dawley , Testículo/metabolismoRESUMEN
This study was designed to (1) assess the in vitro biocompatibility of a chitosan-collagen composite scaffold (C3) constructed by blending commercial chitosan and tilapia scale collagen with oral mucosa keratinocytes, (2) histologically and immunohistochemically characterize an ex vivo-produced oral mucosa equivalent constructed using the C3 (EVPOME-C), and (3) compare EVPOME-C with oral mucosa constructs utilizing AlloDerm® (EVPOME-A), BioMend® Extend™ (EVPOME-B), and native oral mucosa. C3 scaffold had a well-developed fibril network and a sufficiently small porosity to prevent keratinocytes from growing inside the scaffold after cell-seeding. The EVPOME oral mucosa constructs were fabricated in a chemically defined culture system. After culture at an air-liquid interface, EVPOME-C and EVPOME-B had multilayered epithelium with keratinization, while EVPOME-A had a more organized stratified epithelium. Ki-67 and p63 immunolabeled cells in the basal layer of all EVPOMEs suggested a regenerative ability. Compared with native oral mucosa, the keratin 15 and 10/13 expression patterns in all EVPOMEs showed a less-organized differentiation pattern. In contrast to the ß1-integrin and laminin distribution in EVPOME-A and native oral mucosa, the subcellular deposition in EVPOME-C and EVPOME-B indicated that complete basement membrane formation failed. These findings demonstrated that C3 has a potential application for epithelial tissue engineering and provides a new potential therapeutic device for oral mucosa regenerative medicine.