RESUMEN
Ultrafine bubbles (UFBs), which are bubbles with diameters of less than 1 µm, are widely recognized for their ability to exist stably in liquid as a result of the effects of Brownian motion. In this study, we focused on hydrogen, known for its antioxidant potential, and explored the function of H2-filled UFBs, which encapsulate hydrogen, to determine their potential use as oral carriers for the delivery bioactive gases to living organisms. To this end, rats were orally administered ethanol to induce hepatic oxidative stress, and the effects of drinking H2-filled UFBs (H2 NanoGAS®) water for two weeks were evaluated to assess the reduction of oxidative stress. Continuous alcohol consumption was found to significantly increase the blood lipid peroxidation levels in the control group, confirming the induction of oxidative stress. An increase in blood lipid peroxidation was significantly inhibited by the consumption of concentrated H2 NanoGAS® (C-HN) water. Furthermore, the measurement of mitochondrial activity in the liver revealed that drinking H2 NanoGAS® water helped to maintain at a normal level and/or boosted the functional activity of the electron transport system in mitochondria affected by ethanol intake. To our knowledge, this study is the first to provide evidence for the use of orally ingested UFBs as carriers for the delivery gases to tissues, thereby exerting their physiological activity in the body. Our findings highlight the potential for the application of UFBs to various physiologically active gases and their utilization in the medical field in the future.
Asunto(s)
Etanol , Hidrógeno , Peroxidación de Lípido , Hígado , Estrés Oxidativo , Animales , Estrés Oxidativo/efectos de los fármacos , Etanol/administración & dosificación , Hidrógeno/farmacología , Hidrógeno/administración & dosificación , Masculino , Peroxidación de Lípido/efectos de los fármacos , Hígado/metabolismo , Hígado/efectos de los fármacos , Administración Oral , Ratas , Ratas Wistar , Agua , Antioxidantes/farmacología , Antioxidantes/administración & dosificaciónRESUMEN
Caco-2 cell monolayers are widely employed as an in vitro model of the intestinal barrier, capable of accurately predicting the absorption of conventional small-molecule drugs. However, this model may not be applicable to all drugs, and the accuracy of absorption prediction is typically poor for high molecular weight drugs. Recently, human induced pluripotent stem (iPS) cell-derived small intestinal epithelial cells (hiPSC-SIECs), exhibiting properties similar to those of the small intestine when compared with Caco-2 cells, have been developed and are considered a novel candidate model for in vitro evaluation of intestinal drug permeability. Therefore, we evaluated the utility of human hiPSC-SIECs as a new in vitro model to predict the intestinal absorption of middle-molecular weight drugs and peptide drugs. Firstly, we showed that the hiPSC-SIEC monolayer allowed faster transport of peptide drugs (insulin and glucagon-like peptide-1) than the Caco- 2 cell monolayer. Second, we revealed that hiPSC-SIECs require divalent cations (Mg2+ and Ca2+) to maintain barrier integrity. Third, we demonstrated that experimental conditions established for Caco-2 cells are not persistently applicable to hiPSC-SICEs when analyzing absorption enhancers. Comprehensively clarifying the features of hiPSC-SICEs is essential to establish a new in vitro evaluation model.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células CACO-2 , Células Epiteliales , Intestino Delgado/metabolismo , Absorción Intestinal , Péptidos/metabolismo , Mucosa Intestinal/metabolismoRESUMEN
Owing to their unique physicochemical properties and diverse biological effects, ultrafine bubbles (UFBs) have recently been expected to be utilized for industrial and biological purposes. Thus, this study investigated the biological safety of UFBs in water for living beings in drinking the water with a view to future use in health sciences. In this study, we used H2-filled UFBs (NanoGAS®) that can hold hydrogen in the aqueous phase for a long time. Mice were randomly assigned to one of three groups: those receiving NanoGAS® water, reverse osmosis water, or natural mineral water, and they ingested it ad libitum for one month or three months. As a result, subchronic drinking of NanoGAS® water does not affect either the common blood biochemical parameters or the health of the organs and mucosal membranes. Our results, for the first time, scientifically demonstrated the biological safety of H2-filled UFBs water for subchronic oral consumption.
Asunto(s)
Ingestión de Líquidos , Hidrógeno , Agua , Animales , Ratones , Agua/química , Hidrógeno/administración & dosificación , GasesRESUMEN
Endocytosis is an essential biological process for nutrient absorption and intercellular communication; it can also be used to accelerate the cellular internalization of drug delivery carriers. Clarifying the cellular uptake mechanisms of unidentified endogenous and exogenous molecules and designing new effective drug delivery systems require an accurate, specific endocytosis analysis methodology. Therefore, we developed a method to specifically evaluate cellular internalization via three main endocytic pathways: clathrin- and caveolae-mediated endocytosis, and macropinocytosis. We first revealed that most known endocytosis inhibitors had no specific inhibitory effect or were cytotoxic. Second, we successfully established an alternative method using small interfering RNA to knock down dynamin-2 and caveolin-1, which are necessary for clathrin- and caveolae-mediated endocytosis, in HeLa cells. Third, we established another method to specifically analyze macropinocytosis using rottlerin on A431 cells. Finally, we validated the proposed methods by testing the cellular internalization of a biological molecule (insulin) and carriers (nanoparticles and cell-penetrating peptides). Through this study, we established versatile methods to precisely and specifically evaluate endocytosis of newly developed biopharmaceuticals or drug delivery systems.
Asunto(s)
Endocitosis , Pinocitosis , Humanos , Células HeLa , ARN Interferente Pequeño/genética , Clatrina/genética , CaveolasRESUMEN
Antibody drugs that target amyloid ß (Aß) are considered possible treatments for Alzheimer's disease; however, most have been dropped from clinical trials. We hypothesized that administration route for antiAß antibody (AntiAß) might affect its therapeutic potential and thus compared delivery of antibodies to the brain and their effect on cognitive dysfunction and amyloid disposition via intravenous (i.v.) and intranasal routes with and without the cell-penetrating peptide, L-penetratin. We demonstrated that intranasal administration with L-penetratin more efficiently delivered human immunoglobulin G (IgG), a model molecule for AntiAß, to the brain compared with i.v. injection. We found that multiple intranasal treatments with Alexa 594-labeled AntiAß (A594-AntiAß) with L-penetratin significantly improved learning by mice with aged amyloid precursor protein (APP) knock-in (App KI mice). Further, intranasal administration of A594-AntiAß increased the amount of soluble Aß (1-42) in the brain, suggesting suppression of Aß aggregation in insoluble form and involvement of activated microglia in Aß clearance. Thus, administration route may be critical for efficient delivery of AntiAß to the brain, and the nose-to-brain delivery with L-penetratin can maximize its therapeutic efficacy.
Asunto(s)
Enfermedad de Alzheimer , Péptidos de Penetración Celular , Anciano , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/farmacología , Precursor de Proteína beta-Amiloide/uso terapéutico , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G/metabolismo , Inyecciones Intravenosas , RatonesRESUMEN
We previously found that coadministering peptides and proteins with the cell-penetrating peptide L-penetratin intranasally significantly increased transport to the brain and enhanced pharmacological effects. The present study aimed to clarify the mechanisms of nose-to-brain drug delivery enhancement by L-penetratin coadministration. First, we compared the concentrations of Exendin-4 in plasma and brain after intranasal and subcutaneous administration and suggested that coadministration with L-penetratin facilitated the direct nose-to-brain transport of Exendin-4. Second, we demonstrated that L-penetratin did not stimulate the transport of Cy7-labeled Exendin-4 and insulin through the trigeminal nerves but shifted their distribution to the olfactory mucosal pathway. Third, we investigated the distribution of insulin into the deeper regions of the brain after delivery via the olfactory pathway and suggested that insulin had entered the olfactory bulb, bottom part of the brain, and perivascular space through the cerebrospinal fluid and had diffused throughout the brain. We further demonstrated that intranasally delivered insulin with L-penetratin specifically accumulated on the hippocampus neuronal cells. Thus, this study suggested that administrating peptide drugs intranasally with L-penetratin allows direct transport to the olfactory bulb, bottom part of the brain, and perivascular space of the cerebral artery. This technique also potentially allows targeting of specific brain areas.
RESUMEN
Andrographolide (AG), a well-known traditional medicinal plant in Southeast Asia, is widely used for treatment of many chronic diseases. Interestingly, AG has been reported to have inhibitory effects on osteoclast function and anti-inflammatory properties. Because of these therapeutic properties, this study aimed to develop and optimize the formulation of AG using PLGA nanocarriers and gelatin-based hydrogel to prolong the retention time in the joint. We investigated the in vitro release pattern of the AG nanoparticles formulation which prepared by emulsion solvent evaporation method and embedded into gelatin-based hydrogel. The result showed that the AG loaded ester terminated end group PLGA polymer gradually released AG from the PLGA nanoparticles when compared with AG solution. Importantly, the combined use of gelatin-based hydrogel with AG from the PLGA nanoparticles significantly delayed the AG release more than 1 month. Furthermore, we selected the DiR fluorescence dye to represents AG and monitored the retention time by IVIS imaging. The optimal formulation was administered as intra-articular drug delivery systems in in vivo study. The results successfully displayed a long-term sustained release for implantation (≈2 months) and injection (≥2 months) providing a novel strategy for the local management of osteoarthritis disease.
Asunto(s)
Nanopartículas del Metal , Nanopartículas , Preparaciones de Acción Retardada , Diterpenos , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Gelatina , Hidrogeles , Ácido Láctico , Tamaño de la Partícula , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , PlataRESUMEN
The extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can be used as carriers for therapeutic molecules and drugs to target disordered tissues. This aimed to compare the protocols used for isolation of MSC-derived EVs by comparing EV collection conditions and three commercial purification kits. We also determined appropriate fluorescent dyes for labeling EVs. MSC-derived EVs were efficiently secreted during cell growth and highly purified by the phosphatidyl serine-based affinity kit. Although the EV membrane was more efficiently labeled with the fluorescent dye PKH67 compared to other probes, the efficiency was not enough to accurately analyze the endothelial cellular uptake of EVs. Results verified the easy protocol for isolating and fluorescently labeling EVs with commercial reagents and kits, but meanwhile, further modification of the protocol is required in order to scale up the amount of EVs derived from MSCs using fluorescent probes.
Asunto(s)
Portadores de Fármacos/química , Vesículas Extracelulares , Colorantes Fluorescentes/química , Células Madre Mesenquimatosas , Coloración y Etiquetado , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Células HeLa , Humanos , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/metabolismoRESUMEN
PURPOSE: The poor permeability of new drug candidates across intestinal epithelial membranes complicates their development in oral form. This study investigated the potential of cell-penetrating peptides (CPPs) to improve the intestinal permeation and absorption of low-permeable low-molecular-weight (low-MW) drugs. METHODS: The in vitro epithelial permeation of six different drugs (metformin, risedronate, zanamivir, methotrexate [MTX], tacrolimus, and vincristine [VCR]) across Caco-2 cell monolayers was examined in the presence and absence of L- or D-penetratin, and the correlation between permeation enhancement efficiency and the properties of tested drugs was analyzed. In addition, a rat closed ileal loop absorption study was conducted to determine the in vivo effects of penetratin. RESULTS: MTX and VCR efficiently permeated Caco-2 monolayers in the presence of L- and D-penetratin, suggesting that CPPs enhanced the epithelial permeation of drugs with relatively high molecular weight and resultant limited intrinsic permeability. The in vivo rat closed ileal loop absorption study revealed the stimulatory effect of L- and D-penetratin on the intestinal absorption of MTX and VCR. CONCLUSIONS: CPPs are useful as oral absorption enhancers for low-permeable drugs.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Animales , Células CACO-2 , Humanos , Técnicas In Vitro , Mucosa Intestinal/efectos de los fármacos , Masculino , Peso Molecular , Permeabilidad , Ratas , Ratas Sprague-DawleyRESUMEN
We previously reported that oral and intestinal absorption of insulin in rats and mice is significantly enhanced in vivo by coadministration with cell-penetrating peptides (CPPs). To evaluate the clinical use of CPPs as absorption enhancers, it is imperative to clarify the mechanisms associated with the permeation-stimulatory effect of CPPs in vitro. The confirmation experiment revealed a discrepancy between in vivo and in vitro effects of CPPs, such as D-octaarginine (D-R8) and L-penetratin, on epithelial permeation of insulin. The present study was designed to determine the factors that work in vivo but are deficient in an in vitro system consisting of Caco-2 cells. The effects of D-R8 and L-penetratin on permeation of insulin through the Caco-2 cell monolayer were partially boosted in fasted-state simulated intestinal fluid (FaSSIF). Contrary to expectation, the effects of CPPs on cellular uptake of insulin and the binding ratio of CPPs to insulin analyzed by surface plasmon resonance in normal buffer and FaSSIF were similar. Also, the effects of CPPs, especially D-R8, on cellular uptake of insulin, were stronger in Caco-2 cell monolayers with microfold cell (M cell)-like properties. These results suggested a key role of intestinal lipids and M cells in the stimulatory effect of CPPs on net epithelial permeation of insulin in vivo.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Péptidos de Penetración Celular/metabolismo , Insulina/metabolismo , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Secuencia de Aminoácidos , Linfoma de Burkitt/metabolismo , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Péptidos de Penetración Celular/administración & dosificación , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Humanos , Insulina/administración & dosificación , Insulina/genética , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Leptin is an endogenous hormone that regulates the appetite, energy metabolism, and glucose intake in the central nervous system (CNS) and is a potential therapeutic agent for obesity. In the normal healthy condition, peripherally secreted leptin is transported across the blood-brain barrier (BBB) to the target brain site, in particular the hypothalamus. However, it was reported that the progression of obesity causes diminished permeation of leptin across the BBB. The present study therefore aimed to effectively deliver leptin to the brain via intranasal coadministration with penetratin, an amphipathic cell-penetrating peptide (CPP), for potential treatment and prevention of obesity. The single administration study with normal rats demonstrated that leptin coadministered with L-penetratin was efficiently absorbed into the systemic circulation and accumulated in the anterior part of brain. Furthermore, chronic delivery of leptin via repeated intranasal coadministrations with L-penetratin suppressed the appetite and the body weight increase of the rats and lowered their plasma triglyceride levels. Analysis of brain samples after repeated administration suggested that Stat3 phosphorylation via leptin receptor stimulation potentially contributed to the therapeutic effect of leptin in the CNS. Thus, the present study suggests that intranasal coadministration with CPPs will become a promising strategy for delivering leptin to treat and prevent the progression of obesity.
Asunto(s)
Péptidos de Penetración Celular , Leptina , Administración Intranasal , Animales , Encéfalo/metabolismo , Péptidos de Penetración Celular/uso terapéutico , Obesidad/tratamiento farmacológico , RatasRESUMEN
To obtain the therapeutic effect of biological medicines, such as proteins and nucleic acids, these medicines must achieve their intracellular target, such as the cytoplasm, and pass through biological membrane barriers. Endocytosis is an attractive route for the intracellular delivery of such drugs, and various endocytosis inhibitors have been used as tools to study the involvement of endocytosis in the cell internalization of delivery carriers. However, the specificity of these inhibitors has been insufficiently studied, and our preliminary tests could not detect the expected effect of the well-known endocytosis inhibitors. Therefore, the present study aimed to optimize the experimental conditions to precisely analyze cellular internalization via endocytosis. We first found that incubation of model molecules, such as transferrin (Tf) and cholera toxin subunit B (CTB), in cell culture medium (DMEM) could efficiently induce their internalization to HeLa cells compared to that in transport buffer (HBSS). Moreover, we clarified that cell surface wash with glycine-hydrochloric acid buffer before confocal microscopy and flow cytometry strengthened the intracellular fluorescence of Tf, CTB, and dextran tagged with fluorescent probes possibly via the neutralization of endosomal pH. Even under the optimized condition, however, the specificity of endocytosis inhibitors was disputable. The present study suggested the importance of the optimization of the study design with endocytosis inhibitors in analyzing cellular internalization.
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Medios de Cultivo/química , Endocitosis/fisiología , Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Glicina/química , Ácido Clorhídrico/química , Transporte Biológico , Tampones (Química) , Técnicas de Cultivo de Célula , Membrana Celular/fisiología , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Microscopía Confocal , Microscopía Fluorescente , Preparaciones Farmacéuticas/administración & dosificaciónRESUMEN
In a recent study, we demonstrated the potential of a cell-penetrating peptide (CPP) penetratin to deliver the peptide drug insulin to the brain via nasal administration, and its pharmacological effect on the mild cognitive dysfunction in senescence-accelerated mouse (SAMP8). However, the therapeutic potential of intranasal insulin administration was attenuated when applied to the aged SAMP8 with severe cognitive dysfunction. The present study, therefore, aimed to overcome the difficulty in treating severe cognitive dysfunction using insulin by investigating potential alternatives, glucagon-like peptide-1 (GLP-1) receptor agonists such as exendin-4. Examination using normal ddY mice demonstrated that the distribution of exendin-4 throughout the brain was dramatically increased by intranasal coadministration with the L-form of penetratin. The activation of hippocampal insulin signaling after the simultaneous nose-to-brain delivery of exendin-4 and an adequate level of insulin were confirmed by analyzing the phosphorylation of Akt. Furthermore, spatial learning ability, evaluated in the Morris water maze test after daily administration of exendin-4 with L-penetratin and supplemental insulin for 4 weeks, suggested therapeutic efficacy against severe cognitive dysfunction. The present study suggests that nose-to-brain delivery of exendin-4 with supplemental insulin, mediated by CPP coadministration, shows promise for the treatment of progressive cognitive dysfunction in SAMP8.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Péptidos de Penetración Celular/metabolismo , Disfunción Cognitiva/tratamiento farmacológico , Portadores de Fármacos/metabolismo , Exenatida/administración & dosificación , Hipoglucemiantes/administración & dosificación , Administración Intranasal , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/patología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Exenatida/farmacocinética , Exenatida/farmacología , Exenatida/uso terapéutico , Péptido 1 Similar al Glucagón/administración & dosificación , Péptido 1 Similar al Glucagón/farmacocinética , Péptido 1 Similar al Glucagón/farmacología , Péptido 1 Similar al Glucagón/uso terapéutico , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Insulina/metabolismo , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Ratones , Transducción de Señal/efectos de los fármacosRESUMEN
Cell-penetrating peptides (CPPs) have great potential to efficiently deliver drug cargos across cell membranes without cytotoxicity. Cationic arginine and hydrophobic tryptophan have been reported to be key component amino acids for cellular internalization of CPPs. We recently found that l-arginine could increase the oral delivery of insulin in its single amino acid form. Therefore, in the present study, we evaluated the ability of another key amino acid, tryptophan, to enhance the intestinal absorption of biopharmaceuticals. We demonstrated that co-administration with l-tryptophan significantly facilitated the oral and intestinal absorption of the peptide drug insulin administered to rats. Furthermore, l-tryptophan exhibited the ability to greatly enhance the intestinal absorption of other peptide drugs such as glucagon-like peptide-1 (GLP-1), its analog Exendin-4 and macromolecular hydrophilic dextrans with molecular weights ranging from 4000 to 70,000 g/mol. However, no intermolecular interaction between insulin and l-tryptophan was observed and no toxic alterations to epithelial cellular integrity-such as changes to cell membranes, cell viability, or paracellular tight junctions-were found. This suggests that yet to be discovered inherent biological mechanisms are involved in the stimulation of insulin absorption by co-administration with l-tryptophan. These results are the first to demonstrate the significant potential of using the single amino acid l-tryptophan as an effective and versatile bioavailability enhancer for the oral delivery of biopharmaceuticals.
RESUMEN
In previous studies we showed that the complexation hydrogels based in poly(methacrylic acid-g-ethylene glycol) [P(MAA-g-EG)] rapidly release insulin in the intestine owing to their pH-dependent complexation properties; they also exhibit a high insulin-loading efficiency, enzyme-inhibiting properties, and mucoadhesive characteristics. Cell-penetrating peptides (CPPs), such as oligoarginines [hexa-arginine (R6), comprising six arginine residues], have been employed as useful tools for the oral delivery of therapeutic macromolecules. The aim of our study was to investigate the combination strategy of using P(MAA-g-EG) hydrogels with R6-based CPPs to improve the intestinal absorption of insulin. A high efficiency of loading into crosslinked P(MAA-g-EG) hydrogels was observed for insulin (96.1±1.4%) and R6 (46.6±3.8%). In addition, immediate release of the loaded insulin and R6 from these hydrogels was observed at pH 7.4 (80% was released in approximately 30 min). Consequently, a strong hypoglycemic response was observed (approximately 18% reduction in blood glucose levels) accompanied by an improvement in insulin absorption after the co-administration of insulin-loaded particles (ILP) and R6-loaded particles (ALP) into closed rat ileal segments compared with that after ILP administration alone. These results indicate that the combination of P(MAA-g-EG) hydrogels with CPPs may be a promising strategy for the oral delivery of various insulin preparations as an alternative to conventional parenteral routes.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Portadores de Fármacos/administración & dosificación , Hidrogeles/administración & dosificación , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Oligopéptidos/administración & dosificación , Administración Oral , Animales , Glucemia/análisis , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Liberación de Fármacos , Glicol de Etileno/química , Glicol de Etileno/farmacocinética , Hidrogeles/química , Hidrogeles/farmacocinética , Hipoglucemiantes/sangre , Hipoglucemiantes/química , Hipoglucemiantes/farmacocinética , Íleon/metabolismo , Insulina/sangre , Insulina/química , Insulina/farmacocinética , Absorción Intestinal , Masculino , Oligopéptidos/química , Oligopéptidos/farmacocinética , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/farmacocinética , Ratas WistarRESUMEN
To overcome the difficulty in delivery of biopharmaceuticals such as peptides and proteins to the brain, several approaches combining the ligands and antibodies targeting the blood-brain barrier (BBB) have been tried. However, these are inefficient in terms of their permeability through the BBB and structural modification of bioactive drugs. In the present study, we therefore examined the usefulness of a noncovalent method using the cell-penetrating peptides (CPPs) such as octaarginine (R8) as a suitable brain delivery strategy for biopharmaceuticals. A safety examination using microvascular endothelial model bEnd.3 cells clarified that R8 was the safest among the CPPs tested in this study. The cellular uptake study demonstrated that coincubation with R8 enhanced the uptake of model peptide drug insulin by bEnd.3 cells in a concentration-dependent and a temperature-independent manner. Furthermore, an in vivo study with rats showed that the accumulation of insulin in the deeper region of the brain, i.e., hippocampus, significantly increased after the intravenous coadministration of insulin with D-R8 without altering the insulin disposition in plasma. Thus, the present study provided the first evidence suggesting that the noncovalent method with CPPs is one of the strategic options for brain delivery of biopharmaceuticals via intravenous injection.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/administración & dosificación , Péptidos de Penetración Celular/administración & dosificación , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Oligopéptidos/administración & dosificación , Animales , Transporte Biológico , Proteínas Portadoras/farmacocinética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacocinética , Productos del Gen tat/química , Hipoglucemiantes/sangre , Hipoglucemiantes/farmacocinética , Insulina/sangre , Insulina/farmacocinética , Masculino , Ratones , Oligopéptidos/farmacocinética , Ratas Sprague-Dawley , Distribución TisularRESUMEN
This present study aimed to determine the optimal oral insulin delivery conditions that would maximize the utility of cell-penetrating peptides (CPPs) by using a noncovalent strategy. We first compared the effectiveness of two potential CPPs, penetratin and its analog PenetraMax, as absorption enhancers for insulin. The combined effect was evaluated under in vivo oral administration conditions. Both D-forms of CPPs were highly effective for increasing the oral absorption of insulin, and D-PenetraMax showed a more rapid onset of absorption enhancement effects compared with those of D-penetratin. However, synergistic absorption enhancement effects after combination treatment were not observed. Next, we tried a theoretical approach to establish optimized oral insulin delivery conditions. A surface plasmon resonance (SPR)-based analysis demonstrated that binding between insulin and penetratin (2 mM) might be saturated at 100-500 µM penetratin, while the bound concentration of penetratin could increase in accordance with an increased concentration of mixed insulin. To test this hypothesis, we investigated the effectiveness of different insulin doses in the gastric pH-neutralized mice. The results showed that the dissociation of noncovalent complexes of insulin and CPPs at the low gastric pH was prevented in these mice. Our findings clearly suggested that a noncovalent strategy with CPPs represents an effective approach for the L-form of CPP to increase the concentration of CPP-bound insulin to attain greater absorption of insulin, although this approach may not be appropriate for the D-form of CPP. Our findings will contribute to the development of oral dosage forms of insulin for noncovalent strategies involving CPP.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Sistemas de Liberación de Medicamentos , Hipoglucemiantes/administración & dosificación , Insulina Regular Humana/administración & dosificación , Absorción Intestinal/efectos de los fármacos , Administración Oral , Animales , Animales no Consanguíneos , Disponibilidad Biológica , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/química , Proteínas Portadoras/farmacocinética , Proteínas Portadoras/farmacología , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Péptidos de Penetración Celular/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Famotidina/farmacología , Ácido Gástrico/química , Ácido Gástrico/metabolismo , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/metabolismo , Antagonistas de los Receptores H2 de la Histamina/farmacología , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Insulina Regular Humana/genética , Insulina Regular Humana/farmacocinética , Insulina Regular Humana/farmacología , Ligandos , Masculino , Ratones , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Estereoisomerismo , Resonancia por Plasmón de SuperficieRESUMEN
Recent reports suggest that peptide drugs such as insulin have the potential to serve as therapeutics in neurodegenerative diseases such as Alzheimer's disease. However, the transport of these drugs to the therapeutic target, the brain, is significantly hindered by the blood-brain barrier (BBB). Intranasal administration appears to be an ideal solution for drug delivery to the brain, bypassing the BBB, however the entry of peptide drugs into neuronal and epithelial cells in the olfactory mucosa remains low. In this study, we therefore examined whether intranasal coadministration of cell-penetrating peptides (CPPs) could improve nose-to-brain drug transport. In both mice and rats, we found that direct transport of insulin into the brain was significantly facilitated when coadministered with amphipathic CPP penetratin, and eventually insulin reached the deeper regions of the brain such as the hippocampus. In the mouse line senescence-accelerated mouse prone-8 (SAMP8), spatial learning tests demonstrated that long-term intranasal coadministration of insulin with penetratin improved mild memory loss in the early stages of dementia. In contrast, the severe cognitive dysfunction in the aged SAMP8 mice was preserved despite intranasal coadministration of insulin with penetratin. The immunohistological examination of the hippocampus suggested that enhanced nose-to-brain delivery of insulin had a partial neuroprotective effect but unexpectedly increased amyloid ß plaque deposition. In conclusion, intranasal coadministration of insulin with CPPs has the potential to serve as a therapeutic for mild cognitive dysfunction. To identify suitable pharmacotherapy for dementia with severe pathology, further studies of nose-to-brain delivery of molecularly appropriate biopharmaceuticals are necessary.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Péptidos de Penetración Celular/administración & dosificación , Demencia/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Insulina/administración & dosificación , Insulina/farmacocinética , Administración Intranasal , Animales , Transporte Biológico , Demencia/psicología , Humanos , Memoria , Mucosa Nasal/metabolismo , Aprendizaje EspacialRESUMEN
The stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1) is a receptor-type protein tyrosine phosphatase that is specifically expressed on the apical membrane of the intestinal epithelium. SAP-1 is known to maintain the balance of phosphorylation of proteins together with protein kinases; however, its biological function and impact on pharmacokinetics in the intestine remain unclear. The present study, therefore, aimed at clarifying the relationship between SAP-1 and the intestinal absorption behaviors of typical transporter substrates and macromolecules. The endogenous levels of glucose and total cholesterol in the blood were similar between wild-type and SAP-1-deficient mice (Sap1-/-), suggesting no contribution of SAP-1 to biogenic influx. Moreover, in vitro transport study with everted ileal sacs demonstrated that there was no difference in the absorption of breast cancer resistance protein, P-glycoprotein, and peptide transporter substrates between both mice. However, absorptive clearance of macromolecular model dextrans (FD-4 and FD-10) in Sap1-/- mice was significantly higher than that in wild-type mice, and this was confirmed by the trend of increased FD-4 absorption from colonic loops of Sap1-/- mice. Therefore, the results of this study suggest the partial contribution of SAP-1 to the regulated transport of hydrophilic macromolecules through paracellular tight junctions.
Asunto(s)
Mucosa Intestinal/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Uniones Estrechas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Transporte Biológico , Colesterol/metabolismo , Femenino , Eliminación de Gen , Glucosa/metabolismo , Uniones Intercelulares/metabolismo , Absorción Intestinal , Ratones , Ratones Endogámicos C57BL , Microvellosidades/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genéticaRESUMEN
We have reported that cell-penetrating peptides, such as oligoarginine, act as powerful absorption enhancers for the development of oral insulin delivery systems. However, the minimal essential sequence of oligoarginine that stimulates intestinal insulin absorption remains unclear. Therefore, the present study was conducted to clarify this minimum sequence of oligoarginine and to examine the effect of single cationic amino acid arginine on the intestinal and oral absorption of insulin. The results demonstrated that a remarkable enhancement of intestinal insulin absorption was observed after coadministration of insulin with l-arginine. The efficacy of d-forms of oligoarginine/arginine tended to decrease with a decreasing number of amino acid residues, whereas the effect of l-arginine was the strongest of any of the l-forms of oligoarginine/arginine. Interestingly, the effect of l-arginine was stronger than that of d-arginine at various concentrations, and the effect of other cationic amino acids such as lysine and histidine was relatively lower than that of arginine. In addition, no leakage of lactate dehydrogenase from the intestinal epithelium and no change in the transepithelial electrical resistance of a Caco-2 cell monolayer were detected after administration of l-arginine as the single amino acid, which suggests that there were no undesirable effects of arginine on the integrity of cell membranes and paracellular tight junctions. Oral administration study in mice demonstrated that the stronger hypoglycemic effects were observed after coadministration of insulin with l-arginine. In this study, we found that arginine is a key cationic amino acid for delivering insulin across intestinal epithelial barriers and hopefully accelerating the clinical development of oral insulin delivery systems.