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1.
Eur J Pharm Biopharm ; 85(3 Pt A): 736-43, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24060698

RESUMEN

Recently, cell-penetrating peptides (CPPs) based vehicles have been developed for the delivery of different payloads in animals. Our studies have shown that nasal absorption of insulin and other therapeutic peptides and proteins can be improved significantly by co-administration of the CPP penetratin. Successful development of suitable CPP-based delivery systems, however, will depend not only on the efficiency of CPPs to transport therapeutic agents across the biological barriers of the nasal cavity, but also on the risk of adverse effects such as toxicity and undesired immunogenicity, especially in chronic therapy. In this study, we investigated the bioavailability (BA) of insulin and the adverse effects on the nasal mucosa in rats following a long-term dosing regimen of L-penetratin and the novel penetratin analogue "PenetraMax." Following nasal delivery, a significantly higher BA for insulin (almost 100% relative to subcutaneous (s.c.) injections) was observed for PenetraMax in comparison with the parent penetratin peptide after chronic administrations in rats. Importantly, there was negligible biomarker leakage in nasal lavage fluid and the integrity of the nasal epithelium remained unaffected when PenetraMax was used in long-term multiple administrations. In addition, no significant difference in the release of inflammatory and immunogenicity mediators in plasma was observed after nasal administration of PenetraMax with or without insulin solution. In conclusion, PenetraMax, a novel CPP candidate, can open a new avenue in clinical trials for noninvasive nasal insulin delivery.


Asunto(s)
Proteínas Portadoras/química , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Mucosa Nasal/efectos de los fármacos , Administración Intranasal , Animales , Disponibilidad Biológica , Proteínas Portadoras/toxicidad , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/toxicidad , Portadores de Fármacos/química , Portadores de Fármacos/toxicidad , Sistemas de Liberación de Medicamentos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/toxicidad , Insulina/farmacocinética , Insulina/toxicidad , Masculino , Mucosa Nasal/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Pruebas de Toxicidad Subcrónica
2.
J Pharm Sci ; 102(2): 469-79, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23160942

RESUMEN

Our recent work has shown that the intestinal absorption of insulin can be improved significantly by coadministration of cell-penetrating peptides (CPPs), especially penetratin. However, a relatively high dose of penetratin is required to adequately stimulate the intestinal absorption of insulin. Therefore, in this study, we sought to determine the CPP that most effectively enhanced intestinal insulin absorption. An in situ loop absorption study using 26 penetratin analogues suggested that the chain length, hydrophobicity, and amphipathicity of the CPPs, as well as their basicity, contribute to their absorption-enhancing efficiency. Moreover, a molecular orbital method with self-organizing maps (SOMs) classification suggested that multiple factors, including the molecular weight, basicity, the lowest unoccupied molecular orbital energy, absolute hardness, and chemical potential of CPPs, are associated with their effects on intestinal insulin absorption. Furthermore, the new CPPs proposed by SOM clustering had a marked capacity to interact with insulin, and their ability to enhance insulin absorption was much stronger than that of the original penetratin. Therefore, the peptide sequence that optimally enhances intestinal insulin absorption could be defined by SOM with the molecular orbital method, and our present work emphasizes the utility of such methodologies in the development of effective drug delivery systems.


Asunto(s)
Membrana Celular/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Insulina/metabolismo , Absorción Intestinal/fisiología , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Animales , Membrana Celular/efectos de los fármacos , Humanos , Insulina/administración & dosificación , Insulina/genética , Absorción Intestinal/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
J Control Release ; 143(3): 302-10, 2010 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-20096319

RESUMEN

Penetratin, a 16-residue peptide, is used widely as a highly efficient delivery carrier for a wide range of poorly permeable therapeutic cargoes. The crucial structural features of penetratin remain unclear, as demonstrated by the difficulties encountered in designing new molecules. The efficiency in enhancing nasal insulin absorption was compared between l-penetratin and 20 of its analogues in rats. We also measured lactate dehydrogenase (LDH) leakage as an indicator of cytotoxicity and scored the histopathological irritation. Substitution of a cationic residue (Arg or Lys) with Leu or addition of tetra-arginine to the C- or N-terminus of penetratin caused considerable reduction in the enhancing efficiency properties of the modified analogues. Mutual exchanging of Arg and Lys in corresponding analogues produced nearly inactive analogues, although changing Arg to Lys in the same analogue produced similar penetratin activity. In addition, activity was impaired markedly upon modification of penetratin within amphiphilic (Trp) or hydrophobic (Ile and Phe) residues. Chain size-modified analogues lacked the ability to induce nasal insulin absorption. In contrast, rearrangement of the modified analogues by C,N-half-exchange and reverse analogues produced activity similar to that of the original penetratin. The enhancing activity was inhibited almost completely upon sequence arrangement of the resulting analogues. Surprisingly, a shuffle (Arg, Lys fix) 2 analogue increased insulin absorption significantly, reaching a relative bioavailability value 1.85-times that of original penetratin. This analogue caused negligible release of LDH in nasal lavage fluid and maintained the integrity of the nasal respiratory epithelium. In conclusion, modulation of amino acid sequences by fixing the cationic residue positions can augment penetratin-enhanced nasal absorption and may lead to improvements in nasal insulin absorption.


Asunto(s)
Proteínas Portadoras/química , Insulina/administración & dosificación , Insulina/farmacocinética , Cavidad Nasal/metabolismo , Animales , Péptidos de Penetración Celular , Masculino , Cavidad Nasal/ultraestructura , Mucosa Nasal/metabolismo , Ratas , Ratas Sprague-Dawley
4.
J Control Release ; 142(1): 8-13, 2010 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-19804802

RESUMEN

A novel formulation of biodegradable microparticles was developed for the sustained release of peptide and protein drugs. The microparticles were formed by the aggregation of protein nanoparticles through water-in-oil (W/O) emulsion-lyophilization and subsequent solid-in-oil-in-water (S/O/W) emulsion-solvent evaporation. Amphiphilic copolymers were used as an emulsifier in the W/O emulsion and matrix of the microparticles. Among the various copolymers investigated, poly(lactide-co-glycolide)-grafted dextran (Dex-g-PLGA) was chosen as the best candidate on the basis of the encapsulation efficiency and in vitro release profile, the near zero-order release without a significant initial burst, of human growth hormone (hGH). The release rate of hGH was controllable by changing the composition of Dex-g-PLGA. The in vivo release studies using normal mice revealed that the plasma concentration of hGH was maintained for 1week without a significant initial burst. The enhancement of biological activity of hGH by sustained release was confirmed by measuring the IGF-1 concentration and body weight of hypophysectomized mice. These results suggest the high potential of the newly developed microparticles for the sustained release of biopharmaceuticals.


Asunto(s)
Preparaciones de Acción Retardada/química , Hormona de Crecimiento Humana/administración & dosificación , Nanopartículas/química , Animales , Dextranos/química , Hormona de Crecimiento Humana/sangre , Hormona de Crecimiento Humana/farmacología , Humanos , Ácido Láctico/química , Masculino , Ratones , Ratones Endogámicos BALB C , Nanopartículas/ultraestructura , Tamaño de la Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico
5.
J Control Release ; 132(1): 21-5, 2008 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-18727945

RESUMEN

Cell-penetrating peptides (CPPs) are a useful tool for delivering therapeutic macromolecules across cell membranes. We previously devised an approach using CPPs without intermolecular cross-linking and showed the efficient delivery of insulin from the intestine to the systemic circulation using a typical CPP, oligoarginine. However, this approach required relatively high doses of the CPP. Therefore, this study aimed to identify CPPs that are more effective for the delivery of insulin and do not induce toxic effects on the intestine. In this study, we examined the effects of various types of CPPs including arginine-rich peptides and amphipathic peptides that aid insulin absorption from rat ileal segments. Among these peptides, coadministration of insulin with R8, penetratin, pVEC, and RRL helix significantly increased ileal insulin absorption compared with insulin administration alone. In the case of R8, the D-form of the peptide had stronger absorption enhancing ability than the L-form. In contrast, the other three peptides exerted a more significant effect when the L-forms were applied, and L-penetratin had the strongest ability to enhance intestinal insulin absorption. Meanwhile, in a physical mixture of CPP and insulin, aggregates formed in the solution when high concentrations of CPPs were present. L-penetratin enhanced insulin absorption even when administered in an aggregated solution. We then showed that aggregates of L-penetratin and insulin were broken down in the presence of intestinal degradation enzymes. Thus, among CPPs used in this study, L-penetratin had the strongest ability to improve insulin intestinal absorption.


Asunto(s)
Arginina/farmacología , Insulina/farmacocinética , Absorción Intestinal/efectos de los fármacos , Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular , Relación Dosis-Respuesta a Droga , Íleon/efectos de los fármacos , Íleon/metabolismo , Insulina/metabolismo , Intestino Delgado/enzimología , Isomerismo , Masculino , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Ratas
6.
Tissue Eng ; 12(9): 2565-72, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16995789

RESUMEN

Hepatocyte transplantation is an attractive alternative to orthotopic liver transplantation. However, its application has been limited because of its short-term success only. Here we report a new approach to hepatocyte transplantation resulting in the generation of an auxiliary liver in vivo. Isolated primary hepatocytes were encapsulated in isolated spleens and then transplanted by attaching the spleens to the livers of recipient animals (mice or rats) using biodegradable adhesive. A vascular network was rapidly established, and protein molecules circulated freely between the transplanted spleen and the liver, to which they adhered. In contrast, the spleen, which did not adhere to the liver or adhered elsewhere (adipose tissue or peritoneum), did not become vascularized but shrank and died. Encapsulation of hepatocytes in an isolated spleen enhanced their survival significantly, and co-encapsulation of Engelbreth- Holm-Swarm gel together with the hepatocytes further enhanced it. The encapsulated hepatocytes expressed liver-specific differentiation genes for more than 3 weeks. Plasma albumin concentrations in Nagase analbuminemic rats began to increase 3 days after transplantation. The transplanted hepatic cells migrated into the liver parenchyma, whereas the spleen was absorbed. Thus, we have developed a novel, simple approach for the rapid and efficient formation of functional auxiliary liver using a modified hepatocyte transplantation method.


Asunto(s)
Hepatocitos/trasplante , Hígado Artificial , Bazo , Animales , Trasplante de Células/métodos , Células Inmovilizadas/trasplante , Hepatocitos/citología , Hepatocitos/metabolismo , Trasplante de Hígado , Ratones , Ratas , Ratas Sprague-Dawley , Bazo/citología , Bazo/trasplante , Factores de Tiempo , Adhesivos Tisulares/química
7.
Biochim Biophys Acta ; 1724(1-2): 49-58, 2005 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-15905035

RESUMEN

Coenzyme Q functions as an electron carrier and reversibly changes to either an oxidized (CoQ), intermediate (CoQ.-), or reduced (CoQH2) form within a biomembrane. The CoQH2 form also acts as an antioxidant and prevents cell death, and thus has been successfully used as a supplement. On the other hand, the value of the CoQ/CoQH2 ratio has been shown to increase in a number of diseases, presumably due to an anti-proliferative effect involving CoQ. In the present study, we examined the effect of CoQ and its isoprenoid side chain length variants on the growth of cells having different p53 statuses. Treatment with CoQs having shorter isoprenoid chains, especially CoQ2, induced apoptosis in p53-point mutated BALL-1 cells, whereas treatment with longer isoprenoid chains did not. However, CoQ2 did not induce apoptosis in either a p53 wild-type cell line or a p53 null mutant cell line. These results indicated that the induction of apoptosis by CoQ2 was dependent on p53 protein levels. Moreover, CoQ2 induced reactive oxygen species (ROS) and the phosphorylation of p53. An antioxidant, l-ascorbic acid, inhibited CoQ2-induced p53 phosphorylation and further apoptotic stimuli. Overall, these results suggested that short tail CoQ induces ROS generation and further p53-dependent apoptosis.


Asunto(s)
Apoptosis , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquinona/metabolismo , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Células Cultivadas , Humanos , Fosforilación/efectos de los fármacos , Ubiquinona/farmacología
8.
Cancer Sci ; 96(2): 83-92, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15723652

RESUMEN

Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Sphingosine, sphinganine, and phytosphingosine are structural analogs of sphingolipids and are classified as long-chain sphingoid bases. Sphingosine and sphinganine are known to play important roles in apoptosis. In the present study, we examined the phytosphingosine-induced apoptosis mechanism, focusing on mitochondria in human T-cell lymphoma Jurkat cells. Phytosphingosine significantly induced chromatin DNA fragmentation, which is a hallmark of apoptosis. Enzymatic activity measurements of caspases revealed that caspase-3 and caspase-9 are activated in phytosphingosine-induced apoptosis, but there is little activation of caspase-8 suggesting that phytosphingosine influences mitochondrial functions. In agreement with this hypothesis, a decrease in DeltaPsi(m) and the release of cytochrome c to the cytosol were observed upon phytosphingosine treatment. Furthermore, overexpression of mitochondria-localized anti-apoptotic protein Bcl-2 prevented phytosphingosine apoptotic stimuli. Western blot assays revealed that phytosphingosine decreases phosphorylated Akt and p70S6k. Dephosphorylation of Akt was partially inhibited by protein phosphatase inhibitor OA and OA attenuated phytosphingosine-induced apoptosis. Moreover, using a cell-free system, phytosphingosine directly reduced DeltaPsi(m). These results indicate that phytosphingosine perturbs mitochondria both directly and indirectly to induce apoptosis.


Asunto(s)
Apoptosis/efectos de los fármacos , Esfingosina/análogos & derivados , Esfingosina/farmacología , Caspasas/metabolismo , Citocromos c/metabolismo , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo
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