Poly-L-arginine-Induced Internalization of Tight Junction Proteins Increases the Paracellular Permeability of the Caco-2 Cell Monolayer to Hydrophilic Macromolecules

et al. 2017

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Polycationic compounds, such as poly-L-arginine and poly-L-ornithine (PLO), enhance the nasal absorption of hydrophilic macromolecular drugs. However, the bio availability corresponding to the dose of these enhancers has not been obtained in an open system study, where an administered solution is transferred to the pharynx because they do not exhibit mucoadhesion/retention in the nasal cavity. In this study, we prepared PEGylated-poly-L-ornithine (PEG-PLO) and investigated the effects of PEGylation on in vitro adhesion/retention properties, permeation enhancement efficiency, and cytotoxicity. PEG-PLO bearing 3-4 polyethylene glycol (PEG) chains per PLO molecule was more retentive than unmodified PLO on an inclined plate. The permeability of a model drug, FD-4, across Caco-2 cell sheets was enhanced by PEG-PLO as well as by PLO. PLO showed cytotoxicity at high concentrations, whereas PEG-PLO did not decrease cell viability, even above the concentration giving a sufficient enhancement effect. These findings suggest that PEGylation of polycationic absorption enhancers improves their adhesion/retention and decreases their cytotoxicity, which may lead to enhancers with greater utility.Key words poly-L-ornithine (PLO); polyethylene glycol (PEG); permeation enhancer Bioactive peptides, such as insulin, teriparatide, liraglutide, and somatropin, have been used widely as therapeutic drugs because of their intrinsic and effective activities, however, they have poor permeability across the epithelium because they are hydrophilic macromolecules. Orally administered peptides are often inactivated in the gastrointestinal tract, and thus peptides are usually administered as injectable formulations. However, injection can result in poor compliance because it is painful and burdensome. 1) Therefore, alternative routes of administration are required.The nasal mucosa has a large surface area for absorption because of its villus structure. Drugs absorbed across the nasal mucosa avoid the hepatic first pass effect because of the abundant vasculature under the nasal mucosa.2) The intranasal route is effective for absorbing high molecular weight drugs 3) and is usually not painful. Therefore, intranasal administration is an important alternative route to injection for peptides and proteins.Polycationic compounds, such as poly-L-arginine (PLA), poly-L-lysine, and poly-L-ornithine (PLO), enhance the transmucosal absorption of hydrophilic macromolecules.4,5) PLA enhances the paracellular permeability of rabbit nasal mucosa and Caco-2 cell sheets reversibly.6,7) The absorption of hydrophilic macromolecules across the nasal mucosa in rats is also increased by co-administration with PLA. 8) PLA enhances the permeability by altering the localization of tight junction proteins from the cell-cell junction to the intracellular space without causing cytotoxicity. 9) Therefore, polycationic absorption enhancers are useful in developing efficient transmucosal drug delivery systems.Nevertheless, it is difficult to increase the bioavailability of dr...

Section: Fd-4 Permeation Experimentsmentioning

confidence: 99%

mentioning

confidence: 99%

Preparation and Evaluation of PEGylated Poly-L-ornithine Complex as a Novel Absorption Enhancer

Kamiya

et al. 2017

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“…Therefore, it seems that nasal rhGH absorption is increased by increasing the solubility and the paracellular permeability of rhGH by PLA as the same mechanism identified previously. [13][14][15][16][17] With regard to the effects of the molecular weight of PLA, a delay in the rhGH absorption was observed with an increase in the molecular weight. In particular, T max in 1.0 % (w/v) PLA (150) was 9.6 h, and the rhGH absorption was remarkably delayed compared with that in the solutions with other concentrations and molecular weights of PLA.…”

Section: Discussionmentioning

confidence: 99%

“…11,13) In the studies on the mechanism of enhanced absorption by PLA in the mucosa, it has been found that PLA induces the enhanced paracellular permeability by the dispersion of tight junction proteins (claudin-4, occludin, zonula occludens-1 (ZO-1) and tricellulin) into the cytoplasm through the cell-cell junction. [14][15][16][17] In particular, it was known that occludin was important for macromolecular permeation among tight junction proteins. 18,19) In addition, it has been also demonstrated that adherens junction protein (E-cadherin, β-catenin) is dispersed by PLA from the cell-cell junction.…”

mentioning

confidence: 99%

Development of a Transnasal Delivery System for Recombinant Human Growth Hormone (rhGH): Effects of the Concentration and Molecular Weight of Poly-L-arginine on the Nasal Absorption of rhGH in Rats

Kawashima

et al. 2016

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A novel system for delivering recombinant human growth hormone (rhGH) that is noninvasive and has a simple method of administration is strongly desired to improve the compliance of children. The aim of this study was to investigate the potential for the intranasal (i.n.) co-administration of rhGH with poly-L-arginine (PLA) as a novel delivery system by evaluating the effects of the concentration and molecular weight of PLA on the nasal absorption of rhGH. The influence of the formation of insoluble aggregates and a soluble complex in the dosage formulation on nasal rhGH absorption was also evaluated by size-exclusion chromatography and ultrafiltration. PLA enhanced the nasal absorption of rhGH at each concentration and molecular weight examined. Nasal rhGH absorption increased dramatically when the PLA concentration was 1.0 % (w/v) due to the improved solubility of rhGH in the formulation. A delay in rhGH absorption was observed when the molecular weight of PLA was increased. This appeared to be because the increase in molecular weight caused the formation of a soluble complex. It seems that the PLA concentration affects the absorptionenhancing effect on rhGH, while the molecular weight of PLA affects the time when the maximum plasma rhGH concentration was reached (T max ) of rhGH after i.n. administration, mainly because of the interactions among rhGH, PLA, and additives. Therefore, the transnasal rhGH delivery system using PLA is considered to be a promising alternative to subcutaneous (s.c.) injection if these interactions are sufficiently controlled.Key words poly-L-arginine; nasal absorption; enhancer; recombinant human growth hormone Most peptide and protein medicinal drugs are administered clinically by injection. This invasive administration with a needle and syringe is associated with various problems, such as pain for the patients, the risk of trouble at the injection site, increased medical waste and the potential for complicated operation. Hence, an alternative route of administration is strongly desired. There have been numerous studies on the transmucosal delivery of peptide and protein drugs, such as oral, nasal, and pulmonary administration.1-3) However, these types of drugs are rapidly destroyed by the enzymes present in various organs, especially in the gastrointestinal tract, and have a limited permeation across the mucous membrane. Therefore, these drugs have low bioavailability.Recombinant human growth hormone (rhGH), a single polypeptide chain of 191 amino acids and a molecular mass of 22.1 kDa, is used to treat short stature in children caused by growth hormone deficiency, Turner's syndrome, or chronic renal failure. At present, rhGH is administered daily by subcutaneous (s.c.) injections. Therefore, compliance with rhGH therapy is often low, because children hate the pain caused by such injections. The compliance of patients affects the therapeutic outcome, because it is necessary to regularly administer injections of rhGH for the limited period of growth. 4,5) Hence, a novel rhGH deliver...

“…4,6) Therefore, PLA is considered to be promising as an absorption enhancer for transnasal drug delivery. Although some studies have examined whether PLA can also enhance the absorption of hydrophilic macromolecules across the nasal epithelium, conjunctiva, alveolar epithelium, and Caco-2 cell sheets, [7][8][9][10] there have been no studies looking at PLA᾿s absorption enhancing effect across the intestinal mucosa. The intestinal mucosa is an important tissue particularly for the absorption of orally administered drugs.…”

Section: Effect Of Poly-l-arginine On Intestinal Absorption Of Hydropmentioning

confidence: 99%

Effect of Poly-<small>L</small>-arginine on Intestinal Absorption of Hydrophilic Macromolecules in Rats

Kuwahara

et al. 2013

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We have already reported that poly-l-arginine (PLA) remarkably enhanced the in vivo nasal absorption of hydrophilic macromolecules without producing any significant epithelial damage in rats. In the present study, we examined whether PLA could enhance the absorption of a model hydrophilic macromolecule, fluorescein isothiocyanate-dextran (FD-4), across the intestinal mucosa, as well as the nasal mucosa, by an in situ closed-loop method using the rat intestine. PLA was found to enhance the intestinal absorption of FD-4 in a concentration-dependent manner within the concentrations investigated in this study, but segment-specific differences were found to be associated with this effect (ileum>jejunum>duodenum≧colon). The factors responsible for the segment-specific differences were also investigated by intestinal absorption studies using aprotinin, a trypsin inhibitor, and an analysis of the expression of occludin, a tight junction protein. In the small intestine, the differences in the effect of PLA on the absorption of FD-4 may be related to the enzymatic degradation of PLA. In the colon, the reduced effect of PLA on the absorption of FD-4 may be related to the smaller surface area for absorption and the higher expression of occludin compared with other segments.Key words poly-l-arginine; intestinal absorption; enhancer; fluorescein isothiocyanate-dextran (FD-4); occludin; aprotininWe have already reported that poly-l-arginine (PLA), a polycation, remarkably enhanced the in vivo nasal absorption of hydrophilic macromolecules in rats and increased their in vitro permeation across the rabbit nasal epithelium without producing any significant epithelial damage. [1][2][3][4][5] This effect is caused by the opening of tight junctions (TJ) in the epithelial cells. PLA plays an important role in opening the TJ in these cells, and the reaction induced by PLA appears to be reversible.4,6) Therefore, PLA is considered to be promising as an absorption enhancer for transnasal drug delivery. Although some studies have examined whether PLA can also enhance the absorption of hydrophilic macromolecules across the nasal epithelium, conjunctiva, alveolar epithelium, and Caco-2 cell sheets, [7][8][9][10] there have been no studies looking at PLA᾿s absorption enhancing effect across the intestinal mucosa. The intestinal mucosa is an important tissue particularly for the absorption of orally administered drugs.The intestinal absorption of hydrophilic macromolecules is well known to be typically very poor.11) The small intestine has an extensive surface area for absorption, but various digestive enzymes exist in the gastrointestinal tract. The property of intestinal mucosa differs from that of the nasal mucosa in some respects. Therefore, in order to improve the absorption of hydrophilic macromolecules, various strategies such as the use of absorption enhancers, protease inhibitors and chemical modification have been examined in the intestinal mucosa as well as the nasal mucosa. [12][13][14][15] It was recently reported that the TJ ba...