Effect of cell-penetrating peptides on the nasal absorption of insulin

doi:10.1016/j.jconrel.2008.09.076

Journal of Controlled Release

Volume 133, Issue 2, 19 January 2009, Pages 103-108

https://doi.org/10.1016/j.jconrel.2008.09.076 Get rights and content

Abstract

The goal of this study was to evaluate whether cell-penetrating peptides (CPPs) affect the nasal absorption of insulin. L- or D-forms of penetratin, or the L- or D-forms of octaarginine (L- or D-R8), was used as first time for nasal insulin delivery. Furthermore, the concentration of lactate dehydrogenase (LDH) in nasal lavage fluid was determined and a histopathological study of nasal respiratory epithelium was conducted. CPPs dramatically increased nasal insulin absorption, and it was more pronounced for L- and D-penetratin than L- or D-R8. L-penetratin was the most effective promoter of insulin absorption compared with others CPPs. A dose-dependent relationship of L-penetratin and insulin bioavailability was statically significant. The pharmacological availability and bioavailability of nasally administered insulin was up to 76.7% and 50.7% relative to the subcutaneous route, respectively. In contrast, increasing the D-penetratin concentration decreased the efficiency of nasal insulin absorption. There was no significant difference in the release of LDH in nasal lavage fluid and the integrity of nasal respiratory epithelium when L-penetratin was present. In conclusion, these data demonstrate that L-penetratin markedly increased the permeability of insulin across the nasal membrane without causing detectable damage to the integrity of cells in the nasal respiratory mucosa.

Introduction

Nasal administration has attracted a lot of interest as an alternative route for the systemic delivery of insulin [1]. Several potential advantages, including the large surface area of nasal mucosa available for insulin absorption, a porous endothelial membrane, and a highly vascularized subepithelial layer that passes directly into the systemic circulation, thereby avoiding the first passage of metabolism in the liver, contribute to the attainment of adequate bioavailability of medication and rapid onset of action comparable to injections. The ready accessibility of nasal administration also makes it possible for patients on long-term therapy to self medicate. In addition, the possibility of obtaining pharmacokinetic profiles that mimic the ‘pulsatile’ endogenous secretion of insulin in healthy volunteers provides opportunities for the design of an optimal replacement therapy [2]. With these advantages, the nasal administration of insulin for systemic medication has been widely investigated in recent years, and many projects are now under clinical development [3]. In contrast, there are some disadvantages with nasal insulin delivery, including the low permeability of the nasal mucosa to large molecules, enzymatic degradation, and rapid mucociliary clearance. These factors decrease the available insulin for efficient absorption and cause low bioavailability [4]. Low permeability through the nasal mucosa is one of the major limitations to attaining sufficient nasal bioavailability of therapeutic protein and peptide [5]. Several approaches are currently used to improve the permeation of insulin through the nasal mucosa, including using absorption enhancers such as surfactants [6] and cylodextrins [7], as well as carrier systems [8] and mucoadhesive polymers [9]. The use of cell-penetrating peptide (CPPs) as novel high-capacity delivery vectors for different bioactive molecules has been an emerging field since 1994 when the first CPP, penetratin [10], was discovered. Several CPPs, such as transactivator of transcription (Tat) [11] and polyarginine [12], efficiently internalize various molecular cargoes of drugs that are poorly absorbed through the cell membrane [13]. CPPs are used to deliver a large variety of drugs inside cells, such as proteins [14], DNA [15], oligonucleotides [16], and drug carrier systems including liposomes [17] and nanoparticles [18]. The aim of this study was to evaluate the effects of CPPs on the nasal absorption of insulin. A recent study reported that oligomers of the Tat peptide mediate efficient gene delivery across the nasal respiratory epithelium [19]. Our laboratory demonstrated that the coadministration of insulin with CPPs significantly enhances intestinal insulin absorption without causing marked damage to cellular integrity [20]. The ultimate goal of our research is to determine whether CPPs can enhance the nasal absorption of the model peptide drug insulin. In this study, we evaluated the in vivo bioavailability of insulin in rats after intranasal coadministration with various CPPs and examined the toxic liability of this treatment on nasal preparations by quantitatively assessing leached lactate dehydrogenase (LDH), and histopathological examination of the nasal epithelium after pretreatment with effective doses of CPPs.

Section snippets

Materials

Recombinant human insulin (26 IU/mg) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). L-R8, D-R8, L-penetratin, D-penetratin (purity > 95% for each peptide), and sodium taurodeoxycholate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were of analytical grade and commercially available.

Preparation of insulin and CPP solution

Specific amounts of recombinant human insulin were dissolved in 50 μl 0.1N HCl in polypropylene tubes, then the insulin solution was diluted with 1.4 ml

Effect of CPPs on insulin absorption from the nasal cavity

Fig. 1 shows the effect of L-penetratin, D-penetratin, D-R8, and L-R8 on nasal insulin absorption (Fig. 1a) and the resultant hypoglycemic effect (Fig. 1b).

No apparent hypoglycemic response was observed following administration of insulin solution, demonstrating that no insulin absorption occurred in the nasal cavity. In contrast, coadministration of 0.5 mM insulin with CPPs increased insulin absorption. As shown in Fig. 1a, insulin absorption was greater after treatment with penetratin in

Discussion

Several technologies have been designed to improve the cellular uptake of therapeutic molecules, such as CPPs, which have been successfully applied for the in vivo delivery of biomolecules and constitute very promising tools [22]. Penetratin and R8 enter cells through a macropinocytic pathway and efficiently deliver molecular cargoes in a biologically active form into a large variety of cell lines as well as in animal models [16], [23]. Some CPPs require chemical linkage between themselves and

Conclusions

In conclusion, L-penetratin acted as the most effective bioavailability enhancer of nasal insulin among the different CPPs assessed in the present study. The results of LDH leakage and the histopathological study suggest that L-penetratin has great potential as a powerful tool in enhancing insulin delivery across nasal membranes safely, and may be a viable alternative to existing parenteral therapies.

Acknowledgments

The authors are grateful to Dr. K. Asai (Hoshi University) for his technical assistance. This study was supported in part by the Research Program on Development of Innovative Technology from the Japan Science and Technology Agency and The Ministry of Education, Science, Sport and Culture of Japan.

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Effect of cell-penetrating peptides on the nasal absorption of insulin

Abstract

Introduction

Section snippets

Materials

Preparation of insulin and CPP solution

Effect of CPPs on insulin absorption from the nasal cavity

Discussion

Conclusions

Acknowledgments

Adv. Drug Deliv. Rev.

J. Control. Release

Int. J. Pharm.

J. Control. Release

J. Biol. Chem.

Adv. Drug Deliv. Rev.

Int. J. Pharm.

J. Control. Release

J. Control. Release

Adv. Drug Deliv. Rev.

Biol. Chem.

J. Control. Release

Int. J. Pharm.

Adv. Drug Deliv. Rev.

FEBS Lett.

Anal. Biochem.