Exploration of the Key Factors for Optimizing the <i>in Vivo</i> Oral Delivery of Insulin by Using a Noncovalent Strategy with Cell-Penetrating Peptides

Abstract

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. Key words oral delivery; insulin; cell-penetrating peptide; penetratin; intermolecular interactionInsulin is the most important therapeutic agent for diabetes, even though there are now many other oral antidiabetic medicines available. Therefore, many types of insulin formulations, including rapid-and long-acting insulin as well as mixed type insulin, have been developed to date. 1-3) Diabetes patients can appropriately choose injectable types of insulin formulations; however, more convenient forms of insulin that can be administered via noninvasive routes, such as oral administration, have not yet been developed because of poor absorption through mucosal regions. [4][5][6][7] To further improve the QOL and compliance of patients, we aimed to develop a noninvasive form of insulin by using pharmaceutical technologies and drug delivery systems. Moreover, the oral route of insulin absorption has another advantage as insulin absorbed from the intestine first passes through the liver, similarly to endogenous insulin secreted to the portal vein; therefore, oral insulin can circumvent the risk of hypoglycemia.A major hurdle in the development of formulations for administration via noninvasive routes is the digestion and degradation in the harsh gastrointestinal environment. To protect biologics, such as insuli...

Application of Permeation Enhancers in Oral Delivery of Macromolecules: An Update

“…CPPs such as penetratin and its analog, PentraMax TM , continue to be researched for oral peptide delivery. There is evidence that these CPPs act by altering membrane barrier integrity [30], endocytosis [30], and physical complexation [31]. Although a few CPPs have progressed to clinical evaluation, the majority relate to the intracellular delivery of small molecules and not to oral delivery of macromolecules [32].…”

Stereochemistry and Intermolecular Interactions Influence Carrier Peptide-Mediated Insulin Delivery

“…However, that study also concluded that the effect of D-penetratin was superior to the effect of the L-form, which do not comply with our results. 20 Another study by the same research group reported that the stereochemistry effect depended on the region of rat intestines investigated; 21 direct correlations may thus not be evident between different methodologies. This difference in the effect of stereochemistry for the different peptides suggests that the grouping of residues and the positioning of the cationic residues in the amino acid sequence influence the carrier peptide efficacy.…”

“…25 The insulin−carrier peptide interactions did not, however, depend on the stereochemistry as observed from the SAXS investigation, consistent with a study of insulin− penetratin interactions using surface plasmon resonance. 20 Other factors might therefore influence the carrier peptidemediated insulin permeation, which account for the stereochemistry-dependent enhancement of insulin permeation by Dpenetratin and D-penetramax (Figure 3C).…”

Oral delivery of proteins and peptides: Challenges, status quo and future perspectives

“…The low molecular weight protamine (LMWP) with a sequence of VSRRRRRRGGRRRRC is the one of CPPs, which can increase the intestinal cell membrane permeability and oral relative bioavailability of exenatide-Zn 2+ by 29-fold 213 . Both Penetratin® and its analog PenetraMax® exerted absorption enhanced ability for oral insulin in D-form by non-covalent approach; but there is no synergistic effect observed when using the combination of these two CPPs 214 . Although CPPs have exerted excellent capacity in improving membrane permeability, they have not yet been validated in the clinic studies of oral delivery of peptides due to complex GI environment.…”