Safety of the cell-penetrating peptide penetratin as an oral absorption enhancer.

Novel delivery technology using cell-penetrating peptides (CPPs) have recently shown their potential and are emerging as promising candidates for an oral protein and peptide delivery systems. As with for the development of any absorption enhancer that is meant to function across an epithelial layer covering a surface highly exposed to pathogens such as the intestines, concern arises about the safety of such enhancers. The purpose of this study was to investigate the effect of 7 d of consecutive oral administrations of CPPs and a typical enterotoxin, lipopolysaccharide (LPS) to mice to determine the degree, if any, of damage caused to the hepatic tissue. Following the 7-d dosing regimen, we could not detect significantly increased levels of the liver enzymes alanine aminotransferase and aspartate aminotransferase in plasma of mice treated with CPP and LPS compared to the controls, whereas heightened levels were observed in animals receiving the bile salt. In conclusion, the repeated use of CPPs as an oral absorption enhancer for macromolecules was found to be a safe strategy.

In vivo proof of concept of oral insulin delivery based on a co-administration strategy with the cell-penetrating peptide penetratin.

Oral delivery of insulin is blocked by low intestinal absorption caused by the poor permeability of insulin across cellular membranes and the susceptibility to enzymatic degradation in the gastrointestinal tract. Cell-penetrating peptides (CPPs) have been investigated for a number of years as oral absorption enhancers for hydrophilic macromolecules. Penetratin, a cationic and amphipathic CPP, effectively enhances insulin absorption and we were able to alleviate the enzymatic barrier by using the enzymatic resistant D-form of penetratin. In this study, mice were dosed orally with a physical mixture of insulin and penetratin. Blood glucose concentrations were measured and a pharmacological availability (PA) of 18.2% was achieved in mice dosed with insulin and D-penetratin. Following the promising data, we investigated the degradation parameters of insulin and penetratin in rat intestinal fluid. As expected, L-penetratin was degraded rapidly whereas D-penetratin had a halflife of 67±7min in 10-fold diluted gastrointestinal fluid. Insulin degradation was slowed by the presence of penetratin in intestinal fluid. The half-life of insulin increased from 24.9±4.5min to 55.6±14min and 90.5±11.8min in the presence of L- and D-penetratin respectively. In conclusion, both Land D-penetratin acted as oral absorption enhancers at select CPP concentrations for insulin and the current study is the first solid evidence of pharmacological activity of oral insulin delivery systems based on non-covalent intermolecular interactions with penetratin.

One-month subchronic toxicity study of cell-penetrating peptides for insulin nasal delivery in rats.

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.

Noninvasive insulin delivery: the great potential of cell-penetrating peptides.

Insulin, a potent therapeutic peptide used in the treatment of diabetes, is administered to patients via subcutaneous injections because of the poor pharmacokinetics associated with alternative routes of administration such as oral, nasal and pulmonary delivery. Noninvasive nasal and oral formulations are appealing to patients who need consecutive daily treatments of insulin. However, to achieve mucosal absorption of insulin via oral or nasal administration, two barriers must be overcome: the impermeability of insulin through the epithelial membranes and local digestion and enzymatic degradation. Cell-penetrating peptides (CPPs), which efficiently bring exogenous proteins and nucleic acids into cells, have great potential to facilitate insulin permeation from the intestinal lumen or nasal cavity into systemic circulation via efficient uptake by epithelial cells. In fact, the coadministration of insulin with the peptide penetratin, a typical CPP, increased intestinal and nasal insulin bioavailability to 35 and 50%, respectively. In this review, the authors describe recent findings using this novel CPP-based formulation for noninvasive delivery of insulin.

The effect of chemical modification and nanoparticle formulation on stability and biodistribution of siRNA in mice.

Instability and inadequate biodistribution of double-stranded RNA are major drawbacks to the clinical use of RNA interference. This work compares chemical modification and nanoparticle formulation as strategies to improve the systemic delivery of small interfering RNA (siRNA). Variable levels of chemical modified siRNA, either naked or within nanoparticle, were intravenously injected into mice to study temporal stability and biodistribution detected by direct radioactive labeling or by northern blotting. Naked siRNA showed rapid renal clearance, with circulatory half-life of <5 minutes that could be extended to >30 minutes by cholesterol conjugation. The integrity of the chemically stabilized siRNA was maintained in blood for at least 30 minutes, whereas, unmodified siRNA duplex was degraded within 1 minute. Intact chemically modified siRNA could also be detected in all analyzed organs at 30 minutes but disappeared at 24 hours, except for heavy locked nucleic acid (LNA)-modified and cholesterol-conjugated siRNA in the lungs. Chitosan, liposomal, or JetPEI formulation greatly improved the stability and biodistribution of siRNA. Interestingly, high siRNA accumulation of the chitosan/siRNA formulation within the kidney was observed 24 hours postadministration. This comparative study highlights improvements to siRNA stability and pharmacokinetics, key determinants for development of clinically relevant RNAi therapeutics.