HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins.

Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not. Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identify VPS39-a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex-as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+ and Lamp1+ endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+ late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.

A first-in-class, first-in-human, phase I trial of CIGB-552, a synthetic peptide targeting COMMD1 to inhibit the oncogenic activity of NF-κB in patients with advanced solid tumors.

CIGB-552 is a synthetic peptide that interacts with COMMD1 and upregulates its protein levels. The objectives of this phase I study were safety, pharmacokinetic profile, evaluation of the lymphocytes CD4+ and CD8+ and preliminary activity in patients with advanced tumors. A 3 + 3 dose-escalation design with seven dose levels was implemented. Patients were included until a grade 3 related adverse event occurred and the maximum tolerated dose was reached. The patients received subcutaneous administration of CIGB-552 three times per week for 2 weeks. Single-dose plasma pharmacokinetics was characterized at two dose levels, and tumor responses were classified by RECIST 1.1. Twenty-four patients received CIGB-552. Dose-limiting toxicity was associated with a transient grade 3 pruritic maculopapular rash at a dose of 7.0 mg. The maximum tolerated dose was defined as 4.7 mg. Ten patients were assessable for immunological status. Seven patients had significant changes in the ratio CD4/CD8 in response to CIGB-552 treatment; three patients did not modify the immunological status. Stable disease was observed in five patients, including two metastatic soft sarcomas. We conclude that CIGB-552 at dose 4.7 mg was well tolerated with no significant adverse events and appeared to provide some clinical benefits.

Hybrid Cyclic-Linear Cell-Penetrating Peptides Containing Alternative Positively Charged and Hydrophobic Residues as Molecular Transporters.

The cell membrane properties create a significant obstacle in intracellular delivery of cell-impermeable and negatively charged molecules. Herein, we report the synthesis and biological evaluation of a novel series of hybrid cyclic-linear peptides containing alternative positive and hydrophobic amino acids on the ring and side chain [(RW)5]K(RW)X (X = 1-5) to compare their molecular transporter efficiency. The peptides were synthesized through Fmoc solid-phase peptide synthesis. In vitro cytotoxicity of the peptides showed that the peptides did not exhibit any significant cytotoxicity at the concentration of 10 µM in human leukemia carcinoma cell line (CCRF-CEM), human ovarian adenocarcinoma cells (SK-OV-3), human epithelial embryonic kidney healthy (HEK-293), and human epithelial mammary gland adenocarcinoma cells (MDA-MB-231) after 3 h incubation. The cellular uptake of a fluorescence-labeled phosphopeptide (F'-GpYEEI) and anti-human immunodeficiency virus (HIV) drugs (lamivudine (F'-3TC), emtricitabine (F'-FTC), Stavudine (F'-d4T)), where F' is carboxyfluorescein, was measured in the presence of the peptides in CCRF-CEM and SK-OV-3 cells. Among all peptides, [(RW)5K](RW)5 (10 µM) was the most efficient transporter that improved the cellular uptake of F'-GpYEEI (2 µM) by 18- and 11-fold in CCRF-CEM and SK-OV-3, respectively, compared with F'-GpYEEI alone. Fluorescence-activated cell sorting (FACS) analysis results indicated that the cellular uptake of fluorescence-labeled peptide (F'-[(RW)5K](RW)5) was only partially inhibited by chlorpromazine as an endocytosis inhibitor after 3 h incubation in MDA-MB-231 cells. These data suggest the potential of this series of hybrid cyclic-linear peptides as cell-penetrating peptides and molecular transporters.

PAR1 (Protease-Activated Receptor 1) Pepducin Therapy Targeting Myocardial Necrosis in Coronary Artery Disease and Acute Coronary Syndrome Patients Undergoing Cardiac Catheterization: A Randomized, Placebo-Controlled, Phase 2 Study.

OBJECTIVE: Arterial thrombosis leading to ischemic injury worsens the prognosis of many patients with cardiovascular disease. PZ-128 is a first-in-class pepducin that reversibly inhibits PAR1 (protease-activated receptor 1) on platelets and other vascular cells by targeting the intracellular surface of the receptor. The TRIP-PCI (Thrombin Receptor Inhibitory Pepducin in Percutaneous Coronary Intervention) trial was conducted to assess the safety and efficacy of PZ-128 in patients undergoing cardiac catheterization with intent to perform percutaneous coronary intervention. Approach and Results: In this randomized, double-blind, placebo-controlled, phase 2 trial, 100 patients were randomly assigned (2:1) to receive PZ-128 (0.3 or 0.5 mg/kg), or placebo in a 2-hour infusion initiated just before the start of cardiac catheterization, on top of standard oral antiplatelet therapy. Rates of the primary end point of bleeding were not different between the combined PZ-128 doses (1.6%, 1/62) and placebo group (0%, 0/35). The secondary end points of major adverse coronary events at 30 and 90 days did not significantly differ but were numerically lower in the PZ-128 groups (0% and 2% in the PZ-128 groups, 6% and 6% with placebo, p=0.13, p=0.29, respectively). In the subgroup of patients with elevated baseline cardiac troponin I, the exploratory end point of 30-day major adverse coronary events + myocardial injury showed 83% events in the placebo group versus 31% events in the combined PZ-128 drug groups, an adjusted relative risk of 0.14 (95% CI, 0.02-0.75); P=0.02. CONCLUSIONS: In this first-in-patient experience, PZ-128 added to standard antiplatelet therapy appeared to be safe, well tolerated, and potentially reduced periprocedural myonecrosis, thus providing the basis for further clinical trials. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT02561000.

Cell-Penetrating Doxorubicin Released from Elastin-Like Polypeptide Kills Doxorubicin-Resistant Cancer Cells in In Vitro Study.

Elastin-like polypeptides (ELPs) undergo a characteristic phase transition in response to ambient temperature. Therefore, it has been be used as a thermosensitive vector for the delivery of chemotherapy agents since it can be used to target hyperthermic tumors. This novel strategy introduces unprecedented options for treating cancer with fewer concerns about side effects. In this study, the ELP system was further modified with an enzyme-cleavable linker in order to release drugs within tumors. This system consists of an ELP, a matrix metalloproteinase (MMP) substrate, a cell-penetrating peptide (CPP), and a 6-maleimidocaproyl amide derivative of doxorubicin (Dox). This strategy shows up to a 4-fold increase in cell penetration and results in more death in breast cancer cells compared to ELP-Dox. Even in doxorubicin-resistant cells (NCI/ADR and MES-SA/Dx5), ELP-released cell-penetrating doxorubicin demonstrated better membrane penetration, leading to at least twice the killing of resistant cells compared to ELP-Dox and free Dox. MMP-digested CPP-Dox showed better membrane penetration and induced more cancer cell death in vitro. This CPP-complexed Dox released from the ELP killed even Dox-resistant cells more efficiently than both free doxorubicin and non-cleaved ELP-CPP-Dox.

Cell Penetrating Peptide-Based Self-Assembly for PD-L1 Targeted Tumor Regression.

Cell penetrating peptides (CPPs) are peptides that can directly adapt to cell membranes and then permeate into cells. CPPs are usually covalently linked to the surface of nanocarriers to endow their permeability to the whole system. However, hybrids with lipids or polymers make the metabolism much more sophisticated and even more difficult to determine. In this study, we present a continuous sequence of 18 amino acids (FFAARTMIWY(d-P)GAWYKRI). It forms nanospheres around 170 nm, which increase slightly after loading with siRNA and DOX. Notably, it can be internalized by cancer cells mainly through electronic interactions and PD-L1-mediated endocytosis. Compared with poly-l-lysine and polyethyleneimine, it has a much higher efficiency (about four times) of gene transduction while lowering toxicity. In the treatment of cancer, it causes apoptosis (21%) and inhibits the expression of SURVIVIN protein in vitro. In vivo, it shows good biocompatibility as there are no changes in mice's body weight. When administering peptide-siRNA-DOX, tumor growth is inhibited the most (about three times). These results above prove the sequence to be a good candidate for gene therapy and drug delivery.

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.

Cancer targeting peptides.

Despite continuing advances in the development of biomacromolecules for therapeutic purposes, successful application of these often large and hydrophilic molecules has been hindered by their inability to efficiently traverse the cellular plasma membrane. In recent years, cell-penetrating peptides (CPPs) have received considerable attention as a promising class of delivery vectors due to their ability to mediate the efficient import of a large number of cargoes in vitro and in vivo. However, the lack of target specificity of CPPs remains a major obstacle to their clinical development. To address this issue, researchers have developed strategies in which chemotherapeutic drugs are conjugated to cancer targeting peptides (CTPs) that exploit the unique characteristics of the tumor microenvironment or cancer cells, thereby improving cancer cell specificity. This review highlights several of these strategies that are currently in use, and discusses how multi-component nanoparticles conjugated to CTPs can be designed to provide a more efficient cancer therapeutic delivery strategy.

CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis.

CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47-55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membrane and subsequently internalized into the cytosol. An investigation of the cellular internalization mechanism revealed that most CGA-N9 molecules entered the yeast cells, even at 4°C and in the presence of sodium azide (NaN3), both of which block all energy-dependent transport mechanisms. In addition, peptide internalization was affected by the endocytic inhibitors 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), cytochalasin D (CyD) and heparin; chlorpromazine (CPZ) also had some effect on CGA-N9 internalization. Similar results were obtained in the MIC assays, whereby the anticandidal activity of CGA-N9 was blocked to different degrees in the presence of EIPA, CyD, heparin or CPZ. Therefore, most CGA-N9 passes through the C. tropicalis cell membrane via direct cell penetration, whereas the remainder enters through macropinocytosis and sulfate proteoglycan-mediated endocytosis, with a slight contribution from clathrin-mediated endocytosis.

Biodistribution of TAT or QLPVM coupled to receptor targeted liposomes for delivery of anticancer therapeutics to brain in vitro and in vivo.

Combination therapy has emerged as an efficient way to deliver chemotherapeutics for treatment of glioblastoma. It provides collaborative approach of targeting cancer cells by acting via multiple mechanisms, thereby reducing drug resistance. However, the presence of impermeable blood brain barrier (BBB) restricts the delivery of chemotherapeutic drugs into the brain. To overcome this limitation, we designed a dual functionalized liposomes by modifying their surface with transferrin (Tf) and a cell penetrating peptide (CPP) for receptor and adsorptive mediated transcytosis, respectively. In this study, we used two different CPPs (based on physicochemical properties) and investigated the influence of insertion of CPP to Tf-liposomes on biocompatibility, cellular uptake, and transport across the BBB both in vitro and in vivo. The biodistribution profile of Tf-CPP liposomes showed more than 10 and 2.7 fold increase in doxorubicin and erlotinib accumulation in mice brain, respectively as compared to free drugs with no signs of toxicity.

Octopus-like Flexible Vector for Noninvasive Intraocular Delivery of Short Interfering Nucleic Acids.

Gene therapy is promising for chronic posterior ocular diseases, which are causal factors for severe vision impairment and even blindness worldwide. However, the inherent absorption barriers of the eye restrict intraocular delivery of therapeutic nucleic acids via topical instillation. Safe and efficient nonviral vectors for ocular gene therapy are still unmet clinical desires. Herein, an octopus-like flexible multivalent penetratin (MVP) was designed to facilitate condensation and delivery of therapeutic nucleic acids using multiarm polyethylene glycol (PEG) as a core and conjugating penetratin at each end of the PEG arms as outspread tentacles. Among the MVPs, 8-valent penetratin (8VP) stably compacted nucleic acids into positively charged polyplexes smaller than 100 nm, promoting cellular uptake efficiency (approaching 100%) and transfection rate (over 75%). After being instilled into the conjunctival sac, 8VP enabled rapid (<10 min) and prolonged (>6 h) distribution of nucleic acids in the retina via a noncorneal pathway. In a retinoblastoma-bearing mice model, topical instillation of 8VP/siRNA efficiently inhibited the protein expression of intraocular tumor without toxicity. MVP is advantageous over the commercial transfection reagent in safety and efficiency, and therefore provides a promising vector for noninvasive intraocular gene delivery.

Cell-penetrating peptides derived from Clostridium difficile TcdB2 and a related large clostridial toxin.

Clostridium difficile TcdB (2366 amino acid residues) is an intracellular bacterial toxin that binds to cells and enters the cytosol where it glucosylates small GTPases. In the current study, we examined a putative cell entry region of TcdB (amino acid residues 1753-1851) for short sequences that function as cell-penetrating peptides (CPPs). To screen for TcdB-derived CPPs, a panel of synthetic peptides was tested for the ability to enhance transferrin (Tf) association with cells. Four candidate CPPs were discovered, and further study on one peptide (PepB2) pinpointed an asparagine residue necessary for CPP activity. PepB2 mediated the cell entry of a wide variety of molecules including dextran, streptavidin, microspheres, and lentivirus particles. Of note, this uptake was dramatically reduced in the presence of the Na+/H+ exchange blocker and micropinocytosis inhibitor amiloride, suggesting that PepB2 invokes macropinocytosis. Moreover, we found that PepB2 had more efficient cell-penetrating activity than several other well-known CPPs (TAT, penetratin, Pep-1, and TP10). Finally, Tf assay-based screening of peptides derived from two other large clostridial toxins, TcdA and TcsL, uncovered two new TcdA-derived CPPs. In conclusion, we have identified six CPPs from large clostridial toxins and have demonstrated the ability of PepB2 to promote cell association and entry of several molecules through a putative fluid-phase macropinocytotic mechanism.

Synthesis, characterization, and evaluation of novel cell-penetrating peptides based on TD-34.

In this study, six N-1, N-2, or N-11 derivatives of TD-34 (a cationic cyclic cell-penetrating peptide [CPP], ACSSKKSKHCG) were designed and synthesized including both linear peptides and cyclic peptides, such as DL-1 (KWSSKKSKHCG), DLCC-1 (cyclopeptide, KWSSKKSKHCG), DL-2 (KWSSKKSKHCG-NH2 ), DLCC-2 (cyclopeptide, KWSSKKSKHCG-NH2 ), DL-3 (RWSSKKSKHCG), and DLCC-3 (cyclopeptide, RWSSKKSKHCG). The cyclic peptides were synthesized by disulfide bound linkages formed by N-2 and N-10 cysteine. In vitro penetration experiment was conducted to investigate the transdermal enhancement ability of these derivatives, using triptolide (TP) as model drug. The results display that at the presence of DLCC-2, the accumulative penetration amount of TP increased 1.71-fold (P < .05) within 12 hours, displaying better transdermal enhancing ability than TD-34. Meanwhile, DL-3 and DLCC-3 slightly decreased the transdermal delivery of TP, and the presence of DL-1 and DLCC-1 shows no obvious effect. In order to clarify the factors on the transdermal ability of peptides, the solubility of TP in phosphate buffer saline (PBS) at the presence of different peptides and the mechanism of transdermal delivery of CPPs was investigated. The result shows that most of these peptides have no significant effect on the solubility of TP except DLCC-3 (the solubility of TP slightly increased). And in order to investigate transdermal absorption route of DLCC-2, polyarginine linked to rhodamine b (Rh b) derivative is used. The result proved that the transdermal route of polyarginine is via hair follicle, which may change the transdermal route of its cargo molecule (TP). Our group previously proved that polyarginine and TD-34 have similar transdermal enhancing mechanism (changing the transdermal route of their cargo molecule); it is reasonably speculated that the transdermal route of DLCC-2 is the same as polyarginine and then changes the transdermal absorption route of TP. Furthermore, such results have laid a solid foundation for further investigation of CPPs and paved a way for both designing and synthesizing of new drug delivery system for therapy molecules.

Machine-Learning-Based Prediction of Cell-Penetrating Peptides and Their Uptake Efficiency with Improved Accuracy.

Cell-penetrating peptides (CPPs) can enter cells as a variety of biologically active conjugates and have various biomedical applications. To offset the cost and effort of designing novel CPPs in laboratories, computational methods are necessitated to identify candidate CPPs before in vitro experimental studies. We developed a two-layer prediction framework called machine-learning-based prediction of cell-penetrating peptides (MLCPPs). The first-layer predicts whether a given peptide is a CPP or non-CPP, whereas the second-layer predicts the uptake efficiency of the predicted CPPs. To construct a two-layer prediction framework, we employed four different machine-learning methods and five different compositions including amino acid composition (AAC), dipeptide composition, amino acid index, composition-transition-distribution, and physicochemical properties (PCPs). In the first layer, hybrid features (combination of AAC and PCP) and extremely randomized tree outperformed state-of-the-art predictors in CPP prediction with an accuracy of 0.896 when tested on independent data sets, whereas in the second layer, hybrid features obtained through feature selection protocol and random forest produced an accuracy of 0.725 that is better than state-of-the-art predictors. We anticipate that our method MLCPP will become a valuable tool for predicting CPPs and their uptake efficiency and might facilitate hypothesis-driven experimental design. The MLCPP server interface along with the benchmarking and independent data sets are freely accessible at www.thegleelab.org/MLCPP .

An l- to d-Amino Acid Conversion in an Endosomolytic Analog of the Cell-penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape.

Cell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from l- to d-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of l- to d-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the d-peptide, once in the endosome, is significantly more prone to escape than its l-counterpart. Moreover, the d-peptide is retained in the cytosol of cells for several days, whereas the l-peptide is degraded within hours. Notably, while the l-peptide is relatively innocuous to cells, the d-peptide exerts a prolonged anti-proliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties.

Penetration in 3D tumor spheroids and explants: Adding a further dimension to the structure-activity relationship of cell-penetrating peptides.

Drug delivery into tumors and metastases is a major challenge in the eradication of cancers such as epithelial ovarian carcinoma. Cationic cell-penetrating peptides (CPPs) are a promising group of delivery vehicles to mediate cellular entry of molecules that otherwise poorly enter cells. However, little is known about their penetration behavior in tissues. Here, we investigated penetration of cationic CPPs in 3D ovarian cancer spheroids and patient-derived 3D tumor explants. Penetration kinetics and distribution after long-term incubation were imaged by confocal microscopy. In addition, spheroids and tumor explants were dissociated and cell-associated fluorescence determined by flow cytometry. CPPs with high uptake activity showed enhanced sequestration in the periphery of the spheroid, whereas less active CPPs were able to penetrate deeper into the tissue. CPPs consisting of d-amino acids were advantageous over l-amino acid CPPs as they showed less but long lasting cellular uptake activity, which benefitted penetration and retention over time. In primary tumor cultures, in contrast to nonaarginine, the amphipathic CPP penetratin was strongly sequestered by cell debris and matrix components pointing towards arginine-rich CPPs as a preferred choice. Overall, the data show that testing in 3D models leads to a different choice of the preferred peptide in comparison to a standard 2D cell culture.

An opportunistic route to success: Towards a change of paradigm to fully exploit the potential of cell-penetrating peptides.

About 25years ago it was demonstrated that certain peptides possess the ability to cross the plasma membrane. This led to the development of cell-penetrating peptides (CPPs) as vectors to mediate the cellular entry of (macro-)molecules that do not show cell entry by themselves. Nonetheless, in spite of an early bloom of promising pre-clinical studies, not a single CPP-based drug has been approved, yet. It is a paradigm in CPP research that the peptides are taken up by virtually all cells. In exploratory research and early preclinical development, this assumption guides the choice of the therapeutic target. However, while this indiscriminatory uptake may be the case for tissue culture experiments, in an organism this is clearly not the case. Biodistribution analyses demonstrate that CPPs only target a very limited number of cells and many tissues are hardly reached at all. Here, we review biodistribution analyses of CPPs and CPP-based drug delivery systems. Based on this analysis we propose a paradigm change towards a more opportunistic approach in CPP research. The application of CPPs should focus on those pathophysiologies for which the relevant target cells have been shown to be reached in vivo.

Noncovalent Strategy with Cell-Penetrating Peptides to Facilitate the Brain Delivery of Insulin through the Blood-Brain Barrier.

To overcome the difficulty in delivery of biopharmaceuticals such as peptides and proteins to the brain, several approaches combining the ligands and antibodies targeting the blood-brain barrier (BBB) have been tried. However, these are inefficient in terms of their permeability through the BBB and structural modification of bioactive drugs. In the present study, we therefore examined the usefulness of a noncovalent method using the cell-penetrating peptides (CPPs) such as octaarginine (R8) as a suitable brain delivery strategy for biopharmaceuticals. A safety examination using microvascular endothelial model bEnd.3 cells clarified that R8 was the safest among the CPPs tested in this study. The cellular uptake study demonstrated that coincubation with R8 enhanced the uptake of model peptide drug insulin by bEnd.3 cells in a concentration-dependent and a temperature-independent manner. Furthermore, an in vivo study with rats showed that the accumulation of insulin in the deeper region of the brain, i.e., hippocampus, significantly increased after the intravenous coadministration of insulin with D-R8 without altering the insulin disposition in plasma. Thus, the present study provided the first evidence suggesting that the noncovalent method with CPPs is one of the strategic options for brain delivery of biopharmaceuticals via intravenous injection.

Combination Strategy with Complexation Hydrogels and Cell-Penetrating Peptides for Oral Delivery of Insulin.

In previous studies we showed that the complexation hydrogels based in poly(methacrylic acid-g-ethylene glycol) [P(MAA-g-EG)] rapidly release insulin in the intestine owing to their pH-dependent complexation properties; they also exhibit a high insulin-loading efficiency, enzyme-inhibiting properties, and mucoadhesive characteristics. Cell-penetrating peptides (CPPs), such as oligoarginines [hexa-arginine (R6), comprising six arginine residues], have been employed as useful tools for the oral delivery of therapeutic macromolecules. The aim of our study was to investigate the combination strategy of using P(MAA-g-EG) hydrogels with R6-based CPPs to improve the intestinal absorption of insulin. A high efficiency of loading into crosslinked P(MAA-g-EG) hydrogels was observed for insulin (96.1±1.4%) and R6 (46.6±3.8%). In addition, immediate release of the loaded insulin and R6 from these hydrogels was observed at pH 7.4 (80% was released in approximately 30 min). Consequently, a strong hypoglycemic response was observed (approximately 18% reduction in blood glucose levels) accompanied by an improvement in insulin absorption after the co-administration of insulin-loaded particles (ILP) and R6-loaded particles (ALP) into closed rat ileal segments compared with that after ILP administration alone. These results indicate that the combination of P(MAA-g-EG) hydrogels with CPPs may be a promising strategy for the oral delivery of various insulin preparations as an alternative to conventional parenteral routes.

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

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.