Ulrik Lytt Rahbek

Intestinal mucosa permeability following oral insulin delivery using core shell corona nanolipoparticles

Chitosan nanoparticles (NC) have excellent capacity for protein entrapment, favorable epithelial permeability, and are regarded as promising nanocarriers for oral protein delivery. Herein, we designed and evaluated a class of core shell corona nanolipoparticles (CSC) to further improve the absorption through enhanced intestinal mucus penetration. CSC contains chitosan nanoparticles as a core component and pluronic F127-lipid vesicles as a shell with hydrophilic chain and polyethylene oxide PEO as a corona. These particles were developed by hydration of a dry pluronic F127-lipid film with NC suspensions followed by extrusion. Insulin nested inside CSC was well protected from enzymatic degradation. Compared with NC, CSC exhibited significantly higher efficiency of mucosal penetration and, consequently, higher cellular internalization of insulin in mucus secreting E12 cells. The cellular level of insulin after CSC treatment was 36-fold higher compared to treatment with free insulin, and 10-fold higher compared to NC. CSC significantly facilitated the permeation of insulin across the ileum epithelia, as demonstrated in an ex vivo study and an in vivo absorption study. CSC pharmacological studies in diabetic rats showed that the hypoglycemic effects of orally administrated CSC were 2.5-fold higher compared to NC. In conclusion, CSC is a promising oral protein delivery system to enhance the stability, intestinal mucosal permeability, and oral absorption of insulin.

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.

Evaluation of alkylmaltosides as intestinal permeation enhancers: comparison between rat intestinal mucosal sheets and Caco-2 monolayers.

Alkylmaltosides are a class of non-ionic surfactant currently in clinical trials to improve nasal permeation of peptide drugs, however few studies have detailed their potential effects on intestinal permeation enhancement. Tetradecyl maltoside (TDM, C(14)), was examined in Caco-2 monolayers and in isolated rat jejunal and colonic mucosae mounted in Ussing chambers. Dodecyl maltoside (DDM, C(12)) was examined in mucosae. Parameters measured included critical micelle concentration (CMC), transepithelial electrical resistance (TEER), and apparent permeability coefficients (P(app)) of paracellular and transcellular flux markers. TDM and DDM decreased TEER and increased the P(app) of [(14)C]-mannitol and FD-4 across Caco-2 monolayers and colonic mucosae in the concentration range of 0.01-0.1% w/v, concentrations much higher than the CMC. Remarkably, neither agent had any effect on the TEER or fluxes of jejunal mucosae. Histopathology, cell death assays (MTT and LDH) and sub-lethal high content cytotoxicity analyses (HCA) were carried out with TDM. Exposure of colonic mucosae to high concentrations of TDM had no major effects on gross histology and ion transport function was retained. In Caco-2, HCA data at sub-lethal concentrations of TDM was consistent with the action of a mild non-ionic surfactant. In conclusion, alkylmaltosides are effective non-toxic permeation enhancers in isolated colonic tissue and their inclusion in oral peptide formulations directed to that intestinal region warrants further study.

The role of citric acid in oral peptide and protein formulations: relationship between calcium chelation and proteolysis inhibition.

The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CAs calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CAs capacity to chelate calcium decreased ~10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10mM CA. The capacity of CA to chelate luminal calcium does not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulins low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral bioavailability since this is a weak property of CA.

Colonic absorption of salmon calcitonin using tetradecyl maltoside (TDM) as a permeation enhancer

Calcitonin is used as a second line treatment of postmenopausal osteoporosis, but widespread acceptance is somewhat limited by subcutaneous and intranasal routes of delivery. This study attempted to enable intestinal sCT absorption in rats using the mild surfactant, tetradecyl maltoside (TDM) as an intestinal permeation enhancer. Human Caco-2 and HT29-MTX-E12 mucus-covered intestinal epithelial monolayers were used for permeation studies. Rat in situ intestinal instillation studies were conducted to evaluate the absorption of sCT with and without 0.1 w/v% TDM in jejunum, ileum and colon. TDM significantly enhanced sCT permeation across intestinal epithelial monolayers, most likely due to combined paracellular and transcellular actions. In situ, TDM caused an increased absolute bioavailability of sCT in rat colon from 1.0% to 4.6%, whereas no enhancement increase was observed in ileal and jejunal instillations. Histological analysis suggested mild perturbation of colonic epithelia in segments instilled with sCT and TDM. These data suggest that the membrane composition of the colon is different to the small intestine and that it is more amenable to permeation enhancement. Thus, formulations designed to release payload in the colon could be advantageous for systemic delivery of poorly permeable molecules.

Chemically modified peptides and proteins - critical considerations for oral delivery.

abstract Numerous approaches have been explored to date in the pursuit of delivering peptides or proteins via the oral route. One such example is chemical modification, whereby the native structure of a peptide or protein is tailored to provide a more efficient uptake across the epithelial barrier of the gastrointestinal tract via incorporation of a chemical motif or moiety. In this regard, a diverse array of concepts have been reported, ranging from the exploitation of endogenous transport mechanisms to incorporation of physicochemical modifications in the molecule, which promote more favorable interactions with the absorptive membrane at the cell surface. This review provides an overview of the modification technologies described in the literature and offers insights into some pragmatic considerations pertaining to their translation into clinically viable concepts.

Acylation of Glucagon-Like Peptide-2: Interaction with Lipid Membranes and In Vitro Intestinal Permeability

Background Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation as well as increasing enzymatic stability without disrupting biological potency. Acylation has furthermore been shown to increase interactions with the lipid membranes of mammalian cells. The extent to which such interactions hinder or benefit delivery of acylated peptide drugs across cellular barriers such as the intestinal epithelia is currently unknown. The present study investigates the effect of acylating peptide drugs from a drug delivery perspective. Purpose We hypothesize that the membrane interaction is an important parameter for intestinal translocation, which may be used to optimize the acylation chain length for intestinal permeation. This work aims to characterize acylated analogues of the intestinotrophic Glucagon-like peptide-2 by systematically increasing acyl chain length, in order to elucidate its influence on membrane interaction and intestinal cell translocation in vitro. Results Peptide self-association and binding to both model lipid and cell membranes was found to increase gradually with acyl chain length, whereas translocation across Caco-2 cells depended non-linearly on chain length. Short and medium acyl chains increased translocation compared to the native peptide, but long chain acylation displayed no improvement in translocation. Co-administration of a paracellular absorption enhancer was found to increase translocation irrespective of acyl chain length, whereas a transcellular enhancer displayed increased synergy with the long chain acylation. Conclusions These results show that membrane interactions play a prominent role during intestinal translocation of an acylated peptide. Acylation benefits permeation for shorter and medium chains due to increased membrane interactions, however, for longer chains insertion in the membrane becomes dominant and hinders translocation, i.e. the peptides get ‘stuck’ in the cell membrane. Applying a transcellular absorption enhancer increases the dynamics of membrane insertion and detachment by fluidizing the membrane, thus facilitating its effects primarily on membrane associated peptides.

Applicability and Limitations of Cell-Penetrating Peptides in Noncovalent Mucosal Drug or Carrier Delivery Systems

Our recent studies show that cell-penetrating peptides (CPPs) have potential to improve the intestinal absorption of peptide and protein drugs safely and effectively when used in the noncovalent drug--CPP approach. To clarify the applicability and limitations of this strategy, the present study examined the effects of cargo size on the absorption-stimulatory effect of CPPs. Different sizes of hydrophilic macromolecular dextran (4.4, 10, and 70 kDa) and polystyrene-based nanoparticles (20, 100, and 200 nm) were chosen as the model cargos in this study. In an in situ rat intestinal absorption study, CPPs (octaarginine, Tat, penetratin, and PenetraMax) increased the intestinal absorption of dextran, and the efficiency varied according to the molecular size of dextran. Among the CPPs, D-penetratin showed an enhancing effect even when coadministered with the largest dextran (70 kDa). By contrast, an in vitro study of Caco-2 cell uptake showed that the ability of CPPs to deliver nanoparticles into epithelial cells was dependent on their particle size and that the relatively poor internalization of 200-nm nanoparticles could be facilitated by coincubation with CPPs. These findings suggest that the intrinsic uptake properties of macromolecules and particulate cargos determine the effectiveness of their intestinal mucosal delivery using the noncovalent CPP method.

Acylation of salmon calcitonin modulates in vitro intestinal peptide flux through membrane permeability enhancement.

Acylation of peptide drugs with fatty acid chains has proven beneficial for prolonging systemic circulation, as well as increasing enzymatic stability and interactions with lipid cell membranes. Thus, acylation offers several potential benefits for oral delivery of therapeutic peptides, and we hypothesize that tailoring the acylation may be used to optimize intestinal translocation. This work aims to characterize acylated analogues of the therapeutic peptide salmon calcitonin (sCT), which lowers blood calcium, by systematically increasing acyl chain length at two positions, in order to elucidate its influence on intestinal cell translocation and membrane interaction. We find that acylation drastically increases in vitro intestinal peptide flux and confers a transient permeability enhancing effect on the cell layer. The analogues permeabilize model lipid membranes, indicating that the effect is due to a solubilization of the cell membrane, similar to transcellular oral permeation enhancers. The effect is dependent on pH, with larger effect at lower pH, and is impacted by acylation chain length and position. Compared to the unacylated peptide backbone, N-terminal acylation with a short chain provides 6- or 9-fold increase in peptide translocation at pH 7.4 and 5.5, respectively. Prolonging the chain length appears to hamper translocation, possibly due to self-association or aggregation, although the long chain acylated analogues remain superior to the unacylated peptide. For K(18)-acylation a short chain provides a moderate improvement, whereas medium and long chain analogues are highly efficient, with a 12-fold increase in permeability compared to the unacylated peptide backbone, on par with currently employed oral permeation enhancers. For K(18)-acylation the medium chain acylation appears to be optimal, as elongating the chain causes greater binding to the cell membrane but similar permeability, and we speculate that increasing the chain length further may decrease the permeability. In conclusion, acylated sCT acts as its own in vitro intestinal permeation enhancer, with reversible effects on Caco-2 cells, indicating that acylation of sCT may represent a promising tool to increase intestinal permeability without adding oral permeation enhancers.

A multi-chamber microfluidic intestinal barrier model using Caco-2 cells for drug transport studies

This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene ‘click chemistry’ aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9–10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices.