Sam Maher

Safety and efficacy of sodium caprate in promoting oral drug absorption: from in vitro to the clinic.

A major challenge in oral drug delivery is the development of novel dosage forms to promote absorption of poorly permeable drugs across the intestinal epithelium. To date, no absorption promoter has been approved in a formulation specifically designed for oral delivery of Class III molecules. Promoters that are designated safe for human consumption have been licensed for use in a recently approved buccal insulin spray delivery system and also for many years as part of an ampicillin rectal suppository. Unlike buccal and rectal delivery, oral formulations containing absorption promoters have the additional technical hurdle whereby the promoter and payload must be co-released in high concentrations at the small intestinal epithelium in order to generate significant but rapidly reversible increases in permeability. An advanced promoter in the clinic is the medium chain fatty acid (MCFA), sodium caprate (C(10)), a compound already approved as a food additive. We discuss how it has evolved to a matrix tablet format suitable for administration to humans under the headings of mechanism of action at the cellular and tissue level as well as in vitro and in vivo efficacy and safety studies. In specific clinical examples, we review how C(10)-based formulations are being tested for oral delivery of bisphosphonates using Gastro Intestinal Permeation Enhancement Technology, GIPET (Merrion Pharmaceuticals, Ireland) and in a related solid dose format for antisense oligonucleotides (ISIS Pharmaceuticals, USA).

Intestinal permeation enhancers for oral peptide delivery

Intestinal permeation enhancers (PEs) are one of the most widely tested strategies to improve oral delivery of therapeutic peptides. This article assesses the intestinal permeation enhancement action of over 250 PEs that have been tested in intestinal delivery models. In depth analysis of pre-clinical data is presented for PEs as components of proprietary delivery systems that have progressed to clinical trials. Given the importance of co-presentation of sufficiently high concentrations of PE and peptide at the small intestinal epithelium, there is an emphasis on studies where PEs have been formulated with poorly permeable molecules in solid dosage forms and lipoidal dispersions.

Overcoming poor permeability: translating permeation enhancers for oral peptide delivery

Demand for oral alternatives to parenteral delivery has led to renewed interest in excipient-like intestinal permeation enhancers that improve oral drug bioavailability. Oral delivery of macromolecules including peptides and proteins is limited by pre-systemic degradation and poor penetration across the gut wall. Research on oral absorption enhancers that increase gut permeability was first undertaken 50 years ago, yet clinical success has yet to be achieved. Development has been hampered by lack of adequate reproducible efficacy as well as perceived safety concerns. We review some selected permeation-enhancing excipients that are key components of peptide formulations in advanced clinical development and assess why translation of such technologies is close to fruition.

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.

Evaluation of intestinal absorption enhancement and local mucosal toxicity of two promoters. I. Studies in isolated rat and human colonic mucosae

The effects of two absorption promoters, (sodium caprate (C(10)) and melittin), on intestinal permeability and viability were measured in intact rat and human colonic epithelia mounted in Ussing chambers. Apical-side addition of C(10) (10 mM) and melittin (10-50 microM) rapidly reduced the transepithelial electrical resistance (TEER) and increased the apparent permeability coefficient (Papp) of [(14)C]-mannitol and FITC-dextran-4 kDa (FD4) across colonic mucosae from both species. Effects of C(10) on flux were greater than those of melittin at the concentrations selected. C(10) irreversibly decreased TEER, but the effects of melittin were partially reversible. Enhanced permeability of polar sugars (0.18-70 kDa) in colonic mucosae with C(10) was accompanied by significant release of lactate dehydrogenase (LDH) from the luminal surface as well as by inhibition of electrogenic chloride secretion induced by the muscarinic agonist, carbachol (0.1-10 microM). Although melittin did not alter electrogenic chloride secretion in rat or human colonic mucosae, it caused leakage of LDH from rat tissue. Gross histology and electron microscopy of rat and human colonic mucosae demonstrated that each permeation enhancer can induce colonic epithelial damage at concentrations required to increase marker fluxes. C(10) led to more significant mucosal damage than melittin, characterised by sloughing and mucosal erosion. Overall, these results indicate that while C(10) and melittin increase transport of paracellular flux markers across isolated human and rat colonic mucosae in vitro, these effects are associated with some cytotoxicity.

Formulation strategies to improve oral peptide delivery

Delivery of peptides by the oral route greatly appeals due to commercial, patient convenience and scientific arguments. While there are over 60 injectable peptides marketed worldwide, and many more in development, most delivery strategies do not yet adequately overcome the barriers to oral delivery. Peptides are sensitive to chemical and enzymatic degradation in the intestine, and are poorly permeable across the intestinal epithelium due to sub-optimal physicochemical properties. A successful oral peptide delivery technology should protect potent peptides from presystemic degradation and improve epithelial permeation to achieve a target oral bioavailability with acceptable intra-subject variability. This review provides a comprehensive up-to-date overview of the current status of oral peptide delivery with an emphasis on patented formulations that are yielding promising clinical data.

Melittin as a Permeability Enhancer II: In Vitro Investigations in Human Mucus Secreting Intestinal Monolayers and Rat Colonic Mucosae

PurposeMelittin has shown potential as a non-cytotoxic absorption enhancer in Caco-2 monolayers. Our objectives were to assess in vitro efficacy and cytotoxicity of melittin in two intestinal permeability models and investigate the potential mechanism by which melittin might enhance gastrointestinal absorption.Materials and methodsThe effects of melittin were examined in the mucus-secreting intestinal cell monolayers, HT29-MTX-E12 (E12), using transepithelial electrical resistance (TER), transmission electron microscopy (TEM) and the MTT viability assay. The effects of melittin on TER, permeability and short circuit current (Isc) were also investigated in rat colon mucosae mounted in Ussing chambers. Ion transporting capacity of tissue was measured in response to secretagogues as surrogate markers of cytotoxicity. Melittin stability was examined by a means of a hemolytic assay. The mechanism by which melittin decreases TER across the rat mucosa was examined with a range of enzymatic inhibitors.ResultsApical addition of melittin resulted in a reversible non-cytotoxic concentration-dependent decrease in TER across E12 monolayers, which was independent of the presence of mucus. Apical addition of melittin reduced TER and increased the permeability of [14C]-mannitol across rat colonic mucosae. The melittin-induced drop in TER in rat colon was significantly attenuated by W7 suggesting partial mediation by calmodulin.ConclusionsThe rapid and reversible nature of melittin’s permeation enhancing properties and its limited cytotoxicity in polarized intestinal epithelia, suggests a potential drug delivery role for the peptide in oral formulations of poorly absorbed drugs.

Evaluation of intestinal absorption and mucosal toxicity using two promoters. II. Rat instillation and perfusion studies.

We compared the effectiveness of two absorption promoters, sodium caprate (C(10)) and melittin, in increasing the bioavailability (F) of poorly absorbed paracellular flux markers across the intestinal mucosae of rats in situ, together with examination of their effects on morphology. C(10) (100 mM) and melittin (50 microM) significantly increased absorption of FITC-dextran-4 kDa (FD4) following jejunal and colonic instillations. F of FD4 following jejunal instillations with C(10) was increased from 0.07% to 2.3%, while it was increased from 0.07% to 0.53% in the presence of melittin. F of FD4 following colonic instillations with C(10) was increased from 1% to 33% while melittin increased it from 1% to 7%. F of FD70 was unchanged in colonic instillations in the presence of either of the two agents, indicating size limitations of the permeability enhancement effects. In rat jejunal perfusions, C(10) (50 mM) and melittin (50 microM) significantly increased [(14)C]-mannitol permeability by 9- and 1.9-fold respectively. C(10) was more effective than melittin in increasing fluxes in all models. Histology of intestinal sections exposed to either promoter showed mild mucosal damage at those concentrations effective at promoting absorption. Electron microscopy revealed epithelial cell damage induced by both enhancers accompanied by truncation of microvilli, and sloughing. Overall, both melittin and C(10) improved bioavailability of polar sugars across the jejunum and colon of rats in situ, which was associated with some degree of mucosal damage.

Chitooligosaccharide elicits acute inflammatory cytokine response through AP-1 pathway in human intestinal epithelial-like (Caco-2) cells.

Chitooligosaccharides (COSs) are bioactive carbohydrate derivatives that have numerous health benefits, including stimulation of the immune system. The objectives of this study were to evaluate the effect of chitooligosaccharide (COS) on expression of a specific panel of cytokine genes involved in inflammation and to delineate the signal transduction pathway underlying the COS mediated inflammatory response. Human intestinal epithelial-like (Caco-2) cells were treated with COS (5000-10,000Da) and expression of a panel of eighty-four cytokine genes was analyzed by quantitative real-time PCR. COS induced up-regulation of a total of 11 genes including CCL20 and IL8 and concurrent down-regulation of 10 genes including pro-inflammatory mediators CCL15, CCL25 and IL1B. To further establish the signal transduction pathway of COS mediated response in Caco-2 cells, two major inflammatory signal transduction pathways (NF-κB and AP-1) were investigated. COS had inhibitory effect (P<0.01) on TNF-α induced NF-κB binding activity while stimulatory effect (P<0.001) on AP-1 binding activity. COS also inhibited the expression of RELA (P<0.01) and IKBKB (P<0.01) genes of NF-κB pathway while stimulate the expression of JUN (P<0.05) gene of AP-1 pathway. In conclusion, COS elicits an acute inflammatory cytokine response in Caco-2 cells and hence it has the potential to stimulate the immune system in the gut epithelium.

The mycotoxin patulin increases colonic epithelial permeability in vitro.

The gastrointestinal lumen is directly exposed to dietary contaminants, including patulin, a mycotoxin produced by moulds. Patulin is known to increase permeability across intestinal Caco-2 monolayers. This study aimed to determine the effect of patulin on permeability, ion transport and morphology in isolated rat colonic mucosae. Mucosal sheets were mounted in Ussing chambers and voltage clamped. Apical addition of patulin (100-500 μM) rapidly reduced transepithelial electrical resistance (TEER) and increased permeability to [(14)C] mannitol (2.9-fold). Patulin also inhibited carbachol-induced electrogenic chloride secretion and histological evidence of mucosal damage was observed. To examine potential mechanisms of action of patulin on colonic epithelial cells, high-content analysis of Caco-2 cells was performed and this novel, quantitative fluorescence-based approach confirmed its cytotoxic effects. With regard to time course, the cytotoxicity determined by high content analysis took longer than the almost immediate reduction of electrical resistance in isolated mucosal sheets. These data indicate patulin is not only cytotoxic to enterocytes but also has the capacity to directly alter permeability and ion transport in intact intestinal mucosae. These data corroborate and extend findings in intestinal cell culture monolayers, and further suggest that safety limits on consumption of patulin may be warranted.