Development and validation of a porcine artificial colonic mucus model reflecting the properties of native colonic mucus in pigs.

Colonic mucus plays a key role in colonic drug absorption. Mucus permeation assays could therefore provide useful insights and support rational formulation development in the early stages of drug development. However, the collection of native colonic mucus from animal sources is labor-intensive, does not yield amounts that allow for routine experimentation, and raises ethical concerns. In the present study, we developed an in vitro porcine artificial colonic mucus model based on the characterization of native colonic mucus. The structural properties of the artificial colonic mucus were validated against the native secretion for their ability to capture key diffusion patterns of macromolecules in native mucus. Moreover, the artificial colonic mucus could be stored under common laboratory conditions, without compromising its barrier properties. In conclusion, the porcine artificial colonic mucus model can be considered a biorelevant way to study the diffusion behavior of drug candidates in colonic mucus. It is a cost-efficient screening tool easily incorporated into the early stages of drug development and it contributes to the implementation of the 3Rs (refinement, reduction, and replacement of animals) in the drug development process.

Physiological properties, composition and structural profiling of porcine gastrointestinal mucus.

The gastrointestinal mucus is a hydrogel that lines the luminal side of the gastrointestinal epithelium, offering barrier protection from pathogens and lubrication of the intraluminal contents. These barrier properties likewise affect nutrients and drugs that need to penetrate the mucus to reach the epithelium prior to absorption. In order to assess the potential impact of the mucus on drug absorption, we need information about the nature of the gastrointestinal mucus. Today, most of the relevant available literature is mainly derived from rodent studies. In this work, we used a larger animal species, the pig model, to characterize the mucus throughout the length of the gastrointestinal tract. This is the first report of the physiological properties (physical appearance, pH and water content), composition (protein, lipid and metabolite content) and structural profiling (rheology and gel network) of the porcine gastrointestinal mucus. These findings allow for direct comparisons between the characteristics of mucus from various segments and can be further utilized to improve our understanding of the role of the mucus on region dependent drug absorption. Additionally, the present work is expected to contribute to the assessment of the porcine model as a preclinical species in the drug development process.

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.

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.

In vivo in vitro correlations for a poorly soluble drug, danazol, using the flow-through dissolution method with biorelevant dissolution media.

The purpose of the study was to design dissolution tests that were able to distinguish between the behaviour of danazol under fasted and fed conditions, by using biorelevant media. In vitro dissolution of 100mg danazol capsules was performed using the flow-through dissolution method. Flow rates were 8, 16 or 32 ml/min, corresponding to total volumes dissolution medium of 960, 1920 and 3840 ml, respectively. The media used contained bile salt and phospholipid levels relevant for either fasted or fed conditions in vivo. Crude and inexpensive bile components, Porcine Bile Extract and soybean phospholipids, were used as the bile source. The effect of adding different concentrations and molar ratios of monoglycerides and fatty acids to the fed state media was investigated. In vivo release profiles under fasted and fed conditions were obtained from a previous study by deconvolution [Sunesen, V.H., Vedelsdal, R., Kristensen, H.G., Christrup, L., Müllertz, A. 2005. Effect of liquid volume and food intake on the absolute bioavailability of danazol, a poorly soluble drug, Eur. J. Pharm. Sci. 24, 297-303]. In the fasted state, the physiologically most relevant correlation with in vivo results was achieved with a medium containing 6.3 mM bile salts and 1.25 mM phospholipids (8 ml/min). A medium containing 18.8 mM bile salts, 3.75 mM phospholipids, 4.0 mM monoglycerides and 30 mM fatty acids (8 ml/min) gave the closest correlation with fed state in vivo results. By using the flow-through dissolution method it was possible to obtain correlations with in vivo release of danazol under fasted and fed conditions. Both hydrodynamics and medium composition were important for the dissolution of danazol. In the fed state an IVIVC could only be obtained by including monoglycerides and fatty acids in the medium.