Werner Rubas

Comparison of the permeability characteristics of a human colonic epithelial (Caco-2) cell line to colon of rabbit, monkey, and dog intestine and human drug absorption.

The in vitro permeabilities of Caco-2 monolayers and permeabilities in tissue sections from colon of monkey, rabbit, and dog were compared using a series of compounds. The selected compounds differed in their physicochemical properties, such as octanol/water partition coefficient, water solubility, and molecular weight. Their structure included steroids, carboxylic acids, xanthins, alcohols, and polyethylene glycols. A linear permeability relationship was established between Caco-2 and colon tissue from both rabbit and monkey. The results suggest that Caco-2 is twice as permeable as rabbit and five times as permeable as monkey colon. However, no clear relationship could be established between Caco-2 monolayers and dog colon permeability. A relationship between permeability in Caco-2 monolayers and human absorption was found. The results suggest that within certain limits, permeability of Caco-2 monolayers may be used as a predictive tool to estimate human drug absorption.

In Vitro Evaluation of Dexamethasone-β-D-Glucuronide for Colon-Specific Drug Delivery

Dexamethasone-β-D-glucuronide is a potential prodrug for colonic delivery of the antiinflammatory corticosteroid dexamethasone. Previous studies [T. R. Tozer et al., Pharm. Res. 8:445–454 (1991)] indicated that a glucoside prodrug of dexamethasone was susceptible to hydrolysis in the upper gastrointestinal tract. Resistance of dexamethasone-β-D-glucuronide to hydrolysis in the upper gastrointestinal tract was therefore assessed. Conventional, germfree, and colitic rats were used to examine enzyme levels along the gastrointestinal tract to compare the stability of two model substrates (p-nitrophenyl-β-D-glucoside and -β-D-glucuronide) and to evaluate the prodrug dexamethasone-β-D-glucuronide. Hydrolytic activity was examined in the luminal contents, mucosa, and underlying muscle/connective tissues in all three types of rats. Enzymatic activity (β-D-glucosidase and β-D-glucuronidase) was greatest in the lumen of cecum and colon of conventional rats. In contrast, germ-free rats exhibited relatively high levels of β-D-glucosidase activity (about 80% of total activity in the conventional rats) in the proximal small intestine (PSI) and the distal small intestine (DSI). Rats with induced colitis (acetic acid) showed reduced levels of luminal β-D-glucuronidase activity in the large intestine; however, β-D-glucosidase activity was relatively unchanged relative to that of the conventional rat. Mucosal β-D-glucuronidase activity was significantly lower in the colitic rats compared with that in the conventional animals. Despite reduced luminal levels of β-D-glucuronidase activity in the colitic rats, there was still a sharp gradient of activity between the small and the large intestines. Permeability of the glucoside and glucuronide prodrugs of dexamethasone through a monolayer of Caco-2 cells was relatively low compared to that of dexamethasone. The results indicate that dexamethasone-β-D-glucuronide should be relatively stable and poorly absorbed in the upper gastrointestinal tract. Once the compound reaches the large intestine, it should be hydrolyzed to dexamethasone and glucuronic acid. Specificity of colonic delivery in humans should be even greater due to lower levels of β-D-glucuronidase activity in the small intestine compared with that in the laboratory rat.

Validation of Excised Bovine Nasal Mucosa as In Vitro Model to Study Drug Transport and Metabolic Pathways in Nasal Epithelium

The present work aims at the validation of excised bovine nasal mucosa as an in vitro model to address transport and metabolism pathways relative to the nasal mucosal uptake of therapeutic peptides. Preservation of the viability of the excised tissue in the course of in vitro studies of up to 3 h was demonstrated by (i) positive viability staining, (ii) constant transepithelial electrical resistance (42 +/- 12 Omega cm(2)), (iii) constant rates of metabolic turnover, and (iv) linear permeation profiles of therapeutic peptides and (3)H-mannitol. Using 1-leucine-4-methoxy-2-naphthylamide as a model substrate, we observed no difference between bovine and human nasal aminopeptidase activity. By a series of therapeutic peptides, no direct correlation was found between their effective permeability coefficients (from 0. 1 x 10(-5) to 5 x 10(-5) cm s(-1)) and their respective molecular masses (from 417 to 3,432 Da), indicating that other factors dominate nasal permeability. For instance, the permeabilities of metabolically labile peptides were concentration dependent and saturable, as demonstrated for two short thymopoietin fragments, Arg-Lys-Asp (TP3) and Arg-Lys-Asp-Val (TP4). By permeation studies using gonadorelin and two gonadorelin derivatives, buserelin and Hoe 013, without and in the presence of the chemical enhancer bacitracin, we also verified the ability of the model to assess chemical enhancer effects and their reversibility. In conclusion, our work demonstrates the potential of the investigated in vitro model, excised bovine nasal mucosa, to explore mechanistic aspects of nasal transport and metabolism of therapeutic peptides.

Permeability Characteristics of Various Intestinal Regions of Rabbit, Dog, and Monkey

The in vitro permeability of a series of both hydrophilic and lipophilic compounds, as defined by the octanol/water partition coefficient, was measured in four segments of rabbit, monkey, and dog intestine using a side-by-side diffusion cell. A linear relationship was established for tissue resistance to hydrophilic compound diffusion in jejunum and colon among rabbit, monkey, and dog. The results suggest that rabbit jejunum is twice as permeable as monkey and dog jejunum. The colonic tissues of monkey, rabbit, and dog demonstrate similar permeabilities. Measuring the permeabilities of different tissues with compounds of similar physicochemical properties allows comparison of tissue restriction to transport. Thus, in vitro permeability measurements may be used to investigate physiological differences of various intestinal tissue segments that influence tissue permeability. Investigating the permeability of different intestinal segments from various species could allow the identification of an appropriate in vitro intestinal permeability model that will lead to the prediction of intestinal absorption in humans, eliminating the need for extensive and often misleading in vivo animal testing.

Research ArticlesValidation of Excised Bovine Nasal Mucosa as In Vitro Model to Study Drug Transport and Metabolic Pathways in Nasal Epithelium

The present work aims at the validation of excised bovine nasal mucosa as an in vitro model to address transport and metabolism pathways relative to the nasal mucosal uptake of therapeutic peptides. Preservation of the viability of the excised tissue in the course of in vitro studies of up to 3 h was demonstrated by (i) positive viability staining, (ii) constant transepithelial electrical resistance (42 +/- 12 Omega cm(2)), (iii) constant rates of metabolic turnover, and (iv) linear permeation profiles of therapeutic peptides and (3)H-mannitol. Using 1-leucine-4-methoxy-2-naphthylamide as a model substrate, we observed no difference between bovine and human nasal aminopeptidase activity. By a series of therapeutic peptides, no direct correlation was found between their effective permeability coefficients (from 0. 1 x 10(-5) to 5 x 10(-5) cm s(-1)) and their respective molecular masses (from 417 to 3,432 Da), indicating that other factors dominate nasal permeability. For instance, the permeabilities of metabolically labile peptides were concentration dependent and saturable, as demonstrated for two short thymopoietin fragments, Arg-Lys-Asp (TP3) and Arg-Lys-Asp-Val (TP4). By permeation studies using gonadorelin and two gonadorelin derivatives, buserelin and Hoe 013, without and in the presence of the chemical enhancer bacitracin, we also verified the ability of the model to assess chemical enhancer effects and their reversibility. In conclusion, our work demonstrates the potential of the investigated in vitro model, excised bovine nasal mucosa, to explore mechanistic aspects of nasal transport and metabolism of therapeutic peptides.

Gastrointestinal lymphatic absorption of peptides and proteins

There is no doubt that intact peptides and proteins do cross the gastrointestinal wall into the lymphatics. Transfer from the lumen into the lymph system occurs in both lymphoid (PP) and non-lymphoid tissue (villous). Contribution by the paracellular pathway may be low. Transfer into lymph vessels via non-lymphoid tissue depends upon the lipid pathway, vehicle effects, sieving mechanisms of the blood vessels, and the application site. The best lymphatic access has been achieved from the proximal small intestine, while rectal application has also been proven to be suitable. Utilizing formulations composed of a long chain and unsaturated fatty acid in combination with a surfactant favors transfer into lymph. The most promising results were achieved with combinations resembling chylomicrons, attempting to direct the compound into chylomicrons. For smaller substances such as peptides, the physicochemical characteristics are one of the key factors for lymphatic uptake. Substances which are highly lipophilic favor lymphatic passage. Assessment of solubility in peanut oil and/or in the viscous isotropic phase of the digested lipids is a useful tool to predict the lymph absorption potential. In order to utilize the sieving mechanism, conversion of a substance into a drug-polymer complex such as dextran or cyclodextran together with co-application of an absorption promoter (bifunctional system) has been shown to be feasible and suitable for lymphatic delivery. Endocytotic processes if present at all play a minor role in non-lymphoid tissue uptake. The most prominent uptake mechanism for particles and microspheres in lymphoid tissue is phagocytosis. The extent depends on surface property, the amount administered, and the suspension vehicle. Hydrophobic surfaces and aqueous suspending vehicles appear best. Transcytosis through PP, also called the M-cell route, seems to be most suited for highly potent compounds such as lymphokines and antigens (vaccines). The reasons are: (a) limited number of PP, thus, the overall surface area is relatively small, and therefore the total absorption potential is limited, and (b) PP tissue is rich in lymphocytes, thus, substances which interact with lymphocytes are best targeted to PP when using the oral route. Oral delivery to local lymph nodes by means of carrier systems (i.e. poly(lactide-co-glycolide) microspheres) via the M-cell route appears very promising. Migration, however, into and through the mesenteric lymph appears limited to microspheres less than 5 μm in diameter. Though both cell types, M cells and enterocytes, share the same common glycoproteins and glycolipids a number of microorganisms are able to bind selectively to a receptor on the M-cell surface and thereby enter the host. Utilizing the microorganisms ligand could be beneficial for specific targeting to PP, bypassing lysosomal degradation in absorptive cells. Moreover, transport of a membrane-bound macromolecule by M cells is about 50 times more efficient than a soluble, non-adherent macromolecule.

Transport of biologically active interferon-gamma across human skin in vitro.

AbstractPurpose. Several studies have suggested epidermal uptake of cytokines, such as interferons, can be facilitated using topical liposomal formulations. We have evaluated the in vitro transport of biologically active recombinant human interferon-γ (rhIFN-γ) into and through split-thickness human skin to assess this possibility. Methods. Skin samples were exposed to rhIFN-γ under various conditions involving hydrated and dry surface conditions in the presence and absence of liposomes. A new low-level ELISA and an anti-viral bioassay were used to quantitate transported rhIFN-γ. Immunohistochemical staining for ICAM-1 expression by keratinocytes was used to visualize the extent and distribution of rhIFN-γ transport. Results. Apparent steady-state transport of rhIFN-γ occurred within the first 5 hours of exposure with approximately 10% of transported rhIFN-γ demonstrating bioactivity. While the permeability of rhIFN-γ across human skin under drying conditions was enhanced by the presence of liposomes, no augmentation of permeability was observed when the skin was kept hydrated. Liposomal formulations of rhIFN-;γ had greater transport rates than aqueous formulations when the applied formulations were allowed to dry after dosing. Conclusions. Our results demonstrate the transport of biologically active rhIFN-γ across human skin in vitro and suggest a role for stratum corneum hydration as one possibility for the augmented cytokine transport.

The effect of chemical modifications on octanol/water partition (log D) and permeabilities across Caco-2 monolayers

Abstract In the past octanol/water partition coefficients (log D 7.4 ) have been used extensively to predict intestinal absorption. More recently flux measurements across confluent monolayers of Caco-2 cells, a human colonic carcinoma cell line, have been introduced to estimate oral absorption. Recently we reported that dexamethasone-β- d -glucuronide (sodium salt) had a permeability coefficient that was one order of magnitude higher than dexamethasone-β- d -glucoside despite a two orders of magnitude lower log D 7.4 , i.e., the compound displaying the free acid moiety had a higher permeability than the uncharged dexamethasone-β- d -glucoside [1]. This finding is inconsistent with the dogma that a higher log D 7.4 should translate into a higher permeability coefficient. Therefore, the objective of the present study was to examine whether structures unrelated to steroids containing free acid moieties and their respective ethyl esters (uncharged) demonstrate similar characteristics. Using reversed-phase HPLC we have determined the octanol/water partition coefficient (log D 7.4 ) and permeability coefficients across Caco-2 monolayers of a limited series of bioactive compounds based on a benzodiazepine-2,5-dione scaffold, displaying a free acid moiety and the respective ethyl ester. Our results indicate: i . ester prodrugs have approximately two orders of magnitude higher octanol/water partition coefficient (log D 7.4 ) than the parents (free acids) and ii . the permeability coefficients of the prodrugs were lower than for the parents. We conclude log D 7.4 may not be a good predictor of the permeability coefficient and oral absorption.

Structural Elements Which Govern the Resistance of Intestinal Tissues to Compound Transport

The series of cyclized RGD peptides in this study demonstrated a very low partition into octanol as judged by HPLC. Thus, these molecules are likely to move predominantly through the paracellular pathway. Permeability across Caco-2 monolayers was determined using reversed phase HPLC and found to be restricted by molecular weight and possibly charge-charge interactions between the solute and charged moieties within the paracellular shunt. When normalized for molecular weight, molecules with a net charge between -1 and -2 demonstrated the highest permeabilities, which suggests an optimal net charge with respect to permeability.

Mechanism of Dextran Transport Across Rabbit Intesi Tissue and a Human Colon Cell-Line (CACO-2)

The in vitro permeabilities of 14C labeled dextrans (10, 40, and 70 kD) were calculated from mass transport across Peyers patches and non-patch tissues derived from rabbit jejunum, and a human colon cell line (Caco-2) grown as a monolayer on polycarbonate filters. Size distribution of dextrans did not change upon transport as judged from size exclusion chromatography. Permeabilities decreased in a size-dependent manner. Ranking of permeabilities for dextran 10 and 40 kD were: Caco-2 > non-patch tissue > Peyers patches; while dextran 70 kD demonstrated no difference among the barriers. Tissue resistance, expressed as 1/(permeability.tissue thickness) was virtually the same in Peyers patches and non-patch tissue, suggesting that tissue thickness and not interaction determines the difference in permeability. ATP depletion with ouabain, Na(+)-azide and 2-deoxy-D-glucose, and low temperature (4 degrees C) did not result in reduced permeabilities suggesting passive transport. The results suggest that the investigated intestinal barriers transport dextrans in a similar fashion independent of their source. However, comparison of the ratios dextran 10 kD/mannitol and PEG 900/mannitol between rabbit tissue and Caco-2 monolayers suggests Caco-2 monolayers may serve as a model to study absorption potential of potentially harmful compounds in coeliac disease, gastroenteritis, and colon carcinoma.