Pharmacokinetics of recombinant hirudin in healthy horses.

REASONS FOR PERFORMING STUDY: Recombinant (r)-hirudin is a specific inhibitor of thrombin that is independent of the activity of antithrombin. OBJECTIVES: To evaluate pharmacokinetic properties and coagulatory changes of r-hirudin in healthy horses. METHODS: Two clinically healthy horses received a single i.v. bolus of 0.4 mg/kg bwt r-hirudin and 6 clinically healthy horses received the same dose subcutaneously (subcut.) q. 12 h for 3 days. Coagulation times and r-hirudin plasma concentration were determined over 720 mins and 3 days after i.v. and subcut. administration, respectively. RESULTS: In all horses, treatment with r-hirudin was not associated with systemic or local side effects. After i.v. injection, the 2 horses showed an elimination half-life of 58 and 80 mins, respectively. After subcut. administration, maximum plasma concentration of r-hirudin occurred at 128 +/- 55 mins and declined with a terminal half-life of 561 +/- 364 mins. Maximum response of activated partial thromboplastin time (aPTT) occurred 1.5 h after administration of r-hirudin. A prolongation of 1.9 +/- 0.2 times the pretreatment value was noted. CONCLUSIONS: Pharmacokinetics of r-hirudin in healthy horses were similar to those in man and other animal species. POTENTIAL RELEVANCE: The results of this study indicate that r-hirudin can be used in horses, but further studies should be performed in order to prove its effectiveness in diseased horses.

No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides.

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism. We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb(3+)-containing liposomes. Upon chelation of Tb(3+) by an aromatic carboxylic acid, the fluorescence of Tb(3+) increases many fold. The CPP TAT(44-57)-SAM and TAT(37-53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol. In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44-57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44-57). Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44-57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively.

Dynamics of tight and adherens junctions under EGTA treatment.

The dynamics of tight junctions (TJs) and adherens junctions (AJs) under EGTA treatment were investigated in Madin Darby canine kidney (MDCK) cells. Detailed information about the behavior of TJ and AJ proteins during the opening and resealing of TJs and AJs is still scarce. By means of the "calcium chelation" method, the distribution and colocalization of junctional proteins were studied with confocal laser scanning microscopy using a deconvolution algorithm for high-resolution images. Colocalization was analyzed for pairs of the following proteins: ZO-1, occludin, claudin-1, E-cadherin and F-actin. Significant differences were found for the analyzed pairs in control cells compared to EGTA-treated cells with respect to the position of the colocalization maxima within the cell monolayers as well as with respect to the amount of colocalized voxels. Under EGTA treatment, colocalization for ZO-1/occludin, ZO-1/claudin-1, claudin-1/occludin, E-cadherin/occludin and E-cadherin/claudin-1 dropped below 35% of the control value. Only for the ZO-1/E-cadherin pair, the amount of colocalized voxels increased and a shift to a more basal position was observed. During the opening of TJs and AJs, ZO-1 colocalized with E-cadherin in the lateral membrane region, whereas in controls, ZO-1 colocalized with occludin and claudin-1 in the junctional complex. The combination of deconvolution with colocalization analysis of confocal data sets offers a powerful tool to investigate the spatial relationship of TJ and AJ proteins during assembly and disassembly of cell-cell contacts.

Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features.

ECV304 cells reported as originating from human umbilical vein endothelial cells by spontaneous transformation have been used as a model cell line for endothelia over the last decade. Recently, deoxyribonucleic acid fingerprinting revealed an identical genotype for ECV304 and T24 cells (urinary bladder carcinoma cell line). In order to resolve the apparent discrepancy between the identical genotype and the fact that ECV304 cells phenotypically show important endothelial characteristics, a comparative study was performed. Immortalized porcine brain microvascular endothelial cells/C1-2, and Madin Darby canine kidney cells were included as typical endothelial and epithelial cells, respectively. Various methods, such as confocal laser scanning microscopy. Western blot, and protein activity tests, were used to study the cell lines. ECV304 and T24 cells differ in criteria, such as growth behavior, cytoarchitecture, tight junction arrangement. transmembrane electrical resistance, and activity of gamma-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells. Desmoglein and cytokeratin, both known as epithelial markers, were found in ECV304 as well as in T24 tells. However, differences were found for the two cell lines with respect to the type of cytokeratin: in ECV304 cells mainly cytokeratin 18 (45 kDa) is found, whereas in T24 cells cytokeratin 8 (52 kDa) is predominant. As we could demonstrate, the ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in vitro model for endothelia.

Evaluation of particle uptake in human blood monocyte-derived cells in vitro. Does phagocytosis activity of dendritic cells measure up with macrophages?

This work focuses on microparticles as potential antigen delivery systems to target professional antigen-presenting cells. Surface modified polystyrene microparticles were administered to human-derived macrophages (MPhis) and dendritic cells (DCs) in vitro to evaluate the phagocytosis activity of each cell type. To discriminate between internalised particles and those closely attached to the outside of the cells, particle internalisation was verified by confocal laser scanning microscopy. Especially positively charged particles tend to stick to the outer cell membrane and may lead to false positive results when measured by conventional microscopy. In contrast, fluorescence microscopy in combination with an extracellular fluorescence quenching agent (trypan blue) allows the unequivocal assessment of particle uptake for screening purposes. For this assay, the fluorescent label needs to be in direct contact to the quenching agent and cannot be localised inside the particle core. Different types of microparticles varying in size, surface-material and zeta potential resulted in vast differences regarding their uptake by MPhis and DCs as well as the maturation of DCs. Negatively-charged carboxylated and bovine serum albumin-coated particles were phagocytosed by MPhis to a relatively small extent. Interestingly, phagocytosis of these particles was still significantly lower in DCs while positively charged poly-L-lysine (PLL) coated particles induced high phagocytosis activity in both cell types. By comparing our results with literature data, we conclude that phagocytosis activity of DCs and MPhis largely depends on particle size and surface charge and is also influenced by the character of bulk and coating material. PLL can be directed to DCs and MPhis with comparable efficiency and, in addition, induce maturation of DCs.

Physicochemical properties in pharmacokinetic lead optimization.

The ADME (absorption, distribution in the body, metabolism and elimination from the body) profile of a drug determines its pharmacokinetics in the body. Modern drug design includes the modeling of pharmacokinetically favorable behavior. The pharmacokinetic parameters of most interest concern intestinal absorption, blood-brain barrier (BBB) passage and metabolism. Traditionally, experimental parameters such as partition coefficients and chromatographic capacity factors have been used for the estimation of intestinal absorption or BBB passage of newly synthesized compounds. Several studies have shown a sigmoidal relationship between intestinal absorption and lipophilicity. The latter is usually expressed by the apparent partition coefficient log D in a biphasic system at physiological pH or by the affinity to a lipophilic phase determined by chromatographic techniques. In contrast, structure-based descriptors need no experimental investigation of the compound studied. The most relevant descriptors give information on hydrogen-bonding characteristics and molecular volume. In recent years, attempts have been made to recognize substrates for multidrug resistance proteins by their structure characteristics without crucial success. There is evidence that multidrug resistance is not only driven by direct protein-substrate recognition, but also by the behavior of the compound in the lipid environment of the protein.

Differences in the intracellular distribution of acid-sensitive doxorubicin-protein conjugates in comparison to free and liposomal formulated doxorubicin as shown by confocal microscopy.

PURPOSE: To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX) METHODS: LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. RESULTS: The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus. CONCLUSIONS: Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.

Novel peptide conjugates for tumor-specific chemotherapy.

One of the major problems in cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their unselectivity for tumor versus normal cells. In the present work, we show that coupling of anthracyclines to peptides is a promising approach to obtain selectivity. The peptide-drug conjugate was designed to bind to specific receptors expressed on the tumor cells with subsequent internalization of the ligand-receptor complex. Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family, was chosen as model peptide because NPY receptors are overexpressed in a number of neuroblastoma tumors and the thereof derived cell lines. Daunorubicin and doxorubicin, two widely used antineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers that differ in stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicin and a stable amide bond at the 3'-amino position of daunorubicin and doxorubicin. Receptor binding of these three conjugates ([C(15)]-NPY-Dauno-HYD, [C(15)]-NPY-Dauno-MBS, and [C(15)]-NPY-Doxo-MBS) was determined at the human neuroblastoma cell line SK-N-MC, which selectively expresses the NPY Y(1) receptor subtype, and cytotoxic activity was evaluated using a XTT-based colorimetric cellular cytotoxicity assay. The different conjugates were able to bind to the receptor with affinities ranging from 25 to 51 nM, but only the compound containing the acid-sensitive bond ([C(15)]-NPY-Dauno-HYD) showed cytotoxic activity comparable to the free daunorubicin. This cytotoxicity is Y(1) receptor-mediated as shown in blocking studies with BIBP 3226, because tumor cells that do not express NPY receptors were sensitive to free daunorubicin, but not to the peptide-drug conjugate. The intracellular distribution was investigated by confocal laser scanning microscopy. We found evidence that the active conjugate [C(15)]-NPY-Dauno-HYD releases daunorubicin, which is localized close to the nucleus, whereas the inactive conjugate [C(15)]-NPY-Dauno-MBS is distributed distantly from the nucleus and does not seem to release the drug within the cell.

Induction of cell cycle-dependent cytotoxicity and apoptosis by new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine in PC-3 human prostate cancer cells.

Fluorodeoxyuridine (5-FdUrd) is an antineoplastic agent with clinical activity against different types of solid tumours. To enhance the effectiveness of this drug, we have synthesised new heterodinucleoside phosphate dimers of 5-FdUrd. These dimers were compared to 5-FdUrd for their cytotoxic effect and the cell cycle dependence of cytotoxicity, as well as for their capacity to induce apoptosis and inhibit thymidylate synthetase (TS) in androgen-independent human PC-3 prostate tumour cells. Incubation of the cells with the dimers N(4)-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuri din e (dCpam-5-FdUrd) and 2'-deoxy-5-flourouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N(4)-octa decylc ytidine (5-FdUrd-5-FdC18) resulted in a marked cytotoxicity with IC(50) values of 4 microM, similar to 5-FdUrd. In contrast to 5-FdUrd, 100% toxicity was achieved with concentrations of 100-200 microM 5-FdUrd-5-FdC18. Flow cytometric analysis revealed an increase in the cell population in S-phase after treatment with 5-FdUrd, 5-FdUrd-5-FdC18, and dCpam-5-FdUrd from 36 to 63%, 50%, and 77%, respectively. dCpam-5-FdUrd was more potent than 5-FdUrd in arresting the cell cycle. Significant S-phase arrest was indicated by a decreased proportion of cells in G1- and G2/M-phases. Cell cycle arrest and inhibition of cell proliferation were followed by apoptosis, as shown by a 6- to 8-fold increased binding of Apo2.7 antibody, a 9- to 11-fold increase in caspase-3 activity, DNA fragmentation, and by cell morphology showing the appearance of apoptotic bodies. Importantly, 5-FdUrd-5-FdC18 increased the number of apoptotic cells to 160% compared to 5-FdUrd under the same conditions. As with 5-FdUrd, the two dimers also inhibited TS in a time- and concentration-dependent manner, although requiring 100-fold higher concentrations. In conclusion, dCpam-5-FdUrd and 5-FdUrd-5-FdC18 exert stronger cytotoxicity and induce more S-phase arrest and apoptosis than does 5-FdUrd in PC-3 cells, suggesting their potential role in the treatment of human prostate cancer.

P-Glycoprotein in cell cultures: a combined approach to study expression, localisation, and functionality in the confocal microscope.

Madin Darby canine kidney (MDCK) cells transfected with the multidrug resistance mdr1 gene, MDR1-MDCK (Pastan et al., 1988, Proc. Natl. Acad. Sci. USA 85 4486-4470), were used in a combined approach to study expression, localisation and functionality of the P-glycoprotein (P-gp) membrane transporter in the same cell culture preparations. Cells were characterised with regard to their growth curve, transepithelial electrical resistance (TEER), and cytoarchitecture. Efflux of the P-gp substrate rhodamine123 (rho123) was monitored with confocal laser scanning microscopy (CLSM). The transfected cells grew in multilayers. After reaching confluence they exhibited a complete tight junction (TJ) network. P-gp was strongly expressed at the uppermost apical surface of the multilayer already after 4 days in culture. The lower cell layers were not clearly polarised. P-gp-mediated transport could be followed by efflux of the fluorescent rho123 from the cells into the apical extracellular space. Verapamil, a P-gp inhibitor, significantly decreased efflux. For MDCK parent cells the rho123 assay was negative up to about day 20, and only at later times (day 25) low P-gp activity was detected. These results clearly show that despite the fact that the transfected cells form irregular layers, they provide a good model for screening of P-gp substrates and inhibitors.

Cell cultures as tools in biopharmacy.

A survey is given on a few selected cell culture models that are used for transport studies. They are characterised for growth, transcellular electrical resistance and cytoarchitecture. The importance of standardisation in view of their use as transport models is documented. Their potential for studies on passive permeation and P-glycoprotein-mediated transport is explored and related to published data. Transport studies are presented that were performed in a two-chamber set-up, the Costar "vertical diffusion system". A series of non-homologous compounds showed similar permeability data (P(app)) in the different cell cultures. The origin of the cell type had no remarkable influence on passive transcellular permeation. MDCK cells, an epithelial cell line of canine kidney origin, are perfectly suited to screen for passive permeation. They have low expression of transporter proteins and low metabolic activity. In general, they probably represent the best-known epithelial cell line with respect to genetics as well as lipid and protein composition. MDCK cells are easy to handle. Transport experiments can be done between 7 and 14 days after seeding, when the stationary growth phase is reached. To screen for P-glycoprotein substrates, efflux and uptake studies were performed with mdr1-transfected MDCK cells (MDR1-MDCK) in a one-chamber system in the presence or absence of verapamil or cyclosporin A as inhibitor. Evidence is presented why the transfected cells, which express large amounts of P-glycoprotein, are not suitable for two-chamber transport studies.

Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC.

Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein-coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK-N-MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY were resistant to acid removal of cell surface-bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand-receptor complex is collected in pits and caveolae followed by endocytosis.

Permeation studies in vitro and in vivo of potential radiopharmaceuticals with affinity to neuro receptors.

PURPOSE: To check the influence of structural characteristics on their permeation through the blood-brain barrier (BBB), a set of radioactive [99mTc]chelates bearing amine groups was synthesized and tested in vitro as well as in vivo. METHODS: Compounds with different log P and pKa values were obtained by complex forming reactions of [99mTc]pertechnetate with varying substituents. Transport was studied in rats and mice, as well as in an ECV304 cell culture model. RESULTS: In vitro higher permeation was found for compounds with electron attracting substituents in beta-position to the amine group (pKa values 7.4 to 8.3) than for those with more basic amine groups (pKa values > 8.9) even for similar log DH 7.4. In vivo brain uptake between 0.8 and 4.8% of the injected dose (ID) per organ was found for the former, whereas <0.4% ID were present for the latter. CONCLUSIONS: Three structurally diverse classes of [99mTc]chelates showed distinct patterns with regard to brain uptake in vivo and BBB permeability in vitro which could not be predicted by their lipophilicity alone. The close correlation between the data from rats and mice and those obtained with cell cultures render the ECV304 cells an attractive model for the screening of new compounds.

Lipoproteins from Borrelia burgdorferi applied in liposomes and presented by dendritic cells induce CD8(+) T-lymphocytes in vitro.

Borrelia burgdorferi (Bb) is the tick-borne etiologic agent of Lyme borreliosis, which has many aspects of autoimmune diseases. Bb is unable to recycle synthesized membrane lipids and lipoproteins. Consequently, a large amount of liposome-like vesicle (Bb-blebs) is shed from the outer bacterial membrane. The influence of Bb-blebs on the cellular immune response is not yet known. As a Bb-blebs model, we established standardized Bb-liposomes, produced from freshly extracted lipids and lipoproteins of live Bb. Bb-liposomes were incorporated via nonendocytotic mechanisms by different human cell types, namely dendritic cells (DC), lymphocytes, and fibroblasts, as visualized by immunofluorescence and transmission electron microscopy. Bb-liposomes were localized in the cytosol and in the nucleus of the cells. With this in mind, we generated in vitro Bb-specific T-cells from nonadherant peripheral blood mononuclear cells by use of Bb-liposomes loaded autologous DC. More than 95% of those T-cells were CD8(+) and they killed autologous Bb-liposome-loaded T-cell blasts. These results suggest that Bb-blebs may be responsible for the autoimmune-like appearance of Lyme disease.

Formation of multilayers in the caco-2 cell culture model: a confocal laser scanning microscopy study.

PURPOSE: To introduce confocal laser scanning microscopy (CLSM) combined with digital image restoration to characterise Caco-2 cells under different culture conditions, and thus to define additional valid criteria for the optimisation of culture models. METHODS: Growth curves were established and transepithelial electrical resistance (TEER) measured for cells grown in EMEM or DMEM medium on Cyclopore membranes. Cytoskeleton, cell nuclei and tight junctions (TJ) were investigated by CLSM. RESULTS: Cultures reached a plateau of approximately 4.5 x 10(5) cells/cm2 after approximately 10 days. At the same time TEER reached 750 omega cm2. An irregular, fairly complete network of TJ was present at confluence (approximately 2 d). Between 15 and 30 days a regular TJ network was established. Cells formed mixed mono- and multilayers under most conditions with two exceptions: flat monolayers were observed on polycarbonate filters with EMEM and with the Biocoat intestinal epithelium differentiation environment system. In multilayers TJ were found in the upper as well as in the lower cell layers although the regular vertical polarity was disturbed. CONCLUSIONS: CLSM represents an important tool to investigate the cytoarchitecture of Caco-2 cells. 3D-analysis of confocal data gives important clues on the characteristics of cell layers and thus helps to validate optimisation strategies.

Cell-cycle arrest and p53-independent induction of apoptosis in vitro by the new anticancer drugs 5-FdUrd-P-FdCydOct and dCydPam-P-FdUrd in DU-145 human prostate cancer cells.

PURPOSE: Current therapies have limited impact on the progression of metastatic hormone-refractory prostate cancer. Therefore, we investigated the utility of new heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5-FdUrd) in p53-mutated and androgen-independent DU-145 human prostate tumour cells. METHODS: The effects of the dimers were assessed in vitro by a cell proliferation assay for cytotoxicity, flow cytometry for cell cycle distribution, confocal laser scanning microscopy for the detection of apoptotic bodies, poly(ADP-ribose) polymerase cleavage for caspase 3 activity and by a thymidylate synthetase assay. RESULTS: The new dimers N4-palmitoyl-2'-deoxycytidylyl-(3'-->5')-5-fluoro-2'-deoxyuridine (dCydPam-P-FdUrd) and 2'-deoxy-5-fluorouridylyl-(3'-->5')-2'-deoxy-5-fluoro-N4-octade cylcytidine (5-FdUrd-P-FdCydOct) caused marked cytotoxicity with IC50 values of 3-4 microM. 5-FdUrd-P-FdCydOct at 200 microM was capable of eradicating 100% of tumour cells whereas 10% of the cells were resistant to 5-FdUrd. Cytotoxicity was caused by a dramatic S-phase arrest, resulting in an increase of this cell population from 34% to 85% with 5-FdUrd-P-FdCydOct and to 81% with dCydPam-P-FdUrd. S-phase arrest was followed by apoptosis, as shown by 85% of the cells staining positive for Apo 2.7 antibody, a six- to eight-fold increased caspase 3 activity and DNA fragmentation. Thymidylate synthase activity was inhibited by 50% at 0.6-0.7 microM dimer concentration. The dimers were hydrolysed in vitro by phosphodiesterase I and human serum to the corresponding nucleosides and nucleoside monophosphates. CONCLUSIONS: The new dimers dCydPam-P-FdUrd and 5-FdUrd-P-FdCydOct are effective prodrugs of 5-FdUrd and have potential value for the treatment of p53-mutated and hormone-independent human prostate carcinomas.

Immobilized artificial membrane (IAM)-HPLC for partition studies of neutral and ionized acids and bases in comparison with the liposomal partition system.

PURPOSE: To study the partitioning of model acids ((RS)-warfarin and salicylic acid), and bases (lidocaine, (RS)-propranolol and diazepam), with immobilized artificial membrane (IAM)-HPLC, as compared to partitioning in the standardized phosphatidylcholine liposome/buffer system. METHODS: The pH-dependent apparent partition coefficients D were calculated from capacity factors (k'IAM) obtained by IAM-HPLC, using a 11-carboxylundecylphosphocholine column. For lipophilic compounds k'IAM, values were determined with organic modifiers and extrapolation to 100% water phase (k'IAMw) was optimized. Temperature dependence was explored (23 to 45 degrees C), and Gibbs free energy (deltaG), partial molar enthalpy (deltaH) and change in entropy (deltaS) were calculated. Equilibrium dialysis was used for the partitioning studies with the liposome/buffer system. RESULTS: For extrapolation of k'IAMw, linear plots were obtained both with the respective dielectric constants and the mole fractions of the organic modifier. All tested compounds showed a similar pH-D diagram in both systems; however, significant differences were reproducibly found in the pH range of 5 to 8. In all cases, deltaG and deltaH were negative, whereas deltaS values were negative for acids and positive for bases. CONCLUSIONS: In both partitioning systems, D values decreased significantly with the change from the neutral to the charged ionization state of the solute. The differences found under physiological conditions, i.e. around pH 7.4, were attributed to nonspecific interactions of the drug with the silica surface of the IAM column.

Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells.

PURPOSE: To determine the localization of the human intestinal H+/peptide cotransporter (hPepT1) and its function in intestinal epithelial cells after adenoviral transduction. METHODS: Caco-2 cells grown on Transwell membrane filters were transduced with a recombinant replication-deficient adenovirus carrying the hPepT1 gene. The transport of Gly-Sar across both apical and basolateral membranes was measured after adenoviral transduction as a function of pH, temperature, inhibitors, and substrate concentration. The localization of hPepT1 was examined by immunocytochemistry using confocal laser scanning microscopy. RESULTS: The apical-to-basolateral and basolateral-to-apical transport of Gly-Sar in Caco-2 cells after viral transduction was increased 3.3 and 3.5-fold, respectively. The similar magnitude of Gly-Sar permeability from either direction indicates involvement of identical transport pathways in both membranes. This was further confirmed by immunocytochemistry showing that hPepT1 was localized in the apical and basolateral membrane of Caco-2 cells after adenoviral transduction. In both directions, Gly-Sar transport was enhanced in the presence of a pH gradient. In addition, the basolateral-to-apical Gly-Sar transport was dependent on temperature, multiplicity of infection (MOI), and Gly-Sar concentration. It was inhibited in the presence of excess Gly-Pro and cephalexin. CONCLUSIONS: Caco-2 cell monolayers represent an appropriate model to study gene expression in intestinal epithelial cells. Transport characteristics of Gly-Sar from the basolateral to the apical side in adenovirus-transduced Caco-2 cells are in agreement with those from the apical to the basolateral side, indicating that hPepT1 is also expressed in the basolateral membrane and displays a similar level of transport enhancement after adenovirus mediated hPepT1 gene expression.

Translocation of human calcitonin in respiratory nasal epithelium is associated with self-assembly in lipid membrane.

We studied the mechanisms involved in the translocation of human calcitonin (hCT) through excised bovine nasal mucosa (net mucosal-to-serosal permeability approximately 10(-)5 cm s-1). To determine structural requirements for the suggested vesicular internalization two carboxyfluorescein-labeled (fl) hCT fragments, the C-terminal fragment [Nalpha-fl]hCT(9-32) and the N-terminal fragment [Lys(fl)18]hCT(1-24) were synthesized. In presence of the endocytosis inhibitor cytochalasin D mucosal-to-serosal and serosal-to-mucosal hCT permeabilities were equal. Pathway visualization by confocal laser scanning microscopy showed punctated fluorescence indicating vesicular internalization of both hCT and [Nalpha-fl]hCT(9-32). In contrast, the N-terminal fragment lacking the beta-sheet forming C-terminus (25-32) was not internalized. Circular dichroism showed that, when interacting with neutral and negatively charged liposomes, hCT adopts beta-sheet conformation. In a concentrated aqueous solution, beta-sheet formation induces hCT self-assembly and fibrillation. High partitioning of hCT into lipid bilayer membranes was reflected by an apparent partition coefficient log D(pH 7.4) = 2.5 (liposome-buffer equilibrium dialysis). We propose that the high lipid partitioning and beta-sheet formation result in C-terminus-restricted supramolecular self-assembly of hCT and [Nalpha-fl]hCT(9-32) in lipid membranes. Vesicular internalization is suggested to be associated with self-assembly induced perturbation of the lipid bilayer. Condensed hCT self-assemblies may explain the high capacity of net mucosal-to-serosal hCT permeation, which compares favorably with the low transport capacity of receptor-mediated endocytosis.

MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy.

PURPOSE: Madin Darby Canine Kidney (MDCK) cells were grown in culture, and age-related morphological changes in the cytoskeleton and tight junction (TJ) network were used to define stages in view of establishing an optimal in vitro model for the epithelial barrier. METHODS: Growth curves and transepithelial electrical resistance (TEER) were determined, and the cytoskeleton (actin, alpha-tubulin, vimentin) and TJ (Zonula occludens proteins ZO1, ZO2) were investigated with immunofluorescent methods by confocal laser scanning microscopy (CLSM) and digital image restoration. RESULTS: TEER measurements indicated that TJ were functional after one day. Values then remained constant. Four morphological stages could be distinguished. Stage I (0-1 day): Sub confluent cultures with flat cells; TJ established after cell-to-cell contacts are made. Stage II (2-6 days): Confluent monolayers with a complete TJ network, which remains intact throughout the later stages. Stage III (7-14 days): Rearrangement in the cytoskeleton; constant cell number; volume and surface area of cells reduced (cobble-stone appearance). Stage IV (> or = 15 days): Dome formation, i.e. thickening and spontaneous uplifting of the cell monolayer. CONCLUSIONS: Based on the structural characteristics of stage III cell cultures, which are closest to the in vivo situation, we expect them to represent an optimal in vitro model to study drug transport and/or interactions with drugs and excipients.