Differentiated intestinal epithelial cell lines asin vitro models for predicting the intestinal absorption of drugs

The oral absorption of a compound is a critical factor for the future of the compound as a drug. This absorption is mainly controlled by the passage across, the intestinal epithelium. Thus, the prediction of the intestinal absorption by means of anin vitro model may represent a powerful tool for the early selection of molecules during the process of drug development. In the present study, the differentiated human intestinal epithelial cell line HT29-18-C₁, was grown on permeable filters in dual chambers. These cells formed tight monolayers that were used to measurein vitro the transepithelial permeability coefficient (P _c) of various molecules. The results were compared within vivo data of oral absorption. A threshold value ofin vitro permeability of 2×10^–6 cm/s was found. Molecules having a permeability coefficient higher than this value were absorbed orally more than 80%, while drugs withP _c values lower than 2×10^–6 cm/s were poorly absorbed. By mathematical simulation, it was found that thisP _c value, when extrapolated to the surface area and volume of the small intestine, corresponds to an absorption of 80% for a compound with a transit time through the small intestine of 5 h. This demonstrates the predictive utility of the threshold value of the permeability coefficient derived from thein vitro model of intestinal epithelium.Abbreviations P _c transepithelial permeability coefficient - MTX methotrexate     

In vitro and in vivo translational models for rare liver diseases

A challenge in developing effective treatments is the modeling of the human disease using in vitro and in vivo systems. Animal models have played a critical role in the understanding of disease pathophysiology, target validation, and evaluation of novel therapeutic agents. However, as the success rate from entry into clinical testing to drug approval remains low, it is critical to have high quality and well-validated models reflective of the disease condition. Additional experimental models are being developed based on functional in vitro 3D tissue models such as organoids and 3D bioprinted tissues. Because these 3D tissue models mimic closer the architecture, cell composition and physiology of native tissues, they are now being used as screening platforms in drug discovery and development and for tissue transplant in regenerative medicine. Here we review the current state-of-art of in vitro and in vivo translational models for the development of therapies for rare diseases of the liver.     

Collagen-based cell migration models in vitro and in vivo

Fibrillar collagen is the most abundant extracellular matrix (ECM) constituent which maintains the structure of most interstitial tissues and organs, including skin, gut, and breast. Density and spatial alignments of the three-dimensional (3D) collagen architecture define mechanical tissue properties, i.e. stiffness and porosity, which guide or oppose cell migration and positioning in different contexts, such as morphogenesis, regeneration, immune response, and cancer progression. To reproduce interstitial cell movement in vitro with high in vivo fidelity, 3D collagen lattices are being reconstituted from extracted collagen monomers, resulting in the re-assembly of a fibrillar meshwork of defined porosity and stiffness. With a focus on tumor invasion studies, we here evaluate different in vitro collagen-based cell invasion models, employing either pepsinized or non-pepsinized collagen extracts, and compare their structure to connective tissue in vivo, including mouse dermis and mammary gland, chick chorioallantoic membrane (CAM), and human dermis. Using confocal reflection and two-photon-excited second harmonic generation (SHG) microscopy, we here show that, depending on the collagen source, in vitro models yield homogeneous fibrillar texture with a quite narrow range of pore size variation, whereas all in vivo scaffolds comprise a range from low- to high-density fibrillar networks and heterogeneous pore sizes within the same tissue. Future in-depth comparison of structure and physical properties between 3D ECM-based models in vitro and in vivo are mandatory to better understand the mechanisms and limits of interstitial cell movements in distinct tissue environments.     

Injectable gel with synthetic collagen-binding peptide for enhanced osteogenesis in vitro and in vivo

A synthetic peptide denoted as collagen-binding motif (CBM) was identified from osteopontin (OPN), a multisubunit extracellular matrix (ECM) protein, by enzymatic digestion with chymotrypsin. The aim of this study was to examine the feasibility of identified CBM peptide as an active component of gel type scaffold material in osteogenesis. The binding of CBM peptide to collagen was specific and presented high affinity. Cell adhesion and growth on CBM peptide-immobilized gel were significantly increased as compared with those on gel with control peptide or without peptide. The CBM peptide-immobilized gel increased osteoblastic differentiation, followed by marked bone formation in the rabbit calvarial defect sites at 4 weeks. Taken together, the injectable gel with synthetic CBM peptide has a potential to induce osteogenesis in vitro and in vivo, suggesting its clinical application in bone regeneration procedure.     

MNPs-IHSPN nanoparticles in multi-application with absorption of bio drugs in vitro

The aim of this project is to investigate the method of using a common buffer to determine the degree of stabilization and secretion of two drug molecules that have been analyzed in vitro. First, magnetic nanoparticles were synthesized and their structure was identified by instruments such as XPS (X-ray photoelectron spectroscopy) and FT-IR (Fourier transform infrared spectroscopy). The main purpose of this study was to investigate the stabilization and release of methotrexate on the surface of magnetic nanoparticles. The two temperatures were 37 and 25°, respectively. After reaction with the biomolecules, the adsorption rate for both drug molecules was about 60–80. PBS buffer was also used for diffusion of biomolecules and the results were analyzed by spectrophotometer analysis. With these results, the adsorption of cysteine and MTX was more than 60% and its release rate in MNPS-IHSPN was up to 90%, which means that high-strength stabilization and release by magnetic nanoparticles under external magnetic field and in vitro confirmed. The result of this project for the exchange of drugs by the surface of magnetic nanoparticles to repair damaged cells in the body of living organisms can be generalized.     

In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo

One approach to targeted therapies for cardiovascular disease relies on isolating ligands that enhance the tissue-specific uptake of genes or drugs by heart cells. To obtain heart-targeting ligands, phage display biopanning was used to isolate a 20-mer peptide that binds to isolated primary cardiomyocytes. The isolated phage, PCM.1, displays the peptide WLSEAGPVVTVRALRGTGSW, and binds these cells 180 times better than a control phage from the library. Furthermore, phage displaying this peptide preferentially bind to cardiomyocytes when compared with a panel of other cell types. A BLAST search revealed that this peptide contains a 12 amino acid segment with sequence identity to a peptide in tenascin-X, an extracellular matrix protein. Synthetic peptides containing the complete 20-mer or a 12-mer tenascin peptide partially blocked phage binding to the cardiomyocytes. We developed a quantitative real-time PCR assay to assess uptake of this phage by tissues in vivo. Using this assay, preferential localization of the PCM.1 phage in heart was observed compared to the uptake of this phage by other tissues or other phage by heart. Furthermore, PCM.1 phage was associated with cardiomyocytes isolated from mice treated with a phage in vivo. These results demonstrate the utility of biopanning on isolated cells for identifying specific binding peptides that can target a tissue in vivo.     

Comparison of in vitro models for the prediction of compound absorption across the human intestinal mucosa

Several in vitro assays have been developed to evaluate the gastrointestinal absorption of compounds. Our aim was to compare 3 of these methods: 1) the bio-mimetic artificial membrane permeability assay (BAMPA) method, which offers a high-throughput, noncellular approach to the measurement of passive transport; 2) the traditional Caco-2 cell assay, the use of which as a high-throughput tool is limited by the long cell differentiation time (21 days); and 3) The BioCoat high-throughput screening Caco-2 Assay System, which reduces Caco-2 cell differentiation to 3 days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations (r = 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat HTS Caco-2 Assay System does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21-day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption.     

Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

The proline-rich designer antibacterial peptide dimer A3-APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic-resistant Gram-negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half-life of approximately 100 min as documented by reversed-phase chromatography. Indeed, after a 30-min incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however, with degradation products present corresponding to amino-terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 min. Nevertheless, the major early metabolite, a full single-chain fragment, was detectable until 90 min, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1-Arg20 metabolite, when administered three times at 20 mg/kg to mice infected with a sublethal dose (over LD(50)) of an extended spectrum beta-lactamase-producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3-APO, injected subcutaneously twice over a 3-wk period, did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development.     

Esterification and absorption of cholesterol: in vitro and in vivo observations in the rat

Activity of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) in isolated rat enterocytes was reduced by approx. 75% following a single oral dose of Sandoz compound 58-035 (30 mg.kg-1). Despite this, the formation of [14C]cholesteryl esters from [1-14C]oleic acid remained unaffected in ACAT-inhibited cell preparations. The increase in serum cholesterol concentrations observed after overnight cholesterol/cholic acid (1%/0.5%) feeding to rats was abolished by pre-treatment with Sandoz compound 58-035 (30 mg.kg-1). These results can be reconciled with a previously proposed model for the transmembrane movement of cholesterol which implicates ACAT-independent esterification and hydrolysis as a transport mechanism for the movement of cholesterol across the enterocyte apical membrane.     

Preparation and in vitro and in vivo evaluation of quercetin-loaded mixed micelles for oral delivery

Quercetin (QT) is a plant polyphenol with various pharmacological properties. However, the low water solubility limits its therapeutic efficacy. In the present study, QT-loaded sodium taurocholate-Pluronic P123 (QT-loaded ST/P123) mixed micelles were developed and characterized, and the effect of the formulation on improving the water solubility of QT was investigated. QT-loaded ST/P123 mixed micelles were prepared by thin film hydration-direct dissolution and optimized by uniform design. The optimal formulation possessed high drug loading (12.6%) and entrapment efficiency (95.9%) in small (16.20 nm) spherically-shaped micelles. A low critical micelle concentration indicated that the micelles were stable, and they showed a sustained release pattern, as determined in vitro in simulated gastric fluid and intestinal fluid. Pharmacokinetic evaluation showed the C_max and AUC_0–24 were 1.8-fold and 1.6-fold higher than the QT suspension. The present results indicate that QT-loaded ST/P123 micelles are potential candidates to improve the solubility and oral bioavailability of QT.     

Correlation of cellulose synthesis in vivo and in vitro during the encystment of Acanthamoeba

Acanthamoeba castellanii differentiates when placed in a starvation medium. The mature cysts formed are characterized by a cellulosic wall synthesized from endogenous sources during encystment. A particulate enzyme system whose specific activity increases some 30-fold during encystment catalyzes the formation of an alkali-soluble and an alkali-insoluble β-(1 → 4)-glucan (cellulose). The activity in vitro of this enzyme extracted from populations of cells during encystment correlates with the formation in vivo of the mature cyst and the alkali-insoluble β-glucan of the cyst wall. The conclusion is based on the following observations:

1.

1. Both alkali-soluble and alkali-insoluble β-glucans similar to the enzymatic products of the isolated β-glucan synthetase occur in cyst walls.     

In vitro stability and ex vivo absorption of thymol monoglucosides in the porcine gut

Thymol α-D-glucopyranoside (TαG) and thymol β-D-glucopyranoside (TβG) are believed to have different kinetic behaviours in the porcine gut than its parent aglycon thymol. However, recently, it was shown that concentrations of both glucosides decreased rapidly in the stomach and proximal small intestine following oral supplementation to piglets as did thymol. Yet, the stability of thymol glucosides in gut contents and their absorption route remains obscure. Therefore, a series of in vitro incubations were performed, simulating the impact of pH, digestive enzymes, bacterial activity and mucosal extracts on stability of these glucosides. Their absorption mechanisms were investigated using the Ussing chamber model in the presence or the absence of inhibitors of sodium-dependent glucose linked transporter 1 and lactase phlorizin hydrolase. Both glucosides remained intact at physiological pH levels in the presence of digestive enzymes. Recoveries from TαG and TβG were below 90% when incubated with small intestinal homogenates from the distal jejunum or from all sampled sites, respectively. However, no aglycon could be detected in these samples. Bacterial inoculum of the small intestine, on the other hand, hydrolysed TβG quickly with up to 44% of free aglycon appearing. TαG proved more resistant to porcine gastro-intestinal bacterial glucosidases with only trace amounts (<1%) of free thymol at the end of the incubations. Electrophysiological measurements in Ussing chambers did not suggest active transport of the glucosides. Mucosal TαG and TβG concentrations were unchanged between start and end of the absorption measurements. Additionally, no TαG and only a very limited amount of TβG were retrieved from the serosal side. Tissue associated concentrations, although marginal (<1% of luminal concentration), were mainly as intact glucoside or as aglycon for TαG and TβG, respectively. Addition of both inhibitors significantly increased the amount of intact glucosides retrieved from the mucosal tissues as compared to controls. In conclusion, bacterial hydrolysis was identified as the most important source of TβG loss, whereas TαG seemed less prone to degradation or absorption in these in vitro and ex vivo models.     

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs

Three-dimensional cell culture models, such as spheroids, can be used in the process of the development of new anticancer agents because they are able to closely mimic the main features of human solid tumors, namely their structural organization, cellular layered assembling, hypoxia, and nutrient gradients. These properties imprint to the spheroids an anticancer therapeutics resistance profile, which is similar to that displayed by human solid tumors. In this review, an overview of the drug resistance mechanisms observed in 3D tumor spheroids is provided. Furthermore, comparisons between the therapeutics resistance profile exhibited by spheroids, and 2D cell cultures are presented. Finally, examples of the therapeutic approaches that have been developed to surpass the drug resistance mechanisms exhibited by spheroids are described.