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PubMed1992

Evaluation of the hepatotoxicological effects of a drug in an in vivo/in vitro model.

Both in vivo and in vitro models have certain disadvantages for the study of the chronic hepatotoxicity of drugs. The aim of this work was to evaluate a new approach based on an in vivo/in vitro model. After chronic in vivo treatment of rats with Vincamine and Vindeburnol (an eburnamenine derivative which exhibits hepatotoxic properties in man) liver cells were isolated, and functional and metabolic disorders (metabolic utilization of fructose and protein biosynthesis) were studied to determine injury. The results showed no modification of blood parameters, but a direct relationship between the dose of Vindeburnol administered in vivo and the metabolic disorders observed in vitro, evidencing the high sensitivity and reliability of this model.

Animals↗

PubMed1992

Comparison of gentamicin dosing regimens using an in-vitro model.

An in-vitro model which simulates in-vivo pharmacokinetics was used to compare the efficacy against Pseudomonas aeruginosa of dosing regimens of gentamicin which achieve different peak/trough concentrations but use the same total dose over 24 h. First exposure to gentamicin produced a rapid bactericidal effect which was proportional to the initial peak concentration. Subsequent doses of gentamicin produced a smaller bactericidal effect. Regrowth occurred with all dosing regimens, even after very high initial concentrations (26 mg/L). The time to reach bacterial counts above starting values was prolonged in relation to peak concentrations. Regrowth was also demonstrated in continuous infusion experiments which maintained very high concentrations (26 mg/L), although an inhibitory effect was evident compared with single dose experiments and the experiments mimicking in-vitro pharmacokinetics. There was little evidence of a post-antibiotic effect. The data supports the use of larger initial and longer interval bolus dosing compared with current recommendations.

Drug Administration Schedule↗

PubMed1989

Modulation of arterial endothelial permeability: studies on an in vitro model.

1. An in vitro model of the arterial endothelial barrier was established in which transfer of trypan blue-labelled albumin across confluent monolayers of pig aortic endothelial cells grown on polycarbonate membranes was measured. 2. A range of inflammatory mediators, i.e. histamine, bradykinin, platelet activating factor and thrombin, had no effect on the transfer of labelled albumin across aortic endothelial monolayers. 3. Calcium ionophore A23187 and the phorbol ester, phorbol myristate acetate (PMA), each induced concentration-dependent increases in transfer of labelled albumin. These increases were associated with changes in cell shape, consistent with endothelial contraction. Ionophore A23187 caused some detachment of cells. 4. The ability of PMA to increase transfer of labelled albumin probably results from activation of protein kinase C since it was not shared by the inactive analogue, 4 alpha-phorbol 12,13-didecanoate. 5. Neither a combination of superoxide dismutase and catalase nor the cyclo-oxygenase inhibitor, flurbiprofen, affected resting or PMA-induced increases in albumin transfer. Oxygen-derived free radicals and prostaglandins appear not to be involved in the response to PMA. 6. Each of three procedures designed to elevate adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels, i.e. dibutyryl cyclic AMP, forskolin and (+/-)-isoprenaline, reduced the ability of PMA to promote increased transfer of labelled albumin but had no effect on resting transfer. The effect of (+/-)-isoprenaline was abolished by the beta-adrenoceptor blocking agent, propranolol. 7. Elevation of cyclic GMP content by use of 8 bromo cyclic GMP or atriopeptin II had no effect on resting or PMA-induced transfer of labelled albumin. 8. Arterial endothelial barrier function can be compromised by agents that promote endothelial contraction. Agents that increase endothelial cyclic AMP levels, and so reduce entry of high molecular weight substances into the arterial wall, may warrant evaluation as potential anti-atherogenic drugs.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium: in vitro-in vivo correlation with permeability data from rats and humans.

The diverse secretory and absorptive functions of the intestinal epithelium are conducted by a mixed population of absorptive cells and mucus-producing goblet cells as the major cell types. In order to approach the main characteristics in an in vitro model, a coculture system of absorptive Caco-2 cells and mucus-secreting HT29-MTX cells was developed and the permeability of a range of different drugs was tested. Variable goblet cell frequency can be achieved, preserving a significant barrier to drug transport and maintaining the differentiated features of both cell types. Absorption rates for actively transported drugs are rather underestimated in the cell culture model when compared to in vivo data. However, a good correlation with fraction absorbed in humans was attained separating the range of passively transported drugs into two groups of well-absorbable compounds with Peff > or = 10 x 10(-6) cm/s and drugs that are absorbed 40-70% with Peff = 0.1-1 x 10(-5) cm/s. A permeability of Peff < 0.1 x 10(-5) cm/s is suggested for low absorbable drugs.

Animals↗

PubMed1988

The effect of antibiotics on bacterial colonisation of vascular cannulae in a novel in-vitro model.

An in-vitro model for studying semi-quantitatively the bacterial colonisation of the external and internal surfaces of peripheral intravascular cannulae is described. Using this model, we studied the effect of ciprofloxacin, teicoplanin and fusidic acid on cannula colonisation by Staphylococcus epidermidis. Exposure of colonised cannulae to sub-MICs of ciprofloxacin and fusidic acid reduced bacterial attachment, whereas sub-MIC levels of teicoplanin had little effect. Pre-exposure of S. epidermidis to sub-MICs of ciprofloxacin and fusidic acid also reduced slime production and colonisation. In comparison, pre-exposure of S. epidermidis to teicoplanin 1.0 mg/L did not influence colonisation, whereas at 0.1 mg/L it was reduced. The model allowed investigation of bacterial colonisation of cannulae and offers a screening system for the assessment of potential agents for the prophylaxis and treatment of these infections.

Anti-Bacterial Agents↗

PubMed1993

Endotoxin-induced arterial endothelial barrier dysfunction assessed by an in vitro model.

Using an in vitro model in which albumin transfer across monolayers of bovine aortic endothelial cells (BAEC) was measured, we have shown that lipopolysaccharide (LPS) induces a concentration-dependent increase in endothelial permeability. This increase was biphasic, having an early peak at 2 h and rising again by 24 h. Both peaks were abolished by polymixin B (PMB) but were unaffected by N omega-monomethyl-L-arginine, N omega-nitro-L-arginine methyl ester or dexamethasone. Furthermore, LPS did not stimulate nitric oxide production by BAEC following 24 h exposure. Thus, the LPS-induced increase in permeability may account for the vascular leakage of septic shock, but the L-arginine-nitric oxide system does not appear to be involved.

Albumins↗

PubMed1994

Efficacies of different vancomycin dosing regimens against Staphylococcus aureus determined with a dynamic in vitro model.

A dynamic in vitro model was used to assess four different vancomycin dosing regimens against Staphylococcus aureus. These regimens achieved peak drug concentrations of 48 micrograms/ml (single dose) and 30 micrograms/ml (dosed every 12 h) and constant concentrations of 16 and 8 micrograms/ml. Analysis of the area under the bacterial concentration-time curve, area under the first moment of the bacterial concentration-time curve, and bacterial elimination rate constant showed no difference in the rate or extent of bacterial killing. The optimal dosing method may be that which achieves the lowest area under the curve while concentrations are maintained above the MBC.

Dose-Response Relationship, Drug↗

PubMed2002

Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model.

In an in vitro model of cerebral ischemia (oxygen glucose deprivation, OGD) we investigated whether erythropoietin (EPO) plays a critical role in ischemic preconditioning. We found that EPO time and dose-dependently induced protection against OGD in rat primary cortical neurons. Protection was significant at 5 min and reached a maximum at 48 hr after EPO application. Protection was blocked by the coapplication of a soluble Epo receptor (sEpoR) or an antibody against EpoR (anti-EpoR). Medium transfer from OGD-treated astrocytes to untreated neurons induced protection against OGD in neurons, which was attenuated strongly by the application of sEpoR and anti-EpoR. In contrast, medium transfer from OGD-treated neurons to untreated neurons induced protection against OGD that did not involve EPO. In astrocytes the OGD enhanced the nuclear translocation of hypoxia-inducible factor 1 (HIF-1), the major transcription factor regulating EPO expression. Consequently, transcription of EPO-mRNA was increased in astrocytes after OGD. Cultured neurons express EpoR, and the Janus kinase-2 (JAK-2) inhibitor AG490 abolished EPO-induced tolerance against OGD. Furthermore, EPO-induced neuroprotection as well as phosphorylation of the proapoptotic Bcl family member Bad was reduced by the phosphoinositide-3 kinase (PI3K) inhibitor LY294002. The results suggest that astrocytes challenged with OGD provide paracrine protective signals to neurons. We provide evidence for the following signaling cascade: HIF-1 is activated rapidly by hypoxia in astrocytes. After HIF-1 activation the astrocytes express and release EPO. EPO activates the neuronal EPO receptor and, subsequently, JAK-2 and thereby PI3K. PI3K deactivates BAD via Akt-mediated phosphorylation and thus may inhibit hypoxia-induced apoptosis in neurons. Our results establish EPO as an important paracrine neuroprotective mediator of ischemic preconditioning.

Animals↗

PubMed1996

Role of blood components in ocular silicone oil emulsification. Studies on an in vitro model.

PURPOSE: To develop an in vitro model for silicone oil emulsification and to explore the blood components involved in this process. METHODS: The capacity of various blood components to support silicone oil (1000 CS) emulsification was studied by applying 0.5 ml oil on top of 0.5 ml saline containing various blood components. Each tube was sonicated for 150 seconds and centrifuged at 5000 g for 20 minutes. Three phases were noted in the tube: At the top was clear silicone oil, in the middle was emulsified silicone oil, and at the bottom was aqueous solution. The tubes were photographed, and the percentage of the phase length containing emulsified silicone oil (middle) of the total length of the three phases was calculated from the projected image of each tube. RESULTS: Emulsified silicone oil in plasma or serum was initiated after 100 seconds of sonication and quickly reached maximum (approximately 80%) at 120 seconds. The size of these oil droplets prepared in vitro was 0.0467 +/- 0.028 mm, closely resembling that observed in oil samples removed from a patient's anterior chamber (0.038 +/- 0.018 mm). Under these conditions, silicone oil emulsified in the presence of whole blood cells occurred only at a concentration of 120 micrograms protein/ml; in the presence of red blood cell membranes, it occurred at a concentration of 60 micrograms protein/ml. Lipoprotein-deficient serum failed to support emulsification; however, samples of high-density lipoprotein and low-density lipoprotein supported this process. Purified high-density lipoprotein-apolipoproteins supported oil emulsification. The addition of phosphatidylcholine further enhanced this process, but phosphatidylcholine alone failed to support emulsification. CONCLUSIONS: A simple and fast in vitro model to study factors affecting silicone oil emulsification was developed. Using this model, red blood cell membranes, plasma lipoproteins, and purified HDL-apolipoproteins supported silicone oil emulsification. Lipids did not, but they had the capacity to enhance the apolipoprotein-supported emulsification.

Blood Cells↗

PubMed1999

Studies on bioavailability of some bulk and trace elements in Mexican tortilla using an in vitro model.

An in vitro gut model was used to investigate the bioavailability of calcium, magnesium, cadmium, chromium, iron, manganese, and lead in the Mexican (maize) tortilla. The samples (4 g) were digested in quartz tubes using concentrated nitric acid (12 mL) at 65 degrees C (2 h) and then at 120 degrees C (4 h). Total element concentration was determined by atomic absorption spectrometry. Enzymatic hydrolysis was carried out in two steps: first, with pepsin in a hydrochloric acid medium (pH 1.8) and then after neutralization with sodium bicarbonate (pH 6.0), with the mixture of pancreatine, amylase, and bile salts extract. Elements under study were determined in the supernatant by atomic absorption spectrometry. The bioavailable fraction of each element in tortilla was evaluated as the percentage of total element content found in the solution after enzymolysis. The obtained results showed relatively low bioavailability of the selected elements (from 2% to 32%), which possibly may be ascribed to the presence of dietary fiber in tortilla.

Biological Availability↗

PubMed2005

Apical diffusion of calcium hydroxide in an in vitro model.

An in vitro agar model was developed to study the effect of intracanal medicaments on periapical tissues and was used to study the diffusion of three calcium hydroxide (Ca(OH)2) medicaments of varying viscosity through simulated root canals with various sizes of apical foramina. Experimental medicaments were added to pipette tips used to represent tooth roots, which were fixed in syringes containing brain heart infusion agar and calcium-reactive dye. OH and Ca concentrations were measured in the agar at 30 minutes and 24 hours. Ca concentration and pH increased with larger aperture sizes, and higher pH and Ca diffusion was produced by a 10% Ca(OH)2 solution than was produced by Pulpdent or a Ca(OH)2 paste. The results suggest that the properties of the Ca(OH)2-containing vehicle could affect the action of the medicament in the periapical tissues.

Analysis of Variance↗

PubMed2000

In vitro model of atherosclerosis using coculture of arterial wall cells and macrophage.

In order to determine the precise mechanism of the interactions between different types of cells, which are common phenomena in tissues and organs, the importance of coculture techniques are becoming increasingly important. In the area of cardiology, artificial arteries have been developed, based on the understanding of physiological communication of the arterial smooth muscle cells (SMC), endothelial cells (EC), and the extracellular matrix (ECM). In the study of atherosclerosis, the modification of low-density lipoprotein (LDL), which result in the recruitment and accumulation of white blood cells, especially, monocytes/macrophages, and foam cell formation, are hypothesized. Although there are well known animal models, an in vitro model of atherogenesis with a precisely known atherogenesis mechanism has not yet been developed. In this paper, an arterial wall reconstruction model using rabbit primary cultivated aortic SMCs and ECs, was shown. In addition, human peripheral monocytes were used and the transmigration of monocytes was observed by scanning electron and laser confocal microscopy. Monocyte differentiation into macrophages was shown by immunohistochemistry and comprehensive gene expression analysis. With the modified form of LDL, the macrophages were observed to accumulate lipids with a foamy appearance and differentiate into the foam cells in the ECM between the ECs and SMCs in the area of our coculture model.

Animals↗

PubMed1986

A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process.

A model of in vitro mechanical injury of confluent human endothelial cells (EC) in culture was developed. Human EC were obtained from umbilical veins and grown to confluence. Application on the EC monolayer of a calibrated disk of cellulose polyacetate paper resulted in removal of the EC, leaving a continuous subendothelial extracellular matrix (ECM) on the culture dish. The regeneration time depended on the original size of the lesion. Regeneration was similar with EC grown on different substrates such as human fibronectin, human subendothelial ECM, bovine collagen type I or surfaces coated with Transglutine, a surgical glue containing adhesive proteins. A human brain extract containing growth factor activity accelerated significantly the repair of the lesion, especially at low serum concentration. This simple in vitro model of mechanical injury allows the quantitative study of the effects of matrices, growth factors and pharmacological agents on the repair process.

Cells, Cultured↗

PubMedDate not supplied

Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium.

An in vitro model of the human follicle associated epithelium (FAE) was characterized and the influence of nanoparticle properties on the transcellular transport across the in vitro model was investigated. The model was established by co-culturing Caco-2 and Raji cells, with Caco-2 cells alone as control. The conversion of Caco-2 cells to follicle associated epithelium (FAE) like cells was monitored by following the surface expression of beta1-integrins (immunofluorescence) and nanoparticle transport (flow cytometry). The influence of the nanoparticle concentration at the apical side, temperature, size and surface properties of nanoparticles on transport was evaluated, as well as the influence of transport conditions. The conversion of Caco-2 cells into FAE-like cells occurred. The transport was concentration, temperature and size-dependent. Aminated nanoparticles were more efficiently transported than carboxylated nanoparticles, suggesting a role of nanoparticle surface functional groups and hydrophobicity, possibly leading to a different pattern of protein adsorption at their surface. In conclusion, this in vitro model is a promising tool to study the role of M cells in transintestinal nanoparticle transport, as well as to evaluate new drug delivery systems.

B-Lymphocytes↗

PubMed1993

Development and application of in vitro models of hematopoiesis to drug development.

In vitro models of hematopoiesis are used increasingly in investigative hematopathology. Such models complement in vivo animal testing and have been shown to be predictive for hematotoxicity associated with anticancer and antiviral agents in humans. In vitro models of hematopoiesis consist of short-term cloning assays for various hematopoietic progenitor cells and long-term functional assays for the marrow hematopoietic microenvironment. In our laboratories, the cloning assays have been used as investigative tools to study the pathogenetic mechanisms of drug-induced blood disorders and as screening systems to investigate the possible hematotoxic potential of candidate drugs in various animal species. Data in support of these applications are presented in this paper.

Animals↗

PubMed1999

Neuroprotective interaction effects of NMDA and AMPA receptor antagonists in an in vitro model of cerebral ischemia.

An in vitro model of ischemia was developed and characterized using the acute rat hippocampal slice preparation. Neuroprotective concentrations of several competitive and noncompetitive glutamate subtype-selective antagonists (CGS-19755, MK-801, YM90K and GYKI-52466) were initially determined in anoxia-enhanced agonist-induced excitotoxicity experiments. Concentrations which proved to be effective in these studies were subsequently tested for their effectiveness against an ischemic episode. Ischemia was defined as a 30-min exposure to aglycemic media ending in 5 min of concurrent anoxia, a protocol which was arrived at by empirically determining the effect of various hypoglycemic and anoxic insults on the ability of hippocampal slices to retain their electrophysiological viability. Exposure to such an ischemic episode resulted in a loss of viability by most slices, an effect which was strongly dependent on extracellular calcium. AMPA antagonists applied alone produced no neuroprotective effect in the present model of in vitro ischemia, while NMDA antagonists applied alone had a modest neuroprotective effect. In contrast, the coapplication of 10 microM MK-801 and 300 microM GYKI-52466, noncompetitive NMDA and AMPA receptor antagonists, respectively, resulted in almost complete neuroprotection. This protection was comparable to that obtained by withholding extracellular calcium, indicating that the toxic effects of glutamate receptor overstimulation can be accounted for solely by calcium influx. The effect of this combination treatment on the survival rate of hippocampal slices was synergistic, that is greater than the sum of the effects of the individual compounds. The results indicate that neuroprotection against acute ischemic insults may require a combination therapy approach.

Animals↗

PubMed1996

[Place of in vitro models in preclinical evaluation of anticancer drugs].

In vitro models have been intensively developed for several years for selecting new anticancer agents. The National Cancer Institute has even chosen as a primary screen of new molecules a panel of 60 human tumor cell lines. However, it may seem hazardous to rely too much on in vitro models for the discovery and selection of new anticancer drugs: (1 because no metabolism of the compounds occurs in cell culture; (2 because an in vitro cell line cannot be representative of an in situ tumor cell population; (3 because antiproliferative activity is only part of antitumor activity; (4 because the toxicologic properties of the molecules are not taken into account by in vitro systems; (5 because cell cultures do not allow any selectivity study between tumor cells and normal cells. With examples drawn from three different therapeutic classes, anthracyclines, taxoids and camptothecin derivatives, we show that in vitro tests are insufficiently predictive of antitumor potential. The excess of confidence allowed to these models may lead to premature decisions which are not after that justified by clinical trials.

Animals↗

PubMed2001

Development of an in vitro model for assessing the in vivo stability of lanthanide chelates.

An in vitro model was developed to evaluate the in vivo stability of lanthanide polyaminocarboxylate complexes. The ligand-to-metal ratios for the chelates EDTA, CDTA, DTPA, MA-DTPA (monoamide-DTPA) and DOTA with the lanthanides lanthanum, samarium, and lutetium were optimized to achieve > or = 98% complexation yield for the resultant radiolanthanide complexes. The exchange of the radiolanthanides from their EDTA, CDTA, DTPA, MA-DTPA and DOTA complexes with Ca(2+) was determined by in vitro adsorption and in vitro column studies using hydroxyapatite (HA), an in vitro bone model. In vitro serum stability of these radiolanthanide complexes was used as an additional indicator of in vivo stability, although the mechanism of instability in serum will be different than with bone. The in vitro studies were consistent with the expected findings that the smallest lanthanide (Lu) formed the most stable complexes. In vivo studies were done to validate the in vitro model. Biodistribution studies in normal CF-1 mice showed that in vivo stability of the complex (i.e., the more lanthanide remaining in complex form) could be assessed by a combination of the urinary, bone and liver uptake. For example, biodistribution studies demonstrate that high urinary excretion correlated with complex stability, while high liver plus bone uptake correlated with complex instability. The urinary excretion of the EDTA complexes decreased from (177)Lu to (140)La indicating a loss in stability in the direction of (140)La, consistent with the in vitro studies. The more stable a lanthanide complex is, the lower its exchange with HA in vitro will be, and the lower its combined bone plus liver uptake and higher its urinary excretion will be in vivo. This investigation indicates that the in vivo stability can be determined by a screening method that measures the degree of exchange from the lanthanide chelate with hydroxyapatite (HA) and its serum stability.

Adsorption↗