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PubMed2003

Effect of novel benzoylphenylurea derivatives on DNA polymerase alpha activity using the synthesome-based in vitro model system.

Six benzoylphenylurea (BPU) derivatives have been synthesized in Japan and extensively evaluated by the U.S. National Cancer Institute. They demonstrated potent antitumor activity in vitro against several cancer cell lines as well as in vivo against several tumor models. One of these agents, NSC639829, has now entered clinical trials. Studies have shown that these compounds are effective inhibitors of in vitro tubulin polymerization. The parent compound, NSC624548 (HO-221), has been shown to inhibit calf thymus DNA polymerase alpha activity. In this study we examined the effects of four BPU derivatives (NSC624548, NSC639828, NSC639829, and NSC654259) on the activity of the synthesome-associated DNA polymerase alpha, Escherichia coli DNA polymerase I, and calf thymus DNA polymerase alpha. Among the compounds tested, only NSC624548 and NSC639828 inhibited the activities of E. coli DNA polymerase I and calf thymus DNA polymerase alpha. Excess DNA polymerase I or DNA polymerase alpha dramatically reduced the inhibition produced by these compounds. NSC624548 and NSC639828 also showed inhibitory effects of the synthesome-associated DNA polymerase alpha similar to that produced upon using the purified E. coli and calf thymus enzymes. All of the four compounds did not show inhibitory effect on DNA polymerase delta. The similar pattern of inhibition these compounds exert on both the purified calf thymus and the synthesome-associated DNA polymerase alpha offers further support for the validity of the DNA synthesome as a novel in vitro model system for studying anticancer drug action.

Animals↗

PubMedDate not supplied

Human hepatocytes as a key in vitro model to improve preclinical drug development.

Over past decades, numerous in vitro and/or ex vivo models have been developed to investigate drug metabolism. In the order of complexity we found the isolated perfused liver, hepatocytes in co-culture with epithelial cells, hepatocytes in suspension and in primary culture and subcellular hepatic microsomal fractions. Because they can be easily prepared from both animals (pharmacological and toxicological species) and humans (whole livers as well as biopsies obtained during surgery) hepatocytes in primary culture provide the most powerful model to better elucidate drug behavior at an early stage of preclinical development such as: the characterization of main biotransformation reactions, the identification of phase I and phase II isozymes involved in such reactions, the evaluation of inter-species differences allowing the selection of a second toxicological animal species more closely related to man on the basis of metabolic profiles, the detection of the inducing and/or inhibitory effects of a drug on metabolic enzymes, the prediction of drug interactions, the estimation of inter-individual variability in biotransformation reactions. The use of hepatocytes, and in particular those obtained from humans, at an early stage of drug development allows the obtention of more predictive preclinical data and a better knowledge of drug behavior in humans before the first administration of the drug in healthy volunteers.

Animals↗

PubMed1996

Effect of antisense oligonucleotides on cytokine release from human keratinocytes in an in vitro model of skin.

ISIS 1082, a phosphorothioate oligonucleotide 21 nucleotides in length targeted to the translation initiation codon of herpes simplex virus (HSV) type 1 and 2 virion capsid protein, has been shown to inhibit HSV-1 replication in vitro. The effects of ISIS 1082, its phosphodiester congener, ISIS 1049, and analogs consisting of 2' methoxy and 2' propoxy phosphodiesters and phosphorothioates on IL-1 alpha release and viability were evaluated in a three-dimensional in vitro skin model consisting of neonatal keratinocytes and fibroblasts. This in vitro system displays many of the functional and metabolic properties of a differentiated epidermis and can be induced to specifically release IL-1 alpha in response to a mixture of lipopolysaccharide and phorbol myristate acetate. Incubation of the skin model with 250 to 1000 microM concentrations of ISIS 1082 and its 2' methoxy and propoxy phosphorothioate analogs resulted in a concentration-dependent increase of cytokine release with minimal effects on cellular viability, as measured by the Neutral Red assay. This response was confirmed in primary keratinocytes, which were also shown to secrete IL-1 alpha into media supernatants after incubation with phosphorothioate oligomers. These data suggest that the IL-1 alpha released from keratinocytes in response to ISIS 1082 may contribute to the inflammatory and immune cell response seen in vivo.

Antiviral Agents↗

PubMedDate not supplied

In vitro model of the pathogenesis of celiac disease.

The in vitro challenge of duodenal mucosa with gliadin is a useful model to reproduce the immunological features of celiac disease (CD) and allows the study of early pathogenetic events in this disease. With this model it was shown that antigens such as ICAM-1 and HLA-DR are upregulated as early as 1-2 h after gliadin challenge in patients with CD. After 24 h the lamina propria contained CD4+ T cells expressing the IL-2 receptor alpha-chain, which is a sign of activation. Intraepithelial lymphocytes increased in number and showed proliferative activity. After in vitro stimulation with gliadin, endomysial antibodies were found in the supernatant of the cultured mucosa from patients with CD following a gluten-free diet. This supported the notion that endomysial antibodies are at least in part produced locally. The model was also successfully used to identify toxic constituents of gliadin. Presently, organ culture is not commonly used for diagnostic purposes.

Celiac Disease↗

PubMed2002

Antifungal activity of amphotericin B, fluconazole, and voriconazole in an in vitro model of Candida catheter-related bloodstream infection.

The activity of five simulated antifungal regimens for eradication of catheter-related bloodstream Candida infection was evaluated with an in vitro pharmacodynamic model. Single-lumen central venous catheters were colonized with Candida species by sequentially incubating central venous catheters in plasma and then in growth medium (RPMI plus morpholinepropanesulfonic acid) containing a standardized suspension (10(5) CFU/ml) of Candida albicans, Candida glabrata, or slime-producing Candida parapsilosis. Colonized central venous catheters were then placed in a one-compartment pharmacodynamic model where five antifungal regimens (plus control) were simulated: amphotericin B, 1.0 mg/kg every 24 h; amphotericin B, 0.5 mg/kg every 24 h; fluconazole, 400 mg every 24 h; fluconazole, 800 mg every 24 h; and voriconazole, 4 mg/kg every 12 h. During exposure to the simulated clinical regimens, samples were serially removed from the model over 48 h for quantitation of viable organisms. All antifungal regimens suppressed fungal counts by both peripheral and catheter sampling versus control (P = 0.001). Overall, antifungal activity ranked amphotericin B (1 mg/kg) > amphotericin B (0.5 mg/kg) > or = voriconazole > fluconazole (800 mg) > or = fluconazole (400 mg). No regimen, however, completely eradicated (by culture and electron microscopy) central venous catheter colonization. Regrowth was noted in the model during therapy against C. glabrata and C. parapsilosis but was not associated with an increase in the MICs for the isolates. Lack of in vitro antifungal activity against biofilm-encased organisms appeared to be the primary reason for mycological failure of antifungal regimens in the model.

Amphotericin B↗

PubMed1982

Effect of the ratio of surface area to volume on the penetration of antibiotics in to extravascular spaces in an in vitro model.

The penetration of cephapirin into extravascular spaces of various volumes and ratios of surface area to volume (SA/V) was studied in an in vitro kinetic model. The SA/V ratio was found to be an important determinant of the kinetics of these spaces. The greater the surface area in relation to the volume of the extravascular space, the more closely its kinetics mimicked those of the intravascular space-that is, the higher the absolute peak concentration of drug achieved in the extravascular space, the greater the peak-to-trough fluctuation and the more quickly the peak concentration of drug was reached. Extravascular spaces with similar SA/V ratios demonstrated similar kinetics. Data from various models of extravascular fluid in humans and animals were reviewed, and the kinetics of these extravascular spaces were also found to be determined, at least in part, by the SA/V ratios of the spaces.

Cephalosporins↗

PubMed1985

Bactericidal activity of phenoxymethylpenicillin in an in-vitro model simulating tissue kinetics.

The antibacterial efficacy of phenoxymethylpenicillin (Pen-V-K) against strains of Staphylococcus aureus was assessed in an in-vitro kinetic model. Simulation was based on human serum levels and tissue water curves obtained after a single oral dose of 392.2 mg of the drug. Differences in bacterial elimination kinetics were noted depending upon the type of curve (serum or tissue water) being simulated.

Humans↗

PubMed2005

Drug delivery and in vitro models of the blood-brain barrier.

An understanding of the physiology of the blood-brain barrier (BBB) is crucial when addressing complex issues such as drug delivery, pathogenesis of chronic neurological diseases and bio-defense. Rational central nervous system (CNS) drug design cannot entirely and exclusively rely upon the physicochemical properties of putative neurotherapeutics, since lipophilicity alone is a poor predictor for drug penetration into the CNS. This is particularly true for three large families of CNS drugs: antineoplastics, antivirals and anti-epileptics. For these drugs, in contrast to peripheral acting drugs (eg, antihistamines), negligible penetration across the BBB is preferable in order to avoid CNS side effects. Studies performed using small animals such as rodents cannot be directly extrapolated to human brain tissue, as demonstrated by both clinical and in vitro studies. Furthermore, most of the promising CNS drugs that proved effective in vitro have failed in clinical trials due to misleading predictive permeability data extrapolated from models that were not capable of fully reproducing the functional properties of the BBB in vivo. Therefore, a great effort has been made to develop new in vitro models able to reproduce the physiological, anatomical and functional characteristics of the BBB allowing for a better prediction of drug penetration across the BBB, and enabling the design of new pharmaceutical strategies to bypass the shielding of brain parenchyma. Herein we provide a detailed review and discussion of currently employed in vitro BBB models along with probable future developments.

Animals↗

PubMed1993

An in vitro model for studies on bacterial interactions in the avian caecum.

An in vitro intermittent-flow model was developed for studying bacterial interactions in the avian caecum. The model provides a closer simulation of caecal conditions than others described previously but does not require elaborate instrumentation. In preliminary trials, growth of caecal bacteria from an adult chicken was shown to be inhibitory to both Salmonella infantis and entero-haemorrhagic Escherichia coli.

Animals↗

PubMed1999

A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation and the effect of aspirin.

We have developed a versatile in vitro chamber model with a double purpose: first, to be able to study mechanisms of bio-incompatibility, and, second, to test biomaterials at all levels of interactions, in whole blood. The use of biomaterials in the form of microscope slides as walls in the chamber makes it possible to analyse both the biomaterial surface with regard to protein and cell binding, as well as the molecular events taking place in the fluid. Incubation of blood in the chamber, for 60 min at 37 degrees C resulted in the rapid binding of complement and coagulation proteins and of leukocytes and platelets to polyvinylchloride (PVC) slides. The cells formed a layer which more or less covered the underlying surface. Unlike complement activation, as reflected by soluble C3a and C5b-9, the thrombin-antithrombin formation was completely nullified in cell-depleted plasma. Despite the fact that thrombin-antithrombin generation was also negligible in platelet-rich plasma, inhibition of platelet aggregation on the material surface with aspirin resulted in suppressed generation of thrombin antithrombin complexes. Taken together, the coagulation activation in the chamber was dependent on the presence of blood cells which suggests that bound/aggregated platelets initiate a sequence of events involving leukocytes that results in coagulation activation.

Anticoagulants↗

PubMed1985

Modulation of histamine release by fatty acids. A new in vitro model investigating adverse drug reactions in various species.

Histamine release caused by drugs and/or their solvents is a well known phenomenon. In this study, both in vivo (anaesthetized and conscious dogs) and in vitro (isolated rat peritoneal, human and guinea-pig lung mast cells) models were used. Cremophor E1 and six derivatives of 12-hydroxystearic acid were compared for their histamine releasing abilities. Although the three types of isolated mast cells responded similarly, histamine release being observed with DH (the diester of 12-hydroxystearic acid with polyethylene glycol), TN (12-hydroxystearic acid polymerized with ethylene oxide) and ME (the monoester of 12-hydroxystearic acid esterified with polyethylene glycol), the anaesthetized dog exhibited elevated blood histamine levels and clinical symptoms after administration of all the solubilizing agents, except TN and ME. The reasons for this discrepancy are not known. The addition of the drugs (Althesin or propanidid) to their solubilizing agents caused histamine release, which was not observed with the solubilizing agent alone. This is the first demonstration of an in vitro model, which copies the clinical situation, i.e. solvent does nothing but the solvent plus drug combination causes histamine release and hence adverse reactions.

Alfaxalone Alfadolone Mixture↗

PubMed2004

Adenosine acting via A1 receptors, controls the transition to status epilepticus-like behaviour in an in vitro model of epilepsy.

Adenosine has powerful inhibitory effects in the central nervous system. In this study, we aim to understand how adenosine controls the progression of seizure-like events (SLEs) in a seizure-prone region of the brain, the entorhinal cortex. We chose to use a low Mg(2+) model of epilepsy in an in vitro slice preparation where, in the entorhinal cortex, SLEs progress into a type of epileptiform activity called late recurrent discharges (LRDs) that bear resemblance to status epilepticus. Adenosine, acting via its A1 receptor, exerted powerful inhibitory effects to prevent the spontaneous progression to LRDs while the potent A1 receptor antagonist, DPCPX, accelerated the progression in a concentration dependent manner. The spontaneous progression from SLEs to LRDs was associated with a decline in total cellular ATP levels and studies with metabolic inhibitors indicated a key role for the production of endogenous adenosine from ATP. We therefore hypothesise that when ATP becomes rate limiting, extracellular adenosine levels fall, the normal inhibitory brake is removed and the progression from SLEs to LRDs or status epilepticus-like activity can ensue. Moreover, under these conditions, inhibition of the adenine nucleotide salvage pathways reversed the status epilepticus-like activity. Our findings suggest a powerful role for adenosine for the control of the progression to status epilepticus-like activity in an epilepsy model that is refractory to most anti-epileptic drugs. On this basis, manipulation of adenine nucleotide metabolism may represent a potential therapeutic approach for the treatment of status epilepticus.

Adenosine↗

PubMed2003

Regulation of persistent activity by background inhibition in an in vitro model of a cortical microcircuit.

We combined in vitro intracellular recording from prefrontal cortical neurons with simulated synaptic activity of a layer 5 prefrontal microcircuit using a dynamic clamp. During simulated in vivo background conditions, the cell responded to a brief depolarization with a sequence of spikes that outlasted the depolarization, mimicking the activity of a cell recorded during the delay period of a working memory task in the behaving monkey. The onset of sustained activity depended on the number of action potentials elicited by the cue-like depolarization. Too few spikes failed to provide enough NMDA drive to elicit sustained reverberations; too many spikes activated a slow intrinsic hyperpolarization current that prevented spiking; an intermediate number of spikes produced sustained activity. When high dopamine levels were simulated by depolarizing the cell and by increasing the amount of NMDA current, the cell exhibited spontaneous 'up-states' that terminated by the activation of a slow intrinsic hyperpolarizing current. The firing rate during the delay period could be effectively modulated by the standard deviation of the inhibitory background synaptic noise without significant changes in the background firing rate before cue onset. These results suggest that the balance between fast feedback inhibition and slower AMPA and NMDA feedback excitation is critical in initiating persistent activity and that the maintenance of persistent activity may be regulated by the amount of correlated background inhibition.

Action Potentials↗

PubMed1998

Impact of pharmacokinetics on the postantibiotic effect exhibited by Pseudomonas aeruginosa following tobramycin exposure: application of an in-vitro model.

The postantibiotic effect (PAE) exhibited by Pseudomonas aeruginosa after exposure to single doses of tobramycin was investigated under various pharmacokinetic conditions using an established in-vitro kinetic model. At equal doses of the antibiotic stimulating an intravenous bolus condition, the effects of varying elimination half-life on PAE were assessed. The PAE was longer when the rate of antibiotic elimination was lower. However, after correcting for the different degrees of antibiotic exposure using the area under the concentration-time curve above the MIC (AUC > MIC), a coherent PAE versus antibiotic exposure profile was obtained. The effects of increasing tobramycin dose and exposure time on PAE were investigated in another series of experiments; PAE was assessed during antibiotic exposure when the exponentially decreasing concentrations were above the MIC, at the MIC and below the MIC. A longer PAE was achieved at higher doses and changes were dependent on both the degree and time of exposure. For all the doses tested, the PAE was longest when the decreasing antibiotic concentrations were near or at the MIC. Shorter PAEs were detected at sub-MIC concentrations and diminished rapidly as antibiotic concentrations continued to decline. Such a decrease in PAE was counteracted by the longer exposure time, so that the total time for which the organism was under the influence of the antimicrobial effects, i.e. the sum of exposure time and PAE, remained steady at sub-MIC concentrations. Under these simulated pharmacokinetic conditions, present data support a substantial impact of pharmacokinetics on PAE. Along with MIC, AUC > MIC can be a useful pharmacokinetic parameter for PAE assessments. Should PAE be a relevant factor in antibiotic chemotherapy, both time and degree of antibiotic exposure would have to be considered.

Anti-Bacterial Agents↗

PubMedDate not supplied

Estradiol is a neuroprotective factor in in vivo and in vitro models of brain injury.

Many clinical studies suggest that estrogen enhances memory and cognition and protects against neurodegenerative diseases and injury associated with stroke or stress. These results are strongly supported by experiments performed in animal models using both in vivo and in vitro methods. We present here data from our lab that establishes that physiological levels of estradiol exert profound protective actions against ischemic injury. Further we will present evidence that these effects may be mediated through estrogen receptors that may influence the bcl-2 family of genes.

Animals↗

PubMed1980

Desmosome development in an in vitro model.

A model has been devised to study the in vitro formation of desmonsomes. The model is based on the differential labeling of two subpopulations of a desmosome-forming human cancer line (C4I). The labeled subpopulations are dispersed, preincubated separately on a shaking water bath for 24 h to allow the internalization of desmosome fragments and the repair of the cell surface, and then mixed, and allowed to aggregate. Aliquots of the mixed suspension are fixed at various intervals. The time between mixing and fixation represents the maximum age of any junction between dissimilarly labeled cells. The beginnings of desmosome formation were observed within a few minutes after the beginning of aggregation. Close apposition of cell membranes was seen immediately after mixing, followed within 15 min by the appearance of a submembrane density in one or both of the interacting cells. Intracytoplasmic filament formation takes place at between 15 and 30 min. Desmosome formation is complete by 90 min. The process is accompanied by a progressive widening of the extracellular space and the desification and organization of the extracellular material and the submembrane plaques.

Cell Adhesion↗

PubMed1985

[Bactericidal activity of aspoxicillin in an in vitro model simulating human serum levels].

Bactericidal activities of aspoxicillin (ASPC) against E. coli (2 strains) and K. pneumoniae (1 strain) were compared with those of piperacillin (PIPC) using in vitro kinetic models simulating human serum levels. In the model of intravenous injection, both drugs exhibited bactericidal action against the strains of E. coli. The activity of ASPC was found to superior to PIPC and ASPC could decrease viable cell counts from 10(7) cells/ml to 10(8) cells/ml. On the other hand, the bactericidal activity of ASPC against K. pneumoniae was weaker than that of PIPC. In the model of intravenous drip infusion, ASPC showed a high level of bactericidal activity comparable to that observed in the intravenous model against E. coli. Interestingly, the bactericidal activity of ASPC against K. pneumoniae in this model was similar to that of PIPC, though the MIC value of ASPC was higher than that of PIPC. In the intravenous model, the effects of ASPC and PIPC on the morphology of E. coli KC-14 were examined with a phase contrast microscope. Exposure to PIPC caused only an elongation of the cells, but the treatment with ASPC resulted in the formation of spheroplast-like structure of the cells, which were finally subjected to bacteriolysis.

Amoxicillin↗

PubMed2004

Antifungal drug response in an in vitro model of dermatophyte nail infection.

Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.

Antifungal Agents↗