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Biomedical subjects

C M Lehr

Publications and source records attributed to C M Lehr.

At least 19 recordsLinked to original sources
PubMed2002

Lectin-mediated drug delivery: discrimination between cytoadhesion and cytoinvasion and evidence for lysosomal accumulation of wheat germ agglutinin in the Caco-2 model.

Lectin-mediated drug delivery may become a promising strategy to improve the efficacy of poorly permeable drugs by utilising active high-capacity transport pathways of epithelial tissues. This requires the elucidation of the basic mechanisms of lectin uptake prior to their practical use. We studied the interaction between the dietary lectin wheat germ agglutinin (WGA) and Caco-2 cells (single cells and monolayers) by a newly established assay design that is able to discriminate between cellular binding and uptake as well as by confocal microscopy: (i) All binding sites available for WGA at the cell membrane were occupied within 10 min of incubation. (ii) Cytoadhesion was followed by immediate uptake. After 20 min, 60% (single cells) or 30% (monolayers) of the membrane bound lectin were internalised. However, regardless of cell arrangement, 80% of the surface bound lectin was taken up into the cells during the course of the experiment. (iii) About 50% of the internalised lectin accumulated within the lysosomes after 1 h. This was confirmed by assays in the presence of monensin, an inhibitor of endosomal acidification, and by colocalisation with lysosomal cathepsin followed by semiquantitative image analysis. Further analysis by immunocytochemistry suggested that the trans-Golgi complex and the caveoli were not involved. Due to cytoadhesion, cytoinvasion and partial lysosomal accumulation, WGA-mediated drug delivery may provide for improved intracellular availability of conjugated drugs or colloidal carrier systems.

Caco-2 Cells↗
PubMed2001

Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA.

The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1 (N,N-di-(beta-steaorylethyl)-N,N-dimethylammonium chloride) or cetylpyridinium chloride as charge carrier. The resulting particles were approximately 100 nm in size and showed zeta potentials around +40 mV at pH 7.4. DNA binding was tested by agarose gel electrophoresis. The resulting SLN-DNA complexes were further characterised by AFM and zeta potential measurements. Only the SLN batch SII-13, composed of 4% Compritol, 4% Tween/Span and 1% EQ1, was able to form stable complexes with DNA. Typical complexes were 300 to 800 nm in size. Cytotoxicity and transfection efficiency was tested in vitro on Cos-1 cells. Cationic SLN produced by modification with EQ1 were well tolerated, with LD50 values >3 mg/ml in the LDH release assay and >0.6 mg/ml in the WST-1 assay. Further, SLN-DNA complexes containing between 10 and 200 weight equivalents of SII-13 (matrix lipid) efficiently transfected the galactosidase expression plasmid pCMVbeta in the absence and presence of the endosomolytic agent chloroquine.

Animals↗
PubMed2001

Interrelation of permeation and penetration parameters obtained from in vitro experiments with human skin and skin equivalents.

In a comparative study, two different in vitro cutaneous test systems were examined: (1) The Franz diffusion cell (FD-C), a test system to study drug permeation through the skin and to obtain data like steady state flux and lag time as well as permeability and diffusion coefficients. (2) The Saarbruecken penetration model (SB-M), a test system to investigate drug penetration into different skin layers and after varying incubation times to acquire values about the quasi steady state drug amounts in the stratum corneum (SC). Three drug concentrations (0.9, 0.45 and 0.225%) of a lipophilic model drug preparation, flufenamic acid in wool alcohols ointment, were applied on the skin's surface using 'infinite dose' conditions. Trypsin-isolated SC, heat-separated epidermis, full-thickness skin and reconstructed human skin (RHS) served as skin membranes in the FD-C, while the SB-M experiments were only carried out using full-thickness skin. Increasing steady state flux data and m(ss) values (steady state drug amount in the SC) were detectable after the application of rising drug amounts. Concerning the permeability of the used skin membranes in establishing barrier properties, the following rank order was observed: RHS>SC> or =epidermis>full skin. The flux data of the FD-C experiments for isolated SC, separated epidermis and RHS were linearly related with the m(ss) values of the SB-M investigations, allowing a direct comparison of permeation with penetration parameters. Concerning the drug amount in the SC, previous investigations succeeded in the establishment of an in vivo/in vitro correlation. Based on the results presented here, the prediction of drug amounts present in the SC after different incubation times in vivo is now possible after penetration as well as permeation experiments using the lipophilic model drug preparation, flufenamic acid in wool alcohols ointment.

Epidermis↗
PubMed2001

Design of rolipram-loaded nanoparticles: comparison of two preparation methods.

The aim of the present work was to investigate the preparation of nanoparticles as a potential drug carrier and targeting system for the treatment of inflammatory bowel disease. Rolipram was chosen as the model drug to be incorporated within nanoparticles. Pressure homogenization-emulsification (PHE) with a microfluidizer or a modified spontaneous emulsification solvent diffusion method (SESD) were used in order to select the most appropriate preparation method. Poly(epsilon-caprolactone) has been used for all preparations. The drug loading has been optimized by varying the concentration of the drug and polymer in the organic phase, the surfactants (polyvinyl alcohol, sodium cholate) as well as the volume of the external aqueous phase. The rolipram encapsulation efficiency was high (>85%) with the PHE method in all cases, whereas with the SESD method encapsulation efficiencies were lower (<40%) when lower surfactant concentrations and reduced volume of aqueous phase were used. Release profiles were characterized by a substantial initial burst release with the PHE method (25-35%) as well as with the SESD method (70-90%). A more controlled release was obtained after 2 days of dissolution with the PHE method (70-90%), no further significant drug release was observed with the SESD method.

Algorithms↗
PubMed2001

Lectin-functionalized liposomes for pulmonary drug delivery: effect of nebulization on stability and bioadhesion.

The generation of respirable aerosols of a functionalized colloidal carrier has been investigated in this study. Lectin-functionalized liposomes, which proved to show improved cell association (using A549 cell line and primary human alveolar cells) even in the presence of a commercial lung surfactant preparation, have been developed. The stability of non-functionalized liposomes during nebulization using a jet nebulizer (Pari II provocation nebulizer, operated using an air flow of 30 l/min) was firstly investigated, and the experimental and formulation conditions were optimized and applied for the preparation of lectin-functionalized liposomes. The incorporation of cholesterol enhanced the stability of the liposomes during nebulization (from 15-20% leakage of a hydrophilic marker to 8% upon cholesterol incorporation) and upon incubation with lung surfactant preparation. Nebulization of the functionalized liposomes did not significantly influence their physical stability. Their enhanced cell binding capability (compared to non-functionalized liposomes) was also maintained. A drop in cell association compared to fresh functionalized liposomes was detected after nebulization, nevertheless, the binding was still significantly higher than that of the non-functionalized liposomes. The deposition of the liposomal preparation in lung periphery, proved by the deposition of the liposomal preparation on the lower stages of an ASTRA type cascade impinger and a mean median aerodynamic diameter (MMAD) of 2.85 microm, makes it a potential candidate as a macromolecule-drug carrier for local and/or systemic administration.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Systemic delivery of cetrorelix to rats by a new aerosol delivery system.

PURPOSE: To study the pulmonary absorption and tolerability of various formulations of the decapeptide cetrorelix acetate in rats by a new aerosol delivery system (ASTA-ADS) for intratracheal application. METHODS: Using the ASTA-ADS, cetrorelix liquid formulations (aqueous solutions for ultrasonic nebulization) were firstly selected and subsequently delivered as nebulized aerosol to orotracheally cannulated rats. The pharmacologic effect (decrease of testosterone serum level) of four cetrorelix formulations was determined in rats by enzyme linked immunosorbant assay, and pharmacokinetic data were determined after measurement of cetrorelix serum level by radioimmunoassay. Histological examination of the lung was performed at the end of the experiments, and in a supplementary experiment the respiratory parameters (resistance and compliance) of rats were monitored by a validated pulmonary monitoring system during the aerosol application of the same formulations. RESULTS: After an exposure time of 5 min, the applied formulations reduced the testosterone concentration in serum to subnormal levels (< or =1 ng/ml) over a period of 24 h. Comparing the plasma concentration after intratracheal aerosolization with data of intravenous administration, the mean calculated bioavailabilities for the four formulations using the corrected dose (delivered--exhaled amount) were between 48.4 +/- 27.0% and 77.4 +/- 44.0%. The histologic examination of the lungs revealed different tolerability of the various tested formulations ranging from locally intolerable to well tolerated. The measurement of the lung function parameters did not reveal any compound or formulation related changes. CONCLUSIONS: Our studies show that cetrorelix can be effectively administered as aerosol and that intratracheal aerosolization via the ASTA-ADS provides results that are well comparable to other application routes, as demonstrated by statistical comparison of the newly obtained data with previous results from intratracheal instillation of cetrorelix solutions in rats.

Administration, Inhalation↗
PubMed2001

Size-dependent bioadhesion of micro- and nanoparticulate carriers to the inflamed colonic mucosa.

PURPOSE: The size-dependent deposition of microparticles and nanoparticles after oral administration to rats using an experimental model colitis was examined. Local delivery of an entrapped drug could reduce side effects and would be a distinct improvement compared with existing colon delivery devices. METHODS: Ulcerative colitis was induced in Lewis rats with trinitrobenzenesulfonic acid. Fluorescent polystyrene particles with a size of 0.1, 1, or 10 microm were administered for 3 days. The animals then were sacrificed and their guts resected. Particle distribution in the colon was imaged by confocal laser scanning microscopy and quantified by fluorescence spectrophotometry. RESULTS: In the inflamed tissue, an increased adherence of particles was observed at the thicker mucus layer and in the ulcerated regions. A size dependency of the deposition was found, and an increased number of attached particles to the colon was determined compared with the control group. For 10-micorm particles, only fair deposition was observed (control group: 1.4 +/- 0.6%; colitis: 5.2 +/- 3.8% of administered particle mass). One-micrometer particles showed higher binding (control group: 2.0 +/- 0.8%; colitis: 9.1 +/- 4.2%). Highest binding was found for 0.1-microm particles (control group: 2.2 +/- 1.6%; colitis: 14.5 +/- 6.3%). The ratio of colitis/control deposition increased with smaller particle sizes. CONCLUSIONS: The use of submicron-sized carriers holds promise for the targeted delivery of drugs to the inflamed colonic mucosal areas in inflammatory bowel disease.

Adhesives↗
PubMed2001

Development of a new aerosol delivery system for systemic pulmonary delivery in anaesthetized and orotracheal intubated rats.

Over the last decade, the systemic absorption of a broad range of therapeutics after pulmonary application has been demonstrated in animals as well as in humans. The most common method used in the laboratory is the intratracheal instillation of drugs in solution. This method is, however, unsatisfactory, because of discrepancies in particle distribution, clearance, kind of injury and bioavailability between instillation and inhalative application. On the other hand, a precise determination of the amount of drug applied by aerosol, and of the aerosol volume retained within the lungs is rather difficult, and is not possible for use with small animals such as mice or rats. We describe a system which allows the delivery of aerosols directly into the animal's lungs, and calculation of the amount of drug retained in the lungs. Our system was tested in vitro and in vivo and was shown to allow precise and efficient pharmacokinetic and toxicological studies to be carried out.

Absorption↗
PubMed2001

Oral endotracheal intubation of rats for intratracheal instillation and aerosol drug delivery.

As reported in the literature, oral endotracheal intubation of rats is considered to be very difficult. Specialised equipment and complicated techniques have been described to perform this procedure. In our experiment we adopted a simple method, which allowed-without any complicated equipment-the insertion of a relatively wide tube into the trachea of rats, allowing drug administration.

Administration, Inhalation↗
PubMed2001

Lectin-functionalized liposomes for pulmonary drug delivery: interaction with human alveolar epithelial cells.

In this study the interaction of lectin-functionalized liposomes with two different alveolar epithelial cell culture models was evaluated. Plant lectins were coupled to liposomes exploiting the avidin/biotin technology. In contrast to lectin-free liposomes, lectin functionalized liposomes specifically bound to A549 cells, a tumor-derived cell line. Using this cell line, temperature-dependent binding assays as well as confocal laser scanning microscopy (CLSM) revealed that the lectin liposomes were only bound but not taken up by these cells. In contrast to these findings, confocal images of human alveolar epithelial cells in primary culture incubated together with lectin liposomes indicated binding as well as cellular uptake. Fluorescein-isothiocyanate (FITC)-labeled dextrans (Mw 40,000 Da), encapsulated in lectin-functionalized liposomes and incubated with monolayers of primary cultured human alveolar epithelial cells appeared to be localized intracellularly by CLSM. This suggests that lectin-mediated bioadhesion and uptake of liposomal carriers may provide a useful technology for improved delivery of hydrophilic macromolecules to the alveolar epithelium.

Cells, Cultured↗
PubMed2001

[In vitro models of intestinal and alveolar epithelium cultures in pharmaceutical research].

The progress of modern bio- and information-technology has made an enormous impact on the development of new drugs: At the one hand, computer-aided drug design and automated high-throughput screening has enormously facilitated the chemical synthesis of new drug candidates. At the other hand, the number of macromolecular biopharmaceuticals, such as peptides, proteins, antisense agents or gene vectors is continuously increasing. Whether or not such new entities can indeed be developed to safe and efficient medicines, is largely determined by the question, if these molecules are able to reach their actual target (receptor) within the patient"s body. Often, biological barriers, such as the mucosal epithelium of the gastrointestinal tract cannot be passed. In vitro test systems based on human epithelial cells may help to determine those candidate drugs which are well absorbed after oral application. Moreover, such cell culture models are helpful tools for the discovery for new delivery routes for those molecules, which cannot be administered by oral application. In this context, there is an increasing interest in the pulmonary delivery of drugs, as well as in the development of cell culture systems to model the blood-air barrier represented by the alveolar epithelium.

Animal Testing Alternatives↗
PubMed2001

Reconstructed skin equivalents for assessing percutaneous drug absorption from pharmaceutical formulations.

Excised human skin has so far been considered to be one of the most suitable in vitro methods to evaluate the penetration of dermatologically applied substances. The limited supply and the relatively high donor variability stimulated many research groups to use animal skin as a substitute for human skin. Since nowadays reconstructed skin equivalents are commercially available, we examined these cultures for their suitability as a percutaneous absorption model for different pharmaceutical formulations. One such equivalent is EpiDerm (EPI-606, MatTek corporation, Ashland Massachusetts) which was investigated using the lipophilic model drug flufenamic acid. Permeation studies with the Franz diffusion cell were undertaken to evaluate the model for the establishment of a new in vitro method to study the percutaneous absorption of different dosage forms. The drug was applied in two pharmaceutical formulations to the intact surface of the skin disk: dissolved in wool alcohol ointment (0.1125 %), and dissolved in Soerensen phosphate buffer pH 7.4 (0.1125% solution). HPLC was used for the analysis of drug content. It was shown that the model forms a barrier towards diffusion by comparing the permeation across the tissue-free inserts to the equivalents. Flux values were calculated and the permeation across the skin equivalent from the solution was noted to be almost forty times higher than from the ointment. Two different batches of the skin equivalent showed no statistically significant difference. Finally the permeability of the reconstructed skin was compared to human epidermis, and a five times higher flux value was found for the skin equivalent model. Our results suggest that reconstructed skin equivalents based on human keratinocytes have potential as a pharmaceutical test system to study dermal drug transport from topical formulations.

Administration, Cutaneous↗
PubMed2001

[Marker transport across biological barriers in vitro: comparison of cell culture models for the gastrointestinal barrier, the blood-brain barrier and the alveolar epithelium of the lung].

In order to respond to the flood of new active ingredients currently being generated by combinatorial chemistry or molecular biological synthesis, selection procedures able to filter out rapidly and economically those drug candidates with the highest development potential are required. This necessitates the measurement of fundamental biopharmaceutical parameters very early in the drug development process. Any pharmaceutically active agent must be able to overcome the body's natural protective mechanisms. A broad variety of biological barriers can be simulated in the laboratory by cell monolayer models. Apart from ethical aspects, the advantage of these in vitro test systems is that permeability studies can be performed at high throughput rates under controlled and reproducible conditions. The validity of such a model is ultimately reflected in its ability to accurately predict the behaviour of an active ingredient at the corresponding in vivo barrier.

Animal Testing Alternatives↗
PubMed2001

Biodegradable nanoparticles for targeted drug delivery in treatment of inflammatory bowel disease.

The use of nanoparticles for targeted oral drug delivery to the inflamed gut tissue in inflammatory bowel disease was examined. Such a strategy of local drug delivery would be a distinct improvement compared with existing colon delivery devices for this disease. An experimental colitis was induced by trinitrobenzenesulfonic acid to male Wistar rats. Rolipram, an anti-inflammatory model drug, was incorporated within poly(lactic-coglycolic acid) nanoparticles, which were administered once a day orally for five consecutive days. A clinical activity score and myeloperoxidase activity were determined to assess the inflammation, whereas an adverse effect index reflected the remaining neurotropic effect of rolipram resulting from its systemic absorption. All nanoparticle formulations proved to be as efficient as the drug in solution in mitigating the experimental colitis. The clinical activity score and myeloperoxidase activity decreased significantly after the oral administration of rolipram nanoparticles or solution. During the next 5 days when animals were kept without drug treatment the drug solution group displayed a strong relapse, whereas the nanoparticle groups continued to show reduced inflammation levels. The rolipram solution group had a high adverse effect index, whereas the rolipram nanoparticle groups proved their potential to retain the drug from systemic absorption as evidenced by a significantly reduced index. This new delivery system enabled the drug to accumulate in the inflamed tissue with higher efficiency than when given as solution. The nanoparticle deposition in the inflamed tissue should be given particular consideration in the design of new carrier systems for the treatment of inflammatory bowel disease.

Animals↗
PubMed2000

Charge-dependent interaction of self-emulsifying oil formulations with Caco-2 cells monolayers: binding, effects on barrier function and cytotoxicity.

A positively charged self-emulsifying oil formulation (SEOF), aimed to enhance oral bioavailability of drugs poorly soluble in water, was recently developed. In the present study the Caco-2 cell model was used for the investigation of the charge-dependent interactions of this SEOF with human intestinal epithelial cells. The positively charged emulsions affected the barrier properties of the cell monolayer at high concentrations and reduced the cell viability. However, at the dilution with aqueous phase used in the present study (1:2000), the positively charged SEOF did not induce any detectable cytotoxic effect. The binding of the fluorescent dye DiIC(18)(3) was much higher from the positively charged SEOF, compared to the negatively charged formulation, suggesting an increased closer adhesion of the droplets to the cell surface due to the electrostatic attraction. No transepithelial transport of this compound across Caco-2 cell monolayers was observed with any SEOF formulation.

Amines↗
PubMed2000

Biodegradable microparticles as a two-drug controlled release formulation: a potential treatment of inflammatory bowel disease.

A multiple unit dosage form for oral delivery based on the microencapsulation of anti-inflammatory drugs using different biodegradable polymers, poly(epsilon-caprolactone), polylactic acid and poly(lactic-co-glycolic acid), prepared either by the water-in-oil-in-water (w/o/w) or the solid-in-oil-in-water (s/o/w) solvent evaporation method was developed. Microparticles were characterized for their size, morphology, encapsulation efficiency and drug release. The physical state of drugs and polymers was determined by differential scanning calorimetry (DSC), imaging of the particles was performed by scanning electron microscopy and confocal laser scanning microscopy. Sulfasalazine and betamethasone used for the treatment of inflammatory bowel disease, were chosen as model drugs. The microparticles were spherical with diameters in the range of 91 to 258 microm by the w/o/w-method, and in the range of 102 to 277 microm by the s/o/w-method. The encapsulation efficiency (EE) varied between 11 and 16% for sulfasalazine and 50 and 67% for betamethasone with the w/o/w-method, and between 73 and 79% for sulfasalazine and 60 and 70% for betamethasone with the s/o/w-method. DSC showed no interaction between polymers and drugs, while the drugs were dispersed in the polymer. In vitro release studies showed a controlled release of sulfasalazine and betamethasone from microparticles prepared by the s/o/w-method; a pronounced burst release of sulfasalazine was observed from microparticles prepared by the w/o/w-method.

Betamethasone↗
PubMed2000

Visualization and quantification of polymer distribution in microcapsules by confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) was employed in order to characterize microcapsules. Microcapsules were prepared by complex coacervation: gelatin and arabic gum were labelled with fluorescent markers. In the capsule wall a homogeneous distribution for both gelatin and arabic gum throughout the capsule wall was depicted. By the use of CLSM and a computational image analysis the quantification of the labelled polymer in the wall material was possible. Adding fluorescently labelled casein as a macromolecular model compound to the coacervation process, a gradiental distribution in the wall material was observed with highest concentration of casein at the oil-wall interface. The influence of casein concentration on its deposition behaviour in the capsule wall was imaged successfully and thereafter quantified by computational image analysis.

Capsules↗