Biomedical subjects

P Artursson

Publications and source records attributed to P Artursson.

At least 19 recordsLinked to original sources

PubMed2005

Ketobemidone is a substrate for cytochrome P4502C9 and 3A4, but not for P-glycoprotein.

The role of the major drug-metabolizing cytochrome P450 (CYP) enzymes as well as P-glycoprotein (PGP) was investigated in the disposition of ketobemidone in vitro. Formation of norketobemidone from ketobemidone was studied and compared with the activities of 11 major CYP enzymes in human liver microsomes. The formation of norketobemidone from ketobemidone (1 microM) correlated best with CYP2C9 activity, measured as losartan oxidation (rs = 0.82, n = 19, p < 0.001), but there was also a strong correlation with CYP3A4 activity. Additionally, a good correlation was observed with CYP2C19, CYP2C8 and CYP2B6 at a ketobemidone concentration of 50 microM. Inhibition studies confirmed the involvement of CYP2C9 and CTP3A4 in the formation of norketobemidone. The formation rate of norketobemidone was three times higher in the CYP2C9*1*1 genotype group compared with the CYP2C9*1*2, CYP2C9*1*3 and CYP2C9*3*3 genotypes (p < 0.01). Treatment with verapamil as a PGP inhibitor did not affect the transport of ketobemidone in Caco-2 cells, indicating that PGP is not involved. The data suggest that CYP2C9 and CYP3A4 play a major role in the formation of norketobemidone at clinically relevant concentrations.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers.

Nonviral gene delivery systems based on conventional high-molecular-weight chitosans are efficient after lung administration in vivo, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow dissociation and release of plasmid DNA, resulting in a slow onset of action. We therefore developed highly effective nonviral gene delivery systems with improved physical properties from a series of chitosan oligomers, ranging in molecular weight from 1.2 to 10 kDa. First, we established structure-property relationships with regard to polyplex formation and in vivo efficiency after lung administration to mice. In a second step, we isolated chitosan oligomers from a preferred oligomer fraction to obtain fractions, ranging from 10 to 50-mers, of more homogeneous size distributions with polydispersities ranging from 1.01 to 1.09. Polyplexes based on chitosan oligomers dissociated more easily than those of a high-molecular-weight ultrapure chitosan (UPC, approximately a 1000-mer), and released pDNA in the presence of anionic heparin. The more easily dissociated polyplexes mediated a faster onset of action and gave a higher gene expression both in 293 cells in vitro and after lung administration in vivo as compared to the more stable UPC polyplexes. Already 24 h after intratracheal administration, a 120- to 260-fold higher luciferase gene expression was observed compared to UPC in the mouse lung in vivo. The gene expression in the lung was comparable to that of PEI (respective AUCs of 2756+/-710 and 3320+/-871 pg luciferase x days/mg of total lung protein). In conclusion, a major improvement of chitosan-mediated nonviral gene delivery to the lung was obtained by using polyplexes of well-defined chitosan oligomers. Polyplexes of oligomer fractions also had superior physicochemical properties to commonly used high-molecular-weight UPC.

Animals↗

PubMed2003

Relationship between the physical shape and the efficiency of oligomeric chitosan as a gene delivery system in vitro and in vivo.

BACKGROUND: Chitosans of high molecular weights have emerged as efficient nonviral gene delivery systems, but the properties and efficiency of well-defined low molecular weight chitosans (<5 kDa) have not been studied. We therefore characterized DNA complexes of such low molecular weight chitosans and related their physical shape and stability to their efficiency as gene delivery systems in vitro and in vivo. METHODS: Individual complexes between six different chitosan oligomers (6-, 8-, 10-, 12-, 14- and 24-mers) and fluorescence-labeled T4 DNA were visualized and classified into six physical shapes using video-enhanced fluorescence microscopy. The effects of chitosan chain length, charge ratio (+/-) and solvent properties (pH and ionic strength) on the stability and structure of the complexes were studied. Gene expression in vitro and in vivo were studied using a luciferase reporter gene. RESULTS: Free DNA appeared as extended coils. Chitosan complexes had a variety of physical shapes depending on the experimental conditions. In general, the fraction of complexes that had nonaggregated, globular structures increased with increasing chain length of the chitosan oligomer, increasing charge ratio and reduction of pH (from 6.5 to 3.5). A further increase in charge ratio for globular complexes or a further reduction in pH (to 2.5) increased the fraction of aggregates, indicating a window where pharmaceutically desirable globules are obtained. Gene transfection efficiencies in vitro and in vivo were related to the physical shape and stability of the complexes. Only the 24-mer formed stable complexes that gave a high level of gene expression comparable to that of high molecular weight ultrapure chitosan (UPC) in vitro and in vivo. CONCLUSIONS: Chitosan oligomers form complexes with DNA in a structure-dependent manner. We conclude that the 24-mer, which has more desirable physical properties than UPC, is more attractive as a gene delivery system than the conventional high molecular weight chitosans.

Adjuvants, Pharmaceutic↗

PubMed2003

PEI - a potent, but not harmless, mucosal immuno-stimulator of mixed T-helper cell response and FasL-mediated cell death in mice.

Polyethyleneimine (PEI) is one of the most effective gene delivery systems available today. However, very little is known about its ability to stimulate a systemic immune response and the molecular mechanisms thereof. However, this information is vital for the future development of new gene delivery systems. Here we address this issue by studying gene expression profiles from spleen lymphocytes after in vivo immunization of mice with PEI formulated with a reporter plasmid (PEI+) or the formulation alone (PEI-). PEI- was found to provoke the activation of genes with important immunostimulatory functions, but without the necessary costimulatory signals. PEI+ resulted in: a mixed Th1/Th2 response; activation of both CD8(+) and CD4(+) T cells, with a larger effect on CD4(+); and FasL-mediated antigen-induced cell death. A comparison of the immune responses of PEI+ with that of the clinically used tetanus toxoid-aluminum phosphate vaccine showed that the DNA vaccine provoked a stronger immune response as compared to the protein vaccine. However, many genes involved in other cellular responses such as apoptosis, stress responses and oncogenesis were activated in PEI+, supporting the theory of immunostimulation by danger genes, but also pointing toward possible adverse reactions such as Alzheimer's disease.

Animals↗

PubMed2002

Tetanus antigen modulates the gene expression profile of aluminum phosphate adjuvant in spleen lymphocytes in vivo.

Adjuvants play an important role in stimulation of the immune response to antigens. Very little is known about the molecular mechanisms of this stimulation. Here we address this issue by studying gene expression profiles from spleen lymphocytes after in vivo immunization of mice with a clinically relevant vaccine, tetanus toxoid formulated with aluminum phosphate as adjuvant (TT(ADJ)), or the adjuvant alone (ADJ). The Th1/Th2 response to TT(ADJ) was obtained from a combination of up- and downstream markers to conventional cytokines, which were in good agreement with cytokine protein levels. A clustering algorithm revealed that ADJ elicited expression of 47 genes active in cytotoxic lymphocytes, inflammation, oncogenesis, stress, toxicity and cell cycle regulation. In TT(ADJ) these adjuvant-elicited genes were expressed at lower levels and a compensatory onset of protective and inhibitory genes was observed. We conclude that the antigen, to a larger extent than previously recognized, modulates the molecular mechanism of the aluminum phosphate adjuvant and that the identified genes may serve as predictive biomarkers in the development of new adjuvants and vaccines.

Adjuvants, Immunologic↗

PubMed2001

Experimental and computational screening models for the prediction of intestinal drug absorption.

The aim of this study was to devise experimental protocols and computational models for the prediction of intestinal drug permeability. Both the required experimental and computational effort and the accuracy and quality of the resulting predictions were considered. In vitro intestinal Caco-2 cell monolayer permeabilities were determined both in a highly accurate experimental setting (Pc) and in a faster, but less accurate, mode (Papp). Computational models were built using four different principles for generation of molecular descriptors (atom counts, molecular mechanics calculations, fragmental, and quantum mechanics approaches) and were evaluated for their ability to predict intestinal membrane permeability. A theoretical deconvolution of the polar molecular surface area (PSA) was also performed to facilitate the interpretation of this composite descriptor and allow the calculation of PSA in a simplified and fast mode. The results indicate that it is possible to predict intestinal drug permeability from rather simple models with little or no loss of accuracy. A new, fast computational model, based on partitioned molecular surface areas, that predicts intestinal drug permeability with an accuracy comparable to that of time-consuming quantum mechanics calculations is presented.

Biological Transport↗

PubMed2001

Caco-2 monolayers in experimental and theoretical predictions of drug transport.

This review examines the use of Caco-2 monolayers in the prediction of intestinal drug absorption. First, the different routes of drug transport in Caco-2 monolayers are compared with those seen in vivo. Second, the prediction of drug absorption in vivo from transport experiments in cell monolayers is discussed for different classes of drugs. Finally, the use of Caco-2 monolayers as a reference model in physico-chemical and theoretical predictions of drug absorption is discussed. We conclude that Caco-2 monolayers can be used to identify drugs with potential absorption problems, and possibly also to select drugs with optimal passive absorption characteristics from series of pharmacologically active molecules generated in drug discovery programs.

Biological Transport, Active↗

PubMed2001

CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies.

The aim of this study was to find a cell culture model of the intestinal epithelium for use in studies of CYP3A4-mediated first-pass metabolism of drugs and also for studies of the interplay between CYP3A4 metabolism and P-glycoprotein efflux. For this purpose, the expression of CYP3A4, CYP3A5, and MDR1 mRNA was studied in three cell lines of the normal human intestinal epithelium and three transformed cell lines of colonic (Caco-2) origin. Surprisingly, only transformed cell lines were induced by 1alpha,25-dihydroxy vitamin D3 (D3) to express high amounts of CYP3A4. In contrast to the original findings for this model, the monolayer integrity was maintained during D3 treatment. Levels of CYP3A mRNA expression in Caco-2 and TC7 cells differed dramatically. The highest levels of CYP3A4 and lowest levels of CYP3A5 mRNA expression were observed in D3 treated Caco-2 cells of high passage numbers, resulting in a CYP3A4/3A5 expression ratio greater than fourfold higher than that seen in TC7 cells. Functional studies, using the CYP3A probe testosterone, showed that CYP3A activity was completely absent only in uninduced Caco-2 cells. After D3 induction, high levels of the metabolite were produced in both cell lines (149.4 +/- 12.3 and 86.5 +/- 3.8 pmol 6beta-OH testosterone/min/mg cellular protein from 75 microM testosterone in Caco-2 and TC7 cells, respectively). The maximum velocity (Vmax) and the apparent Michaelis constant (Km) for the 6beta-hydroxylation of testosterone by CYP3A4 in intact Caco-2 monolayers were similar to those obtained from human intestinal microsomes. Only minor changes in P-glycoprotein activity were observed when the metabolically stable P-glycoprotein substrate celiprolol was used. In conclusion, these results show that the features of the generally available Caco-2 cell line from American Type Culture Collection make it suitable for studies of CYP3A4-mediated first-pass metabolism and also for studies of the interplay between CYP3A4 and drug efflux mechanisms.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2001

Transport of lipophilic drug molecules in a new mucus-secreting cell culture model based on HT29-MTX cells.

PURPOSE: A new mucus-secreting in vitro drug absorption model based on monolayers of goblet-cell like sub-clones of the human colon carcinoma cell line HT29 obtained by methotrexate (MTX) treatment was investigated. METHODS: Twelve sub-clones were isolated and characterized by light microscopy (LM), transelectron microscopy (TEM), confocal laser scanning microscopy (CLSM), transepithelial electrical resistance (TEER) and the transport of a paracellular marker FITC-Dextran (Mw 4400) (FD-4). RESULTS: Significant differences of microscopical appearance, TEER-values and permeability of FD-4 between the sub-clones were evident. However, two of them, namely MTX-D1 and MTX-E12. formed tight confluent monolayers with a thick mucus-layer on the apical surface. They were used to compare the apparent permeability coefficient (Papp) of a series of lipophilic drugs, which should be affected by the mucus-layer, namely barbiturates (barbituric acid, barbital, phenobarbital, methylphenobarbital and heptabarbital) and testosterone, as a reference, to mucus-free Caco-2 cells. The permeability of drugs with a partition coefficient (log P) > 1 was decreased in the mucus-producing cell lines. Testosterone, the most lipophilic compound, showed a decrease of up to 43%. CONCLUSIONS: We demonstrated that the mucus layer is a significant barrier to drug absorption for lipophilic drugs. In conclusion, our model may serve as a suitable in-vitro cell culture model to study the influence of the mucus layer on drug diffusion.

Algorithms↗

PubMed2001

Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo.

Chitosan is a natural cationic linear polymer that has recently emerged as an alternative nonviral gene delivery system. We have established the relationships between the structure and the properties of chitosan-pDNA polyplexes in vitro. Further, we have compared polyplexes of ultrapure chitosan (UPC) of preferred molecular structure with those of optimised polyethylenimine (PEI) polyplexes in vitro and after intratracheal administration to mice in vivo. Chitosans in which over two out of three monomer units carried a primary amino group formed stable colloidal polyplexes with pDNA. Optimized UPC and PEI polyplexes protected the pDNA from serum degradation to approximately the same degree, and they gave a comparable maximal transgene expression in 293 cells. In contrast to PEI, UPC was non toxic at escalating doses. After intratracheal administration, both polyplexes distributed to the mid-airways, where transgene expression was observed in virtually every epithelial cell, using a sensitive pLacZ reporter containing a translational enhancer element. However, the kinetics of gene expression differed - PEI polyplexes induced a more rapid onset of gene expression than UPC. This was attributed to a more rapid endosomal escape of the PEI polyplexes. Although this resulted in a more efficient gene expression with PEI polyplexes, UPC had an efficiency comparable to that of commonly used cationic lipids. In conclusion, this study provides insights into the use of chitosan as a gene delivery system. It emphasises that chitosan is a nontoxic alternative to other cationic polymers and it forms a platform for further studies of chitosan-based gene delivery systems.

Cell Line↗

PubMed2001

Correlation of gene expression of ten drug efflux proteins of the ATP-binding cassette transporter family in normal human jejunum and in human intestinal epithelial Caco-2 cell monolayers.

This investigation describes the expression and interindividual variability in transcript levels of multiple drug efflux systems in the human jejunum and compares the expression profiles in these cells with that of the commonly used Caco-2 cell drug absorption model. Transcript levels of ten-drug efflux proteins of the ATP-binding cassette (ABC) transporter family [MDR1, MDR3, ABCB5, MRP1-6, and breast cancer resistance protein (BCRP)], lung resistance-related protein (LRP), and CYP3A4 were determined using quantitative polymerase chain reaction in jejunal biopsies from 13 healthy human subjects and in Caco-2 cells. All genes except ABCB5 were expressed, and transcript levels varied between individuals only by a factor of 2 to 3. Surprisingly, BCRP and MRP2 transcripts were more abundant in jejunum than MDR1 transcripts. Jejunal transcript levels of the different ABC transporters spanned a range of three log units with the rank order: BCRP approximately MRP2 > MDR1 approximately MRP3 approximately MRP6 approximately MRP5 approximately MRP1 > MRP4 > MDR3. Furthermore, transcript levels of 9 of 10 ABC transporters correlated well between jejunum and Caco-2 cells (r2 = 0.90). However, BCRP exhibited a 100-fold lower transcript level in Caco-2 cells compared with jejunum. Thus, the expression of a number of efflux protein transcripts in jejunum are equal to, or even higher than, that of MDR1, suggesting that the roles of these proteins (in particular BCRP and MRP2) in intestinal drug efflux have been underestimated. Also, we tentatively conclude that the Caco-2 cell line is a useful model of jejunal drug efflux, if the low expression of BCRP is taken into account.

ATP-Binding Cassette Transporters↗

PubMed2000

Expression of specific markers and particle transport in a new human intestinal M-cell model.

The aim of this work was to establish a new, simplified in vitro model of the human M-cell. Cocultures of physically separated human intestinal epithelial Caco-2 cells and B-cell lymphoma Raji cells were established. The cocultures were characterized under the criteria of morphology, integrity, expression of M-cell markers and cell adhesion molecules (CAMs), and altered particle transport. Using this construct, the epithelial cells were transformed to cells with an M-cell-like morphology and had altered expression of potential human M-cell markers (alkaline phosphatase down-regulation and Sialyl Lewis A antigen up-regulation). The expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule was altered and there was an increased binding of lectins wheat germ agglutinin and peanut agglutinin with a 40-fold increase in microparticle transport. The particle transport was size-dependent and could be inhibited at 4 degrees C or by replacing the Raji B-cells with Jurkat T-cells. This new coculture model will enable controlled studies of M-cell development and function in vitro.

Alkaline Phosphatase↗

PubMed2000

Virtual screening of intestinal drug permeability.

Lead compounds generated in high throughput drug discovery programmes often have unfavorable biopharmaceutical properties, resulting in a low success rate of such drug candidates in clinical development. Drug companies and researchers would thus like to have methods of predicting biopharmaceutical properties accurately. The intestinal permeability to a lead compound is one such property which is particularly important. Therefore, access to methods to accurately predict biopharmaceutical properties, such as the intestinal permeability of a large series of compounds, is of particular importance. This review deals with new theoretical methods used to predict intestinal drug permeability. There are several possible transport routes across the intestine, but theoretical methods generally deal with only one of them, the passive transcellular route. Therefore, this review will also discuss the relative importance of passive and active drug transport and efflux routes using recent data generated in cell cultures, animal models and human subjects.

Animals↗

PubMed2000

A new method for preparing biodegradable microparticles and entrapment of hydrocortisone in DL-PLG microparticles using supercritical fluids.

An improved process for the production of polymeric microparticles, based on solution-enhanced dispersion by supercritical fluids (SEDS) using a combination of supercritical N(2) and CO(2), was evaluated. The biodegradable polymers, poly(D,L-lactide-co-glycolide): copolymer composition 50:50 (DL-PLG), poly(L-lactide) (L-PLA), poly(D,L-lactide) (DL-PLA) and polycaprolactone, were used for preparation of microparticles by a modified SEDS process. Solutions of the polymers in organic solvents were dispersed and the solvent was extracted with supercritical CO(2) and N(2). The morphology, the size distributions and degree of hydrocortisone entrapment were determined. The combination of supercritical N(2) and CO(2) led to a more efficient dispersion of the polymer solutions than CO(2) alone. This resulted in a reduction of particle size of the microparticles produced from all of the amorphous polymers. Discrete spherical microparticles with a mean volumetric diameter of less than 10 microm were produced from DL-PLG, DL-PLA and L-PLA. Hydrocortisone was successfully entrapped within the DL-PLG microparticles. The modified SEDS process improved the dispersion of amorphous polymer solutions resulting in formation of small spherical microparticles. The SEDS process can be used for incorporation of drugs into the DL-PLG microparticles.

Anti-Inflammatory Agents↗

PubMed1999

Novel lipoamino acid- and liposaccharide-based system for peptide delivery: application for oral administration of tumor-selective somatostatin analogues.

Lipoamino acid and liposaccharide conjugates of somatostatin analogue TT-232 were synthesized to modify the physicochemical properties of the parent peptide. The relative position, the number, and the nature of the lipid and/or saccharide moieties were varied. Experiments in vitro clearly showed that many compounds modified at the N- and/or C-terminus with lipid or sugar moieties retained the biological activity of the parent compound. An interesting construct was synthesized containing lipid and sugar units at opposite ends of the somatostatin analogue, so that the entire molecule could be considered as an amphipathic surfactant.

Administration, Oral↗

PubMed1999

Encapsulation of rotavirus into poly(lactide-co-glycolide) microspheres.

Two small-scale double emulsion techniques for incorporation of formaldehyde-inactivated rotavirus particles (FRRV) into poly(lactide-co-glycolide) (PLG) microspheres were developed and optimised. The effects of high-speed homogenisation versus vortex mixing on the double emulsion stability, microsphere size, entrapment efficiency and in vitro release of FRRV in the second emulsification step were studied. A stable double emulsion was verified only when using vortex mixing in this step. Slow removal of the organic phase allowed measurement of the size of the emulsion droplets and subsequent prediction of the size of the resulting microspheres. Microspheres in the size range of 1-10 microm were prepared using both techniques. The homogenisation technique was sensitive to changes in the operating time, the emulsification energy and the volume of the outer aqueous phase, while the vortex technique was more robust. Rotavirus was released in vitro in a triphasic manner with both techniques. The more robust vortex technique was selected for preparation of PLG microspheres containing rotavirus for in vivo studies. After immunisation of mice with a single intramuscular injection, the PLG-FRRV microspheres elicited an IgG antibody response in serum detected by ELISA equally high as that elicited with FRRV alone. These results indicate that the antigenicity of FFRV was retained after incorporation into PLG microspheres using the vortex technique.

Animals↗

PubMed1999

Theoretical calculation and prediction of intestinal absorption of drugs in humans using MolSurf parametrization and PLS statistics.

A method for modeling and prediction of the intestinal absorption of drugs in humans using theoretically computed molecular descriptors and multivariate statistics has been investigated using 20 diverse drug-like compounds. The program MolSurf was used to compute theoretical molecular descriptors related to physicochemical properties such as lipophilicity, polarity, polarizability and hydrogen bonding. The multivariate Partial Least Squares Projections to Latent Structures (PLS) method was used to delineate the relationship between the intestinal absorption of drugs in humans and the theoretically computed molecular descriptors.Good statistical models were derived. Properties associated with hydrogen bonding had the largest impact on absorption and should be kept to a minimum to promote high absorption. High charge-transfer properties and the presence of surface electrons, i.e. valence electrons, which are not tightly bonded to the molecule, were also found to promote high absorption.

Computer Simulation↗

PubMed1999

Chitosans as absorption enhancers of poorly absorbable drugs. 3: Influence of mucus on absorption enhancement.

Chitosans are potent nontoxic absorption enhancers after nasal administration but their effects on the intestinal epithelium in vivo has not been studied in detail. In this study, the effects of chitosans with varying molecular weights and degrees of acetylation on the absorption of a poorly absorbed model drug (atenolol) were studied in intestinal epithelial cell layers with or without a mucus layer and in an in situ perfusion model of rat ileum. The effects of the chitosans on epithelial morphology and release of lactate dehydrogenase (LDH) into the perfusate were investigated in the in situ model. The chitosans had pronounced effects on the permeability of mucus-free Caco-2 layers and enhanced the permeation of atenolol 10- to 15-fold, with different absorption kinetics for different chitosans, in accordance with previous results. In contrast, enhancement of atenolol absorption through rat ileum was modest. LDH release from the tissues perfused with chitosans did not increase, indicating that the chitosans were used at nontoxic concentrations. Morphological examination of the perfused ileal tissues revealed more mucus discharge from the tissues exposed to chitosans than from controls, which suggested that the discharged mucus may inhibit the binding of chitosan to the epithelial surface and hence decrease the absorption-enhancing effect. This hypothesis was supported by studies with intestinal epithelial HT29-H goblet cells covered with a mucus layer. The binding of chitosan to the epithelial cell surface and subsequent absorption-enhancing effects were significantly reduced in mucus-covered HT29-H cultures. When the mucus layer was removed prior to the addition of chitosan, the cell surface binding and absorption-enhancing effects of the chitosans were increased. We conclude that the modest absorption-enhancing effects of unformulated chitosan solutions in the perfused rat ileum are a result of the mucus barrier in this tissue. This effect may be overcome by increasing the local concentrations of both chitosan and drug, i.e,. through formulation of the chitosan into a particulate dosage form.

Adrenergic beta-Antagonists↗