| 1. |
- Ahlin, Gustav, 1977-, et al.
(författare)
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Genotype-dependent effects of inhibitors of the organic cation transporter, OCT1: : predictions of metformin interactions
- 2011
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Ingår i: The Pharmacogenomics Journal. - : Springer Science and Business Media LLC. - 1470-269X .- 1473-1150. ; 11:6, s. 400-411
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Tidskriftsartikel (refereegranskat)abstract
- Common genetic variants of the liver-specific human organic cation transporter 1 (OCT1; SLC22A1) have reduced transport capacity for substrates such as the antidiabetic drug metformin. The effect of the reduced OCT1 function on drug interactions associated with OCT1 has not been investigated and was, therefore, the focus of the study presented here. HEK293 cells expressing human OCT1-reference or the variants R61C, V408M, M420del and G465R were first used to study the kinetics and inhibition pattern of the OCT1 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). In the second part OCT1-mediated (14)C-metformin uptake was studied in the presence of drugs administered concomitantly with metformin. Transport studies using ASP(+) showed that the function of the variants decreased in the following order: OCT1-reference = V408M = M420del >R61C > >G465R. Variants M420del and R61C were more sensitive to drug inhibition, with IC(50) values up to 23 times lower than those of the OCT1-reference. Uptake studies using (14)C-metformin were in qualitative agreement with those using ASP(+), with the exception that a larger reduction in transport capacity was observed for M420del. Concomitantly administered drugs, such as verapamil and amitriptyline, revealed potential drug-drug interactions at clinical plasma concentrations of metformin for OCT1-M420del.
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| 2. |
- Avdeef, Alex, et al.
(författare)
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Caco-2 permeability of weakly basic drugs predicted with the Double-Sink PAMPA method :
- 2005
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Ingår i: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987. ; 24:4, s. 333-49
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to analyze pH-dependent permeability of cationic drugs in Caco-2 cell monolayers using the method and to correlate the results with those obtained in PAMPA (parallel artificial membrane permeability assay). The pH-dependent permeability of verapamil and propranolol was studied in Caco-2 cell monolayers. The data were subsequently processed using software developed for the PAMPA method. Literature values for an additional nine cationic drugs were also analyzed. Double-Sink PAMPA data were also obtained for the same cationic drugs, to compare with the Caco-2 data. The Algorithm Builder program was then used to develop a predictive model of Caco-2 permeability based on PAMPA permeability and calculated Abraham molecular descriptors. From the relationship between permeability and pH it was shown that in PAMPA only the uncharged form of the drugs permeated across the membrane barrier, while charged and ionized forms of the drugs were significantly permeable in Caco-2. The charged-form permeability, Pi, was therefore determined and subsequently subtracted from all permeability coefficients in Caco-2 prior to the comparison with PAMPA. The resulting intrinsic permeability coefficients (Po) obtained in Caco-2 were successfully related to those derived from the PAMPA model. In this study we have shown that permeability coefficients obtained in PAMPA can predict the passive transcellular permeability in Caco-2.
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| 3. |
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| 4. |
- Bergström, Christel A S, et al.
(författare)
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Absorption classification of oral drugs based on molecular surface properties
- 2003
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 46:4, s. 558-570
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate whether easily calculated and comprehended molecular surface properties can predict drug solubility and permeability with sufficient accuracy to allow theoretical absorption classification of drug molecules. For this purpose, structurally diverse, orally administered model drugs were selected from the World Health Organization (WHO)'s list of essential drugs. The solubility and permeability of the drugs were determined using well-established in vitro methods in highly accurate experimental settings. Descriptors for molecular surface area were generated from low-energy conformations obtained by conformational analysis using molecular mechanics calculations. Correlations between the calculated molecular surface area descriptors, on one hand, and solubility and permeability, on the other, were established with multivariate data analysis (partial least squares projection to latent structures (PLS)) using training and test sets. The obtained models were challenged with external test sets. Both solubility and permeability of the druglike molecules could be predicted with high accuracy from the calculated molecular surface properties alone. The established correlations were used to perform a theoretical biopharmaceutical classification of the WHO-listed drugs into six classes, resulting in a correct prediction for 87% of the essential drugs. An external test set consisting of Food and Drug Administration (FDA) standard compounds for biopharmaceutical classification was predicted with 77% accuracy. We conclude that PLS models of easily comprehended molecular surface properties can be used to rapidly provide absorption profiles of druglike molecules early on in drug discovery.
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| 5. |
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| 6. |
- Englund, Gunilla, et al.
(författare)
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Regional levels of drug transporters along the human intestinal tract : Co-expression of ABC and SLC transporters and comparison with Caco-2 cells
- 2006
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Ingår i: European Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0928-0987 .- 1879-0720. ; 29:3-4, s. 269-277
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Tidskriftsartikel (refereegranskat)abstract
- A vast number of drugs are subjected to active or facilitated transport and multiple transport mechanism may contribute to the net flux during drug absorption. The main objective of this study was to quantify the regional mRNA expression and determine the co-expression of drug transporters from the ABC (Pgp, BCRP, MRP2, MRP3) and SLC (PEPT1, MCT1, OATPB, OCTN2, OCT1) families along the human intestine (duodenum, jejunum, ileum, and colon). A second objective was to compare the transporter expression between the different intestinal regions and Caco-2 cells. Eight out of nine of the investigated transporters exhibited significant regional differences in expression. OATPB was the only transporter that did not show a region-dependency in the expression along the human intestinal canal. The expression of Pgp, BCRP, OCTN2 and MCT1 differed along the small intestine, but the expression differences were greater than five-fold only for Pgp. The rank order of transcript prevalence was identical in the ileum and the jejunum. Between the ileum and colon, seven transcripts were differentially expressed, and MCT1, OCTN2 and MRP3 were expressed at higher levels in the colon than in the small intestine. The expression of transporters in Caco-2 was closest to the expression pattern in the small intestine, although the expression of OATPB, BCRP and MRP2 differed more than five-fold between the Caco-2 cells and ileum. In conclusion, this study provides quantitative data on the expression of transporters from the ABC and SLC families along the human intestine, which can be useful in the interpretation of clinical studies where more than one intestinal transporter contribute to the net transport and in the computer modelling of drug absorption.
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| 7. |
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| 8. |
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| 9. |
- Lazorova, Lucia, et al.
(författare)
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Structural Features Determining the Intestinal Epithelial Permeability and Efflux of Novel HIV-1 Protease Inhibitors
- 2011
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Ingår i: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 100:9, s. 3763-3772
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Tidskriftsartikel (refereegranskat)abstract
- The primary aim of this study was to identify structural features that alter the intestinal epithelial permeability and efflux in a series of novel HIV-1 protease inhibitors (PIs). Eleven PIs were selected containing a tertiary alcohol in a transition-state mimicking scaffold, in which two substituents (R1 and R2) were varied systematically. Indinavir was selected as a reference compound. The apical-to-basolateral permeability was investigated in 2/4/A1 and Caco-2 monolayers. In addition, the basolateral-to-apical permeability was investigated in the Caco-2 monolayers and the efflux ratios were calculated. The absence of active drug transport processes in 2/4/A1 cells allowed identification and modeling of structural elements affecting the passive permeability. For instance, small aromatic R1 substituents and a small (bromo-) R2 substituent were associated with a high passive permeability. Efflux studies in Caco-2 cells indicated that amide-substituted neutral hydrophobic amino acids, such as valine and leucine, in the R1 position, reduced the apical-to-basolateral transport and enhanced the efflux. We conclude that our investigation revealed structural features that alter the intestinal epithelial permeability and efflux in the series of PIs and hope that these results can contribute to the synthesis of PIs with improved permeability and limited efflux properties.
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| 10. |
- Lazorova, Lucia, et al.
(författare)
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Transport of an influenza virus vaccine formulation (iscom) in Caco-2 cells
- 1996
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Ingår i: American Journal of Physiology. - 0002-9513 .- 2163-5773. ; 270:4, s. G554-G564
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Tidskriftsartikel (refereegranskat)abstract
- The influenza virus envelope glycoproteins hemagglutinin and neuraminidase were administered to the apical or basolateral sides of Caco-2 monolayers either as native protein micelles (mic-ag) or after incorporation into the orally active adjuvant formulation, immune stimulating complexes (iscoms) (isc-ag). Biotin-conjugated isc-ag were localized in intracellular vesicles as early as 2 min after administration to the apical side at 37 degrees C. Ten minutes after administration, both intracellular vesicles and intercellular spaces were labeled, and extracellular labeling was observed on the basolateral side of the cells, indicating that isc-ag were transported across the epithelium within 10 min of exposure. Transport of 125I-labeled isc-ag and mic-ag in the apical-to-basolateral and basolateral-to-apical directions across Caco-2 monolayers was comparable at 37 degrees C. Gel chromatography analysis revealed that only 0.55-3.1% of transported isc-ag and mic-ag had a molecular weight of > 5,000, while 21.0-42.3% was eluted at a position corresponding to peptides of approximately 10 amino acids. Although isc-ag and mic-ag were transported and degraded by Caco-2 monolayers in comparable amounts, only transported isc-ag induced a dose-dependent proliferative response in vitro of T cells primed with influenza virus antigen. High-performance gel chromatography and reverse-phase high-performance liquid chromatography indicated that transported antigenic isc-ag consisted of hydrophobic peptides with a molecular weight of < or = 3,000. These results indicate that antigens incorporated into the orally active adjuvant formulation iscom are degraded to antigenic peptides during transport across the intestinal epithelium.
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| 11. |
- Lindmark, T, et al.
(författare)
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Absorption enhancement in intestinal epithelial Caco-2 monolayers by sodium caprate : Assessment of molecular weight dependence and demonstration of transport routes
- 1998
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Ingår i: Journal of drug targeting (Print). - : Informa UK Limited. - 1061-186X .- 1029-2330. ; 5:3, s. 215-223
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Tidskriftsartikel (refereegranskat)abstract
- Sodium caprate (CIO), a medium chain fatty acid, is used clinically to enhance rectal absorption of the low molecular weight (MW) drug ampicillin. The main aim of this study was to investigate whether CIO also enhances the permeability of high MW model drugs in a model of the intestinal epithelium. The second aim was to present visual evidence of the route of enhanced transport across the epithelial cell layer. The studies were performed in Caco-2 monolayers cultured on permeable supports. The effects of non-toxic concentrations (≤ 13 mM) of CIO on drug transport across the monolayers was studied using monodisperse 14C-polyethylene glycols (MW 238-502; 14C-PEGs), 125I-Arg8-vasopressin (MW 1,208), 125I-insulin (MW 6,000) and FITC-labelled dextrans (MW 4,400 and 19,600; FD4 and FD20 respectively) as model drugs. Electron and confocal laser scanning microscopy were used to demonstrate transport routes across the epithelium. 10 mM C10 increased the permeability of all 14C-PEGs to approximately the same extent. 13 mM C10 increased the permeability of 125I-Arg8-vasopressin 10-fold. Only small increases in FD4 and FD20 permeabilities were observed. After C10 exposure, both tight junctions with normal morphology and those with dilatations showed an increased permeability to ruthenium red, indicating that C10 enhanced the paracellular transport of molecules with a MW < 1,000. Confocal microscopy showed that C10 increased the transport of FD4 and FD20 by the paracellular route. In conclusion, nontoxic concentrations of C10 can be used to enhance the permeability of drugs of MW up to approximately 1,200. Enhancement of the absorption of molecules larger than 4,000 is quantitatively insignificant. The enhanced permeability occurred via the paracellular pathway.
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| 12. |
- Linnankoski, Johanna, et al.
(författare)
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Paracellular porosity and pore size of the human intestinal epithelium in tissue and cell culture models
- 2010
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Ingår i: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 99:4, s. 2166-2175
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Tidskriftsartikel (refereegranskat)abstract
- The paracellular space defines the passive permeation of hydrophilic compounds in epithelia. The goal of this study was to characterise the paracellular permeation pathway in the human intestinal wall and differentiated epithelial cell models (MDCKII, Caco-2 and 2/4/A1). The permeabilities of hydrophilic polyethylene glycols (PEG) were investigated in diffusion chambers, and mass spectrometry was used to obtain accurate concentrations for each PEG molecule. The paracellular porosity and the size of the pores in the membranes were estimated from the PEG permeability data using an effusion-based approach. The porosities were found to be low (fraction 10−7–10−5 of the epithelial surface) in all investigated membranes. Two different pore sizes (radii 5–6 and >10 Å) were detected in the human intestinal epithelium and the Caco-2 and MDCKII cells, while only one (about 15 Å) in the 2/4/A1 monolayer. The paracellular porosities of the human small intestine and 2/4/A1 monolayers were larger (>10−7) than that of the MDCKII and Caco-2 cells (<10−7). We report for the first time the quantitative values describing both porosity and pore size of the paracellular space in the human intestine. The cell models deviate from the small intestine either with respect to porosity (Caco-2, MDCKII) or pore size distribution (2/4/A1).
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| 13. |
- Matsson, Elin M., et al.
(författare)
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Combined in Vitro-in Vivo Approach To Assess the Hepatobiliary Disposition of a Novel Oral Thrombin Inhibitor
- 2013
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 10:11, s. 4252-4262
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Tidskriftsartikel (refereegranskat)abstract
- Two clinical trials and a large set of in vitro transporter experiments were performed to investigate if the hepatobiliary disposition of the direct thrombin inhibitor prodrug AZD0837 is the mechanism for the drug-drug interaction with ketoconazole observed in a previous clinical study. In Study 1, [H-3]AZD0837 was administered to healthy male volunteers (n = 8) to quantify and identify the metabolites excreted in bile. Bile was sampled directly from the jejunum by duodenal aspiration via an oro-enteric tube. In Study 2, the effect of ketoconazole on the plasma and bile pharmacokinetics of AZD0837, the intermediate metabolite (AR-H069927), and the active form (AR-H067637) was investigated (n = 17). Co-administration with ketoconazole elevated the plasma exposure to AZD0837 and the active form approximately 2-fold compared to placebo, which may be explained by inhibited CYP3A4 metabolism and reduced biliary clearance, respectively. High concentrations of the active form was measured in bile with a bile-to-plasma AUC ratio of approximately 75, indicating involvement of transporter-mediated excretion of the compound. AZD0837 and its metabolites were further investigated as substrates of hepatic uptake and efflux transporters in vitro. Studies in MDCK-MDRI cell monolayers and P-glycoprotein (P-gp) expressing membrane vesicles identified AZD0837, the intermediate, and the active form as substrates of P-gp. The active form was also identified as a substrate of the multidrug and toxin extrusion 1 (MATE!) transporter and the organic cation transporter 1 (OCT1), in HEK cells transfected with the respective transporter. Ketoconazole was shown to inhibit all of these three transporters; in particular, inhibition of P-gp and MATE1 occurred in a clinically relevant concentration range. In conclusion, the hepatobiliary transport pathways of AZD0837 and its metabolites were identified in vitro and in vivo. Inhibition of the canalicular transporters P-gp and MATE1 may lead to enhanced plasma exposure to the active form, which could, at least in part, explain the clinical interaction with ketoconazole.
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| 14. |
- Nilsson, Harriet, et al.
(författare)
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CFTR and tight junctions in cultured bronchial epithelial cells
- 2010
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Ingår i: Experimental and molecular pathology (Print). - : Elsevier BV. - 0014-4800 .- 1096-0945. ; 88:1, s. 118-127
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Tidskriftsartikel (refereegranskat)abstract
- Airway epithelial salt and water transport takes place through paracellular and transcellular pathways. This transport depends critically on the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR), operating in concert with the paracellular pathway through the tight junctions (TJs). Normal (16HBE14o-), cystic fibrosis (CFBE41o-), and corrected CFBE41o- (CFBE41o pCep4) airway epithelial cell lines were cultured under isotonic conditions. Transepithelial electrical resistance (TEER) was measured as indicator of the tightness of the cultures. Morphology was investigated by immunofluorescence and paracellular permeability by lanthanum nitrate or [14C] mannitol as permeability markers. CFBE41o pCep4 cells developed lower TEER than CFBE41o- cells. Addition of a specific inhibitor of CFTR (CFTRinh-172) to 16HBE14o- and CFBE pCep4 cells resulted in a time-dependent increase in TEER whereas stimulation of CFTR by IBMX and forskolin caused a decrease. Permeability to lanthanum and [14C] mannitol was lower in 16HBE14o- cells exposed to CFTRinh-172 and in CFBE41o- cells compared to untreated 16HBE14o- and CFBE41o pCep4 cells, respectively. 16HBE14o- cells exposed to IBMX and forskolin showed higher permeability to lanthanum but lower permeability to [14C] mannitol compared to control. Immunofluorescence revealed a disorganisation of F-actin and a-tubulin in 16HBE14o- cells exposed to CFTRinh-172, which was not seen in untreated cultures. A higher degree of disorganised F-actin and a-tubulin was also seen in CFBE41o- cells compared to CFBE41o- pCep4 cells. Changes in F-actin and a-tubulin in 16HBE14o- cells exposed to IBMX and forskolin were also seen, although these were less apparent. These results suggest the possibility of an interaction between the activity of CFTR and the TJ protein complex, probably via the cytoskeleton.
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| 15. |
- Nordström, Josefina, et al.
(författare)
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The degree of compression of spherical granular solids controls the evolution of microstructure and bond probability during compaction
- 2013
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Ingår i: International Journal of Pharmaceutics. - : Elsevier BV. - 0378-5173 .- 1873-3476. ; 442:1-2, s. 3-12
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Tidskriftsartikel (refereegranskat)abstract
- The effect of degree of compression on the evolution of tablet microstructure and bond probability during compression of granular solids has been studied. Microcrystalline cellulose pellets of low (about 11%) and of high (about 32%) porosity were used. Tablets were compacted at 50, 100 and 150 MPa applied pressures and the degree of compression and the tensile strength of the tablets determined. The tablets were subjected to mercury intrusion measurements and from the pore size distributions, a void diameter and the porosities of the voids and the intra-granular pores were calculated. The pore size distributions of the tablets had peaks associated with the voids and the intra-granular pores. The void and intra-granular porosities of the tablets were dependent on the original pellet porosity while the total tablet porosity was independent. The separation distance between pellets was generally lower for tablets formed from high porosity pellets and the void size related linearly to the degree of compression. Tensile strength of tablets was higher for tablets of high porosity pellets and a scaled tablet tensile strength related linearly to the degree of compression above a percolation threshold. In conclusion, the degree of compression controlled the separation distance and the probability of forming bonds between pellets in the tablet.
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| 16. |
- Pazesh, Samaneh, et al.
(författare)
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Considerations on the quantitative analysis of apparent amorphicity of milled lactose by Raman spectroscopy
- 2016
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Ingår i: International Journal of Pharmaceutics. - : Elsevier BV. - 0378-5173 .- 1873-3476. ; 511:1, s. 488-504
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Tidskriftsartikel (refereegranskat)abstract
- The main purpose of the study was to evaluate various pre-processing and quantification approaches of Raman spectrum to quantify low level of amorphous content in milled lactose powder. To improve the quantification analysis, several spectral pre-processing methods were used to adjust background effects. The effects of spectral noise on the variation of determined amorphous content were also investigated theoretically by propagation of error analysis and were compared to the experimentally obtained values. Additionally, the applicability of calibration method with crystalline or amorphous domains in the estimation of amorphous content in milled lactose powder was discussed. Two straight baseline pre-processing methods gave the best and almost equal performance. By the succeeding quantification methods, PCA performed best, although the classical least square analysis (CLS) gave comparable results, while peak parameter analysis displayed to be inferior. The standard deviations of experimental determined percentage amorphous content were 0.94% and 0.25% for pure crystalline and pure amorphous samples respectively, which was very close to the standard deviation values from propagated spectral noise. The reasonable conformity between the milled samples spectra and synthesized spectra indicated representativeness of physical mixtures with crystalline or amorphous domains in the estimation of apparent amorphous content in milled lactose.
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| 17. |
- Ragnarsson, Eva, 1971-
(författare)
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Effects of Microparticulate Drug Delivery Systems : Tissue Responses and Transcellular Transport
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the past decade, the development of macromolecular drugs based on peptides, proteins and nucleic acids has increased the interest in microparticulate drug delivery, i.e., the delivery of drug systems in the nanometer and micrometer ranges. However, little is known so far about the effect that microparticulate systems have on various tissues after administration. Additionally, the knowledge of mechanisms responsible for the uptake and transport of microparticles across the human intestine is incomplete and requires further investigation to improve both the safety profiles and the efficiency of these drug delivery systems.This thesis is comprised of two parts. The first one investigates gene expression responses obtained from DNA arrays in local and distal tissues after microparticulate drug delivery. The second part focuses on the mechanisms responsible for the transport of microparticles across epithelial cells lining the intestine.The results presented in the first part demonstrated that gene expression analysis offers a detailed picture of the tissue responses after intramuscular or pulmonary administration of microparticulate drug delivery systems compared to the traditional techniques used for such evaluations. In addition, DNA arrays provided a useful and sensitive tool for the initial characterization and evaluation of both local and distal tissue responses, making it possible to distinguish between gene expression patterns related to each studied delivery system.The results presented in the second part demonstrated that the surface properties of the microparticle were important for the extent of transport across an in vitro model of the follicle-associated epithelium (FAE), comprised of intestinal epithelial cells specialized in particle transport (M cells). Another important finding was that the enteropathogen bacterium, Yersinia pseudotuberculosis, induced microparticle transport across the normal intestinal epithelium, represented by Caco-2 cells and excised human ileal tissue. This transport was most probably mediated by an increased capacity for macropinocytosis in the epithelial cells.
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| 18. |
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| 19. |
- Tavelin, Staffan, et al.
(författare)
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A new principle for tight junction modulation based on occludin peptides
- 2003
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 64:6, s. 1530-1540
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [14C]mannitol as a para-cellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The l- and d-diastereomers of C14-OP90-103 had distinctly different effects. The d-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The l-isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.
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| 20. |
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| 21. |
- Wong, AK, et al.
(författare)
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Determination of transport in the Caco-2 cell assay of compounds varying in lipophilicity using LC-MS : enhanced transport of Leu-enkephalin analogues
- 2002
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Ingår i: European Journal of Pharmaceutical Sciences. - 0928-0987 .- 1879-0720. ; 16:3, s. 113-118
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: To synthesize a number of analogues of Leu-enkephalin with different lipophilicities and to develop an LC-MS method for determining the Caco-2 cell permeability values of these compounds. METHODS: A number of sugar and sugar plus lipoamino acid analogues of Leu-enkephalin were synthesized by solid-phase and solution methods. An LC-MS method was developed for analyzing the Caco-2 cell assay samples and validated against the traditional method using radiolabelled compounds. RESULTS: A sensitive and specific LC-MS assay was developed. Standard curves were linear in the range of 0.025-5 microM. Apparent permeability values determined by LC-MS and liquid scintillation counter were identical, for both a hydrophilic drug, cephalexin and a lipophilic Leu-enkaphalin analogue. Caco-2 permeability values for the analogues of Leu-enkephalin were determined and it was found that attachment of sugar or sugar and lipoamino acid to the Leu-enkephalin peptide resulted in an increase in the apparent permeability values compared to the native peptide, which was not transported across the Caco-2 cell monolayers. CONCLUSIONS: A rapid, generic LC-MS method for analyzing a range of compounds was developed. Attachment of a sugar or sugar and lipoamino acid to Leu-enkephalin improves the apparent permeability across Caco-2 cell monolayers.
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