Biomedical subjects

Lucia Lazorova

Publications and source records attributed to Lucia Lazorova.

7 recordsLinked to original sources

PubMed2006

Regional levels of drug transporters along the human intestinal tract: co-expression of ABC and SLC transporters and comparison with Caco-2 cells.

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.

ATP-Binding Cassette Transporters↗

PubMed2005

Caco-2 permeability of weakly basic drugs predicted with the double-sink PAMPA pKa(flux) method.

The aim of this study was to analyze pH-dependent permeability of cationic drugs in Caco-2 cell monolayers using the pK(a)(flux) 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 pK(a)(flux) 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, P(i), was therefore determined and subsequently subtracted from all permeability coefficients in Caco-2 prior to the comparison with PAMPA. The resulting intrinsic permeability coefficients (P(o)) 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.

Caco-2 Cells↗

PubMed2005

Orally active antiviral tripeptide glycyl-prolyl-glycinamide is activated by CD26 (dipeptidyl peptidase IV) before transport across the intestinal epithelium.

The tripeptide amide glycyl-prolyl-glycinamide (GPG-amide) is a new antiretroviral drug candidate, but its absorption mechanism is unknown. In this investigation, the transport and metabolism of GPG-amide were studied in a model of the human intestinal epithelium, Caco-2 cell monolayers. The results show that when the tripeptide amide came into contact with the apical enterocyte membrane, it was degraded by CD26 (dipeptidyl peptidase IV) to glycylproline and the antiretrovirally active metabolite glycinamide. Glycinamide retained antiretroviral activity in vitro after transport through the Caco-2 cell monolayers. The transport of glycinamide across Caco-2 cell monolayers occurred via passive diffusion with an apparent permeability coefficient of about 2 x 10(-6) cm s(-1), which suggests that it is absorbed by the oral route in sufficient amounts to be considered for oral administration. In conclusion, the tripeptide GPG-amide acts as a prodrug that is activated by CD26 to release the orally active antiretroviral compound glycinamide.

Administration, Oral↗

PubMed2004

Exploring the quantitative relationship between the level of MDR1 transcript, protein and function using digoxin as a marker of MDR1-dependent drug efflux activity.

A limited number of gene expression studies have investigated the quantitative relationships between the amount of transcript, level of protein or activity/function, with disparate conclusions regarding these relationships. Collectively these studies indicate that the relevance of quantitative transcript analysis as a predictor of phenotype has to be evaluated on a gene-by-gene or even a case-by-case basis. The purpose of this study was to define a suitable marker for MDR1-dependent drug efflux, and to quantitatively investigate the relationships between the amount of transcript, protein and drug efflux in the frequently used Caco-2 cell model. The substrate specificity of digoxin, a commonly used marker for MDR1, was investigated using transgenic MDCK II or LLC-PK1 cell lines expressing the efflux proteins MDR1, BCRP and MRP2, since these proteins are localised to the apical part of the enterocyte plasma membrane and exhibit comparatively high transcript levels in the human small intestine. Relationships between levels of transcript, protein and function were investigated quantitatively using real-time RT-PCR, ECL western blot analysis and basolateral-to-apical and apical-to-basolateral efflux ratios. Our results indicate that digoxin is a specific marker for MDR1-dependent drug efflux in the Caco-2 cell drug absorption model and that MDR1 transcript abundance is at least as valid as MDR1 protein abundance as a predictor of MDR1 efflux activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Absorption classification of oral drugs based on molecular surface properties.

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.

Administration, Oral↗

PubMed2003

A new principle for tight junction modulation based on occludin peptides.

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.

Amino Acid Sequence↗

PubMed2002

Determination of transport in the Caco-2 cell assay of compounds varying in lipophilicity using LC-MS: enhanced transport of Leu-enkephalin analogues.

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.

Absorption↗