Birger Brodin

Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories

Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.

In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood–brain barrier models with a focus on their validation regarding a set of well-established blood–brain barrier characteristics. As an ideal cell culture model of the blood–brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described.

In Vitro Blood-Brain Barrier Models—An Overview of Established Models and New Microfluidic Approaches

The societal need for new central nervous system (CNS) medicines is substantial, because of the global increase in life expectancy and the accompanying increase in age-related CNS diseases. Low blood-brain barrier (BBB) permeability has been one of the major causes of failure for new CNS drug candidates. There has therefore been a great interest in cell models, which mimic BBB permeation properties. In this review, we present an overview of the performance of monocultured, cocultured, and triple-cultured primary cells and immortalized cell lines, including key parameters such as transendothelial electrical resistance values, permeabilities of paracellular flux markers, and expression of BBB-specific marker proteins. Microfluidic systems are gaining ground as a new automated technical platform for cell culture and systematic analysis. The performance of these systems was compared with current state-of-the-art models and it was noted that, although they show great promise, these systems have not yet reached beyond the proof-of-concept stage. In general, it was found that there were large variations in experimental protocols, BBB phenotype markers, and paracellular flux markers used. It is the authors opinion that the field may benefit greatly from developing standardized methodologies and initiating collaborative efforts on optimizing culture protocols.

Human peptide transporters: therapeutic applications

Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2, which possess transport activity, have been identified. The transporters are not drug targets per se, but due to uniquely broad substrate specificity they have proven to be relevant in drug therapy at the level of drug transport. Therapeutic agents such as orally active β-lactam antibiotics, bestatin, prodrugs of acyclovir and gancyclovir have oral bioavailabilities, which are largely a result of their interaction with PepT1. The transporters have therefore received considerable attention in relation to drug delivery. The aim of the present review is to highlight structural requirements for binding to peptide transporters, as well as their role in drug delivery and in potential future drug design and targeted tissue delivery of peptides and peptidomimetics.

Intestinal gaboxadol absorption via PAT1 (SLC36A1): modified absorption in vivo following co-administration of L-tryptophan.

Background and purpose:  Gaboxadol has been in development for treatment of chronic pain and insomnia. The clinical use of gaboxadol has revealed that adverse effects seem related to peak serum concentrations. The aim of this study was to investigate the mechanism of intestinal absorption of gaboxadol in vitro and in vivo.

δ-Aminolevulinic acid is a substrate for the amino acid transporter SLC36A1 (hPAT1)

Background and purpose:  δ‐Aminolevulinic acid (ALA) is used in cancer patients for photodynamic diagnosis or therapy. Oral administration of ALA has been used in patients with prostate and bladder cancer. The present aim was to investigate the mechanism of intestinal absorption of ALA and its transport via the amino acid transporter SLC36A1.

hPEPT1 affinity and translocation of selected Gln-Sar and Glu-Sar dipeptide derivatives.

The intestinal di- and tripeptide transporter hPEPT1 is considered responsible for the absorption of di- and tripeptides arising from digestion, along with several drugs and prodrugs. In order to gather information on the binding site of the protein, several structure-affinity relationships have been suggested. However, these are not necessarily predictive of compounds that are actually translocated by hPEPT1. More information on affinity to and translocation via hPEPT1 of side-chain-modified dipeptides may be gained by conducting a study of selected dipeptide derivatives with variety in size, hydrophobicity, and bond type. The aim of the present study was to synthesize new esters and amides based on L-Glu-Sar and investigate the effects that bond type and size of modification of the N-terminal side chain of sarcosine-containing dipeptides have on the affinity to and translocation via hPEPT1. The esters L-Glu(O-i-Bu)-Sar and L-Glu(OCH(2)Ada)-Sar and the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were synthesized, and affinity to and translocation via hPEPT1 were investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay using (14)C-labeled Gly-Sar. Translocation was measured as fluorescence ratios induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All compounds showed high affinity to hPEPT1, but only the amides L-Gln(N,N-dimethyl)-Sar and L-Gln(N-piperidinyl)-Sar were translocated by hPEPT1. hPEPT1 is very susceptible to modifications of the N-terminal amino acid side chain of dipeptidomimetic substrates, in terms of achieving compounds with high affinity for the transporter. However, as affinity is not predictive of translocation, derivatization in this position must be performed with great caution since some of the compounds investigated turn out not to be translocated by the transporter.

Transport Characteristics of L-Carnosine and the Anticancer Derivative 4-Toluenesulfonylureido-Carnosine in a Human Epithelial Cell Line

AbstractPurpose. The aim of the present study was to evaluate whether the transepithelial transport of the anticancer compound 4-toluenesulfonylureido-carnosine (Ts-carnosine) and the dipeptide moiety L-carnosine was due to a hPepT1 carrier-mediated flux. Methods. Transport experiments were conducted using Caco-2 cell monolayers and either reversed-phase HPLC-UV or liquid scintillation counting methods for quantification. pKa, LogD, and LogP were determined using the Sirius GlpKa meter. Results. L-carnosine was transported across the apical membrane with a Km,app of 2.48 ± 1.16 mM and a Vmax of 2.08 ± 0.34 nmol · cm−2 · min−1 and across the basolateral membrane with a Km,app of 7.21 ± 3.17 mM and a Vmax of 0.54 ± 0.10 nmol · cm−2 · min−1, and transepithelially with a Papp of 4.46 · 10−2 ± 6.4 · 10−6 cm · min−10. Ts-carnosine had an affinity (Ki) for hPepT1 of 2.33 ± 0.54 mM; however, the transepithelial transport was low as compared to that of L-carnosine. Conclusions. L-carnosine was transported across both the apical and basolateral membrane of Caco-2 cell monolayers in a carrier-mediated manner however, the transepithelial transport followed apparent simple non-saturable kinetics. Ts-carnosine had an affinity for hPepT1 but a relatively low transepithelial transport. This indicates that the transepithelial transport of L-carnosine and Ts-carnosine is not hPepT1 carrier-mediated and that L-carnosine is not a suitable dipeptide moiety for hPepT1-mediated absorption of sulfonamide-type anticancer compounds.

Prodrugs of purine and pyrimidine analogues for the intestinal di/tri-peptide transporter PepT1: affinity for hPepT1 in Caco-2 cells, drug release in aqueous media and in vitro metabolism

A general drug delivery approach for increasing oral bioavailability of purine and pyrimidine analogues such as acyclovir may be to link these compounds reversibly to stabilized dipeptide pro-moieties with affinity for the human intestinal di/tri-peptide transporter, hPepT1. In the present study, novel L-Glu-Sar and D-Glu-Ala ester prodrugs of acyclovir and 1-(2-hydroxyethyl)-linked thymine were synthesized and their affinities for hPepT1 in Caco-2 cells were determined. Furthermore, the degradation of the prodrugs was investigated in various aqueous and biological media and compared to the corresponding hydrolysis of the prodrug valaciclovir. Affinity studies showed that the L-Glu-Sar prodrugs had high affinity for hPepT1 (K(i) approximately 0.2-0.3 mM), whereas the D-Glu-Ala prodrugs had poor affinity (K(i) approximately 50 mM). The pH-rate profiles of the prodrugs D-Glu[1-(2-hydroxyethyl)thymine]-Ala and L-Glu[acyclovir]-Sar showed specific base catalyzed degradation at pH above 4.5 and 5.5, respectively. This implicates that the degradation rates at pH approximately 7.4 (t(1/2) approximately 3.5 and 5.5 h) are approximately 25 times faster than at upper small intestinal pH approximately 6.0. In 10% porcine intestinal homogenate and 80% human plasma the half-lives of the L-Glu-Sar prodrugs were approximately between 45 and 90 min indicating a limited enzyme catalyzed degradation. In contrast, valaciclovir underwent extensive enzyme catalyzed hydrolysis in 10% porcine intestinal homogenate (t(1/2) approximately 1 min). In conclusion, L-Glu-Sar may potentially function as pro-moiety for purine and pyrimidine analogues, where release of parent compound primarily is controlled by a specific base catalyzed hydrolysis. Acyclovir is quantitatively released at the relevant pH 7.4, whereas the 1-(2-hydroxyethyl)-linked thymine is released instead of the parent compound thymine.

Calu-3 cells grown under AIC and LCC conditions: Implications for dipeptide uptake and transepithelial transport of substances

The aim of the present study was to investigate whether Calu-3 cell culture conditions influence drug and nutrient transport known to occur via carriers or transporters. Calu-3 cell layers, an in vitro model of the lung epithelium, were cultured using air interfaced culture (AIC) or liquid covered culture (LCC) on either polycarbonate or polyester as filter support material. We found that the development of the Calu-3 cell layer barrier function did not depend on the filter material but rather on the culture conditions as follows: (i) the apical uptake of Gly-Sar was significantly larger for cells grown in AIC compared to LCC, (ii) the TEER values for cells grown in LCC were approximately three times larger than for cells grown in AIC, (iii) the transepithelial transport in both AIC and LCC Calu-3 cells was polarized in the apical-basolateral direction of proline, glycine, α-methyl-d-glucoside, glipizide, taurocholic acid and estrone-3-sulfate, whereas inulin, mannitol and Gly-Sar showed no polarized transport. Etoposide showed polarized efflux (basolateral to apical transport) in AIC and LCC Calu-3 layers. These findings provide information about nutrient and drug transport in Calu-3 cells, and this may have implications for selecting culture conditions for transport studies in this in vitro model of the lung epithelium.