Carsten Uhd Nielsen

Prevalence and characteristics of multinucleoside-resistant human immunodeficiency virus type 1 among European patients receiving combinations of nucleoside analogues.

ABSTRACT The prevalence and the genotypic and phenotypic characteristics of multinucleoside-resistant (MNR) human immunodeficiency virus type 1 (HIV-1) variants in Europe were investigated in a multicenter study that involved centers in nine European countries. Study samples (n = 363) collected between 1991 and 1997 from patients exposed to two or more nucleoside analogue reverse transcriptase inhibitors (NRTIs) and 274 control samples from patients exposed to no or one NRTI were screened for two marker mutations of multinucleoside resistance (the Q151M mutation and a mutation with a 2-amino-acid insertion at codon 69, T69S-XX). Q151M was identified in six of the study samples (1.6%), and T69S-XX was identified in two of the study samples (0.5%; both of them T69S-SS), but both patterns were absent among control samples. Non-NRTI (NNRTI)-related changes were observed in viral strains from two patients, which displayed the Q151M resistance pattern, although the patients were NNRTI naive. The patients whose isolates displayed multinucleoside resistance had received treatment with zidovudine and either didanosine, zalcitabine, or stavudine. Both resistance patterns conferred broad cross-resistance to NRTIs in vitro and a poor response to treatment in vivo. MNR HIV-1 is found only among multinucleoside-experienced patients. Its prevalence is low in Europe, but it should be closely monitored since it seriously limits treatment options.

The Calculated Genetic Barrier for Antiretroviral Drug Resistance Substitutions Is Largely Similar for Different HIV-1 Subtypes

Background: The genetic barrier, defined as the number of mutations required to overcome drug-selective pressure, is an important factor for the development of HIV drug resistance. Because of high variability between subtypes, particular HIV-1 subtypes could have different genetic barriers for drug resistance substitutions. This study compared the genetic barrier between subtypes using some 2000 HIV-1 sequences (>600 of non-B subtype) isolated from anti-retroviral-naive patients in Europe. Methods: The genetic barrier was calculated as the sum of transitions (scored as 1) and/or transversions (2.5) required for evolution to any major drug resistance substitution. In addition, the number of minor protease substitutions was determined for every subtype. Results: Few dissimilarities were found. An increased genetic barrier was calculated for I82A (subtypes C and G), V108I (subtype G), V118I (subtype G), Q151M (subtypes D and F), L210W (subtypes C, F, G, and CRF02_AG), and P225H (subtype A) (P < 0.001 compared with subtype B). A decreased genetic barrier was found for I82T (subtypes C and G) and V106M (subtype C) (P < 0.001 vs subtype B). Conversely, minor protease substitutions differed extensively between subtypes. Conclusions: Based on the calculated genetic barrier, the rate of drug resistance development may be similar for different HIV-1 subtypes. Because of differences in minor protease substitutions, protease inhibitor resistance could be enhanced in particular subtypes once the relevant major substitutions are selected.

Di / tri-Peptide Transporters as Drug Delivery Targets: Regulation of Transport Under Physiological and Patho-physiological Conditions

Two human di/tri-peptide transporters, hPepT1 and hPepT2 have been identified and functionally characterized. In the small intestine hPepT1 is exclusively expressed, whereas both PepT1 and PepT2 are expressed in the proximal tubule. The transport via di/tri-peptide transporters is proton-dependent, and the transporters thus belong to the Proton-dependent Oligopeptide Transporter (POT)-family. The transporters are not drug targets per se, however due to their uniquely broad substrate specificity; they have proved to be relevant drug targets at the level of drug transport. Drug molecules such as oral active beta-lactam antibiotics, bestatin, prodrugs of aciclovir and ganciclovir have oral bioavailabilities, which largely are a result of their interaction with PepT1. In the last few years an increasing number of studies concerned with regulation of di/tri-peptide transporter capacity have appeared. Studies on receptor-mediated regulation has shown that both PepT1 and PepT2 is down-regulated by long-term exposure to epidermal growth factor (EGF) due to a decreased gene transcription. PepT1-mediated transport is up-regulated by certain substrates and in response to fasting and starvation at the level of increased gene transcription. PepT1-mediated transport is up-regulated by short-term exposure to receptor agonists such as EGF, insulin, leptin, and clonidine, and down-regulated by VIP. Overall, the regulation of di/tri-peptide transport may be contributed to 1) changes in apical proton-motive force 2) recruitment of di/tri-peptide transporters from vesicular storages 3) changes in gene transcription/mRNA stability. The aim of the present review is to discuss physiological, patho-physiological and drug-induced regulation of di/tri-peptide transporter mediated transport.

In vitro evidence for the brain glutamate efflux hypothesis: brain endothelial cells cocultured with astrocytes display a polarized brain-to-blood transport of glutamate

The concentration of the excitotoxic amino acid, L‐glutamate, in brain interstitial fluid is tightly regulated by uptake transporters and metabolism in astrocytes and neurons. The aim of this study was to investigate the possible role of the blood‐brain barrier endothelium in brain L‐glutamate homeostasis. Transendothelial transport‐ and accumulation studies of 3H‐L‐glutamate, 3H‐L‐aspartate, and 3H‐D‐aspartate in an electrically tight bovine endothelial/rat astrocyte blood‐brain barrier coculture model were performed. After 6 days in culture, the endothelium displayed transendothelial resistance values of 1014 ± 70 Ω cm2, and 14C‐D‐mannitol permeability values of 0.88 ± 0.13 × 10−6 cm s−1. Unidirectional flux studies showed that L‐aspartate and L‐glutamate, but not D‐aspartate, displayed polarized transport in the brain‐to‐blood direction, however, all three amino acids accumulated in the cocultures when applied from the abluminal side. The transcellular transport kinetics were characterized with a Km of 69 ± 15 μM and a Jmax of 44 ± 3.1 pmol min−1 cm−2 for L‐aspartate and a Km of 138 ± 49 μM and Jmax of 28 ± 3.1 pmol min−1 cm−2 for L‐glutamate. The EAAT inhibitor, DL‐threo‐ß‐Benzyloxyaspartate, inhibited transendothelial brain‐to‐blood fluxes of L‐glutamate and L‐aspartate. Expression of EAAT‐1 (Slc1a3), −2 (Slc1a2), and −3 (Slc1a1) mRNA in the endothelial cells was confirmed by conventional PCR and localization of EAAT‐1 and −3 in endothelial cells was shown with immunofluorescence. Overall, the findings suggest that the blood‐brain barrier itself may participate in regulating brain L‐glutamate concentrations.

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.

Transport of amino acids and GABA analogues via the human proton-coupled amino acid transporter, hPAT1 : Characterization of conditions for affinity and transport experiments in Caco-2 cells

The objective of this study was to investigate transepithelial amino acid transport as a function of Caco-2 cell culture time. Furthermore, the objective was to investigate apical uptake characteristics of hPAT1-mediated transport under various experimental conditions. Apical amino acid uptake and transport studies were conducted in Caco-2 monolayers cultured for 4-28 days. Transepithelial transport of the prototypic hPAT1 (SLC36A1) substrates l-proline and glycine were maximal after 21-28 days in culture. Based on proton-dependency and substrate kinetics the major apical uptake and transport of Gly and Pro in Caco-2 cell monolayers is hPAT1-mediated. The apical uptake of Pro is decreased at apical hyperosmolarity conditions. Furthermore we identified the two GABA-analogues, muscimol and THPO as novel hPAT1 substrates. THPO had an affinity for hPAT1 of 11.3mM, whereas muscimol had one of the highest affinities for hPAT1 (1.7mM) reported. Our findings illustrate the suitability of the Caco-2 model for studying hPAT1-mediated transport. Furthermore, the affinity of THPO and muscimol underlines the possible importance of hPAT1 as a transporter for heterocyclic compounds consisting of a 3-isoxazolol moiety, which has been shown to function as a carboxylic acid bioisostere for substrates of the GABA receptor and transport systems.

Phe-Gly dipeptidomimetics designed for the di-/tripeptide transporters PEPT1 and PEPT2: synthesis and biological investigations.

A series of five Phe-Gly dipeptidomimetics containing different amide bond replacements have been synthesized in a facile way from the readily available unsaturated ketoester 1, and their affinities for the di-/tripeptide transporters hPEPT1 (Caco-2 cells) and rPEPT2 (SKPT cells) were tested. The compounds contained the amide bond isosteres ketomethylene (2a), (R)- and (S)-hydroxyethylidene (3a and 4a), and (R)- and (S)-hydroxyethylene (5a and 6a) to provide information on the conformational and stereochemical requirements for hPEPT1 and rPEPT2 affinity. The affinity studies showed that for rPEPT2 there is no significant difference in affinity between the ketomethylene isostere 2a and the natural substrate Phe-Gly (K(i) values of 18.8 and 14.6 microM, respectively). Also the affinities for hPEPT1 are in the same range (K(i) values of 0.40 and 0.20 mM, respectively). This corroborates earlier findings that the amide bond as such is not essential for binding to PEPTX, but the results also reveal possible differences in the binding of ketomethylene isosteres to hPEPT1 and rPEPT2. The trans-hydroxyethylidene and hydroxyethylene isosteres proved to be poor substrates for PEPTX. These results provide new information about the importance of flexibility and of the stereochemistry at the C(4)-position for this class of compounds. Furthermore, the intracellular uptake of 2a-4a in Caco-2 cells was investigated, showing a 3-fold reduction of the uptake of 2a in the presence of the competetive inhibitor Gly-Pro, indicating contribution from an active transport component. No active uptake of 3a and 4a was observed. Transepithelial transport studies also indicated active transport of 2a across Caco-2 monolayers.

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.