Substances that compete for the same saturable intestinal transporters may when dosed together lead to altered permeability and hence influence bioavailability. The aim was to simulate kinetic parameters, i.e. Km and Jmax, for transporter... more
Substances that compete for the same saturable intestinal transporters may when dosed together lead to altered permeability and hence influence bioavailability. The aim was to simulate kinetic parameters, i.e. Km and Jmax, for transporter mediated E1S permeability across Caco-2 cells by a combined experimental modeling approach. 4 classes of transporters were suggested to be involved in the permeability of E1S, i.e. apical influx (TI) and efflux (TIII) as well as basolateral efflux (TII) and influx (TIV). Efflux ratio of E1S was determined to 6.8. E1S is suggested to have highest affinity to TIII. TIV is however suggested to be rate limiting in exsorptive PAPP due to lower Jmax of TIV, compared to TIII. Possible interactions between E1S and the excipients erythrosine and Brij35 on these 4 classes of transporters were also studied. From these studies it is suggested that erythrosine does interact with E1S on apical efflux transporter TIII by competitive inhibition. Furthermore interaction between erythrosine and E1S is suggested on apical influx transporter (TI). Brij35 does not seem to interact with E1S on apical transporters. The present model seem to be a valuable tool to simulate kinetic parameters for compounds being substrates to multiple transporters as well as to estimate kinetic parameters for compounds interacting on the same transporters.
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A key determinant for oral bioavailability of a drug candidate is the intestinal epithelial permeation of the drug candidate. This intestinal permeation may be affected by interactions on membrane transporters expressed in the intestinal... more
A key determinant for oral bioavailability of a drug candidate is the intestinal epithelial permeation of the drug candidate. This intestinal permeation may be affected by interactions on membrane transporters expressed in the intestinal epithelial cells. The purpose of the present study was to investigate whether transporters were involved in the intestinal absorption of an organic anion A275 and to compare the impact of interactions related to transporters in the Caco-2 cell model versus the in vivo rat model of intestinal absorption. In both models, it was investigated whether intestinal permeation of A275 was concentration dependent and affected by inhibitors or competitive organic anions. Interactions related to transporters in intestinal permeation was clearly demonstrated in the Caco-2 cell model but was not directly evident for in vivo rat absorption. However, an observed biphasic in vivo absorption and a large intervariability between rats might mask a dose-dependent absorption of A275. To avoid these suggested interactions, a dose of at least 10 mg/kg, which saturates the intestinal transporters involved in A275 absorption, should be administered, but at doses below that the risk of such drug interactions should be taken into account.
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The discovery of novel ligands for the hPEPT1 transporter is reported. By exploiting a fast and rigorously validated QSAR model in combination with the distance in activity-centered chemical space (DACCS) approach, a database of... more
The discovery of novel ligands for the hPEPT1 transporter is reported. By exploiting a fast and rigorously validated QSAR model in combination with the distance in activity-centered chemical space (DACCS) approach, a database of commercially available compounds (Sigma-Aldrich) was screened for virtual hits. Twelve compounds were then purchased and characterized in an apical [14C]Gly-Sar uptake competition assay. Four compounds displayed affinity in the medium-to-high range. A simple benzophenone derivative displayed high affinity with a sub-millimolar binding constant (Ki=0.24 mM). The results of this study will serve as starting points for future projects, including the design and synthesis of compound libraries that seek to systematically explore the fundamental requirements for binding and transport by hPEPT1.
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The human intestinal proton coupled di/tri-peptide transporter hPEPT1 promotes the oral bioavailability of several drug compounds. The strategy behind the present work is that by linking a suitable di- or tripeptidic promoiety to a drug... more
The human intestinal proton coupled di/tri-peptide transporter hPEPT1 promotes the oral bioavailability of several drug compounds. The strategy behind the present work is that by linking a suitable di- or tripeptidic promoiety to a drug substance, by a hydrolysable ester bond, it may give rise to a prodrug that targets hPEPT1. 29 tripeptides were designed based on known structural requirements for substrates binding hPEPT1. Serine, homoserine, or threonine was incorporated in the tripeptide as hydroxy group donors in order for them to be linked to carboxylic drug substances. Optimisation of the promoiety included a study of 14 unnatural tripeptides whose diversity was expressed by VolSurf descriptors. A total of 29 tripeptides was synthesised by solid phase peptide synthesis and a standard Fmoc protocol. The affinity of the tripeptides to hPEPT1 was determined by measuring the inhibition of [(14)C]Gly-Sar in mature Caco-2 cell monolayers which resulted in K(i) values ranging from 0.22 to 25 mM or above. Translocation through the intestinal membrane, mediated by hPEPT1, was measured by recording the membrane potential relative to that induced by the known substrate Gly-Sar. The change in membrane potential is caused by influx of protons due to the co-transport of substrates and protons by hPEPT1 and is, as such, an indication of translocation. A K(i) value of 0.30 mM combined with efficient translocation indicated that H-Phe-Ser-Ala-OH is a suitable lead promoiety for targeted hPEPT1 prodrug design.
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The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet),... more
The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite, and methylseleninic acid (MeSeA), were applied to Caco-2 cells in a concentration of 10 μM, and fluxes in both directions were studied for 2 h. Fluxes of selenite and MeSeA in the presence of excess reduced glutathione (selenite + GSH and MeSeA + GSH) and flux of MeSeA in the presence of excess cysteine (MeSeA + Cys) were also studied. Selenium absorptive and exsorptive fluxes and accumulation in cell cytosol were analyzed by means of flow injection inductively coupled plasma mass spectrometry (ICP-MS). Absorptive flux of SeMet, MeSeCys, and selenate showed values correlating to complete in vivo absorption, while selenite and MeSeA fluxes correlated to poor in vivo absorption. Speciation analysis of cell lysate and donor and receptor solutions by LC-ICP-MS showed limited transformation of all selenium compounds. Extensive transformation as well as significantly increased absorptive flux was observed when co-administering selenite with glutathione compared to administering selenite alone. These observations are possibly due to formation of selenodiglutathione (GS-Se-SG) which may be absorbed differently than selenite. Concomitant application of GSH or cysteine with MeSeA resulted in extensive transformation of MeSeA, including volatile species, whereas no significant increases in fluxes were observed. In summary, the absorption of selenite selenate and the selenoamino acids is considered complete under physiological conditions, but the absorption mechanisms and metabolism of the compounds are different.
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The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified... more
The rationale for targeting the human di-/tripeptide transporter hPEPT1 for oral drug delivery has been well established by several drug and prodrug cases. The aim of this study was to synthesize novel ketomethylene modified tripeptidomimetics and to investigate their binding affinity for hPEPT1. Three related tripeptidomimetics of the structure H-Phe-ψ[COCH2]-Ser(Bz)-Xaa-OH were synthesized applying the tandem chain extension aldol reaction, where amino acid derived β-keto imides were stereoselectively converted to α-substituted γ-keto imides. In addition, three corresponding tripeptides, composed of amide bonds, were synthesized for comparison of binding affinities. The six investigated compounds were all defined as high affinity ligands (Ki-values <0.5 mM) for hPEPT1 by measuring the concentration dependent inhibition of apical [14C]Gly-Sar uptake in Caco-2 cells. Consequently, the ketomethylene replacement for the natural amide bond and α-side chain modifications appears to offer a promising strategy to modify tripeptidic structures while maintaining a high affinity for hPEPT1.
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Research Interests:
The purpose of the present study was to design and characterize low exudate level wound (LEW) and high exudate level wound (HEW) in vitro models by means of investigating therapeutic substance release from exudate-absorbing formulations.... more
The purpose of the present study was to design and characterize low exudate level wound (LEW) and high exudate level wound (HEW) in vitro models by means of investigating therapeutic substance release from exudate-absorbing formulations. Biatain®Ibu foam dressing was used to characterize in vitro release of ibuprofen within the models and also for in vitro–in vivo correlation (IVIVC) studies. Ibuprofen release was described by zero order rate constants of 0.0147 for 1 day and 0.0038 mg/cm2 h for 3 days in HEW and LEW models, respectively. The release is suggested to be controlled by ibuprofen diffusion from the dressing in the HEW model, whereas fluid absorption is rate-limiting in the LEW model. Ibuprofen release, from Biatain®Ibu foam dressings in vivo, is within the same ranges as in vitro. Thus, it is suggested that, depending on the level of exudate, the in vivo release of ibuprofen depends on ibuprofen diffusion from and absorption of exudates to the dressings. Consequently, both the HEW and LEW in vitro models should be applied in order to fully characterize ibuprofen release from Biatain®Ibu foam dressings. Future studies may show whether these in vitro models can be used to characterize therapeutic substance release from exudate-absorbing formulations in general.
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Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degradation in the GI-tract by various peptidases. The... more
Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degradation in the GI-tract by various peptidases. The aim of the present study was to evaluate the general application of N-methyl amide bioisosteres as peptide bond replacements in tripeptides in order to decrease degradation by peptidases and yet retain affinity for and transport via hPEPT1. Seven structurally diverse N-methyl amide tripeptidomimetics were selected based on a principal component analysis of structural properties of 6859 N-methyl amide tripeptidomimetics. In vitro extracellular degradation of the selected tripeptidomimetics as well as affinity for and transepithelial transport via hPEPT1 were investigated in Caco-2 cells. Decreased apparent degradation was observed for all tripeptidomimetics compared to the corresponding natural tripeptides. However, affinity for and transepithelial transport via hPEPT1 were only seen for Gly-Sar-Sar, AsnΨ[CONCH3]PheΨ[CONCH3]Trp, and Gly-Sar-Leu. This implies that tripeptidomimetics originating from tripeptides with neutral side chains are more likely to be substrates for hPEPT1 than tripeptidomimetics with charged side chains. The results of the present study indicate that the N-methyl amide peptide bond replacement approach for increasing bioavailability of tripeptidomimetic drug candidates is not generally applicable to all tripeptides. Nevertheless, retained affinity for and transport via hPEPT1 were shown for three of the evaluated N-methyl amide tripeptidomimetics.
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The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood–brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation... more
The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the blood–brain barrier (BBB) permeation of cyclic prodrugs of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). The BBB permeation characteristics of these prodrugs and DADLE were determined using an in situ perfused rat brain model and in vitro cell culture model (MDCK-MDR1 cells) of the BBB. The activities of P-gp in these models were characterized using a known substrate (quinidine) and known inhibitors [cyclosporine A (CyA), GF-120918, PSC-833] of P-gp. Cyclic peptide prodrugs exhibited very poor permeation in both models. Inclusion of GF-120918, CyA, or PSC-833 in the brain perfusion medium or the cell culture medium significantly increased the permeation of these cyclic prodrugs. The order of potency of these P-gp inhibitors, as measured using the cyclic prodrugs as substrates, was, by in vitro MDCK-MDR1 cells: GF-120918 = CyA ≥ PSC-833; and by in situ rat brain perfusion: GF-120918 > CyA = PSC-833. In conclusion, P-gp in the BBB is the major factor restricting the brain permeation of these cyclic prodrugs. MDCK-MDR1 cells can predict the order of potencies of the investigated P-gp inhibitors to enhance the rat BBB permeation of quinidine and the cyclic prodrugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2227–2236, 2009
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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... more
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.
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Purpose. To investigate the effects of the β-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of... more
Purpose. To investigate the effects of the β-turn structure of a peptide on its permeation via the paracellular and transcellular routes across cultured bovine brain microvessel endothelial cell (BBMEC) monolayers, an in vitro model of the blood-brain barrier (BBB). Methods. The effective permeability coefficients (Peff) of the model peptides were determined across BBMEC monolayers. The dimensions of the aqueous pores in the tight junctions (TJs) of the BBMEC monolayers were determined using a series of hydrophilic permeants. This value and the molecular radius of each peptide were used to calculate the theoretical paracellular (PP*) and transcellular (PT*) permeability coefficients for each peptide. Results. A comparison of the theoretical PP* values with the observed Peff values was made for a series of model peptides. For the most hydrophobic peptides (Ac-PheProXaaIle-NH2 and Ac-PheProXaaIleVal-NH2; Xaa = Gly, Ile), it was concluded that the Gly-containing peptide of each pair more readily permeates BBMEC monolayers via the transcellular pathway than the Ile-containing analog. In addition, the Gly-containing peptides, which exhibit more β-turn structure, were shown to be more lipophilic than the Ile-containing peptides as estimated by the log of their l-octanol:HBSS partition coefficients (log Po/w). However, the three hydrophilic peptide pairs (Ac-TyrProXaaAspVal-NH2, Ac-TyrProXaaAsnVal-NH2, and Ac-TyrProXaaIleVal-NH2; Xaa = Gly, Ile) were found to permeate BBMEC monolayers predominantly via the paracellular pathway. No differences were observed in the Peff values of the hydrophilic peptides having higher β-turn structures as compared to the peptides lacking these structural features. In addition, the Ile-containing peptides exhibited significantly higher log Po/w values than the Gly-containing hydrophilic peptides. Conclusions. Hydrophobic peptides that exhibit significant β-turn structure in solution are more lipophilic as measured by log Po/w, and more readily permeate BBMEC monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties of the peptides so as to alter their paracellular flux through BBMEC monolayers.
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The aim of the present study was to improve the synthetic pathway of bioreversible dipeptide derivatives as well as evaluate the potential of using l-Glu-Sar as a pro-moiety for delivering three newly synthesised nucleoside and pyrimidine... more
The aim of the present study was to improve the synthetic pathway of bioreversible dipeptide derivatives as well as evaluate the potential of using l-Glu-Sar as a pro-moiety for delivering three newly synthesised nucleoside and pyrimidine l-Glu-Sar derivatives. l-Glu(trans-2-thymine-1-yl-tetrahydrofuran-3-yl ester)-Sar (I), l-Glu(thymine-1-yl-methyl ester)-Sar (II) and l-Glu(acyclothymidine)-Sar (III) were synthesised and in vitro stability was studied in various aqueous and biological media. Affinity to and translocation via hPEPT1 was investigated in mature Caco-2 cell monolayers, grown on permeable supports. Affinity was estimated in a competition assay, using [14C] labelled Gly-Sar (glycylsarcosine). Translocation was measured as pHi-changes induced by the substrates using the fluorescent probe BCECF and an epifluorescence microscope setup. All dipeptide derivatives released the model drugs quantitatively by specific base-catalysed hydrolysis at pH > 6.0. II was labile in aqueous buffer solution, whereas I and III showed appropriate stability for oral administration. In 10% porcine intestinal homogenate, the half-lives of the dipeptide derivatives indicated limited enzyme catalyzed degradation. All compounds showed good affinity to hPEPT1, but the Compounds I and III showed not to be translocated by hPEPT1. The translocation of the l-Glu-Sar derivative of acyclovir, l-Glu(acyclovir)-Sar was also investigated and showed not to take place. Consequently, l-Glu-Sar seems to be a poor pro-moiety for hPEPT1-mediated transport.
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The present summary highlight chemical strategies applied to improve plasma half-lives and oral bioavailability of peptidic drugs as well as view on intestinal and pancreatic peptidase mediated degradation of peptidic drugs. In general... more
The present summary highlight chemical strategies applied to improve plasma half-lives and oral bioavailability of peptidic drugs as well as view on intestinal and pancreatic peptidase mediated degradation of peptidic drugs. In general chemical strategies used to increase the oral bioavailability of peptidic drugs consisting of more than three amino acids is disappointing. On the other hand chemical approaches to stabilize peptidic drugs against metabolism seem promising for increasing plasma half-lives of parental peptidic drugs as well as for increasing oral bioavailability of di/tripeptidomimetics and dipeptidyl pro-moieties targeting peptide transporters.
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There is a discrepancy in the adverse effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, statins between the clinical reports and the studies using skeletal muscle cell models. In the clinical reports, both hydrophilic and... more
There is a discrepancy in the adverse effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, statins between the clinical reports and the studies using skeletal muscle cell models. In the clinical reports, both hydrophilic and lipophilic statins induce myotoxicity, whereas in in vitro experiments using cell lines of myoblasts, lipophilic, but not hydrophilic, statins exert myotoxicity. We investigated the cause of this discrepancy. Skeletal myofibres, fibroblasts and satellite cells were isolated from rat flexor digitorum brevis (FDB) muscles. Using these primary cultured cells as well as the L6 myoblast cell line, we compared the toxicity of hydrophilic pravastatin and lipophilic fluvastatin. The mRNA expression levels of possible drug transporters for statins were also examined in these cells using reverse transcriptase-PCR. In the skeletal myofibres, both pravastatin and fluvastatin induced vacuolation and cell death, whereas in the mononuclear cells only fluvastatin, but not pravastatin, was toxic. mRNA of the organic anion transporting polypeptides (Oatp) 1a4 and Oatp2b1 were expressed in the skeletal myofibres, but not in mononucleate cells. Estrone-3-sulphate, a substrate for Oatps, attenuated the effects of pravastatin and fluvastatin in skeletal myofibres; p-aminohippuric acid, a substrate for the organic anion transporters (Oats), but not Oatps, failed to do so. The statin transporters Oatp1a4 and Oatp2b1 are expressed in rat skeletal myofibres, but not in satellite cells, fibroblasts or in L6 myoblasts. This is probably why hydrophilic pravastatin affects skeletal muscle, but not skeletal myoblasts.
Research Interests: Skeletal muscle biology, Kinetics, British, Enzyme Inhibitors, Positron Emission Tomography, and 25 moreGene expression, Cell line, Liver, Pharmaceutical, Female, Animals, Feasibility Studies, Male, Cell Death, Intestinal absorption, Rats, Xenopus laevis, Pyridines, Biotransformation, Wistar Rats, Protein isoforms, Benzimidazoles, Oocytes, Hydrogen-Ion Concentration, Small Intestine, Enterocytes, Bile, Molecular Pharmaceutics, Myoblasts, and fibroblasts(Gene expression, Cell line, Liver, Pharmaceutical, Female, Animals, Feasibility Studies, Male, Cell Death, Intestinal absorption, Rats, Xenopus laevis, Pyridines, Biotransformation, Wistar Rats, Protein isoforms, Benzimidazoles, Oocytes, Hydrogen-Ion Concentration, Small Intestine, Enterocytes, Bile, Molecular Pharmaceutics, Myoblasts, and fibroblasts)
(Gene expression, Cell line, Liver, Pharmaceutical, Female, Animals, Feasibility Studies, Male, Cell Death, Intestinal absorption, Rats, Xenopus laevis, Pyridines, Biotransformation, Wistar Rats, Protein isoforms, Benzimidazoles, Oocytes, Hydrogen-Ion Concentration, Small Intestine, Enterocytes, Bile, Molecular Pharmaceutics, Myoblasts, and fibroblasts)
Carriers may mediate the permeation across enterocytes for drug substances being organic anions. Carrier mediated permeation for the organic anions estrone-3-sulfate (ES) and glipizide across Caco-2 cells were investigated kinetically,... more
Carriers may mediate the permeation across enterocytes for drug substances being organic anions. Carrier mediated permeation for the organic anions estrone-3-sulfate (ES) and glipizide across Caco-2 cells were investigated kinetically, and interactions on involved carriers evaluated. Initial uptakes (PUP) at apical and basolateral membranes, apparent permeabilities (PAPP) and corresponding intracellular end-point accumulations (PEPA) of radioactive labeled compounds were studied. Possible effects of other anionic compounds were investigated. Apical PUP and absorptive PAPP for ES were inhibited and its absorptive PEPA prevented in presence of the investigated organic anions and apical PUP was saturable with Km 23 μM. Basolateral PUP and exsorptive PAPP were inhibited, its exsorptive PEPA was prevented, and basolateral PUP and exsorptive PAPP were saturable with Km 44 μM and 38 μM, respectively. BCRP inhibition affected both absorptive an exsorptive PEPA and PAPP for ES. Glipizide apical PUP and absorptive PAPP were not inhibitable. Basolateral PUP for glipizide was inhibitable, its PEPA prevented, and PUP was saturable with Km 56 μM, but exsorptive PAPP was not affected. Carrier mediated exsorption kinetics for ES are seen at both apical and basolateral membranes, resulting in predominant exsorption despite presence of absorptive carrier(s). Carrier mediated basolateral PUP for glipizide was observed, but glipizide PAPP was not described by carrier kinetics. However, glipizide is affecting exsorption for ES, due to interactions on basolateral carrier. The study confirms that estrone-3-sulfate can be used to characterize anionic carrier kinetics. Furthermore it is suggested that estrone-3-sulfate may be used to identify compounds which may interact on anionic carriers.
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The aim of the present study was to develop a computational method aiding the design of dipeptidomimetic pro-moieties targeting the human intestinal di-/tripeptide transporter hPEPT1. First, the conformation in which substrates bind to... more
The aim of the present study was to develop a computational method aiding the design of dipeptidomimetic pro-moieties targeting the human intestinal di-/tripeptide transporter hPEPT1. First, the conformation in which substrates bind to hPEPT1 (the bioactive conformation) was identified by conformational analysis and 2D dihedral driving analysis of 15 hPEPT1 substrates, which suggested that ψ1 ∼ 165°, ω1 ∼ 180°, and ϕ2 ∼ 280° were descriptive of the bioactive conformation. Subsequently, the conformational energy required to change the peptide backbone conformation (ΔEbbone) from the global energy minimum conformation to the identified bioactive conformation was calculated for 20 hPEPT1 targeted model prodrugs with known Ki values. Quantitatively, an inverse linear relationship (r2 = 0.81, q2 = 0.80) was obtained between ΔEbbone and log 1/Ki, showing that ΔEbbone contributes significantly to the experimentally observed affinity for hPEPT1 ligands. Qualitatively, the results revealed that compounds classified as high affinity ligands (Ki < 0.5 mM) all have a calculated ΔEbbone < 1 kcal/mol, whereas medium and low-affinity compounds (0.5 mM < Ki < 15 mM) have ΔEbbone values in the range 1–3 kcal/mol. The findings also shed new light on the basis for the experimentally observed stereoselectivity of hPEPT1.
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Sulfonamides and their derivatives inhibit the catalytic activity of carbonic anhydrases (CA, EC 4.2.1.1). Isozyme IX (CA IX) is a transmembrane isoform with the active site oriented toward the extracellular space. CA IX was recently... more
Sulfonamides and their derivatives inhibit the catalytic activity of carbonic anhydrases (CA, EC 4.2.1.1). Isozyme IX (CA IX) is a transmembrane isoform with the active site oriented toward the extracellular space. CA IX was recently shown to be a drug target, and it is highly overexpressed in hypoxic tumors with limited distribution in normal tissues. The present report deals with the drug design, synthesis, and biological investigation of a group of thioureido sulfonamides, which have been obtained by reaction of isothiocyanate-substituted aromatic sulfonamides with amines. These compounds have potent inhibitory properties against CA IX with KI values in the range of 10–37 nM and Pappvalues > 0.34 × 10−6 cm/s for the absorptive transepithelial transport in Caco-2 cells. In Caco-2 cells, one of these compounds (A6) was shown to be a substrate for efflux transporters such as P-glycoprotein (P-gp). P-gp activity is not likely to be rate-limiting for intestinal absorption, but might be useful when targeting hypoxic tumors expressing both P-gp and CA IX.
Research Interests: Organic Chemistry, Humans, Drug Design, P-glycoprotein, Intestinal absorption, and 11 moreCaco-2 cells, Active site, Carbonic anhydrase, Isoenzymes, Limit Distribution, Bioorganic and medicinal Chemistry, Structure activity Relationship, Caco 2 Cell, Drug Targeting, Catalytic Activity, and Molecular Structure
Five dipeptidomimetic-based model prodrugs containing ketomethylene amide bond replacements were synthesized from readily available alpha,beta-unsaturated gamma-ketoesters. The model drug (BnOH) was attached to the C-terminus or to one of... more
Five dipeptidomimetic-based model prodrugs containing ketomethylene amide bond replacements were synthesized from readily available alpha,beta-unsaturated gamma-ketoesters. The model drug (BnOH) was attached to the C-terminus or to one of the side chain positions of the dipeptidomimetic. The stability, the affinity for the di-/tripeptide transporter hPEPT1, and the transepithelial transport properties of the model prodrugs were investigated. ValPsi[COCH(2)]Asp(OBn) was the compound with highest chemical stability in buffers at pH 6.0 and 7.4, with half-lives of 190 and 43 h, respectively. All five compounds showed high affinity for hPEPT1 (K(i) values &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 1 mM), and PhePsi[COCH(2)]Asp(OBn) and ValPsi[COCH(2)]Asp(OBn) had the highest affinities with K(i) values of 68 and 19 microM, respectively. An hPEPT1-mediated transport component was demonstrated for the transepithelial transport of three compounds, a finding that was corroborated by hPEPT1-mediated intracellular uptake. The results indicate that the stabilized Phe-Asp and Val-Asp derivatives are promising pro-moieties in a prodrug approach targeting hPEPT1.
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The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) of the opioid peptide DADLE... more
The objective of this study was to elucidate the role of P-glycoprotein (P-gp) in restricting the intestinal mucosal permeation of cyclic prodrugs (AOA-DADLE, CA-DADLE, and OMCA-DADLE) of the opioid peptide DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In the Caco-2 cell model, the high Papp,BL-to-AP/Papp,AP-to-BL ratios of AOA-DADLE, CA-DADLE, and OMCA-DADLE (71–117) were significantly decreased by including known P-gp inhibitors, GF-12098, cyclosporine (CyA), or PSC-833, in the incubation media, suggesting that P-gp is restricting the AP-to-BL permeation of these cyclic prodrugs. In the in situ perfused rat ileum model, AOA-DADLE, CA-DADLE, and OMCA-DADLE were shown to exhibit very low permeation into the mesenteric blood (PB = 0.40, 0.56 and 0.42 × 10−7 cm/s, respectively). PSC-833 was found to increase significantly the PB values for all three prodrugs. In contrast, CyA and GF-12918 were either inactive or substantially less active than PSC-833 in increasing the PB values of these prodrugs. These data suggest that, while P-gp plays a role, other factors (e.g., substrate activity for other efflux transporters and/or for metabolic enzymes) may contribute to restricting the permeation of AOA-DADLE, CA-DADLE, and OMCA-DADLE across the rat intestinal mucosa. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:337–348, 2009
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The purpose of these studies was to compare the in vitro absorption of two inorganic chromium(III) compounds: chromium chloride and chromium nitrate, with organic chromium(III)-picolinate; and to investigate if any in vitro metabolism of... more
The purpose of these studies was to compare the in vitro absorption of two inorganic chromium(III) compounds: chromium chloride and chromium nitrate, with organic chromium(III)-picolinate; and to investigate if any in vitro metabolism of chromium(VI) takes place. The in vitro metabolism studies showed that chromium (VI) was reduced by artificial gastric juice. The reduction followed first order kinetics with a half-life of 23 min. The studies also showed that the chromium picolinate complex was stable in artificial gastric juice for 4 hours. By the rat everted gut sac technique, chromium chloride, chromium nitrate and chromium picolinate penetrated the rat jejunum with 165±59,160±26 and 127±36 ng chromium per g rat jejunum, respectively, whereas the permeability coefficients (Papp ) were 0.7±0.3, 1.0±0.4, and 9.6±2.2 μm/min, respectively. Absorption studies on pig intestine in Ussing chambers showed a nearly total adsorption of chromium(III) by the chambers, resulting in unreliable data.
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The aim of this study was to develop a three-dimensional quantitative structure-activity relationship (QSAR) model for binding of tripeptides and tripeptidomimetics to hPEPT1 based on a series of 25 diverse tripeptides. VolSurf... more
The aim of this study was to develop a three-dimensional quantitative structure-activity relationship (QSAR) model for binding of tripeptides and tripeptidomimetics to hPEPT1 based on a series of 25 diverse tripeptides. VolSurf descriptors were generated and correlated with binding affinities by multivariate data analysis. The affinities for hPEPT1 of the tripeptides and tripeptidomimetics were determined experimentally by use of Caco-2 cell monolayers. The Ki-values of the 25 tripeptides and tripeptidomimetics ranged from 0.15 to 25 mM and the structural diversity of the compounds was described by VolSurf descriptors. A QSAR model that correlated the VolSurf descriptors of the tripeptides with their experimental binding affinity for hPEPT1 was established. Structural information on tripeptide properties influencing the binding to hPEPT1 was extracted from the QSAR model. This information may contribute to the drug design process of tripeptides and tripeptidomimetics where hPEPT1 is targeted as an absorptive transporter for improvement of intestinal absorption. To our knowledge, this is the first time a correlation between VolSurf descriptors and binding affinities for hPEPT1 has been reported.
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Page 1. Review 10.1517/13543776.15.2.153 © 2005 Ashley Publications Ltd ISSN 1354-3776 153 Ashley Publications www.ashley-pub.com For reprint orders, please contact: reprints@ashley-pub.com Recent advances in therapeutic applications of... more
Page 1. Review 10.1517/13543776.15.2.153 © 2005 Ashley Publications Ltd ISSN 1354-3776 153 Ashley Publications www.ashley-pub.com For reprint orders, please contact: reprints@ashley-pub.com Recent advances in therapeutic applications of human peptide ...
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The human peptide transporter, hPepT1, situated in the small intestine, may be exploited to increase absorption of drugs or model drugs by attaching them to a dipeptide, which is recognised by hPepT1. A synthetic protocol for this kind of... more
The human peptide transporter, hPepT1, situated in the small intestine, may be exploited to increase absorption of drugs or model drugs by attaching them to a dipeptide, which is recognised by hPepT1. A synthetic protocol for this kind of model prodrugs was developed, in which model drugs containing a hydroxy group were attached to enzymatically stable dipeptides by hydrolysable ester linkages. Furthermore, a number of benzyl alcohols with various substituents in the 4-position of the phenyl ring were coupled to d-Asp–Ala and d-Glu–Ala. Ideally, a prodrug should be stable in the upper small intestine and be converted to the parent drug during or after transport into the blood circulation. Therefore, we investigated the influence of the electronegativity of the substituent in the 4-position of the phenyl ring on stability in aqueous solution at pH 6.0 and 7.4, corresponding to pH in jejunum and blood, respectively. In addition, the influence of the electronegativity of the substituent on stability upon storage was examined. Model prodrugs containing electron donating substituents in the 4-position of the phenyl ring decomposed upon storage, while model prodrugs containing no substituents or electron withdrawing substituents in the 4-position were stable. In aqueous solution (pH 6.0 and 7.4), electron withdrawing substituents in the 4-position decreased the half-life of the model prodrug. These data provide important information on stability of this kind of model prodrugs upon storage and under aqueous conditions. The results may be applied in the rational design of oligopeptide ester prodrugs to obtain prodrugs, which are stable upon storage and have an optimal release profile of the drug.
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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... more
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.
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Various N1-alkoxycarbonyl prodrugs of 5-fluorouracil (5-FU) ranging from 5 to 18 carbon units in the pro-moiety were synthesized. The compounds were physico-chemically characterized by determining the ionization constant (Ka), aqueous... more
Various N1-alkoxycarbonyl prodrugs of 5-fluorouracil (5-FU) ranging from 5 to 18 carbon units in the pro-moiety were synthesized. The compounds were physico-chemically characterized by determining the ionization constant (Ka), aqueous solubility (S), octanol/water partition coefficient (P), and the degradation rate in aqueous solution at different pH values. The in vitro degradation rate of the prodrugs in calf serum, human plasma, and rabbit vitreous humor was also investigated. Drug delivery systems were prepared by dissolving or dispersing 5-FU prodrugs in silicone oil 1000 (SO-1000). The release of 5-FU seemed to follow the square root of time kinetics until more than 60% of the drug had been released. The release rate of 5-FU was found to decrease with increased lipophilicity of the prodrug.
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Purpose: To examine the in vivo pharmacokinetics of intravitreal 5-Fluorouracil (5-FU) following tamponade with 5-FU prodrug silicone oil formulations.Method: Two different alkoxycarbonyl 5-FU prodrugs denoted C12 and C18 were synthesized... more
Purpose: To examine the in vivo pharmacokinetics of intravitreal 5-Fluorouracil (5-FU) following tamponade with 5-FU prodrug silicone oil formulations.Method: Two different alkoxycarbonyl 5-FU prodrugs denoted C12 and C18 were synthesized and formulated as silicone oil suspensions. A total of 26 pigs underwent conventional three-port lens-sparing pars plana vitrectomy. Approximately 1.6 ml of the prodrug-silicone oil formulation was placed in the vitreous cavity. Operated eyes were enucleated between 20 min and 168 hours postoperatively, and analysed for their content of free 5-FU by high performance liquid chromatography.Results: With the C12 prodrug silicone oil formulation, the concentration of free 5-FU in the vitreous water phase 1 hour after surgery was 3.30 ± 1.62 µg/ml. After 4 hours this concentration had declined to 1 µg/ml. With the C18 prodrug, the concentration of free vitreal 5-FU never reached 1 µg/ml during the 7 days these experiments lasted. A mathematical model is presented that can explain the measured data if the clearance of 5-FU from the vitreous water phase follows first order kinetics with a half-life of 20 min.Conclusion: These experiments, and the model analysis, suggest that the elimination half-life of 5-FU in the vitreous cavity of a vitrectomized, silicone oil-filled eye is very fast. The model analysis indicates that an alkoxycarbonyl 5-FU prodrug with a specific release rate constant of 10.7 µg/( ) can maintain an intravitreal 5-FU concentration above 1 µg/ml for 5 days in the porcine eye.
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It has previously been shown that the prodrug Glu(acyclovir)-Sar has a high affinity for PEPT1 in Caco-2 cells. However, affinity does not necessarily lead to translocation by the transporter which is necessary for achieving an increased... more
It has previously been shown that the prodrug Glu(acyclovir)-Sar has a high affinity for PEPT1 in Caco-2 cells. However, affinity does not necessarily lead to translocation by the transporter which is necessary for achieving an increased oral bioavailability. Therefore i.v. and p.o. doses of Glu(acyclovir)-Sar, acyclovir and valacyclovir were given to rats and the collected blood samples were analysed via LC–MS–MS. Furthermore, Caco-2 cell monolayers were exposed apically to Glu(acyclovir)-Sar, acyclovir, and valacyclovir and the concentration of drug and prodrugs in the cell extracts were determined and taken as a measure for intracellular accumulation. In addition, bi-directional transport studies of Glu(acyclovir)-Sar across Caco-2 cell monolayers and in vitro metabolism studies of Glu(acyclovir)-Sar in various media of rat origin were performed. For these purposes HPLC-UV analysis was applied. Oral administration of Glu(acyclovir)-Sar to rats resulted in low bioavailabilities of acyclovir (<2%) and intact prodrug (<5%). Studies performed on Caco-2 cell monolayers showed that in contrast to valacyclovir Glu(acyclovir)-Sar did not result in a detectable amount of acyclovir or Glu(acyclovir)-Sar in the cell extracts. Bi-directional flux across Caco-2 cell monolayers apical to basolateral (FluxA→B) and basolateral to apical (FluxB→A) was measured and the FluxB→A/FluxA→B ratios of approximately 0.8 indicate that apical efflux mechanisms may not explain this lack of intracellular accumulation. These data indicate that Glu(acyclovir)-Sar may not be translocated by PEPT1.
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Aims: Little is known about the physiological regulation of the human intestinal di/tri-peptide transporter, hPepT1. In the present study we evaluated the effects of epidermal growth factor (EGF) and insulin on hPepT1-mediated dipeptide... more
Aims: Little is known about the physiological regulation of the human intestinal di/tri-peptide transporter, hPepT1. In the present study we evaluated the effects of epidermal growth factor (EGF) and insulin on hPepT1-mediated dipeptide uptake in the intestinal cell line Caco-2.Methods: Caco-2 cells were grown on filters for 23–27 days. Apical dipeptide uptake was measured using [14C]glycylsarcosine([14C]Gly-Sar). HPepT1 mRNA levels were investigated using RT-PCR, cytosolic pH was determined using the pH-sensitive fluorescent probe BCECF.Results: Basolateral application of EGF increased [14C]Gly-Sar uptake with an ED50 value of 0.77 ± 0.25 ng mL−1 (n = 3−6) and a maximal stimulation of 33 ± 2% (n = 3−6). Insulin stimulated [14C]Gly-Sar uptake with an ED50 value of 3.5 ± 2.0 ng mL−1 (n = 3−6) and a maximal stimulation of approximately 18% (n = 3−6). Gly-Sar uptake followed simple Michaelis-Menten kinetics. Km in control cells was 0.98 ± 0.11 mM (n = 8) and Vmax was 1.86 ± 0.07 nmol cm−2 min−1 (n = 8). In monolayers treated with 200 ng mL−1 of EGF, Km was 1.11 ± 0.05 mM (n = 5) and Vmax was 2.79 ± 0.05 nmol cm−2 min−1 (n = 5). In monolayers treated with 50 ng mL−1 insulin, Km was 1.03 ± 0.08 mM and Vmax was 2.19 ± 0.06 nmol cm−2 min−1 (n = 5). Kinetic data thus indicates an increase in the number of active transporters, following stimulation. The incrased Gly-Sar uptake was not accompanied by changes in hPepT1 mRNA, nor by measurable changes in cytosolic pH.Conclusions: Short-term stimulation with EGF and insulin caused an increase in hPepT1-mediated uptake of Gly-Sar in Caco-2 cell monolayers, which could not be accounted for by changes in hPepT1 mRNA or proton-motive driving force.
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The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a target for increasing intestinal transport of low permeability compounds by creating prodrugs designed for the transporter. Model ester prodrugs using the... more
The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a target for increasing intestinal transport of low permeability compounds by creating prodrugs designed for the transporter. Model ester prodrugs using the stabilized dipeptides d-Glu-Ala and d-Asp-Ala as pro-moieties for benzyl alcohol have been shown to have affinity for hPepT1. Furthermore, in aqueous solution at pH 5.5 to 10, the release of the model drug seems to be controlled by a specific base-catalyzed hydrolysis, indicating that the compounds may remain relatively stable in the upper small intestinal lumen with a pH of ∼6.0, but still release the model drug at the intercellular and blood pH of ∼7.4. Even though benzyl alcohol is not a low molecular weight drug molecule, these results indicate that the dipeptide prodrug principle is a promising drug delivery concept. However, the physico-chemical properties such as electronegativity, solubility, and log P of the drug molecule may also have an influence on the potential of these kinds of prodrugs. The purpose of the present study is to investigate whether the model drug electronegativity, estimated as Taft substitution parameter (σ*) may influence the acid, water or base catalyzed model drug release rates, when released from series of d-Glu-Ala and d-Asp-Ala pro-moieties. Release rates were investigated in both aqueous solutions with varying pH, ionic strength, and buffer concentrations as well as in in vitro biological media. The release rates of all the investigated model drug molecules followed first-order kinetics and were dependent on buffer concentration, pH, ionic strength, and model drug electronegativity. The electronegativity of the model drug influenced acid, water and base catalyzed release from d-Asp-Ala and d-Glu-Ala pro-moieties. The model drug was generally released faster from d-Asp-Ala- than from the d-Glu-Ala pro-moieties. In biological media the release rate was also dependent on the electronegativity of the model drug. These results demonstrate that the model drug electronegativity, estimated as Taft (σ*) values, has a significant influence on the release rate of the model drug.
Research Interests: Algorithms, Biomedical Engineering, Kinetics, Drug delivery, Prodrugs, and 20 moreHumans, Controlled release, QSAR, Animals, Plasma, First-Order Logic, Solubility, High Pressure Liquid Chromatography, Intestines, Buffers, Solutions, Swine, Ionic Strength, Physico chemical Properties, Aqueous Solution, Drug Carriers, Hydrogen-Ion Concentration, Drug Release, Low molecular weight, and Small Intestine(Humans, Controlled release, QSAR, Animals, Plasma, First-Order Logic, Solubility, High Pressure Liquid Chromatography, Intestines, Buffers, Solutions, Swine, Ionic Strength, Physico chemical Properties, Aqueous Solution, Drug Carriers, Hydrogen-Ion Concentration, Drug Release, Low molecular weight, and Small Intestine)
(Humans, Controlled release, QSAR, Animals, Plasma, First-Order Logic, Solubility, High Pressure Liquid Chromatography, Intestines, Buffers, Solutions, Swine, Ionic Strength, Physico chemical Properties, Aqueous Solution, Drug Carriers, Hydrogen-Ion Concentration, Drug Release, Low molecular weight, and Small Intestine)
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... more
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 (Ki∼0.2–0.3 mM), whereas the d-Glu-Ala prodrugs had poor affinity (Ki∼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∼7.4 (t1/2∼3.5 and 5.5 h) are approximately 25 times faster than at upper small intestinal pH∼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 (t1/2∼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.
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One approach to increase drug stability and to facilitate oral absorption of low bioavailability drugs may be to design oligopeptide ester prodrugs which are stable in the gastrointestinal tract, are transported via the oligopeptide... more
One approach to increase drug stability and to facilitate oral absorption of low bioavailability drugs may be to design oligopeptide ester prodrugs which are stable in the gastrointestinal tract, are transported via the oligopeptide transporter, and finally release the parent drug molecule into the blood circulation and/or by its site of action. In these kinds of prodrugs the ester linkage may be broken by pH dependent and/or enzyme catalyzed hydrolysis. The objective of the present study was to investigate the degradation mechanism and rate of the model compounds Glu(OBzl)–Sar, d-Glu(OBzl)–Ala and Asp(OBzl)–Sar in aqueous solution and in relevant biological media and to compare these results with those of our previous study of d-Asp(OBzl)–Ala. Furthermore, the resulting aqueous stability and in vitro metabolism data are related to our previous affinity data to evaluate if Glu–Sar, d-Glu–Ala, and Asp–Sar have potential as pro-moieties in these kinds of prodrugs. The degradation rates follow first-order kinetics, show maximun stability at pH 4–5 with maximum half-lives for Asp(OBzl)–Sar, Glu(OBzl)–Sar, and d-Glu(OBzl)–Ala of 115 h, 30 days and 152 days, respectively. The stability was dependent on buffer concentration, temperature, pH, and ionic strength. In biological media such as 80% human plasma, human gastric juice and intestinal fluid, and 10% rat jejunal homogenate at 37°C, the half-lives were greater than 1 h except for the hydrolysis of Glu(OBzl)–Sar in 10% rat jejunal homogenate, where the half-life was approximately 16 min. All the stabilized dipeptides may have potential as drug carriers targeting hPepT1.
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The model prodrug d-Asp(OBzl)–Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous... more
The model prodrug d-Asp(OBzl)–Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous stability of d-Asp(OBzl)–Ala and its in vitro metabolism in different gastrointestinal media arising from rats and humans, as well as in human plasma. The second major aim of the study was to evaluate our previous study in Caco-2 cell culture, by determining the effective intestinal permeability (Peff) of d-Asp(OBzl)–Ala in situ using the single-pass rat perfusion model. The aqueous stability studies show water, general buffer, as well as specific acid and base catalysis of d-Asp(OBzl)–Ala. The degradation of the model prodrug was independent of ionic strength. The half-lives in rat jejunal fluid and homogenate were >3 h. In human gastric and intestinal fluids, the half-lives were >3 h and 2.3±0.03 h, respectively. Using the rat single-pass perfusion technique, the effective jejunal permeability (Peff) of d-Asp(OBzl)–Ala was determined to be high (1.29±0.5·10−4 cm/s). The 32 times higher Peff value found in the perfusion model compared to Caco-2 cells is most likely due to a higher functional expression of the oligopeptide transporter. Rat jejuna Peff was reduced by approximately 50% in the presence of well known oligopeptide transporter substrates, such as Gly–Sar and cephalexin. It may be that d-Asp(OBzl)–Ala is primarily absorbed intact by the rat jejunal oligopeptide transporter, since the stability in the intestinal homogenate and fluids was rather high (t1/2>2.3 h).
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Page 1. Review 2002 © Ashley Publications Ltd ISSN 1354-3776 1329 Ashley Publications www.ashley-pub.com 1. Introduction 2. Structure and function of peptide transporters 3. Therapeutic applications of human peptide transporters 4.... more
Page 1. Review 2002 © Ashley Publications Ltd ISSN 1354-3776 1329 Ashley Publications www.ashley-pub.com 1. Introduction 2. Structure and function of peptide transporters 3. Therapeutic applications of human peptide transporters 4. Patents in the area 5. Expert opinion ...
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A large amount of absorptive intestinal membrane transporters play an important part in absorption and distribution of several nutrients, drugs and prodrugs. The present paper gives a general overview on intestinal solute carriers as well... more
A large amount of absorptive intestinal membrane transporters play an important part in absorption and distribution of several nutrients, drugs and prodrugs. The present paper gives a general overview on intestinal solute carriers as well as on trends and strategies for targeting drugs and/or prodrugs to these carriers in order to increasing oral bioavailability and distribution. A number of absorptive intestinal transporters are described in terms of gene and protein classification, driving forces, substrate specificities and cellular localization. When targeting absorptive large capacity membrane transporters in the small intestine in order to increase oral bioavailabilities of drug or prodrug, the major influence on in vivo pharmacokinetics is suggested to be dose-dependent increase in bioavailability as well as prolonged blood circulation due to large capacity facilitated absorption, and renal re-absorption, respectively. In contrast, when targeting low-capacity transporters such as vitamin transporters, dose independent saturable absorption kinetics are suggested. We thus believe that targeting drug substrates for absorptive intestinal membrane transporters could be a feasible strategy for optimizing drug bioavailability and distribution.
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The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a drug delivery target via increasing the intestinal transport of low permeability compounds by designing peptidomimetic prodrugs. Model ester prodrugs using the... more
The human intestinal di/tri-peptide carrier, hPepT1, has been suggested as a drug delivery target via increasing the intestinal transport of low permeability compounds by designing peptidomimetic prodrugs. Model ester prodrugs using the stabilized dipeptides d-Glu–Ala and d-Asp–Ala as pro-moieties for benzyl alcohol have been shown to maintain affinity for hPepT1. The primary aim of the present study was to investigate if modifications of the benzyl alcohol model drug influence the corresponding d-Glu–Ala and d-Asp–Ala model prodrugs’ affinity for hPepT1 in Caco-2 cells. A second aim was to investigate the transepithelial transport and hydrolysis parameters for d-Asp(BnO)–Ala and d-Glu(BnO)–Ala across Caco-2 cell monolayers. In the present study, all investigated d-Asp–Ala and d-Glu–Ala model prodrugs retained various degrees of affinity for hPepT1 in Caco-2 cells. These affinities are used to establish a QSAR of our benzyl alcohol modified model prodrugs, aided at elucidating the observed differences in model prodrug affinity for hPepT1; additionally, these data suggest that the hydrophobicity of the side-chain model drug is the major determinant in the compounds affinity for hPepT1. Transepithelial transport studies performed using Caco-2 cells of d-Asp(BnO)–Ala and d-Glu(BnO)–Ala showed that the Km for transepithelial transport was not significantly different for the two compounds. The maximal transport rate of the carrier-mediated flux component does not differ between the two model prodrugs either. The transepithelial transport of d-Asp(BnO)–Ala and d-Glu(BnO)–Ala follows simple kinetics, and the release of benzyl alcohol is pH-dependent, but unaffected by 1 mM of the esterase inhibitor Paraoxon in 80% human plasma and Caco-2 cell homogenate.
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Abstract: The apical membrane of small intestinal enterocytes possess an uptake system for di- and tripeptides. The physiological function of the system is to transport small peptides resulting from digestion of dietary protein. Moreover,... more
Abstract: The apical membrane of small intestinal enterocytes possess an uptake system for di- and tripeptides. The physiological function of the system is to transport small peptides resulting from digestion of dietary protein. Moreover, due to the broad substrate specificity of the system, it is also capable of transporting a number of orally administered peptidomimetic drugs. Absorbed peptides may be hydrolysed in the cells due to the high peptidase activity present in the cytosol. Peptidomimetic drugs may, if resistant to the cellular enzyme activity, pass the basolateral membrane via a basolateral peptide transport mechanism and enter the systemic circulation. As the number of new peptide and peptidomimetic drugs are rapidly increasing, the peptide transport system has gained increasing attention as a possible drug delivery system for small peptides and peptide-like compounds. In this paper we give an updated introduction to the transport system and discuss the substrate characteristics of the di/tri-peptide transporter system with special emphasis on chemically modified substrates and prodrugs.
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An analytical procedure for detection of ivermectin in plasma samples was modified and used for measuring ivermectin concentrations in dung samples. The residues were quantified by high-performance liquid chromatography and fluorescence... more
An analytical procedure for detection of ivermectin in plasma samples was modified and used for measuring ivermectin concentrations in dung samples. The residues were quantified by high-performance liquid chromatography and fluorescence detection after extraction, purification, and derivatization into fluorescent reaction products. The concentrations of ivermectin in dung were measured after dosing heifers with 0.2 mg kg−1 by subcutaneous injection or 0.5 mg kg−1 by pour-on. Despite the 2.5 time higher pour-on dose, this formulation did not result in excretion of detectable concentrations of ivermectin over a longer period of time than did the injection treatment. Dung pats with ivermectin residues were exposed on grazing pastures until they were no longer colonised by flies and beetles. In the experiments performed under temperate and tropical conditions, the aging of the pats did not lead to a significant lowering of the concentrations of ivermectin.Flies or beetles colonising the pats some time after deposition may therefore not be exposed to decreased levels of ivermectin.
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Abstract Heifers were treated with the recommended doses of ivermectin: 0.2 mg/ kg bw by subcutaneous injection or 0.5 mg/kg bw by pour-on. An analytic procedure is described and used for the detection of ivermectin residues ex- creted in... more
Abstract Heifers were treated with the recommended doses of ivermectin: 0.2 mg/ kg bw by subcutaneous injection or 0.5 mg/kg bw by pour-on. An analytic procedure is described and used for the detection of ivermectin residues ex- creted in dung. A large amount of the higher pour-on dose was ...