A general drug delivery approach for increasing oral bioavailability of purine and pyrimidine ana... 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 (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.
Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denat... more Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS-lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.
American Journal of Physiology-Renal Physiology, 2004
The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides i... more The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides in the proximal tubule; however, knowledge of regulation of PEPT2 by growth factors and hormones is limited. In the present study, we examined the effects of epidermal growth factor (EGF) on PEPT2 transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 exp...
Little is known about the physiological regulation of the human intestinal di/tri-peptide transpo... more 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. 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. 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. 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.
ABSTRACT Abstract The thermodynamic phase behavior and lipid-membrane structure of fully hydrated... more ABSTRACT Abstract The thermodynamic phase behavior and lipid-membrane structure of fully hydrated uni- and multilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) surface-modified by a glycosphingolipid, sulfogalactosylceramide (sulfatide), have been investigated by means of differential scanning calorimetry and fluorescence polarization spectroscopy. The calorimetric and spectroscopic scans of the two-component liposomes demonstrate a distinct influence of increasing amounts of sulfatide on the lipidmembrane thermodynamics. This is manifested as a broad gel-fluid phasecoexistence region and a low-temperature two-phase region composed of highly ordered lipids. In addition, the pre-transition is abolished for small concentrations of sulfatide. A well-defined heat capacity peak, reflecting a thermotropic chain melting transition is observed for liposomes composed of pure sulfatide. On basis of the calorimetric and spectroscopic data a phase diagram has been established in the full temperature and composition plane. Dynamic light scattering measurements of liposome sizes reveal that the incorporation of sulfatide into the DPPC membrane matrix leads to a stabilization of the characteristic size of the extruded liposomes. Furthermore, incubation with insulin does not affect the liposome size and the aggregational behavior of the two-component sulfatide-DPPC liposomes. In accordance with this, the heat capacity curves demonstrate an insignificant influence on both the lipid membrane phase behavior and the thermal unfolding characteristics of insulin.
The aim of this study was to develop a structure-property model for membrane partitioning of olig... more The aim of this study was to develop a structure-property model for membrane partitioning of oligopeptides using statistical design methods and multivariate data analysis. A set of 20 tetrapeptides with optional N-methylations at residues 2 and 4 was designed by a D-optimal design procedure. After synthesis and purification, the membrane partitioning abilities of the peptides were tested in two chromatographic systems with phospholipids as the stationary phase: immobilized artificial membrane chromatography (IAM) and immobilized liposome chromatography (ILC). The relationship between these measures and three different sets of calculated descriptors was analyzed by partial least-squares projection to latent structures (PLS). The descriptors used were the molecular surface area, Molsurf parameters, and Volsurf parameters. All three models were of good statistical quality and supported that a large hydrogen-bonding potential and the presence of a negative charge impair membrane partitioning, whereas hydrophobic parameters promote partitioning. The findings are in accordance with what has been found for absorption of known drugs and have implications for the design of peptide-like drugs with good oral bioavailability.
A general drug delivery approach for increasing oral bioavailability of purine and pyrimidine ana... 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 (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.
Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denat... more Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS-lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.
American Journal of Physiology-Renal Physiology, 2004
The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides i... more The renal peptide transporter PEPT2 plays an important role in absorption of di- and tripetides in the proximal tubule; however, knowledge of regulation of PEPT2 by growth factors and hormones is limited. In the present study, we examined the effects of epidermal growth factor (EGF) on PEPT2 transport capacity and expression in the rat proximal tubule cell line SKPT0193 cl.2 (SKPT), which expresses rat PEPT2 (rPEPT2) in the apical membrane. Treatment of SKPT cells with EGF during cell culture growth caused a dose-dependent decrease in rPEPT2 transport capacity and expression, as determined by studies of apical uptake of [14C]glycylsarcosine, rPepT2 mRNA levels, and immunostaining of SKPT cells with a rPEPT2-specific antibody. On the contrary, apical uptake of glucose and lysine was increased in EGF-treated cells, indicating that EGF was not acting generally to decrease apical nutrient uptake mechanisms in the proximal tubule cells. Our findings indicate that EGF decreases rPEPT2 exp...
Little is known about the physiological regulation of the human intestinal di/tri-peptide transpo... more 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. 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. 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. 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.
ABSTRACT Abstract The thermodynamic phase behavior and lipid-membrane structure of fully hydrated... more ABSTRACT Abstract The thermodynamic phase behavior and lipid-membrane structure of fully hydrated uni- and multilamellar liposomes composed of dipalmitoylphosphatidylcholine (DPPC) surface-modified by a glycosphingolipid, sulfogalactosylceramide (sulfatide), have been investigated by means of differential scanning calorimetry and fluorescence polarization spectroscopy. The calorimetric and spectroscopic scans of the two-component liposomes demonstrate a distinct influence of increasing amounts of sulfatide on the lipidmembrane thermodynamics. This is manifested as a broad gel-fluid phasecoexistence region and a low-temperature two-phase region composed of highly ordered lipids. In addition, the pre-transition is abolished for small concentrations of sulfatide. A well-defined heat capacity peak, reflecting a thermotropic chain melting transition is observed for liposomes composed of pure sulfatide. On basis of the calorimetric and spectroscopic data a phase diagram has been established in the full temperature and composition plane. Dynamic light scattering measurements of liposome sizes reveal that the incorporation of sulfatide into the DPPC membrane matrix leads to a stabilization of the characteristic size of the extruded liposomes. Furthermore, incubation with insulin does not affect the liposome size and the aggregational behavior of the two-component sulfatide-DPPC liposomes. In accordance with this, the heat capacity curves demonstrate an insignificant influence on both the lipid membrane phase behavior and the thermal unfolding characteristics of insulin.
The aim of this study was to develop a structure-property model for membrane partitioning of olig... more The aim of this study was to develop a structure-property model for membrane partitioning of oligopeptides using statistical design methods and multivariate data analysis. A set of 20 tetrapeptides with optional N-methylations at residues 2 and 4 was designed by a D-optimal design procedure. After synthesis and purification, the membrane partitioning abilities of the peptides were tested in two chromatographic systems with phospholipids as the stationary phase: immobilized artificial membrane chromatography (IAM) and immobilized liposome chromatography (ILC). The relationship between these measures and three different sets of calculated descriptors was analyzed by partial least-squares projection to latent structures (PLS). The descriptors used were the molecular surface area, Molsurf parameters, and Volsurf parameters. All three models were of good statistical quality and supported that a large hydrogen-bonding potential and the presence of a negative charge impair membrane partitioning, whereas hydrophobic parameters promote partitioning. The findings are in accordance with what has been found for absorption of known drugs and have implications for the design of peptide-like drugs with good oral bioavailability.
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Papers by Sven Frokjaer