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Per Artursson - One of the best experts on this subject based on the ideXlab platform.
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Ph Dependent passive and active transport of acidic drugs across caco 2 cell monolayers
European Journal of Pharmaceutical Sciences, 2005Co-Authors: Annalena Ungell, Sibylle Neuhoff, Ismael Zamora, Per ArturssonAbstract:The aim of this study was to investigate Ph-Dependent passive and active transport of acidic drugs across Caco-2 cells. Therefore, the bidirectional Ph-Dependent transport of two acidic drugs, indomethacin and salicylic acid, across Caco-2 cells was studied in the Physiological Ph range of the gastrointestinal tract. The transport of both drugs decreased with increased Ph, as expected from the Ph-partition hypothesis. Net absorption occurred when the basolateral Ph exceeded the apical Ph. Concentration dependence and transporter inhibition studies indicated passive transport for indomethacin and a mixture of Ph-Dependent passive and active influx for salicylic acid. Unexpectedly, active and passive drug transport results were indistinguishable in temperature dependency studies. The transport of salicylic acid (apical Ph 5.0; basolateral Ph 7.4) was partly blocked by inhibitors of the proton-Dependent transporters MCT1 (SLC16A1) and OATP-B (SLC21A9, SLCO2B1). This study shows that the asymmetry in bidirectional transport of acidic drugs is affected by both passive and active components in the presence of Ph gradients across Caco-2 cells. Thus, combined studies of concentration-dependency and transport-inhibition are preferred when acidic drug transport is studied in a Ph gradient. The findings of this in vitro study can be extrapolated to in vivo situations involving an acidic microclimate.
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accuracy of calculated Ph Dependent aqueous drug solubility
European Journal of Pharmaceutical Sciences, 2004Co-Authors: Christel A S Bergstrom, Kristina Luthman, Per ArturssonAbstract:New effective experimental techniques in medicinal chemistry and Pharmacology have resulted in a vast increase in the number of Pharmacologically interesting compounds. However, the number of new drugs undergoing clinical trial has not augmented at the same pace, which in part has been attributed to poor absorption of the compounds.The main objective of this thesis was to investigate whether computer-based models devised from calculated molecular descriptors can be used to predict aqueous drug solubility, an important property influencing the absorption process. For this purpose, both experimental and computational studies were performed. A new small-scale shake flask method for experimental solubility determination of crystalline compounds was devised. This method was used to experimentally determine solubility values used for the computational model development and to investigate the Ph-Dependent solubility of drugs. In the computer-based studies, rapidly calculated molecular descriptors were used to predict aqueous solubility and the melting point, a solid state characteristic of importance for the solubility. To predict the absorption process, drug permeability across the intestinal epithelium was also modeled.The results show that high quality solubility data of crystalline compounds can be obtained by the small-scale shake flask method in a microtiter plate format. The experimentally determined Ph-Dependent solubility profiles deviated largely from the profiles predicted by a traditionally used relationship, highlighting the risk of data extrapolation. The in silico solubility models identified the non-polar surface area and partitioned total surface areas as potential new molecular descriptors for solubility. General solubility models of high accuracy were obtained when combining the surface area descriptors with descriptors for electron distribution, connectivity, flexibility and polarity. The used descriptors proved to be related to the solvation of the molecule rather than to solid state properties. The surface area descriptors were also valid for permeability predictions, and the use of the solubility and permeability models in concert resulted in an excellent theoretical absorption classification. To summarize, the experimental and computational models devised in this thesis are improved absorption screening tools applicable to the lead optimization in the drug discovery process.
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Ph Dependent bidirectional transport of weakly basic drugs across caco 2 monolayers implications for drug drug interactions
Pharmaceutical Research, 2003Co-Authors: Sibylle Neuhoff, Annalena Ungell, Ismael Zamora, Per ArturssonAbstract:Purpose. The purpose of this study was to investigate the Ph-Dependent passive and active transport of weakly basic drugs across the human intestinal epithelium. Methods. The bidirectional Ph-Dependent transport of weak bases was studied in Caco-2 cell monolayers in the Physiologic Ph range of the gastrointestinal tract. Results. A net secretion of atenolol and metoprolol was observed when a Ph gradient was applied. However, the bidirectional transport of both compounds was equal in the nongradient system. Hence, at lower apical than basolateral Ph a change in passive transport caused by an imbalance in the concentration of the uncharged drug species resulted in a “false” asymmetry (efflux ratio). Furthermore, a mixture of Ph-Dependent passive and active efflux was found for the P-glycoprotein (P-gp, MDR1, ABCB1) substrates, talinolol and quinidine, but not for the neutral drug, digoxin. However, the clinically important digoxin-quinidine interaction depended on the presence of a Ph gradient. Hence, the degree of interaction depends on the amount of quinidine available at the binding site of the P-gp. Conclusions. Active efflux of weak bases can only be accounted for when the fraction of unionized drug species is equal in all compartments because the transport is biased by a Ph-Dependent passive component. However, this component may take part in vivo and contribute to drug-drug interactions involving P-gp.
Patricia A. Jennings - One of the best experts on this subject based on the ideXlab platform.
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a single mutation disrupts the Ph Dependent dimerization of glycinamide ribonucleotide transformylase
Journal of Molecular Biology, 1998Co-Authors: Christine A Mullen, Patricia A. JenningsAbstract:Monomeric GART reversibly associates into a dimeric form as a function of decreasing solution Ph. The transition is consistent with a three-proton transfer reaction with an apparent pKa near 7. We now report that a single mutation, which replaces a glutamic acid at position 70 in the dimer interface with alanine (E70A), disrupts the Ph-Dependent dimerization of GART based on dynamic light scattering and gel filtration studies. A comparison of data obtained from UV-absorbance difference spectroscopy for both the wild-type and mutant forms of GART indicates that a tyrosine residue(s) undergoes a change in solvent exposure over the Ph range 6.55 to 8.19. A conformational change in tertiary structure that accompanies dimerization accounts for 60% of the observed optical difference, while the remaining 40% can be attributed to a Ph-Dependent process unrelated to dimerization. In addition, fluorescence studies of the mutant protein indicate that a Ph-Dependent change in tryptoPhan fluorescence exhibited by the wild-type protein is unrelated to quaternary structural changes and is likely a result of simple fluorescence quenching by nearby protonated histidine side-chains. Taken together, our results indicate that a single amino acid change at the dimer interface is sufficient to interrupt the highly specific, Ph-Dependent assembly reaction of GART, although Ph-Dependent conformational changes present in the wild-type protein also occur in E70A GART. This work is a first application of structure-based site-directed mutagenesis to the analysis of this Ph-Dependent assembly reaction.
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glycinamide ribonucleotide transformylase undergoes Ph Dependent dimerization
Journal of Molecular Biology, 1996Co-Authors: Christine A Mullen, Patricia A. JenningsAbstract:Abstract Glycinamide ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (Ph 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (Ph 7.5). We undertook a study of the Ph-Dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state. In the Ph range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering. The data give a best fit to a cooperative three-proton transfer mechanism: 2M + 3H + ⇌MH 2 2+ + MH + ⇌DH 3 3+ A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization. Fluorescence studies show that the environment of one or both of the tryptoPhan residues distal to the dimer interface are also perturbed by dimerization. Fitting of the normalized titration curves yields an apparent p K a =7.16(±0.02) and a subnanomolar K D for the transition. Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the Ph-Dependent dimerization. There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization. Our study indicates that under Physiological Ph conditions, GART exists as a mixture of monomer and dimer in solution. Taken together, the fact that the monomer-dimer transition displays a sharp Ph dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle Ph changes in the cell.
Sibylle Neuhoff - One of the best experts on this subject based on the ideXlab platform.
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Ph Dependent passive and active transport of acidic drugs across caco 2 cell monolayers
European Journal of Pharmaceutical Sciences, 2005Co-Authors: Annalena Ungell, Sibylle Neuhoff, Ismael Zamora, Per ArturssonAbstract:The aim of this study was to investigate Ph-Dependent passive and active transport of acidic drugs across Caco-2 cells. Therefore, the bidirectional Ph-Dependent transport of two acidic drugs, indomethacin and salicylic acid, across Caco-2 cells was studied in the Physiological Ph range of the gastrointestinal tract. The transport of both drugs decreased with increased Ph, as expected from the Ph-partition hypothesis. Net absorption occurred when the basolateral Ph exceeded the apical Ph. Concentration dependence and transporter inhibition studies indicated passive transport for indomethacin and a mixture of Ph-Dependent passive and active influx for salicylic acid. Unexpectedly, active and passive drug transport results were indistinguishable in temperature dependency studies. The transport of salicylic acid (apical Ph 5.0; basolateral Ph 7.4) was partly blocked by inhibitors of the proton-Dependent transporters MCT1 (SLC16A1) and OATP-B (SLC21A9, SLCO2B1). This study shows that the asymmetry in bidirectional transport of acidic drugs is affected by both passive and active components in the presence of Ph gradients across Caco-2 cells. Thus, combined studies of concentration-dependency and transport-inhibition are preferred when acidic drug transport is studied in a Ph gradient. The findings of this in vitro study can be extrapolated to in vivo situations involving an acidic microclimate.
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Ph Dependent bidirectional transport of weakly basic drugs across caco 2 monolayers implications for drug drug interactions
Pharmaceutical Research, 2003Co-Authors: Sibylle Neuhoff, Annalena Ungell, Ismael Zamora, Per ArturssonAbstract:Purpose. The purpose of this study was to investigate the Ph-Dependent passive and active transport of weakly basic drugs across the human intestinal epithelium. Methods. The bidirectional Ph-Dependent transport of weak bases was studied in Caco-2 cell monolayers in the Physiologic Ph range of the gastrointestinal tract. Results. A net secretion of atenolol and metoprolol was observed when a Ph gradient was applied. However, the bidirectional transport of both compounds was equal in the nongradient system. Hence, at lower apical than basolateral Ph a change in passive transport caused by an imbalance in the concentration of the uncharged drug species resulted in a “false” asymmetry (efflux ratio). Furthermore, a mixture of Ph-Dependent passive and active efflux was found for the P-glycoprotein (P-gp, MDR1, ABCB1) substrates, talinolol and quinidine, but not for the neutral drug, digoxin. However, the clinically important digoxin-quinidine interaction depended on the presence of a Ph gradient. Hence, the degree of interaction depends on the amount of quinidine available at the binding site of the P-gp. Conclusions. Active efflux of weak bases can only be accounted for when the fraction of unionized drug species is equal in all compartments because the transport is biased by a Ph-Dependent passive component. However, this component may take part in vivo and contribute to drug-drug interactions involving P-gp.
Etelka Tombacz - One of the best experts on this subject based on the ideXlab platform.
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adsorption of organic acids on magnetite nanoparticles Ph Dependent colloidal stability and salt tolerance
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2013Co-Authors: Etelka Tombacz, Ildiko Y Toth, Daniel Nesztor, Erzsebet Illes, Angela Hajdu, Marta Szekeres, L VekasAbstract:Abstract The adsorption of different organic acids and their influence on the Ph-Dependent charging, salt tolerance and so the colloidal stability of magnetite nanoparticles are compared. Adsorption isotherms of citric acid – CA, gallic acid – GA, poly(acrylic acid) – PAA, poly(acrylic-co-maleic acid) – PAM and humic acid – HA were measured. The Ph-Dependent charge state of MNPs was characterized by electroPhoretic mobility and their aggregation by dynamic light scattering. The salt tolerance was tested in coagulation kinetic experiments. Although the adsorption capacities, the type of bonding (either H-bonds or metal ion-carboxylate complexes) and so the bond strengths are significantly different, the following general trends have been found. Small amount of organic acids at Ph s the positive charges, and so promotes the aggregation and sedimentation of nanoparticles. Greater amounts of organic acid, above the charge neutralization, cause the sign reversal of particle charge, and at high overcharging promote stabilization and dispersing. The thicker layer of PAA, PAM and HA provides better electrosteric stability than CA and GA. GA undergoes surface polymerization, thereby improving stabilization. The organic acids studied here eliminate completely the Ph sensitivity of amPhoteric magnetite, but only the polyanionic coverage provides significant increase in resistance against coagulating effects of salts at neutral Ph commonly prevailing in natural waters.
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the effect of humic acid adsorption on Ph Dependent surface charging and aggregation of magnetite nanoparticles
Journal of Colloid and Interface Science, 2006Co-Authors: Erzsebet Illes, Etelka TombaczAbstract:The Ph-Dependent adsorption of humic acid (HA) on magnetite and its effect on the surface charging and the aggregation of oxide particles were investigated. HA was extracted from brown coal. Synthetic magnetite was prepared by alkaline hydrolysis of iron(II) and iron(III) salts. The Ph-Dependent particle charge and aggregation, and coagulation kinetics at Ph approximately 4 were measured by laser Doppler electroPhoresis and dynamic light scattering. The charge of pure magnetite reverses from positive to negative at Ph approximately 8, which may consider as isoelectric point (IEP). Near this Ph, large aggregates form, while stable sols exist further from it. In the presence of increasing HA loading, the IEP shifts to lower Ph, then at higher loading, magnetite becomes negatively charged even at low Phs, which indicate the neutralization and gradual recharging positive charges on surface. In acidic region, the trace HA amounts are adsorbed on magnetite surface as oppositely charged patches, systems become highly unstable due to heterocoagulation. Above the adsorption saturation, however, the nanoparticles are stabilized in a way of combined steric and electrostatic effects. The HA coated magnetite particles form stable colloidal dispersion, particle aggregation does not occur in a wide range of Ph and salt tolerance is enhanced.
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the effect of humic acid adsorption on Ph Dependent surface charging and aggregation of magnetite nanoparticles
Journal of Colloid and Interface Science, 2006Co-Authors: Erzsebet Illes, Etelka TombaczAbstract:The Ph-Dependent adsorption of humic acid (HA) on magnetite and its effect on the surface charging and the aggregation of oxide particles were investigated. HA was extracted from brown coal. Synthetic magnetite was prepared by alkaline hydrolysis of iron(II) and iron(III) salts. The Ph-Dependent particle charge and aggregation, and coagulation kinetics at Ph ∼ 4 were measured by laser Doppler electroPhoresis and dynamic light scattering. The charge of pure magnetite reverses from positive to negative at Ph ∼ 8, which may consider as isoelectric point (IEP). Near this Ph, large aggregates form, while stable sols exist further from it. In the presence of increasing HA loading, the IEP shifts to lower Ph, then at higher loading, magnetite becomes negatively charged even at low Phs, which indicate the neutralization and gradual recharging positive charges on surface. In acidic region, the trace HA amounts are adsorbed on magnetite surface as oppositely charged patches, systems become highly unstable due to heterocoagulation. Above the adsorption saturation, however, the nanoparticles are stabilized in a way of combined steric and electrostatic effects. The HA coated magnetite particles form stable colloidal dispersion, particle aggregation does not occur in a wide range of Ph and salt tolerance is enhanced.
Sarah R Dennison - One of the best experts on this subject based on the ideXlab platform.
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Linearized esculentin-2EM shows Ph Dependent antibacterial activity with an alkaline optimum
Molecular and Cellular Biochemistry, 2021Co-Authors: Erum Malik, Frederick Harris, David A. Phoenix, Timothy J. Snape, Jaipaul Singh, Leslie H. G. Morton, Sarah R DennisonAbstract:Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses Ph Dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as Ph was increased from 6 to 8 (↑ 15.5–26.9%), whilst similar increases in Ph enhanced the ability of the peptide to penetrate (↑ 2.3–5.1 mN m^− 1 ) and lyse (↑ 15.1–32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1–23) in the N → C direction, with − increasing overall from circa − 0.8 to − 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of PhosPhatidylglycerol to induce α-helical structure in the peptide as compared to PhosPhatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of Ph Dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms.
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Ph Dependent antimicrobial peptides and proteins their mechanisms of action and potential as therapeutic agents
Pharmaceuticals, 2016Co-Authors: Erum Malik, Sarah R Dennison, Frederick Harris, David Andrew PhoenixAbstract:Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise Ph Dependent antimicrobial mechanisms, and here we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic, although a number of anionic examples are known. Some of these molecules exhibit high Ph optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low Ph optima, which reflects the acidic Ph generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure/function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low Ph. The Ph Dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by Ph Dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of Ph Dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many Ph Dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era.