Kinetic Design for Establishing Long-Term Stationary Cytosol Concentrations During Drug Transport across P-gp Expressing Confluent Cell Monolayers to Facilitate Measuring Cytosol Concentration, Fitting Drug Molar Partition Coefficients into the Cytosolic Monolayer of the Plasma Membrane, and Kinetically Identifying Drug Uptake Transporters

2021 ◽  
pp. 41-65
Author(s):  
Joe Bentz
Keyword(s):  
Plasma Membrane ◽  
Partition Coefficients ◽  
Drug Transport ◽  
Drug Uptake ◽  
Cell Monolayers ◽  
Confluent Cell ◽  
Uptake Transporters

scholarly journals Cell Adherence and Drug Delivery from Particle Based Mesoporous Silica Films

2019 ◽  
Author(s):  
Emma Björk ◽  
Bernhard Baumann ◽  
Florian Hausladen ◽  
Rainer Wittig ◽  
mika lindén
Keyword(s):  
Drug Delivery ◽  
Laser Scanning ◽  
Single Cells ◽  
Silicon Wafers ◽  
Laser Scanning Microscopy ◽  
Drug Uptake ◽  
C2c12 Cells ◽  
Confocal Laser ◽  
Particle Sizes

Spatially and temporally controlled drug delivery is important for implant and tissue engineering applications, as the efficacy and bioavailability of the drug can be enhanced, and can also allow for drugging stem cells at different stages of development. Long-term drug delivery over weeks to months is however difficult to achieve, and coating of 3D surfaces or creating patterned surfaces is a challenge using coating techniques like spin- and dip-coating. In this study, mesoporous films consisting of SBA-15 particles grown onto silicon wafers using wet processing were evaluated as a scaffold for drug delivery. Films with various particle sizes (100 – 900 nm) and hence thicknesses were grown onto OTS-functionalized silicon wafers using a direct growth method. Precise patterning of the areas for film growth could be obtained by local removal of the OTS functionalization through laser ablation. The films were incubated with the model drug DiO, and murine myoblast cells (C2C12 cells) were seeded onto films with different particle sizes. Confocal laser scanning microscopy (CLSM) was used to study the cell growth, and a vinculin-mediated adherence of C2C12 cells on all films was verified. The successful loading of DiO into the films was confirmed by UV-vis and CLSM. It was observed that the drugs did not desorb from the particles during 24 hours in cell culture. During adherent growth on the films for 4 h, small amounts of DiO and separate particles were observed inside single cells. After 24 h, a larger number of particles and a strong DiO signal were recorded in the cells, indicating a particle mediated drug uptake. A substantial amount of DiO loaded particles were however attached on the substrate after 24 making the films attractive as a long-term reservoir for drugs on e.g. medical implants.<br>

scholarly journals Inverse mechanistic modeling of transdermal drug delivery for fast identification of optimal model parameters

2020 ◽  
Author(s):  
Thijs Defraeye ◽  
Flora Bahrami ◽  
Rene M Rossi
Keyword(s):  
Drug Delivery ◽  
Inverse Modeling ◽  
Transdermal Drug Delivery ◽  
Drug Transport ◽  
Mechanistic Model ◽  
Mechanistic Modeling ◽  
Drug Uptake ◽  
Added Value ◽  
Model Parameters ◽  
Transdermal Drug Delivery Systems

Transdermal drug delivery systems are a key technology to administer drugs with a high first-pass effect in a non-invasive and controlled way. Physics-based modeling and simulation are on their way to become a cornerstone in the engineering of these healthcare devices since it provides a unique complementarity to experimental data and insights. Simulations enable to virtually probe the drug transport inside the skin at each point in time and space. However, the tedious experimental or numerical determination of material properties currently forms a bottleneck in the modeling workflow. We show that multiparameter inverse modeling to determine the drug diffusion and partition coefficients is a fast and reliable alternative. We demonstrate this strategy for transdermal delivery of fentanyl. We found that inverse modeling reduced the normalized root mean square deviation of the measured drug uptake flux from 26 to 9%, when compared to the experimental measurement of all skin properties. We found that this improved agreement with experiments was only possible if the diffusion in the reservoir holding the drug was smaller than the experimentally-measured diffusion coefficients suggested. For indirect inverse modeling, which systematically explores the entire parametric space, 30 000 simulations were required. By relying on direct inverse modeling, we reduced the number of simulations to be performed to only 300, so a factor 100 difference. The modeling approach's added value is that it can be calibrated once in-silico for all model parameters simultaneously by solely relying on a single measurement of the drug uptake flux evolution over time. We showed that this calibrated model could accurately be used to simulate transdermal patches with other drug doses. We showed that inverse modeling is a fast way to build up an accurate mechanistic model for drug delivery. This strategy opens the door to clinically-ready therapy that is tailored to patients.

Abstract 274: Spatiotemporal Regulation of β Adrenergic Signaling In Heart failure

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Yang K Xiang ◽  
Federica Barbagallo ◽  
Bing Xu ◽  
Qin Fu
Keyword(s):  
Heart Failure ◽  
Plasma Membrane ◽  
Sarcoplasmic Reticulum ◽  
Cardiac Myocytes ◽  
Cardiac Disease ◽  
Spatiotemporal Regulation ◽  
Disease Therapy ◽  
Functional Implications ◽  
Underlying Mechanisms

Our long-term goal is to understand mechanisms that govern spatiotemporal regulation of cAMP/PKA signaling in cardiac myocytes under physiological and pathophysiological conditions, and their implication in cardiac disease therapy. Here we use a series of biosensors to measure cAMP/PKA activity under βAR subtype regulation. In failing cardiac myocytes, the cAMP and PKA activity are shifted from the plasma membrane to the intracellular sarcoplasmic reticulum and the myofilaments. Meanwhile, β2AR displays an increased role in signaling to the myofilaments in failing myocytes when compared to the control myocytes. Moreover, we show that an increased βAR association with phosphodiesterases promotes the alteration in spatiotemporal propagation of cAMP/PKA signaling in failing myocytes. These observations and the underlying mechanisms and functional implications will be discussed.

scholarly journals Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein

2020 ◽  
Vol 117 (47) ◽  
pp. 29609-29617
Author(s):  
Andaleeb Sajid ◽  
Sabrina Lusvarghi ◽  
Megumi Murakami ◽  
Eduardo E. Chufan ◽  
Biebele Abel ◽  
...  
Keyword(s):  
Drug Transport ◽  
Atp Hydrolysis ◽  
Binding Pocket ◽  
Drug Binding ◽  
Drug Uptake ◽  
Cancer Drug ◽  
Binding Domains ◽  
Cancer Drug Resistance ◽  
P Glycoprotein ◽  
Cell Transforming

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.

scholarly journals Long-Term Exposure to Glucose and Lipids Inhibits Glucose-Induced Insulin Secretion Downstream of Granule Fusion With Plasma Membrane

Diabetes ◽  
2007 ◽  
Vol 56 (7) ◽  
pp. 1888-1897 ◽  
Author(s):  
C. S. Olofsson ◽  
S. Collins ◽  
M. Bengtsson ◽  
L. Eliasson ◽  
A. Salehi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion

p-Nitrophenylphosphatase Activity Catalyzed by Plasma Membrane (Ca2++Mg2+)ATPase: Correlation with Structural Changes Modulated by Glycerol and Ca2+

2001 ◽  
Vol 21 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Gutemberg G. Alves ◽  
Luis Maurício T. R. Lima ◽  
Maely P. Fávero-Retto ◽  
Adriana P. Lemos ◽  
Carlos E. Peres-Sampaio ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synergistic Effect ◽  
Phosphatase Activity ◽  
Structural Changes ◽  
Center Of Mass ◽  
Binding Domain ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Substrate Binding Domain

The plasma membrane (Ca2++Mg2+)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca2+ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca2+ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca2+-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca2+ is related to opposite long-term hydration effects on the substrate binding domain and the Ca2+ binding domain.

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