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John C. Conboy - One of the best experts on this subject based on the ideXlab platform.
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Phosphatidylglycerol Asymmetry and Translocation in Lipid Membranes
Biophysical Journal, 2015Co-Authors: John C. ConboyAbstract:Our current understanding of the structure and dynamics of cellular membranes emerged in the early 1970 's. However, there is still much we do not know about the underlying lipid dynamics in cellular membranes, specifically the process of lipid translocation or Flip-Flop. The Flip-Flop rates of only a few lipid species, principally phosphatidylcholines (PCs), have been measured. Anionic lipids, such as phosphatidylseryine (PS), phosphatidylinositol (PI), and phosphatidylglycerol (PG), are known to be play active roles in membrane function, but almost nothing is known of the rates of translocation of these species. While PS and PI are the major anionic lipids of eukaryotic cellular membranes, PG is mainly found in prokaryotic membranes and comprises about 10% of the phospholipid content of the mitochondria membrane in eukaryotes. In the work presented here, the native Flip-Flop rates of 1,2-diasterol-sn-glycero-3-[phospho-(1′-rac-glycerol)] (DSPG) Flip-Flop in mixed DSPG: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) bilayers have been investigated. Using methods of classical surface chemistry coupled with nonlinear optical methods, we have developed a novel analytical approach, using sum-frequency vibrational spectroscopy (SFVS), to selectively probe lipid compositional asymmetry in a planar supported lipid bilayer. This new method allows for the detection of lipid Flip-Flop Kinetics and compositional asymmetry without the need for a fluorescent or spin-labeled lipid species by exploiting the coherent nature of SFVS. Using SFVS, the rates of DSPG Flip-Flop in a DSPC matrix have been examined for the first time. Analysis of the dynamics provides an assessment of the underlying energetic barrier to PG translocation. The results will be discussed in the framework of the protein-free energetic barriers to PG Flip-Flop and the role of electrostatics in this process.
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Free energy and entropy of activation for phospholipid Flip-Flop in planar supported lipid bilayers.
The journal of physical chemistry. B, 2010Co-Authors: Timothy C. Anglin, Michael P. Cooper, Katherine Chandler, John C. ConboyAbstract:Basic transition state theory is used to describe the activation thermodynamics for phospholipid Flip-Flop in planar-supported lipid bilayers (PSLBs) prepared by the Langmuir−Blodgett/Langmuir−Schaeffer method. The Kinetics of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) Flip-Flop were determined as a function of temperature and lateral surface pressure using sum-frequency vibrational spectroscopy (SFVS). From the temperature and lateral pressure dependent DSPC Flip-Flop Kinetics, a complete description of the activation thermodynamics for Flip-Flop in the gel state, including free energy of activation (ΔG‡), area of activation (Δa‡), and entropy of activation (ΔS‡), was obtained. The free energy barrier for Flip-Flop of DSPC was determined to be ΔG‡ = 105 ± 2 kJ/mol at 40 °C at a deposition surface pressure of 30 mN/m. The free energy barrier was found to consist of large opposing entropic and enthalpic contributions. The influence of alkyl chain length on the activation thermodynamics of Flip-Flop ...
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Lateral Pressure Dependence of the Phospholipid Transmembrane Diffusion Rate in Planar-Supported Lipid Bilayers
Biophysical journal, 2008Co-Authors: Timothy C. Anglin, John C. ConboyAbstract:The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) Flip-Flop Kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The Kinetics of lipid Flip-Flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10−4 s−1 to 1.03 × 10−4 s−1) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed Kinetics, the area of activation for native phospholipid Flip-Flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 A2/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid Flip-Flop are discussed.
Timothy C. Anglin - One of the best experts on this subject based on the ideXlab platform.
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Free energy and entropy of activation for phospholipid Flip-Flop in planar supported lipid bilayers.
The journal of physical chemistry. B, 2010Co-Authors: Timothy C. Anglin, Michael P. Cooper, Katherine Chandler, John C. ConboyAbstract:Basic transition state theory is used to describe the activation thermodynamics for phospholipid Flip-Flop in planar-supported lipid bilayers (PSLBs) prepared by the Langmuir−Blodgett/Langmuir−Schaeffer method. The Kinetics of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) Flip-Flop were determined as a function of temperature and lateral surface pressure using sum-frequency vibrational spectroscopy (SFVS). From the temperature and lateral pressure dependent DSPC Flip-Flop Kinetics, a complete description of the activation thermodynamics for Flip-Flop in the gel state, including free energy of activation (ΔG‡), area of activation (Δa‡), and entropy of activation (ΔS‡), was obtained. The free energy barrier for Flip-Flop of DSPC was determined to be ΔG‡ = 105 ± 2 kJ/mol at 40 °C at a deposition surface pressure of 30 mN/m. The free energy barrier was found to consist of large opposing entropic and enthalpic contributions. The influence of alkyl chain length on the activation thermodynamics of Flip-Flop ...
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Lateral Pressure Dependence of the Phospholipid Transmembrane Diffusion Rate in Planar-Supported Lipid Bilayers
Biophysical journal, 2008Co-Authors: Timothy C. Anglin, John C. ConboyAbstract:The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) Flip-Flop Kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The Kinetics of lipid Flip-Flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10−4 s−1 to 1.03 × 10−4 s−1) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed Kinetics, the area of activation for native phospholipid Flip-Flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 A2/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid Flip-Flop are discussed.
William J. Jusko - One of the best experts on this subject based on the ideXlab platform.
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Pharmacokinetic/Pharmacodynamic Modeling of GLP-1 in Healthy Rats
Pharmaceutical Research, 2012Co-Authors: Yanguang Cao, Wei Gao, William J. JuskoAbstract:Purpose To provide a mechanism-based model to quantitatively describe GLP-1 pharmacoKinetics (PK) and pharmacodynamics (PD) in rats. Methods Intravenous (IV), infusion (IF), subcutaneous (SC), and intraperitoneal (IP) doses of GLP-1 were administered after glucose challenge in healthy Sprague–Dawley rats. Blood was analyzed for GLP-1, glucose, and insulin. The PK-PD modeling was performed with ADAPT 5. The concentration-response curve was generated and analyzed in comparison with other incretin-related therapeutics. Results The PK of GLP-1 was described using a two-compartment model with a zero-order input accounting for endogenous GLP-1 synthesis. For SC and IP dosing, sequential zero-order and first-order absorption models reasonably described the rapid absorption process and Flip-Flop Kinetics. In dynamics, GLP-1 showed insulinotropic effects (3-fold increase) after IV glucose challenge in a dose-dependent manner. The concentration-response curve was bell-shaped, which was captured using a biphasic two-binding site Adair model. Receptor binding of GLP-1 exhibited high capacity and low affinity Kinetics for both binding sites (K_D = 9.94 × 10^3 pM, K_2 = 1.56 × 10^−4 pM^−1). Conclusions The PK of GLP-1 was linear and bi-exponential and its PD showed glucose-dependent insulinotropic effects. All profiles were captured by the present mechanistic model and the dynamic analysis yields several implications for incretin-related therapies.
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PharmacoKinetics of Methylprednisolone after Intravenous and Intramuscular Administration in Rats
Biopharmaceutics & drug disposition, 2007Co-Authors: Anasuya Hazra, Nancy A. Pyszczynski, Debra C. Dubois, Richard R. Almon, William J. JuskoAbstract:Methylprednisolone (MPL) pharmacoKinetics was examined in adrenalectomized (ADX) and normal rats to assess the feasibility of intramuscular (i.m.) dosing for use in pharmacodynamic studies. Several study phases were pursued. Parallel group studies were performed in normal and ADX rats given 50 mg/kg MPL (i.v. or i.m.) and blood samples were collected up to 6 h. Data from studies where normal rats were dosed with 50 mg/kg MPL i.m. and killed over either 6 or 96 h were combined to determine muscle site and plasma MPL concentrations. Lastly, ADX rats were dosed with 50 mg/kg MPL i.m. and killed over 18 h to assess hepatic tyrosine aminotransferase (TAT) dynamics. MPL exhibited bi-exponential Kinetics after i.v. dosing with a terminal slope of 2.1 h−1. The i.m. drug was absorbed slowly with two first-order absorption rate constants, 1.26 and 0.219 h−1 indicating Flip-Flop Kinetics with overall 50% bioavailability. The Kinetics of MPL at the injection site exhibited slow, dual absorption rates. Although i.m. MPL showed lower bioavailability compared with other corticosteroids in rats, TAT dynamics revealed similar i.m. and i.v. response profiles. The more convenient intramuscular dosing can replace the i.v. route without causing marked differences in pharmacodynamics. Copyright © 2007 John Wiley & Sons, Ltd.
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Pharmacokinetic and pharmacodynamic modeling of recombinant human erythropoietin after multiple subcutaneous doses in healthy subjects.
European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 2005Co-Authors: Wojciech Krzyzanski, William J. Jusko, Mary C Wacholtz, Neil Minton, Wing K CheungAbstract:A pharmacokinetic and pharmacodynamic (PK/PD) model for recombinant human erythropoietin (Epoetin alfa) in healthy subjects was developed to describe the time profiles of changes in serum Epoetin alfa and the pharmacological responses of percent reticulocytes, total red blood cell counts, and hemoglobin after single and multiple subcutaneous administration of Epoetin alfa. Data used in the development of the model were obtained from a clinical study carried out in healthy volunteers in which Epoetin alfa was administered either as 150 IU/kg three-times-a-week (t.i.w.) or fixed 40,000 IU weekly (q.w.) doses for 4 weeks. A dual-absorption rate model (fast zero-order and slow first-order inputs) with linear disposition Kinetics was used to characterize the pharmacoKinetics of erythropoietin after subcutaneous administration. A new catenary cell production and lifespan loss model was used to fit the pharmacodynamic data yielding estimates of SC50, Smax, and other pharmacodynamic parameters. Flip-Flop Kinetics was apparent in the pharmacoKinetics as the absorption rate was slower (k(a) = 0.7 day(-1)) than the elimination rate (CL/V(d) = 1.2-9.2 day(-1)). In the pharmacodynamics, an SC50 of 58 mIU/mL was estimated indicating that low serum erythropoietin concentrations were sufficient to produce pharmacological effects. The established PK/PD model predicts similar pharmacological responses of hemoglobin and total red blood cell counts for the 150 IU/kg t.i.w. and 40,000 IU q.w. regimens in healthy subjects.
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Receptor-Mediated Pharmacokinetic/Pharmacodynamic Model of Interferon-β 1a in Humans
Pharmaceutical Research, 2002Co-Authors: Donald E. Mager, William J. JuskoAbstract:Purpose . An integrated receptor-based pharmacokinetic/pharmaco- dynamic (PK/PD) model of interferon-β 1a (IFN-β 1a) previously developed for monkeys was used to capture the time-course of drug and induced neopterin concentrations after intravenous (IV) and subcutaneous (SC) dosing in humans. Methods . Data were extracted from the literature by digitalization. Single-dose (3 IV doses and 1 SC dose) PK/PD profiles were simultaneously fitted using the basic model and the ADAPT II computer program. Additional submodels incorporating neutralizing antibody formation and negative feedback inhibition were applied to account for drug accumulation and lower than expected neopterin concentrations encountered after multiple-dosing (1 SC dose every 48 hs). Results . The basic model jointly-captured the nonlinear PK behavior of the drug and induced neopterin concentrations after all single doses. Slow and incomplete absorption (F = 0.33) of the SC dose resulted in prolonged drug concentrations reflective of Flip-Flop Kinetics. Despite lower drug concentrations, SC dosing produced a similar neopterin profile as compared with the IV doses; however, with a longer time to peak effect and slightly higher neopterin concentrations at later time points. The PD component of the model represents a modified precursor-dependent indirect response model driven by the amount of internalized drug-receptor complex. The latter stimulated a 6-fold increase in the production of the neopterin precursor (S_max = 5.89). Drug accumulation and lower than expected neopterin concentrations after multiple dosing were also captured after the inclusion of the submodels. Conclusions . The present integrated PK/PD model for IFN-β 1a is mechanistic in nature with receptor-mediated disposition and dynamics and was successfully applied to human clinical data.
Jeu-ming P. Yuann - One of the best experts on this subject based on the ideXlab platform.
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Determination by photoreduction of Flip-Flop Kinetics of spin-labeled stearic acids across phospholipid bilayers.
Biochimica et biophysica acta, 1999Co-Authors: Jeu-ming P. Yuann, R D MorseAbstract:Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (Flip-Flop). The Flip-Flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the Flip-Flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the Flip-Flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar Flip-Flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution Kinetics of stearic acid derivatives in biological systems.
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Determination by photoreduction of Flip-Flop Kinetics of spin-labeled stearic acids across phospholipid bilayers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1999Co-Authors: Jeu-ming P. Yuann, Reef (philip D.ii) MorseAbstract:AbstractSpin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (Flip-Flop). The Flip-Flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the Flip-Flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the Flip-Flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8±4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar Flip-Flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa (∼8.3) of stearic acid at air–water interface. Thus, N-DS are good probes for studying the distribution Kinetics of stearic acid derivatives in biological systems
R D Morse - One of the best experts on this subject based on the ideXlab platform.
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Determination by photoreduction of Flip-Flop Kinetics of spin-labeled stearic acids across phospholipid bilayers.
Biochimica et biophysica acta, 1999Co-Authors: Jeu-ming P. Yuann, R D MorseAbstract:Spin-labeled stearic acid derivatives (N-DS) can be used to determine the rate at which lipid-derived drugs can cross a phospholipid bilayer (Flip-Flop). The Flip-Flop rate of N-DS (where N=5, 6, 7, 9, 10, 12, 16), was measured using vectorial photoreduction of nitroxides to their corresponding hydroxylamine by FMN, a charged, membrane-impermeable flavin, by hydrogen atom transfer from EDTA. From the time difference in the photoreduction rates of N-DS located in the outer and inner half of the bilayer, the Flip-Flop rate of N-DS across the bilayer can be determined. The results show that at pH 8.0 or lower, the photoreduction of 5-DS on one side of the membrane by FMN is slower than the Flip-Flop rate of 5-DS across phospholipid bilayers. For 5-DS at pH 7.0, this rate is at least 33.8+/-4.24 s or faster. Stearic acids with the spin label at different positions along the acyl chain (N=5, 6, 7, 9, 10, 12) have similar Flip-Flop rates in the liposomes at pH 7.0 although 16-DS is slower, probably due to the inaccessibility of the nitroxide moiety to FMN. It is most likely that the fast distribution of 5-DS in cells is due to the fast movement of acidic form, but not the salt form, of 5-DS across membrane bilayers. The oxazolidine (nitroxide moiety) does not seem to affect the pKa ( approximately 8.3) of stearic acid at air-water interface. Thus, N-DS are good probes for studying the distribution Kinetics of stearic acid derivatives in biological systems.