Membrane Transport Rates Research Articles

19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

A Study on Biofouling and Cleaning of Anion Exchange Membranes for Reverse Electrodialysis.

This study covers the modification, (bio)fouling characterization, use, and cleaning of commercial heterogeneous anion exchange membranes (AEMs) to evaluate their feasibility for reverse electrodialysis (RED) applications. A surface modification with poly (acrylic) acid resulted in an improved monovalent perm-selectivity (decreased sulfate membrane transport rate). Moreover, we evaluated the (bio)fouling potential of the membrane using sodium dodecyl sulfate (SDS), sodium dodecyl benzenesulfonate (SDBS), and Aeromonas hydrophila as model organic foulants and a biofoulant, respectively. A detailed characterization of the AEMs (water contact angle, ion exchange capacity (IEC), scanning electron microscopy (SEM), cyclic voltammetry (CV), and Fourier Transform Infrared (FTIR) spectra) was carried out, verifying that the presence of such foulants reduces IEC and the maximum current obtained by CV. However, only SDS and SDBS affected the contact angle values. Cleaning of the biofouled membranes using a sodium hypochlorite aqueous solution allows for (partially) recovering their initial properties. Furthermore, this work includes a fouling characterization using real surface and sea water matrixes, confirming the presence of several types of fouling microorganisms in natural streams. A lower adhesion of microorganisms (measured in terms of total bacteria counts) was observed for the modified membranes compared to the unmodified ones. Finally, we propose a cleaning strategy to mitigate biofouling in AEMs that could be easily applied in RED systems for an enhanced long-term process performance.

Baseline Peritoneal Membrane Transport Characteristics Are Associated with Peritonitis Risk in Incident Peritoneal Dialysis Patients.

The peritoneal equilibration test (PET) is a semi-quantitative measurement that characterizes the rate of transfer of solutes and the water transfer rate across the peritoneum in patients treated with peritoneal dialysis (PD). The results of the PET are used to maximize daily peritoneal ultrafiltration and solute clearances. Previous studies have shown that high transport status is associated with ultrafiltration failure, malnutrition, and reduced survival; however, the way in which peritoneum transport characteristics affect peritonitis risk is unknown. In the current cohort study, we recruited 898 incident-PD patients and used intention-to-treat analysis to test if baseline PET affected the subsequent 3-year peritonitis rate. Among all recruited PD patients, 308 (34.2%) developed peritonitis within three years. Multivariate Cox regression analysis showed that the high-transport group has the greatest peritonitis risk (HR 1.98, 95% CI: 1.08–3.62) even after an adjustment for demographics, comorbid diseases, and biochemical measurements. We concluded that a baseline high peritoneal membrane transport rate is an independent risk factor for peritonitis in incident PD patients.

Amorphous solid dispersions of enzalutamide and novel polysaccharide derivatives: investigation of relationships between polymer structure and performance

Amorphous solid dispersion (ASD) is a widely employed formulation technique for drugs with poor aqueous solubility. Polymers are integral components of ASDs, but mechanisms by which polymers lead to the generation and maintenance of supersaturated solutions, which enhance oral absorption in vivo, are poorly understood. Herein, a diverse group of newly synthesized cellulose derivatives was evaluated for their ability to inhibit crystallization of enzalutamide, a poorly soluble compound used to treat prostate cancer. ASDs were prepared from selected polymers, specifically a somewhat hydrophobic polymer that was extremely effective at inhibiting drug crystallization, and a less effective, but more hydrophilic, crystallization inhibitor, that might afford better release. Drug membrane transport rate was evaluated in vitro and compared to in vivo performance, following oral dosing in rats. Good correlation was noted between the in vitro diffusion cell studies and the in vivo data. The ASD formulated with the less effective crystallization inhibitor outperformed the ASD prepared with the highly effective crystallization inhibitor in terms of the amount and rate of drug absorbed in vivo. This study provides valuable insight into key factors impacting oral absorption from enabling ASD formulations, and how best to evaluate such formulations using in vitro approaches.

Impact of Hypromellose Acetate Succinate Grade on Drug Amorphous Solubility and In Vitro Membrane Transport

The goal of this study was to evaluate the effect of polymer substituent type on drug amorphous solubility as well as drug membrane transport rate. Two grades of hypromellose acetate succinate (HPMC-AS), AS-LF and AS-HF, were studied with 4 model drugs. Experimental techniques used to evaluate the drug-polymer systems included solution 1H nuclear magnetic resonance spectroscopy (NMR) and absorptive dissolution testing. AS-HF substantially reduced the drug amorphous solubility, while AS-LF had only a minor impact. By NMR spectroscopy, AS-HF was found to distribute extensively into the drug-rich phase formed via liquid-liquid phase separation. Polymer distribution into the drug-rich phase accounts for the reduction in the drug amorphous solubility. Absorptive dissolution testing showed that drug mass transport was lower in the presence of AS-HF compared to AS-LF for drug concentrations above the amorphous solubility. Thus, the decrease in drug amorphous solubility by AS-HF reduced drug transport. This study highlights the need to consider the polymer impact on drug amorphous solubility and subsequent drug transport, when optimizing amorphous solid dispersion formulations, with the goal of maximizing drug absorption.

Identification of a selective manganese ionophore that enables nonlethal quantification of cellular manganese

Available assays for measuring cellular manganese (Mn) levels require cell lysis, restricting longitudinal experiments and multiplexed outcome measures. Conducting a screen of small molecules known to alter cellular Mn levels, we report here that one of these chemicals induces rapid Mn efflux. We describe this activity and the development and implementation of an assay centered on this small molecule, named manganese-extracting small molecule (MESM). Using inductively-coupled plasma-MS, we validated that this assay, termed here "manganese-extracting small molecule estimation route" (MESMER), can accurately assess Mn in mammalian cells. Furthermore, we found evidence that MESM acts as a Mn-selective ionophore, and we observed that it has increased rates of Mn membrane transport, reduced cytotoxicity, and increased selectivity for Mn over calcium compared with two established Mn ionophores, calcimycin (A23187) and ionomycin. Finally, we applied MESMER to test whether prior Mn exposures subsequently affect cellular Mn levels. We found that cells receiving continuous, elevated extracellular Mn accumulate less Mn than cells receiving equally-elevated Mn for the first time for 24 h, indicating a compensatory cellular homeostatic response. Use of the MESMER assay versus a comparable detergent lysis-based assay, cellular Fura-2 Mn extraction assay, reduced the number of cells and materials required for performing a similar but cell lethality-based experiment to 25% of the normally required sample size. We conclude that MESMER can accurately quantify cellular Mn levels in two independent cells lines through an ionophore-based mechanism, maintaining cell viability and enabling longitudinal assessment within the same cultures.

Interplay of Supersaturation and Solubilization: Lack of Correlation between Concentration-Based Supersaturation Measurements and Membrane Transport Rates in Simulated and Aspirated Human Fluids.

Supersaturating formulations are increasingly being used to improve the absorption of orally administered poorly water-soluble drugs. To better predict outcomes in vivo, we must be able to accurately determine the degree of supersaturation in complex media designed to provide a surrogate for the gastrointestinal environment. Herein, we demonstrate that relying on measurements based on consideration of the total dissolved concentration leads to underestimation of supersaturation and consequently membrane transport rates. Crystalline and amorphous solubilities of two compounds, atazanavir and posaconazole, were evaluated in six different media. Concurrently, diffusive flux measurements were performed in a side-by-side diffusion cell to determine the activity-based supersaturation by evaluating membrane transport rates at the crystalline and amorphous solubilities. Solubility values were found to vary in each medium because of different solubilization capacities. Concentration-based supersaturation ratios were also found to vary for the different media. Activity-based measurements, however, were largely independent of the medium, leading to relatively constant values for the estimated supersaturation. These findings have important consequences for modeling and prediction of supersaturation impact on the absorption rate as well as for better defining the thermodynamic driving force for crystallization in complex media.

Renal Damaging Effect Elicited by Bicalutamide Therapy Uncovered Multiple Action Mechanisms As Evidenced by the Cell Model

Bicalutamide (Bic) is frequently used in androgen deprivation therapy (ADT) for treating prostate cancer. ADT-induced hypogonadism was reported to have the potential to lead to acute kidney injury (AKI). ADT was also shown to induce bladder fibrosis via induction of the transforming growth factor (TGF)-β level. We hypothesized that Bic can likely induce renal fibrosis. To understand this, a cell model was used to explore expressions of relevant profibrotic proteins. Results indicated that Bic initiated multiple apoptotic and fibrotic pathways, including androgen deprivation, downregulation of the androgen receptor → phosphatidylinositol-3-kinase → Akt pathway, upregulation of the extrinsic apoptotic pathway- tumor necrosis factor α → nuclear factor κB → caspase-3, increased expressions of fibrosis-related proteins including platelet-derived growth factor β, fibronectin and collagen IV, and enhanced cell migration. The endoplasmic reticular stress pathway and smooth muscle actin were unaffected by Bic. Co-treatment with testosterone was shown to have an anti-apoptotic effect against Bic, suggesting a better outcome of Bic therapy if administered with an appropriate testosterone intervention. However, since Bic was found to inhibit the membrane transport and consumption rates of testosterone, a slightly larger dose of testosterone is recommended. In conclusion, these pathways can be considered to be pharmaceutically relevant targets for drug development in treating the adverse effects of Bic.

The relationship between vascular endothelial growth factor (VEGF) in the serum and drained dialysate with the quality of peritoneal dialysis and peritoneal membrane transport rates

Vascular endothelial growth factor (VEGF), a powerful angiogenetic agent crucial for microvascular hyperpermeability and neoangiogenesis in the peritoneum, is associated with increased solute transport rates in chronic peritoneal dialysis (PD) patients. We investigated the correlation between serum and drained dialysate (dd) concentrations of VEGF and the transport characteristics of peritoneal membrane and dialysis quality in 20 patients with end-stage renal failure at the beginning and after six months of PD. The serum VEGF (sVEGF) concentration rose significantly (149.33?}116.71 pg/ mL vs 239.36?}102.23 pg/mL; p=0.012) and ddVEGF concentration increased slightly (38.44?}50.47 pg/mL vs 43.55?}51.10 pg/mL) during the first 6 months of PD. At the beginning of chronic PD, ddVEGF concentrations correlated inversely with the peritoneal equilibrium test (PET) glucose (R=-0.565; p=0.009) and creatinine (R=-0.506; p=0.023) and residual renal function (RRF) (R=-0.691; p=0.001); sVEGF concentrations inversely correlated with PET creatinine (R=-0.457; p=0.043) and residual diuresis (RD) (R=-0.691; p=0.001). After 6 months of treatment, ddVEGF concentrations correlated directly with PETcreatinine (R=0.450; p=0.047), and inversely with RRF (R=-0.552; p=0.012) and residual renal weekly Kt/V (R=-0.488; p=0.029). The sVEGF concentration inversely correlated with RD (R=-0.589; p=0.006). High ddVEGF at the beginning of PD is predictive of adverse alterations of the peritoneal membrane, i.e. increased transport rate of glucose and creatinine. ddVEGF values may help to identify patients who will preserve adequate transport characteristics of the peritoneal membrane and maintain successful long-term PD.

Effects of ketamine/xylazine and isoflurane on rat brain glucose metabolism measured by 18F‐fluorodeoxyglucose‐positron emission tomography

The aim of the present study was to investigate changes in glucose metabolism in male Wistar rats induced by the anesthetics isoflurane and ketamine combined with xylazine via 18 F-fluorodeoxyglucose-positron emission tomography. We analyzed the differential effects of the anesthetics on 18 F-fluorodeoxyglucose uptake and pharmacokinetics in 33 rats using quantification methods: (a) the standardized uptake value, (b) voxel-based analyses, and (c) kinetic analysis. Both anesthetics reduced glucose uptake in the entire brain. The voxel-based analyses detected smaller uptake reductions in the bilateral primary somatosensory system cortex and part of the limbic system in the ketamine-xylazine (KX) group and in the vestibular nucleus in the isoflurane group. Through kinetic analysis, we found that the volume of distribution and the membrane transport rate K1 were reduced in the KX group. Through various methods of 18 F-fluorodeoxyglucose-positron emission tomography quantification, the present study found that anesthesia with the ketamine-xylazine combination induced a global reduction of glucose metabolism compared with isoflurane; this reduction of metabolism was relatively lower in the primary somatosensory cortex and part of the limbic system. The volume of distribution of 18 F-fluorodeoxyglucose and its Glut1-mediated transport across the brain membranes (K1 ) were decreased in the KX group.

Relationship between amorphous solid dispersion in vivo absorption and in vitro dissolution: phase behavior during dissolution, speciation, and membrane mass transport

Enzalutamide is a fast crystallizing, hydrophobic compound that has solubility limited absorption in vivo. Given the low aqueous solubility of this compound, it was of interest to evaluate amorphous formulations in vitro and in vivo. Amorphous solid dispersions (ASD) of enzalutamide were prepared with the hydrophilic polymers, hydroxypropyl methylcellulose acetate succinate (HPMCAS) and copovidone (PVPVA). A side-by-side diffusion cell was developed as an in vitro characterization tool to discriminate enzalutamide ASDs based upon the solute thermodynamic activity achieved during dissolution and its impact on the subsequent membrane transport rates, phase behavior, and drug speciation. The same formulations were then tested in vivo in rats using oral dosing of ASD suspensions. Different levels of plasma exposure were observed between the ASDs, which could be correlated to the phase behaviors of the ASDs following dissolution. Unsurprisingly, ASDs that underwent crystallization show lower plasma exposures. However, differences were also observed between ASDs that dissolved to form nanosized amorphous drug aggregates versus those that dissolved to yield only supersaturated solutions, with the former outperforming the latter in terms of the plasma exposure. These observations highlight the importance of thoroughly understanding the phase behavior of an amorphous formulation following dissolution and the need to discriminate between different types of precipitation, specifically crystallization versus glass liquid phase separation to form nanosized amorphous aggregates.

Electrodialysis Extraction and Electrodeposition of Lead(II) in Systems with Liquid Membranes

The extraction of lead(II) ions from nitric acid solutions through liquid membranes under galvanostatic electrodialysis with metal electrodeposition from the receiving solution of perchloric, nitric, or acetic acids is studied. The solitons of di(2-ethylhexyl)phosphoric acid with additives of tri-n-octylamine in 1,2-dichloroethane are used as liquid membranes. The effect of the current density of the electrodialysis and the composition of the initial aqueous solution and receiving solution (catholyte), as well as the composition of liquid membranes on the rate of membrane transport and electrodeposition of the lead(II) ions, is investigated. The optimal conditions of the process are determined. Fine-grained and highly adherent lead deposits are obtained on a platinum cathode during the deposition from perchloric acid solutions. It is shown that the degree of extraction of lead(II) ions from the initial aqueous solution is higher than 90% in the process, while the degree of electrodeposition amounts to 60% within 5 h of electrodialysis.

Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons: Import of Folded Proteins.

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, including the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that the TOC/TIC translocons accommodated the tightly folded dihydrofolate reductase (DHFR) protein in complex with its stabilizing ligand, methotrexate (MTX). We employed a fluorescein-conjugated methotrexate (FMTX), which has slow membrane transport rates relative to unconjugated MTX, to show that the rate of ligand accumulation inside chloroplasts is faster when bound to DHFR that is actively being imported. Stromal accumulation of FMTX is ATP dependent when DHFR is actively being imported but is otherwise ATP independent, again indicating DHFR/FMTX complex import. Furthermore, the TOC/TIC pore size was probed with fixed-diameter particles and found to be greater than 25.6 Å, large enough to support folded DHFR import and also larger than mitochondrial and bacterial protein translocons that have a requirement for protein unfolding. This unique pore size and the ability to import folded proteins have critical implications regarding the structure and mechanism of the TOC/TIC translocons.

Supersaturation Potential of Ordered Mesoporous Silica Delivery Systems. Part 1: Dissolution Performance and Drug Membrane Transport Rates.

Ordered mesoporous silica materials have shown great potential as oral drug delivery systems for poorly soluble drugs. However, the ability of these delivery systems to generate drug supersaturation has not been widely investigated, and the recently noted phenomenon of incomplete drug release is not well understood. Therefore, the aim of this study was to comprehensively evaluate the release of hydrophobic drug molecules into solution from ordered mesoporous silica, focusing on the extent and duration of drug supersaturation. The dissolution and supersaturation behavior of ritonavir, following loading into mesoporous SBA-15 silica particles, was investigated by undertaking simple in vitro dissolution studies in phosphate buffer pH 6.8 and fasted state simulated intestinal fluid, as well as membrane flux studies using a side-by-side diffusion cell apparatus. It was found that supersaturated ritonavir solutions were generated from ritonavir-loaded mesoporous SBA-15 particles; however, drug release was always incomplete, even under sink conditions. In addition, the percentage drug release was observed to decrease significantly as the theoretical supersaturation ratio and dose of ritonavir-loaded SBA-15 formulation increased. The data obtained suggest an equilibrium exists between drug adsorbed to the SBA-15 silica surface and free drug present in solution. The findings described herein are highly significant in aiding our understanding of ordered mesoporous silica as a supersaturating drug delivery system for bioavailability enhancement.

Cancer antigen 125 concentrations in patients on chronic peritoneal dialysis: Relationship with dialysis quality and membrane transport properties

The aim of this study was to evaluate longitudinal changes in drained dialysate cancer antigen 125 (dCA-125) levels and to assess relationships between dCA-125 and dialysis quality, peritoneal membrane transport rates, dialysate glucose load, peritonitis and use of erythropoiesis stimulating agents (ESA), angiotensin-converting-enzyme inhibitors (ACEi) and statins in patients with end-stage renal failure during the first 6 months of peritoneal dialysis (PD) treatment. This prospective study included 20 patients (11 males and 9 females; mean age 62.90?12.69 years) who were followed-up during the first 6 months of PD using conventional low pH glucose-based dialysis fluids. The concentration of dCA-125 was measured in all patients, and the peritoneal equilibration test (PET), peritoneal dialysis treatment adequacy (Kt/V), normalized protein catabolic rate (nPCR), and total, peritoneal and residual clearances of urea and creatinine were calculated. Information on peritonitis occurrence, the use of ESA, ACEi and statins were collected. Data were analyzed by the Mann-Whitney test, Wilcoxon matched pairs test and Spearman?s rank correlation. The concentration of dCA-125 significantly decreased during the follow-up (p=0.016). After 6 months of PD treatment, the concentration of dCA-125 decreased significantly (p=0.016) in all patients. The decrease was present in all patients, but was statistically significant in patients on ACEi therapy (p=0.006) and in patients not using statins (p=0.005) or ESA (p=0.012). No correlation was found between dCA-125 and glucose load, but a statistically significant negative correlation between dCA-125 and the PET for creatinine was observed (p=0.013). These findings challenge the role of dCA-125 in predicting mesothelial cell integrity in PD patients.

Metabolic Measurements of Nonpermeating Compounds in Live Cells Using Hyperpolarized NMR.

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) has emerged as a technique for enhancing NMR signals by several orders of magnitude, thereby facilitating the characterization of metabolic pathways both in vivo and in vitro. Following the introduction of an externally hyperpolarized compound, real-time NMR enables the measurement of metabolic flux in the corresponding pathway. Spin relaxation however limits the maximum experimental time and prevents the use of this method with compounds exhibiting slow membrane transport rates. Here, we demonstrate that on-line electroporation can serve as a method for membrane permeabilization for use with D-DNP in cell cultures. An electroporation apparatus hyphenated with stopped-flow sample injection permits the introduction of the hyperpolarized metabolite within 3 s after the electrical pulse. In yeast cells that do not readily take up pyruvate, the addition of the electroporation pulse to the D-DNP experiment increases the signals of the downstream metabolic products CO2 and HCO3-, which otherwise are near the detection limit, by 8.2- and 8.6-fold. Modeling of the time dependence of these signals then permits the determination of the respective kinetic rate constants. The observed conversion rate from pyruvate to CO2 normalized for cell density was found to increase by a factor of 12 due to the alleviation of the membrane transport limitation. The use of electroporation therefore extends the applicability of D-DNP to in vitro studies with a wider range of metabolites and at the same time reduces the influence of membrane transport on the observed conversion rates.

Transcript Profiling Identifies Gene Cohorts Controlled by Each Signal Regulating Trans-Differentiation of Epidermal Cells of Vicia faba Cotyledons to a Transfer Cell Phenotype.

Transfer cells (TCs) support high rates of membrane transport of nutrients conferred by a plasma membrane area amplified by lining a wall labyrinth comprised of an uniform wall layer (UWL) upon which intricate wall ingrowth (WI) papillae are deposited. A signal cascade of auxin, ethylene, extracellular hydrogen peroxide (H2O2) and cytosolic Ca2+ regulates wall labyrinth assembly. To identify gene cohorts regulated by each signal, a RNA- sequencing study was undertaken using Vicia faba cotyledons. When cotyledons are placed in culture, their adaxial epidermal cells spontaneously undergo trans-differentiation to epidermal TCs (ETCs). Expressed genes encoding proteins central to wall labyrinth formation (signaling, intracellular organization, cell wall) and TC function of nutrient transport were assembled. Transcriptional profiles identified 9,742 annotated ETC-specific differentially expressed genes (DEGs; Log2fold change > 1; FDR p ≤ 0.05) of which 1,371 belonged to signaling (50%), intracellular organization (27%), cell wall (15%) and nutrient transporters (9%) functional categories. Expression levels of 941 ETC-specific DEGs were found to be sensitive to the known signals regulating ETC trans-differentiation. Significantly, signals acting alone, or in various combinations, impacted similar numbers of ETC-specific DEGs across the four functional gene categories. Amongst the signals acting alone, H2O2 exerted most influence affecting expression levels of 56% of the ETC-specific DEGs followed by Ca2+ (21%), auxin (18%) and ethylene (5%). The dominance by H2O2 was evident across all functional categories, but became more attenuated once trans-differentiation transitioned into WI papillae formation. Amongst the eleven signal combinations, H2O2/Ca2+ elicited the greatest impact across all functional categories accounting for 20% of the ETC-specific DEG cohort. The relative influence of the other signals acting alone, or in various combinations, varied across the four functional categories and two phases of wall labyrinth construction. These transcriptome data provide a powerful information platform from which to examine signal transduction pathways and how these regulate expression of genes encoding proteins engaged in intracellular organization, cell wall construction and nutrient transport.

Delayed fluorescence as an indicator of the influence of the herbicides Irgarol 1051 and Diuron on hard coral Acropora digitifera

We examined the effect of two herbicides (Irgarol 1051 and Diuron) on symbiotic dinoflagellates in the hard coral Acropora digitifera using delayed fluorescence (DF), specifically assessing changes in molecular membrane transport, i.e. inflow and outflow rates, and the binding of the herbicides to target proteins in photosystem II. The DF approach is rapid (e.g. measurement time, 60 s) and non-invasive, and can provide data on the extent of a photosynthetic system and the activity of its electron carriers. The DF of A. digitifera is inhibited 2 h after exposure to 1 μg/L of either Irgarol or Diuron. Analysis of DF inhibition over time by a compartment model suggests that Irgarol exposure results in a relatively higher inflow rate and lower outflow rate than does Diuron exposure. This suggests that Irgarol exposure more strongly inhibits photosynthesis and that the coral symbiotic dinoflagellates recover less from inhibition.

Exploiting the Phenomenon of Liquid-Liquid Phase Separation for Enhanced and Sustained Membrane Transport of a Poorly Water-Soluble Drug.

Recent studies on aqueous supersaturated lipophilic drug solutions prepared by methods including antisolvent addition, pH swing, or dissolution of amorphous solid dispersions (ASDs) have demonstrated that when crystallization is slow, these systems undergo liquid-liquid phase separation (LLPS) when the concentration of the drug in the medium exceeds its amorphous solubility. Following LLPS, a metastable equilibrium is formed where the concentration of drug in the continuous phase corresponds to the amorphous solubility while the dispersed phase is composed of a nanosized drug-rich phase. It has been reasoned that the drug-rich phase may act as a reservoir, enabling the rate of passive transport of the drug across a membrane to be maintained at the maximum value for an extended period of time. Herein, using clotrimazole as a model drug, and a flow-through diffusion cell, the reservoir effect is demonstrated. Supersaturated clotrimazole solutions at concentrations below the amorphous solubility show a linear relationship between the maximum flux and the initial concentration. Once the concentration exceeds the amorphous solubility, the maximum flux achieved reaches a plateau. However, the duration for which the high flux persists was found to be highly dependent on the number of drug-rich nanodroplets present in the donor compartment. Macroscopic amorphous particles of clotrimazole did not lead to the same reservoir effect observed with the nanodroplets formed through the process of LLPS. A first-principles mathematical model was developed which was able to fit the experimental receiver concentration-time profiles for concentration regimes both below and above amorphous solubility, providing support for the contention that the nanodroplet phase does not directly diffuse across the membrane but, instead, rapidly replenishes the drug in the aqueous phase that has been removed by transport across the membrane. This study provides important insight into the properties of supersaturated solutions and how these might in turn impact oral absorption through effects on passive membrane transport rates.

Characterization of Supersaturated Danazol Solutions – Impact of Polymers on Solution Properties and Phase Transitions

Excipients are essential for solubility enhancing formulations. Hence it is important to understand how additives impact key solution properties, particularly when supersaturated solutions are generated by dissolution of the solubility enhancing formulation. Herein, the impact of different concentrations of dissolved polymers on the thermodynamic and kinetic properties of supersaturated solutions of danazol were investigated. A variety of experimental techniques was used, including nanoparticle tracking analysis, fluorescence and ultraviolet spectroscopy and flux measurements to characterize the solution phase behavior. Neither the crystalline nor amorphous solubility of danazol was impacted by common amorphous solid dispersion polymers, polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC) or HPMC-acetate succinate. Consequently, the maximum membrane transport rate was limited only by the amorphous solubility, and not by the presence of the polymers. The polymers were able to inhibit crystallization to some extent at concentrations as low as 1 μg/mL, with the maximum effectiveness being reached at 10 μg/mL. Aqueous danazol solutions formed a drug-rich phase with a mean size of 250 nm when the concentration exceeded the amorphous solubility, and the polymers modified the surface properties of this drug-rich phase. The phase behavior of supersaturated solutions is complex and the kinetics of phase transformations can be substantially modified by polymeric additives present at low concentrations. However, fortunately, these additives do not appear to impact the bulk thermodynamic properties of the solution, thus enabling supersaturated solutions, which provide enhanced membrane transport relative to saturated solutions to be generated.