Belinda S. Akpa

Multi-modal contributions to detoxification of acute pharmacotoxicity by a triglyceride micro-emulsion.

Triglyceride micro-emulsions such as Intralipid® have been used to reverse cardiac toxicity induced by a number of drugs but reservations about their broad-spectrum applicability remain because of the poorly understood mechanism of action. Herein we report an integrated mechanism of reversal of bupivacaine toxicity that includes both transient drug scavenging and a cardiotonic effect that couple to accelerate movement of the toxin away from sites of toxicity. We thus propose a multi-modal therapeutic paradigm for colloidal bio-detoxification whereby a micro-emulsion both improves cardiac output and rapidly ferries the drug away from organs subject to toxicity. In vivo and in silico models of toxicity were combined to test the contribution of individual mechanisms and reveal the multi-modal role played by the cardiotonic and scavenging actions of the triglyceride suspension. These results suggest a method to predict which drug toxicities are most amenable to treatment and inform the design of next-generation therapeutics for drug overdose.

Resuscitation with lipid emulsion: dose-dependent recovery from cardiac pharmacotoxicity requires a cardiotonic effect.

Background:Recent publications have questioned the validity of the “lipid sink” theory of lipid resuscitation while others have identified sink-independent effects and posed alternative mechanisms such as hemodilution. To address these issues, the authors tested the dose-dependent response to intravenous lipid emulsion during reversal of bupivacaine-induced cardiovascular toxicity in vivo. Subsequently, the authors modeled the relative contribution of volume resuscitation, drug sequestration, inotropy and combined drug sequestration, and inotropy to this response with the use of an in silico model. Methods:Rats were surgically prepared to monitor cardiovascular metrics and deliver drugs. After catheterization and instrumentation, animals received a nonlethal dose of bupivacaine to produce transient cardiovascular toxicity, then were randomized to receive one of the four treatments: 30% intravenous lipid emulsion, 20% intravenous lipid emulsion, intravenous saline, or no treatment (n = 7 per condition; 28 total animals). Recovery responses were compared with the predictions of a pharmacokinetic–pharmacodynamic model parameterized using previously published laboratory data. Results:Rats treated with lipid emulsions recovered faster than did rats treated with saline or no treatment. Intravenous lipid emulsion of 30% elicited the fastest hemodynamic recovery followed in order by 20% intravenous lipid emulsion, saline, and no treatment. An increase in arterial blood pressure underlay the recovery in both lipid emulsion–treated groups. Heart rates remained depressed in all four groups throughout the observation period. Model predictions mirrored the experimental recovery, and the model that combined volume, sequestration, and inotropy predicted in vivo results most accurately. Conclusion:Intravenous lipid emulsion accelerates cardiovascular recovery from bupivacaine toxicity in a dose-dependent manner, which is driven by a cardiotonic response that complements the previously reported sequestration effect.

Validity of the lipid sink as a mechanism for the reversal of local anesthetic systemic toxicity: a physiologically based pharmacokinetic model study.

Background:In vitro observations support the lipid sink theory of therapeutic action by confirming the capacity of lipid emulsions to successfully uptake bupivacaine from aqueous media. However, competing hypotheses and some in/ex vivo small animal studies suggest that a metabolic or positive inotropic effect underlies the dramatic effects of lipid therapy. Controlled clinical tests to establish causality and mechanism of action are an impossibility. In an effort to quantitatively probe the merits of a “sink” mechanism, a physiologically based pharmacokinetic model has been developed that considers the binding action of plasma lipid. Methods:The model includes no fitting parameters and accounts for concentration dependence of plasma protein and lipid:anesthetic binding as well as the metabolism of the lipid scavenger. Predicted pharmacokinetics were validated by comparison with data from healthy volunteers administered a nontoxic dose of bupivacaine. The model was augmented to simulate lipid therapy and extended to the case of accidental IV infusion of bupivacaine at levels known to cause systemic toxicity. Results:The model yielded quantitative agreement with available pharmacokinetic data. Simulated lipid infusion following an IV overdose was predicted to yield (1) an increase in total plasma concentration, (2) a decrease in unbound concentration, and (3) a decrease in tissue content of bupivacaine. Conclusions:Results suggest that the timescale on which tissue content is reduced varies from organ to organ, with the concentration in the heart falling by 11% within 3 min. This initial study suggests that, in isolation, the lipid sink is insufficient to guarantee a reversal of systemic toxicity.

Non-invasive mass transfer measurements in complex biofilm-coated structures.

We demonstrate a novel application of 13C pulsed field gradient (PFG) NMR to monitor mass transfer, due to both flow and diffusion, in a 3D complex porous support structure modified by biofilm growth. This enables timescales an order of magnitude larger than previously possible to be accessed with respect to displacement probability distribution (propagator) measurements. The evolution in the propagator shape with observation time to the Gaussian asymptote (constant dispersion coefficient) is consequently well resolved. We also simulated the measured displacement propagators with good agreement between experiment and prediction. The methodology has significant potential for the selective characterization of the transport of nutrients, metabolic products, pollutants and biocides in such complex biofilm‐containing structures. Biotechnol. Bioeng. 2008;101: 602–608.

Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.

The emerging threat of superwarfarins: history, detection, mechanisms, and countermeasures

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half‐lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin‐resistant rodents. By the mid‐1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1–2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K–dependent and –independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

Using spectral and cumulative spectral entropy to classify anomalous diffusion in Sephadex gels

Sephadexgel beads are commonly used to separate mixtures of similar molecules based on trapping and size exclusion from internal submicron diameter cavities. Water, as it freely moves through the porous gel and enclosed chambers of Sephadexbeads, exhibits both normal (Gaussian) and anomalous (non-Gaussian) water diffusion. The apparent diffusion coefficient (ADC) of water in Sephadexgels can be measured using magnetic resonance imaging (MRI) by applying diffusion-weighted pulse sequences. This study investigates the relationship between the ADC of water and the complexity (i.e., size and number of cavities) of a series of Sephadexbeads. We first classified the stochastic movement of water by using the solution to the space and time fractional diffusion equation to extract the ADC and the fractional time and space parameters (, ), which are essentially the order of the respective fractional derivatives in Ficks second law. From the perspective of the continuous time random walk (CTRW) model of anomalous diffusion, these parameters reflect waiting times (trapping) and jump increments (nano-flow) of the water in the gels. The observed MRI diffusion signal decay represents the Fourier transform of the diffusion propagator (i.e., the characteristic function of the stochastic process). In two series of Sephadexgel beads, we observed a strong inverse correlation between bead porosity (which is also responsible for molecular size exclusion) and the fractional order parameters; as the gels become more heterogeneous, the ADC decreases, both and are reduced and the diffusion exhibits anomalous (sub-diffusion) behavior. In addition, as a new measure for the structural complexity in Sephadexgel beads, we propose using the spectral and the cumulative spectral entropy that are derived from the observed characteristic function. We find that both measures of entropy increase with the porosity and tortuosity of the gel in a manner consistent with fractional order diffusional dynamics.

Structural perturbation of a dipalmitoylphosphatidylcholine (DPPC) bilayer by warfarin and its bolaamphiphilic analogue: A molecular dynamics study

Compounds with nominally similar biological activity may exhibit differential toxicity due to differences in their interactions with cell membranes. Many pharmaceutical compounds are amphiphilic and can be taken up by phospholipid bilayers, interacting strongly with the lipid-aqueous interface whether or not subsequent permeation through the bilayer is possible. Bolaamphiphilic compounds, which possess two hydrophilic ends and a hydrophobic linker, can likewise undergo spontaneous uptake by bilayers. While membrane-spanning bolaamphiphiles can stabilize membranes, small molecules with this characteristic have the potential to create membrane defects via disruption of bilayer structure and dynamics. When compared to the amphiphilic therapeutic anticoagulant, warfarin, the bolaamphiphilic analogue, brodifacoum, exhibits heightened toxicity that goes beyond superior inhibition of the pharmacological target enzyme. We explore, herein, the consequences of anticoagulant accumulation in a dipalmitoylphosphatidylcholine (DPPC) bilayer. Coarse-grained molecular dynamics simulations reveal that permeation of phospholipid bilayers by brodifacoum causes a disruption of membrane barrier function that is driven by the bolaamphiphilic nature and size of this molecule. We find that brodifacoum partitioning into bilayers causes membrane thinning and permeabilization and promotes lipid flip-flop - phenomena that are suspected to play a role in triggering cell death. These phenomena are either absent or less pronounced in the case of the less toxic, amphiphilic compound, warfarin.

Differential Effects of Sterols on Ion Channels: Stereospecific Binding vs Stereospecific Response

Numerous ion channels have been shown to be regulated by the level of membrane cholesterol, but the mechanisms responsible for these effects are still not well understood. The key question in the field is how to discriminate between the contributions of the two central mechanisms that might be responsible for the sensitivity of ion channels to cholesterol: specific sterol-protein interactions or regulation of channels by the bilayer physical properties. Comparative analysis of cholesterol and its isomers on the function of an ion channel is a powerful tool to achieve this goal. An increasing number of studies show that cholesterol regulates several types of ion channels in a stereospecific manner, suggesting an involvement of specific sterol-protein interactions. However in this chapter, we present evidence that the stereospecificity of cholesterol-ion channel interactions may be mediated, not by a lack of binding, as has been generally assumed, but by the specificity of the interaction, which results in a functional effect, in the case of native cholesterol, and a lack of functional effect, in the case of a cholesterol isomer. In other words, accumulating evidence suggests that the structural requirements of ion channel cholesterol-binding sites are lax, allowing chiral isomers of cholesterol to bind to the same site in a nonstereospecific way, but the ability of a sterol to confer a functional effect on the channel activity can still be stereospecific. This is an important distinction both conceptually and methodologically. Indeed, our analysis shows that the orientations of cholesterol and its chiral isomer ent-cholesterol within a hydrophobic binding pocket of Kir2.2 are significantly different, and we propose that this difference may underlie distinct functional outcomes.

The emerging threat of superwarfarins

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half‐lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin‐resistant rodents. By the mid‐1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1–2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K–dependent and –independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.