Endovascular drug-eluting stents have changed the practice of medicine, and yet it is unclear how they so dramatically reduce restenosis and how to distinguish between the different formulations available. Biological drug potency is not the sole determinant of biological effect. Physicochemical drug properties also play important roles. Historically, two classes of therapeutic compounds emerged: hydrophobic drugs, which are retained within tissue and have dramatic effects, and hydrophilic drugs, which are rapidly cleared and ineffective. Researchers are now questioning whether individual properties of different drugs beyond lipid avidity can further distinguish arterial transport and distribution. In bovine internal carotid segments, tissue-loading profiles for hydrophobic paclitaxel and rapamycin are indistinguishable, reaching load steady state after 2 days. Hydrophilic dextran reaches equilibrium in several hours at levels no higher than surrounding solution concentrations. Both paclitaxel and rapamycin bind to the artery at 30 -40 times bulk concentration. Competitive binding assays confirm binding to specific tissue elements. Most importantly, transmural drug distribution profiles are markedly different for the two compounds, reflecting, perhaps, different modes of binding. Rapamycin, which binds specifically to FKBP12 binding protein, distributes evenly through the artery, whereas paclitaxel, which binds specifically to microtubules, remains primarily in the subintimal space. The data demonstrate that binding of rapamycin and paclitaxel to specific intracellular proteins plays an essential role in determining arterial transport and distribution and in distinguishing one compound from another. These results offer further insight into the mechanism of local drug delivery and the specific use of existing drug-eluting stent formulations.

Background-Drug-eluting stents deliver potent compounds directly to arterial segments but can become clot laden when deployed. The question arises as to whether thrombi affect drug elution and arterial uptake. Methods and Results-Paclitaxel transport and retention were assessed in clots of different blood components. Diffusivity, affected by clot organization, is fastest in fibrin (Ϸ347 m 2 /s), slower in fibrin-red blood cell clots (34.98 m 2 /s), and slowest in whole-blood clots (3.55 m 2 /s). Blood cells bind and retain paclitaxel such that levels in clot increase linearly with red cell fraction. At physiological hematocrit, clot retains 3 times the amount of paclitaxel in surrounding solutions. Computational models predict that the potential of thrombus to absorb, retain, and release drug or to act as a barrier to drug delivery depends on clot geometry and strut position in clot relative to the vessel wall. Clot between artery and stent can reduce uptake 10-fold, whereas clot overlying the stent can shield drug from washout, increasing uptake. Model assumptions were confirmed and predictions were validated in a novel rat model that introduces thrombosis within stented aortas where nonocclusive thrombus acts as capacitive space for drug and shifts drug levels to decrease tissue uptake 2-fold. Conclusions-Thrombus apposed on stents creates large variations in drug uptake and can act to either increase or decrease wall deposition according to the clot and stent geometry. Arterial deposition of drug from stents deployed in clots will be highly variable and unpredictable unless the clot can be adequately controlled or removed.

Background Local drug delivery has transformed medicine, yet it remains unclear how drug efficacy depends on physicochemical properties and delivery kinetics. Most therapies seek to prolong release, yet, recent studies demonstrate sustained clinical benefit following local bolus endovascular delivery. Objectives The purpose of the current study was to examine the interplay between drug dose, diffusion and binding in determining tissue penetration and effect. Methods We introduce a quantitative framework that balances dose, saturable binding and diffusion and measured the specific binding parameters of drugs to target tissues. Results Model reduction techniques augmented by numerical simulations revealed that the impact of saturable binding on drug transport and retention is determined by the magnitude of a binding potential, Bp, the ratio of the binding capacity to the product of the equilibrium dissociation constant and accessible tissue volume fraction. At low Bp (<1) drugs are predominantly free and transport scales linearly with concentration. At high Bp (>40) drug transport exhibits a threshold dependence on applied surface concentration. Conclusions In this paradigm, drugs and antibodies with large Bp penetrate faster and deeper into tissues when presented at high concentrations. A threshold dependence of tissue transport on the applied surface concentration of paclitaxel and rapamycin may explain the threshold dose dependence of the in vivo biological efficacy of these drugs.

Mitogen-activated protein (MAP) kinase-mediated phosphorylation of specific residues in tyrosine hydroxylase leads to an increase in enzyme activity. However, the mechanism whereby phosphorylation affects enzyme turnover is not well understood. We used a combination of fluorescence resonance energy transfer (FRET) measurements and molecular dynamics simulations to explore the conformational free energy landscape of a 10-residue MAP kinase substrate found near the N terminus of the enzyme. This region is believed to be part of an autoregulatory sequence that overlies the active site of the enzyme. FRET was used to measure the effect of phosphorylation on the ensemble of peptide conformations, and molecular dynamics simulations generated free energy profiles for both the unphosphorylated and phosphorylated peptides. We demonstrate how FRET transfer efficiencies can be calculated from molecular dynamics simulations. For both the unphosphorylated and phosphorylated peptides, the calculated FRET efficiencies are in excellent agreement with the experimentally determined values. Moreover, the FRET measurements and molecular simulations suggest that phosphorylation causes the peptide backbone to change direction and fold into a compact structure relative to the unphosphorylated state. These results are consistent with a model of enzyme activation where phosphorylation of the MAP kinase substrate causes the N-terminal region to adopt a compact structure away from the active site. The methods we employ provide a general framework for analyzing the accessible conformational states of peptides and small molecules. Therefore, they are expected to be applicable to a variety of different systems.Phosphorylation of specific amino acids near the surface of a protein is an almost universal mechanism of protein activation that has been long appreciated (1,2). Yet the mechanism whereby phosphorylation modifies the activity of the protein is not well understood (1). Analysis of phosphorylation sites from different enzymes reveals common themes. In eukaryotes, phosphorylation typically occurs at tyrosine, threonine, or serine side chains, and these phosphorylated residues often form a network of hydrogen bonds with adjacent positively charged arginine residues (1,(3)(4)(5)(6). The network of hydrogen bonds and salt bridges that form can then communicate phosphorylation to distant areas of the protein (6). In the case of yeast glycogen phosphorylase, phosphorylation occurs at a threonine residue located near the N terminus, a region that overlies the active site of the enzyme (3). The enzyme, which normally exists as a homodimer, contains two active catalytic sites, one in each monomer (3). Phosphorylation at this site causes the N-terminal region to fold into a compact structure that wedges between the dimer interface. This structural change helps to reorient the active site in a manner that facilitates enzymatic activation (3). Examples such as this suggest that large scale movements of flexible regions within a protein are an impor...

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Global commitments aim to provide antiretroviral therapy (ART) to 15 million people living with HIV by 2015, and recent studies have demonstrated the potential for widespread ART to prevent HIV transmission. Increasingly, countries are adapting their national guidelines to start ART earlier, for both clinical and preventive benefits. To maximize the benefits of ART in resource-limited settings, six key principles need to guide ART choice: simplicity, tolerability and safety, durability, universal applicability, affordability and heat stability. Currently available drugs, combined with those in late-stage clinical development, hold great promise to simplify treatment in the short term. Over the longer term, newer technologies, such as long-acting formulations and nanotechnology, could radically alter the treatment paradigm. This commentary reviews recommendations made in an expert consultation on treatment scale up in resource-limited settings.