System dynamics of subcellular transport

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.     

The subcellular distribution of small molecules: a meta-analysis

To explore the extent to which current knowledge about the organelle-targeting features of small molecules may be applicable toward controlling the accumulation and distribution of exogenous chemical agents inside cells, molecules with known subcellular localization properties (as reported in the scientific literature) were compiled into a single data set. This data set was compared to a reference data set of approved drug molecules derived from the DrugBank database, and to a reference data set of random organic molecules derived from the PubChem database. Cheminformatic analysis revealed that molecules with reported subcellular localizations were comparably diverse. However, the calculated physicochemical properties of molecules reported to accumulate in different organelles were markedly overlapping. In relation to the reference sets of DrugBank and PubChem molecules, molecules with reported subcellular localizations were biased toward larger, more complex chemical structures possessing multiple ionizable functional groups and higher lipophilicity. Stratifying molecules based on molecular weight revealed that many physicochemical properties' trends associated with specific organelles were reversed in smaller vs larger molecules. Most likely, these reversed trends are due to the different transport mechanisms determining the subcellular localization of molecules of different sizes. Molecular weight can be dramatically altered by tagging molecules with fluorophores or by incorporating organelle targeting motifs. Generally, in order to better exploit structure-localization relationships, subcellular targeting strategies would benefit from analysis of the biodistribution effects resulting from variations in the size of the molecules.     

Rational design of potent and selective EGFR tyrosine kinase inhibitors as anticancer agents

Increasing knowledge of the structure and function of the Epidermal Growth Factor Receptor (EGFR) subfamily of tyrosine kinases, and of their role in the initiation and progression of various cancers has led to the search for inhibitors of signaling molecules that may prove to be important in cancer therapy. The complex nature of EGFR biology allows for potential opportunities for EGFR inhibitors in a number of areas of cancer therapy, including proliferative, angiogenic, invasive, and metastatic aspects. Different approaches have been used to target either the extracellular ligand-binding domain of the EGFR or the intracellular tyrosine kinase region that results in interference with its signaling pathways that modulate cancer-promoting responses. Examples of these include a number of monoclonal antibodies, immunotoxins and ligand-binding cytotoxic agents that target the extracellular ligand binding region of EGFR, and small molecule inhibitors that target the intracellular kinase domain and act by interfering with ATP binding to the receptor. During the past 3 years, significant progress has been made towards the identification of new structural classes of small molecule inhibitors that show high potency and specificity towards EGFR. The search for new small molecules that inhibit kinases has included traditional approaches like the testing of natural products, random screening of chemical libraries, the use of classical structure-activity-relationship studies, and the incorporation of structure-based drug design and combinatorial chemistry techniques. There has been a significant improvement in the development of selective EGFR inhibitors with the use of a structure-based design approach employing a homology model of the EGFR kinase domain. Molecular modeling procedures have been used to generate novel molecules that are complementary in shape and electrostatics to the EGFR kinase domain topography. This review focuses on some examples of the successful use of this method.     

Lipophilicity in trans-bilayer transport and subcellular pharmacokinetics

The aim of subcellular pharmacokinetics in drug design is to model drug disposition and response as a function of the properties of drugs and biosystems involved and the observation time. Biosystems are represented by systems of alternating membranes and aqueous phases that differ in acidity and contain low-molecular cell constituents, enzymes and other proteins. The resulting disposition models are combined with linear free-energy assumptions, drug/receptor binding kinetics and relationships between receptor binding and response to produce model-based quantitativestructure–(time–)activity relationships, QS(T)AR. This review summarizes the present status of subcellular pharmacokinetics with emphasis on passive trans-bilayer transport. In particular, mechanisms of transport are analyzed with respect to amphiphilicity and lipophilicity. The overall rate of transport is strongly governed by amphiphilicity, the tendency of drug molecules to adsorb to the bilayer/water interface. Depending on amphiphilicity, the time needed for a drug to cross a single bilayer ranges from seconds to days. The main advantage of the subcellular pharmacokinetic approach is that the resulting models, once calibrated for a given biosystem, provide a detailed recipe for tailoring the drug properties to ensure optimum disposition. This revised version was published online in August 2006 with corrections to the Cover Date.     

Subcellular drug distribution: mechanisms and roles in drug efficacy,toxicity, resistance,and targeted delivery

Abstract

After administration, drug molecules usually enter target cells to access their intracellular targets. In eukaryotic cells, these targets are often located in organelles, including the nucleus, endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, and peroxisomes. Each organelle type possesses unique biological features. For example, mitochondria possess a negative transmembrane potential, while lysosomes have an intraluminal delta pH. Other properties are common to several organelle types, such as the presence of ATP-binding cassette (ABC) or solute carrier-type (SLC) transporters that sequester or pump out xenobiotic drugs. Studies on subcellular drug distribution are critical to understand the efficacy and toxicity of drugs along with the body’s resistance to them and to potentially offer hints for targeted subcellular drug delivery. This review summarizes the results of studies from 1990 to 2017 that examined the subcellular distribution of small molecular drugs. We hope this review will aid in the understanding of drug distribution within cells.     

The Role of the VPS4A-Exosome Pathway in the Intrinsic Egress Route of a DNA-Binding Anticancer Drug

Purpose This study investigates the subcellular pharmacokinetics of drug efflux in cancer cells and explores the role of the multivesicular body (MVB) in facilitating efflux of doxorubicin, a widely used DNA-targeting anticancer agent, from the nucleus.Methods Human erythroleukemic K562 cells were pulsed with doxorubicin and then chased in drug-free media to allow for efflux. Microscopy and biochemical techniques were used to visualize the subcellular localization of the drug and measure drug content and distribution during the efflux period. To explore the role of the MVB in doxorubicin efflux, K562 cells were transfected with dominant negative mutant forms of VPS4a–GFP chimeras.Results Although the intracellular concentration of drug exceeds the extracellular concentration, nuclear efflux of doxorubicin occurs in living cells at a faster rate than doxorubicin unbinding from isolated nuclei into drug-free buffer. In cells expressing dominant negative VPS4a, doxorubicin accumulates in VPS4a-positive vesicles and drug sequestration is inhibited, directly implicating the MVB pathway in the egress route of doxorubicin in this cell type.Conclusions Cellular membranes are a component of the doxorubicin efflux mechanism in K562 cells. Dominant-negative GFP chimeric mutants can be used to elucidate the role of specific membrane trafficking pathways in subcellular drug transport routes.     

Seeing small molecules in action with bioorthogonal chemistry

Chemical probes that target specific protein families offer powerful tools to accelerate drug discovery. Small molecules modified with uniquely reactive functional groups and detection tags provide novel tools to characterize complex proteomes functionally and also to help determine the specificity of small molecule inhibitors toward various enzyme/protein classes. This review highlights the application of bioorthogonal chemistries in combination with chemical probes, which together are offering unprecedented opportunities to dissect the functions of enzyme/protein families in vivo and enabling more precise target identification of small molecules. Advances in chemical probes and bioorthogonal reactions are poised to reveal new therapeutic targets and to facilitate the discovery and characterization of small molecules aimed at disease.     

Carboxylesterase inhibitors

INTRODUCTION: Carboxylesterases play major roles in the hydrolysis of numerous therapeutically active compounds. This is, in part, due to the prevalence of the ester moiety in these small molecules. However, the impact these enzymes may play on drug stability and pharmacokinetics is rarely considered prior to molecule development. Therefore, the application of selective inhibitors of this class of proteins may have utility in modulating the metabolism, distribution and toxicity of agents that are subjected to enzyme hydrolysis. AREAS COVERED: This review details the development of all such compounds dating back to 1986, but principally focuses on the very recent identification of selective human carboxylesterases inhibitors. EXPERT OPINION: The implementation of carboxylesterase inhibitors may significantly revolutionize drug discovery. Such molecules may allow for improved efficacy of compounds inactivated by this class of enzymes and/or reduce the toxicity of agents that are activated by these proteins. Furthermore, since lack of carboxylesterase activity appears to have no obvious biological consequence, these compounds could be applied in combination with virtually any esterified drug. Therefore, inhibitors of these proteins may have utility in altering drug hydrolysis and distribution in vivo. The characteristics, chemical and biological properties and potential uses of such agents are discussed here.     

Challenges and Opportunities in Absorption, Distribution, Metabolism, and Excretion Studies of Therapeutic Biologics

With the advancement of biotechnology in the last two decades, optimized and novel modalities and platforms of biologic moieties have emerged rapidly in drug discovery pipelines. In addition, new technologies for delivering therapeutic biologics (e.g., needle-free devices, nanoparticle complexes), as well as novel approaches for disease treatments (e.g., stem cell therapy, individualized medicine), continue to be developed. While pharmacokinetic studies are routinely carried out for therapeutic biologics, experiments that elucidate underlying mechanisms for clearance and biodistribution or identify key factors that govern absorption, distribution, metabolism, and excretion (ADME) of biologics often are not thoroughly conducted. Realizing the importance of biologics as therapeutic agents, pharmaceutical industry has recently begun to move the research focus from small molecules only to a blended portfolio consisting of both small molecules and biologics. This trend brings many opportunities for scientists working in the drug disposition research field. In anticipation of these opportunities and associated challenges, this review highlights impact of ADME studies on clinical and commercial success of biologics, with a particular focus on emerging applications and technologies and linkage with mechanistic pharmacokinetic/pharmacodynamic modeling and biomarker research.     

The alluring potential of functionalized carbon nanotubes in drug discovery

Importance of the field: The possibility of carbon nanotube integration into living systems for therapeutic and diagnostic purposes has opened the way to explore their applications in drug delivery and discovery. A wide variety of chemical approaches has been developed to functionalize carbon nanotubes with therapeutic molecules towards different biomedical uses. Areas covered in this review: This review covers the recent advances in the development of functionalized carbon nanotubes to offer improvements for different diseases, in particular for cancer therapy. What the reader will gain: Functionalized carbon nanotubes are able to transport therapeutic agents. Targeted methodologies using carbon nanotube-based conjugates have been investigated to improve the efficacy of some drugs. The capacity of such nanomaterials to seamlessly translocate into cells with alternative various mechanisms and their pharmacokinetic properties is also discussed. Take home message: Although at its infancy, functionalized carbon nanotubes are very promising as a new nanomedicine platform in the field of drug discovery and delivery. They have the capacity to cross biological barriers and can be eliminated via renal and/or fecal excretion. They can transport small drug molecules while maintaining - and in some cases improving - their therapeutic efficacy.     

Relationship between drugs and functional activity of various mammalian P-glycoproteins (ABCB1)

P-glycoprotein (Pgp, ABCB1) is an efflux transporter for a variety of amphipathic agents that can affect the pharmacokinetics of drugs. In order to extrapolate transport and pharmacokinetic data of the drug candidates obtained from in vitro and animal models to those in humans, it is important to understand the functional differences of Pgps from various mammalian species including human, monkey, dog, rat, and mouse. Here, we review differences/similarities in the properties of Pgp from numerous mammalian species commonly used in preclinical studies and discuss their relevance to the pharmacokinetics of potential drug molecules.     

Modern methods for delivery of drugs across the blood-brain barrier

The blood-brain barrier (BBB) is a highly regulated and efficient barrier that provides a sanctuary to the brain. It is designed to regulate brain homeostasis and to permit selective transport of molecules that are essential for brain function. Unfortunately, drug transport to the brain is hampered by this almost impermeable, highly selective and well coordinated barrier. With progress in molecular biology, the BBB is better understood, particularly under different pathological conditions. This review will discuss the barrier issue from a biological and pathological perspective to provide a better insight to the challenges and opportunities associated with the BBB. Modern methods which can take advantage of these opportunities will be reviewed. Applications of nanotechnology in drug transport, receptor-mediated targeting and transport, and finally cell-mediated drug transport will also be covered in the review. The challenge of delivering an effective therapy to the brain is formidable; solutions will likely involve concerted multidisciplinary approaches that take into account BBB biology as well as the unique features associated with the pathological condition to be treated.     

Pharmacological therapies for raising HDL cholesterol beyond synthetic small molecules

Epidemiological studies support the hypothesis that HDL particles possess inherent atheroprotective properties. The protective properties of HDL are primarily attributed to its known involvement in cholesterol mobilization from peripheral tissues and reverse cholesterol transport for hepatic excretion of cholesterol; removal of excess cholesterol load from the arterial wall is essential for prevention or reversal of unstable plaque development. Other known protective properties of HDL include its anti-inflammatory, antioxidant, antithrombotic and vasoprotective activities, which have been demonstrated in preclinical models of disease. Pharmacological therapies aimed at increasing HDL are predicted to offer tremendous clinical benefit for the prevention and treatment of cardiovascular disease. Advances in biologicals as therapeutics provide new opportunities for drug discovery. This review discusses some of the potential benefits of therapeutic approaches designed to raise HDL beyond that of traditional synthetic small molecules.     

Functional Expression of P-glycoprotein and Organic Anion Transporting Polypeptides at the Blood-Brain Barrier: Understanding Transport Mechanisms for Improved CNS Drug Delivery?

Drug delivery to the central nervous system (CNS) is greatly limited by the blood-brain barrier (BBB). Physical and biochemical properties of the BBB have rendered treatment of CNS diseases, including those with a hypoxia/reoxygenation (H/R) component, extremely difficult. Targeting endogenous BBB transporters from the ATP-binding cassette (ABC) superfamily (i.e., P-glycoprotein (P-gp)) or from the solute carrier (SLC) family (i.e., organic anion transporting polypeptides (OATPs in humans; Oatps in rodents)) has been suggested as a strategy that can improve delivery of drugs to the brain. With respect to P-gp, direct pharmacological inhibition using small molecules or selective regulation by targeting intracellular signaling pathways has been explored. These approaches have been largely unsuccessful due to toxicity issues and unpredictable pharmacokinetics. Therefore, our laboratory has proposed that optimization of CNS drug delivery, particularly for treatment of diseases with an H/R component, can be achieved by targeting Oatp isoforms at the BBB. As the major drug transporting Oatp isoform, Oatp1a4 has demonstrated blood-to-brain transport of substrate drugs with neuroprotective properties. Furthermore, our laboratory has shown that targeting Oatp1a4 regulation (i.e., TGF-β signaling mediated via the ALK-1 and ALK-5 transmembrane receptors) represents an opportunity to control Oatp1a4 functional expression for the purpose of delivering therapeutics to the CNS. In this review, we will discuss limitations of targeting P-gp-mediated transport activity and the advantages of targeting Oatp-mediated transport. Through this discussion, we will also provide critical information on novel approaches to improve CNS drug delivery by targeting endogenous uptake transporters expressed at the BBB.     

A cell-based molecular transport simulator for pharmacokinetic prediction and cheminformatic exploration

In the body, cell monolayers serve as permeability barriers, determining transport of molecules from one organ or tissue compartment to another. After oral drug administration, for example, transport across the epithelial cell monolayer lining the lumen of the intestine determines the fraction of drug in the gut that is absorbed by the body. By modeling passive transcellular transport properties in the presence of an apical to basolateral concentration gradient, we demonstrate how a computational, cell-based molecular transport simulator can be used to define a physicochemical property space occupied by molecules with desirable permeability and intracellular retention characteristics. Considering extracellular domains of cell surface receptors located on the opposite side of a cell monolayer as a drug's desired site of action, simulation of transcellular transport can be used to define the physicochemical properties of molecules with maximal transcellular permeability but minimal intracellular retention. Arguably, these molecules would possess very desirable features: least likely to exhibit nonspecific toxicity, metabolism, and side effects associated with high (undesirable) intracellular accumulation; and most likely to exhibit favorable bioavailability and efficacy associated with maximal rates of transport across cells and minimal intracellular retention, resulting in (desirable) accumulation at the extracellular site of action. Simulated permeability values showed good correlations with PAMPA, Caco-2, and intestinal permeability measurements, without "training" the model and without resorting to statistical regression techniques to "fit" the data. Therefore, cell-based molecular transport simulators could be useful in silico screening tools for chemical genomics and drug discovery.     

Interaction of innovative small molecule drugs used for cancer therapy with drug transporters

Multiple new small molecules such as tyrosine kinase, mammalian target of rapamycin (mTOR) and proteasome inhibitors have been approved in the last decade and are a considerable progress for cancer therapy. Drug transporters are important determinants of drug concentrations in the systemic circulation. Moreover, expression of drug transporters in blood-tissue barriers (e.g. blood-brain barrier) can limit access of small molecules to the tumour (e.g. brain tumour). Finally, transporter expression and (up)regulation in the tumour itself is known to affect local drug concentrations in the tumour tissue contributing to multidrug resistance observed for multiple anticancer agents. This review summarizes the current knowledge on: (i) small molecules as substrates of uptake and efflux transporters; (ii) the impact of transporter deficiency in knockout mouse models on plasma and tissue concentrations; (iii) small molecules as inhibitors of uptake and efflux transporters with possible consequences for drug-drug interactions and the reversal of multidrug resistance; and (iv) on clinical studies investigating the association of polymorphisms in genes encoding drug transporters with pharmacokinetics, outcome and toxicity during treatment with the small molecules.     

Novel approaches to antimalarial drug discovery

Major advances in our understanding of malaria parasite biology have been made. Coupled with the completion of the malaria genome, this has presented exciting opportunities for target-based antimalarial drug discovery. However, the unraveling of more validated biological targets will not necessarily translate into the identification of new chemical entities that are effective against drug resistant parasites in the long term. As history has already shown, development of antiplasmodial agents aimed at a single parasite target or specialized process has failed to stem the tide of drug resistance. This review highlights recent starting points and/or approaches to antimalarial drug discovery with particular emphasis on innovative efforts, which are not necessarily based on the identification of new drug targets and attendant inhibitor design. Approaches covered include utilization of validated chemical scaffolds, bioprecursor and carrier prodrugs, double drug development and/or multi-therapeutic strategies, use of metallocenic scaffolds, the medicinal chemistry of antimalarial natural products and in silico drug design.     

Gold nanoparticles: opportunities and challenges in nanomedicine

Importance of the field: Site-specific drug delivery is an important area of research that is anticipated to increase the efficacy of the drug and reduce potential side effects. Owing to this, substantial work has been done developing non-invasive and targeted tumor treatment with nanoscale metallic particles.

Areas covered in this review: This review focuses on the work done in the last few years developing gold nanoparticles as cancer therapeutics and diagnostic agents. However, there are challenges in using gold nanoparticles as drug delivery systems, such as biodistribution, pharmacokinetics and possible toxicity. Approaches to limit these issues are proposed.

What the reader will gain: Different approaches from several different disciplines are discussed. Potential clinical applications of these engineered nanoparticles are also presented.

Take home message: As a result of their unique size-dependent physicochemical and optical properties, adaptability, subcellular size and biocompatibility, these nanosized carriers offer a suitable means of transporting small molecules as well as biomacromolecules to diseased cells/tissues.     

Therapeutic interventions in sepsis: current and anticipated pharmacological agents

Sepsis is a clinical syndrome characterized by a multisystem response to a pathogenic assault due to underlying infection that involves a combination of interconnected biochemical, cellular and organ–organ interactive networks. After the withdrawal of recombinant human-activated protein C (rAPC), researchers and physicians have continued to search for new therapeutic approaches and targets against sepsis, effective in both hypo- and hyperinflammatory states. Currently, statins are being evaluated as a viable option in clinical trials. Many agents that have shown favourable results in experimental sepsis are not clinically effective or have not been clinically evaluated. Apart from developing new therapeutic molecules, there is great scope for for developing a variety of drug delivery strategies, such as nanoparticulate carriers and phospholipid-based systems. These nanoparticulate carriers neutralize intracorporeal LPS as well as deliver therapeutic agents to targeted tissues and subcellular locations. Here, we review and critically discuss the present status and new experimental and clinical approaches for therapeutic intervention in sepsis.