Ryan S. Funk

Lysosomal sequestration (trapping) of lipophilic amine (cationic amphiphilic) drugs in immortalized human hepatocytes (Fa2N-4 cells)

Lipophilic (logP > 1) and amphiphilic drugs (also known as cationic amphiphilic drugs) with ionizable amines (pKa > 6) can accumulate in lysosomes, a process known as lysosomal trapping. This process contributes to presystemic extraction by lysosome-rich organs (such as liver and lung), which, together with the binding of lipophilic amines to phospholipids, contributes to the large volume of distribution characteristic of numerous cardiovascular and central nervous system drugs. Accumulation of lipophilic amines in lysosomes has been implicated as a cause of phospholipidosis. Furthermore, elevated levels of lipophilic amines in lysosomes can lead to high organ-to-blood ratios of drugs that can be mistaken for active drug transport. In the present study, we describe an in vitro fluorescence-based method (using the lysosome-specific probe LysoTracker Red) to identify lysosomotropic agents in immortalized hepatocytes (Fa2N-4 cells). A diverse set of compounds with various physicochemical properties were tested, such as acids, bases, and zwitterions. In addition, the partitioning of the nonlysosomotropic atorvastatin (an anion) and the lysosomotropics propranolol and imipramine (cations) were quantified in Fa2N-4 cells in the presence or absence of various lysosomotropic or nonlysosomotropic agents and inhibitors of lysosomal sequestration (NH4Cl, nigericin, and monensin). Cellular partitioning of propranolol and imipramine was markedly reduced (by at least 40%) by NH4Cl, nigericin, or monensin. Lysosomotropic drugs also inhibited the partitioning of propranolol by at least 50%, with imipramine partitioning affected to a lesser degree. This study demonstrates the usefulness of immortalized hepatocytes (Fa2N-4 cells) for determining the lysosomal sequestration of lipophilic amines.

Mechanisms of amine accumulation in, and egress from, lysosomes

The human body is continuously exposed to small organic molecules containing one or more basic nitrogen atoms. Many of these are endogenous (i.e., neurotransmitters, polyamines and biogenic amines), while others are exogenously supplied in the form of drugs, foods and pollutants. It is well-known that many amines have a strong propensity to specifically and substantially accumulate in highly acidic intracellular compartments, such as lysosomes, through a mechanism referred to as ion trapping. It is also known that cells have acquired the unique ability to sense and respond to amine accumulation in lysosomes in an effort to prevent potential negative consequences associated with hyperaccumulation. We describe here methods that are used to evaluate the dynamics of amine accumulation in, and egress from, lysosomes. Moreover, we highlight specific proteins that are thought to play important roles in these pathways. A theoretical model describing lysosomal amine dynamics is described and shown to adequately fit experimental kinetic data. The implications of this research in understanding and treating disease are discussed.

Cationic amphiphilic drugs cause a marked expansion of apparent lysosomal volume: Implications for an intracellular distribution-based drug interaction

How a drug distributes within highly compartmentalized mammalian cells can affect both the activity and pharmacokinetic behavior. Many commercially available drugs are considered to be lysosomotropic, meaning they are extensively sequestered in lysosomes by an ion trapping-type mechanism. Lysosomotropic drugs typically have a very large apparent volume of distribution and a prolonged half-life in vivo, despite minimal association with adipose tissue. In this report we tested the prediction that the accumulation of one drug (perpetrator) in lysosomes could influence the accumulation of a secondarily administered one (victim), resulting in an intracellular distribution-based drug interaction. To test this hypothesis cells were exposed to nine different hydrophobic amine-containing drugs, which included imipramine, chlorpromazine and amiodarone, at a 10 μM concentration for 24 to 48 h. After exposure to the perpetrators the cellular accumulation of LysoTracker Red (LTR), a model lysosomotropic probe, was evaluated both quantitatively and microscopically. We found that all of the tested perpetrators caused a significant increase in the cellular accumulation of LTR. Exposure of cells to imipramine caused an increase in the cellular accumulation of other lysosomotropic probes and drugs including LyosTracker Green, daunorubicin, propranolol and methylamine; however, imipramine did not alter the cellular accumulation of non-lysosomotropic amine-containing molecules including MitoTracker Red and sulforhodamine 101. In studies using ionophores to abolish intracellular pH gradients we were able to resolve ion trapping-based cellular accumulation from residual pH-gradient independent accumulation. Results from these evaluations in conjunction with lysosomal pH measurements enabled us to estimate the relative aqueous volume of lysosomes of cells before and after imipramine treatment. Our results suggest that imipramine exposure caused a 4-fold expansion in the lysosomal volume, which provides the basis for the observed drug interaction. The imipramine-induced lysosomal volume expansion was shown to be both time- and temperature-dependent and reversed by exposing cells to hydroxypropyl-β-cyclodextrin, which reduced lysosomal cholesterol burden. This suggests that the expansion of lysosomal volume occurs secondary to perpetrator-induced elevations in lysosomal cholesterol content. In support of this claim, the cellular accumulation of LTR was shown to be higher in cells isolated from patients with Niemann-Pick type C disease, which are known to hyperaccumulate cholesterol in lysosomes.

Pediatric pharmacokinetics: human development and drug disposition.

Human development is described by the various anatomic and physiologic changes that occur as the single-celled zygote matures into an adult human being. Concomitant with bodily maturation are changes in the complex interactions between pharmacologic agents and the biologic matrix that is the human body. Profound changes in the manner by which drugs traverse the body during development can have significant implications in drug efficacy and toxicity. Although not a replacement for well-conducted, pediatric, pharmacokinetic studies, an understanding of developmental biology and the mechanisms for drug disposition invariably assists the pediatric clinician with the judicious use of medications in children.

Drug-drug interactions involving lysosomes: mechanisms and potential clinical implications

Introduction: Many commercially available, weakly basic drugs have been shown to be lysosomotropic, meaning they are subject to extensive sequestration in lysosomes through an ion trapping-type mechanism. The extent of lysosomal trapping of a drug is an important therapeutic consideration because it can influence both activity and pharmacokinetic disposition. The administration of certain drugs can alter lysosomes such that their accumulation capacity for co-administered and/or secondarily administered drugs is altered. Areas covered: In this review the authors explore what is known regarding the mechanistic basis for drug-drug interactions involving lysosomes. Specifically, the authors address the influence of drugs on lysosomal pH, volume and lipid processing. Expert opinion: Many drugs are known to extensively accumulate in lysosomes and significantly alter their structure and function; however, the therapeutic and toxicological implications of this remain controversial. The authors propose that drug-drug interactions involving lysosomes represent an important potential source of variability in drug activity and pharmacokinetics. Most evaluations of drug-drug interactions involving lysosomes have been performed in cultured cells and isolated tissues. More comprehensive in vivo evaluations are needed to fully explore the impact of this drug-drug interaction pathway on therapeutic outcomes.

Nicotinamide Phosphoribosyltransferase Attenuates Methotrexate Response in Juvenile Idiopathic Arthritis and In Vitro

Variability in response to methotrexate (MTX) in the treatment of juvenile idiopathic arthritis (JIA) remains unpredictable and poorly understood. Based on previous studies implicating an interaction between nicotinamide phosphoribosyltransferase (NAMPT) expression and MTX therapy in inflammatory arthritis, we hypothesized that increased NAMPT expression would be associated with reduced therapeutic response to MTX in patients with JIA. A significant association was found between increased plasma concentrations of NAMPT and reduced therapeutic response in patients with JIA treated with MTX. Inhibition of NAMPT in cell culture by either siRNA‐based gene silencing or pharmacological inhibition with FK‐866 was found to result in a fourfold increase in the pharmacological activity of MTX. Collectively, these findings provide evidence that NAMPT inhibits the pharmacological activity of MTX and may represent a predictive biomarker of response, as well as a therapeutic target, in the treatment of JIA with MTX.

Folate Depletion and Increased Glutamation in Juvenile Idiopathic Arthritis Patients Treated with Methotrexate

Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply.

Cytokine Biomarkers of Disease Activity and Therapeutic Response after Initiating Methotrexate Therapy in Patients with Juvenile Idiopathic Arthritis

To evaluate the relationship between plasma cytokine levels with disease activity and therapeutic response in patients with juvenile idiopathic arthritis (JIA) after initiating methotrexate (MTX) therapy.

Reverse Translation in Advancing Pharmacotherapy in Pediatric Rheumatology: A Logical Approach in Rare Diseases with Limited Resources

Reverse translation, defined here as the use of clinical observations to drive scientific investigation, has been used in the field of Pediatric Rheumatology for decades. Forced in part by limited resources and rare childhood diseases, clinical questions have driven scientific investigation and have focused basic science research efforts to address the most clinically relevant questions. Here we discuss the application of reverse translational approaches, by us and others, to improve pharmacotherapy in Pediatric Rheumatology. Pediatric Rheumatology is a field focused on treating autoimmune and autoinflammatory diseases in childhood. With 300 pediatric rheumatologists in North America, and entire states without representation, it is appropriate to say that the clinical need in Pediatric Rheumatology is underserved. However, despite the inherent challenges in caring for a rare pediatric patient population, great strides have been made in advancing the field through scientific discovery inspired by the multitude of questions naturally generated while providing routine clinical care. The concept of “reverse translation” is practiced every day, even at times unintentionally, in fields that let their clinical questions guide their research efforts. Furthermore, in subspecialties like pediatric rheumatology, which is based on a strong foundation of clinical diagnosticians, clinical variability becomes a natural driver for scientific discovery. Effective use of pharmacotherapy is critical to disease management in the treatment of the Pediatric Rheumatology patient population. However, clinical response to drug therapy can be highly variable, resulting in a trial-and-error approach to treatment that can have long-term deleterious consequences for the patient. These observations have fueled scientific investigation into factors impacting drug response and represents a reverse translational approach to improve patient outcomes through the early use of optimal drug therapy. We posit that three factors primarily contribute to the observed variation in drug response (Figure 1). These factors include heterogeneity in the underlying disease that can significantly impact how the patient responds to the particular drug therapy, pharmacokinetic variation resulting

Disease modifying anti-rheumatic drugs in juvenile idiopathic arthritis: striving for individualized therapy

ABSTRACT Disease-Modifying Anti-Rheumatic Drug (DMARD) use in the treatment of juvenile idiopathic arthritis (JIA) has experienced a dramatic evolution since the early introduction of methotrexate in the 1970’s. This renaissance has been primarily driven by innovation in drug discovery and development that has resulted in the approval of a number of protein-based therapeutics targeting disease-specific pathways. Despite the expansion in the number of drugs available in the treatment of JIA, interindividual variation in therapeutic response and drug-associated toxicities continue to be a major concern and has driven efforts towards individualized therapy. Recent advances in pediatric drug development and innovative approaches to identifying a priori predictors of drug response hold the promise for an individualized approach to therapy that will yield the highest efficacy and safety potential for each JIA patient.