Predicting clinically significant drug interactions during drug development is a challenge for the pharmaceutical industry and regulatory agencies. Since the publication of the US Food and Drug Administration's (FDA's) first in vitro and in vivo drug interaction guidance documents in 1997 and 1999, researchers and clinicians have gained a better understanding of drug interactions. This knowledge has enabled the FDA and the industry to progress and begin to overcome these challenges. The FDA has continued its efforts to evaluate methodologies to study drug interactions and communicate recommendations regarding the conduct of drug interaction studies, particularly for CYP-based and transporter-based drug interactions, to the pharmaceutical industry. A drug interaction Web site was established to document the FDA's current understanding of drug interactions (http://www.fda.gov/cder/drug/drugInteractions/default.htm). This report provides an overview of the evolution of the drug interaction guidances, includes a synopsis of the steps taken by the FDA to revise the original drug interaction guidance documents, and summarizes and highlights updated sections in the current guidance document, Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling.

Obesity has become a worldwide challenge with significant health and socioeconomic implications. One of the major implications is its impact on drug therapy. In order to gain a better understanding of this impact, we surveyed the regulatory guidances, the newly approved molecular entity drug products, and drug product labels in the Physician's Desk Reference. This review summarizes the findings of the survey along with the existing knowledge on pharmacokinetic and pharmacodynamic changes associated with obesity.

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Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.

Acute pancreatitis is a disease associated with inflammation and tissue damage. One protein that protects against acute injury, including ischemic injury to both the kidney and heart, is renalase, which is secreted into the blood by the kidney and other tissues. However, whether renalase reduces acute injury associated with pancreatitis is unknown. Here, we used both and murine models of acute pancreatitis to study renalase's effects on this condition. In isolated pancreatic lobules, pretreatment with recombinant human renalase (rRNLS) blocked zymogen activation caused by cerulein, carbachol, and a bile acid. Renalase also blocked cerulein-induced cell injury and histological changes. In the cerulein model of pancreatitis, genetic deletion of renalase resulted in more severe disease, andadministering rRNLS to cerulein-exposed WT mice after pancreatitis onset was protective. Because pathological increases in acinar cell cytosolic calcium levels are central to the initiation of acute pancreatitis, we also investigated whether rRNLS could function through its binding protein, plasma membrane calcium ATPase 4b (PMCA4b), which excretes calcium from cells. We found that PMCA4b is expressed in both murine and human acinar cells and that a PMCA4b-selective inhibitor worsens pancreatitis-induced injury and blocks the protective effects of rRNLS. These findings suggest that renalase is a protective plasma protein that reduces acinar cell injury through a plasma membrane calcium ATPase. Because exogenous rRNLS reduces the severity of acute pancreatitis, it has potential as a therapeutic agent.

Ca2؉ -activated Cl ؊ channels are inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P 4 ) (Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996) J. Biol. Chem. 271, 14092-14097), a novel second messenger that is formed after stimulusdependent activation of phospholipase C (PLC). In this study, we show that inositol 1,3,4-trisphosphate (Ins(1,3,4)P 3 ) is the specific signal that ties increased cellular levels of Ins(3,4,5,6)P 4 to changes in PLC activity. We first demonstrated that Ins(1,3,4)P 3 inhibited Ins(3,4,5,6)P 4 1-kinase activity that was either (i) in lysates of AR4 -2J pancreatoma cells or (ii) purified 22,500-fold (yield ‫؍‬ 13%) from bovine aorta. Next, we incubated [ 3 H]inositol-labeled AR4 -2J cells with cell permeant and non-radiolabeled 2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate-hexakis(acetoxymethyl) ester. This treatment increased cellular levels of Ins(1,3,4)P 3 2.7-fold, while [ 3 H]Ins(3,4,5,6)P 4 levels increased 2-fold; there were no changes to levels of other 3 H-labeled inositol phosphates. This experiment provides the first direct evidence that levels of Ins(3,4,5,6)P 4 are regulated by Ins(1,3,4)P 3 in vivo, independently of Ins(1,3,4)P 3 being metabolized to Ins(3,4,5,6)P 4 . In addition, we found that the Ins(1,3,4)P 3 metabolites, namely Ins(1,3)P 2 and Ins(3,4)P 2 , were >100-fold weaker inhibitors of the 1-kinase compared with Ins(1,3,4)P 3 itself (IC 50 ‫؍‬ 0.17 M). This result shows that dephosphorylation of Ins(1,3,4)P 3 in vivo is an efficient mechanism to "switch-off " the cellular regulation of Ins(3,4,5,6)P 4 levels that comes from Ins(1,3,4)P 3 -mediated inhibition of the 1-kinase. We also found that Ins(1,3,6)P 3 and Ins(1,4,6)P 3 were poor inhibitors of the 1-kinase (IC 50 ‫؍‬ 17 and >30 M, respectively). The non-physiological trisphosphates, D/LIns(1,2,4)P 3 , inhibited 1-kinase relatively potently (IC 50 ‫؍‬ 0.7 M), thereby suggesting a new strategy for the rational design of therapeutically useful kinase inhibitors. Overall, our data provide new information to support the idea that Ins(1,3,4)P 3 acts in an important signaling cascade.

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domaincontaining inositol-5-phosphatase SHIP2. We show that in addition to Mg 2؉ , transition metals such as Mn 2؉ , Co 2؉ , and Ni 2؉ are also effective activators of SPsynaptojanin. In contrast, Ca 2؉ and Cu 2؉ are inhibitory. We provide evidence that Mg 2؉ binds the same site occupied by Ca 2؉ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P 2 ). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P 3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SPsynaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P 3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) Ϸ Ins(4,5)P 2 Ϸ Ins(1,4,5)P 3 Ϸ Ins(4,5,6)P 3 > PtdIns(3,5)P 2 Ϸ PtdIns(3,4,5)P 3 Ϸ Ins(1,2,4,5)P 4 Ϸ Ins(1,3,4,5)P 4 Ϸ Ins-(2,4,5,6)P 4 Ϸ Ins(1,2,4,5,6)P 5 . The rank order for SHIP2 is Ins(1,2,3,4,5)P 5 > Ins(1,3,4,5)P 4 > PtdIns(3,4,5)P 4 Ϸ PtdIns(3,5)P 2 Ϸ Ins(1,4,5,6)P 4 Ϸ Ins(2,4,5,6)P 4 . Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.Eukaryotic cells contain both water-soluble inositol phosphates and the corresponding phosphoinositide lipids. These molecules reside in different cellular compartments and regulate the localization and activity of proteins through their interaction with specific binding domains. Inositol and phosphatidylinositol phosphates are key modulators of cellular processes including signal transduction, cell proliferation, and apoptosis, vesicle trafficking, cell motility and cytoskeletal organization, and transcription (1-5). The best known inositolbased signaling pathway is the cell surface receptor-triggered hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-(4,5)P 2 )1 by phospholipase C to yield the second messengers inositol 1,4,5-triphosphate (Ins(1,4,5)P 3 ), a regulator of Ca 2ϩ release, and diacylglycerol, an activator of protein kinase C. The activity of the inositol-based molecules depends on which site(s) on the inositol ring is phosphorylated, and the level of phosphorylation is maintained by the combined action of a host of inositol kinases and phosphatases. Although consider...