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Supplementary Information, Methods, Table 1, Figures 1-7 from 3-Phosphoinositide–Dependent Kinase 1 Potentiates Upstream Lesions on the Phosphatidylinositol 3-Kinase Pathway in Breast Carcinoma

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

Although epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have demonstrated encouraging clinical outcomes for patients with EGFR-mutated non-small cell lung cancer, a considerable number of patients will develop drug resistance and eventually undergo disease progression after taking EGFR-TKIs for a period of time. EGFR

Fruit shape is an important quality and yield trait in melon (Cucumis melo). Although some quantitative trait loci for fruit shape have been reported in in this species, the genes responsible and the underlying mechanisms remain poorly understood. Here, we identified and characterized a gene controlling fruit shape from two melon inbred lines, B8 with long-horn fruit and HP22 with flat-round fruit. Genetic analysis suggested that the shape was controlled by a single and incompletely dominant locus, which we designate as CmFSI8/CmOFP13. This gene was finely mapped to a 53.7-kb interval on chromosome 8 based on bulked-segregant analysis sequencing and map-based cloning strategies. CmFSI8/CmOFP13 encodes an OVATE family protein (OFP) and is orthologous to AtOFP1 and SlOFP20. The transcription level of CmFSI8/CmOFP13 in the ovary of HP22 was significantly higher than that in B8, and sequence analysis showed that a 12.5-kb genomic variation with a retrotransposon insertion identified in the promoter was responsible for elevating the expression, and this ultimately caused the differences in fruit shape. Ectopic overexpression of CmFSI8/CmOFP13 in Arabidopsis led to multiple phenotypic changes, including kidney-shaped leaves and shortened siliques. Taken together, our results demonstrate the involvement of an OFP in regulating fruit shape in melon, and our improved understanding of the molecular mechanisms will enable us to better manipulate fruit shape in breeding.

Introduction Cystinosis is a rare, metabolic, autosomal recessive, genetic lysosomal storage disorder characterized by an accumulation of cystine in various organs and tissues. Cysteamine bitartrate (CB) is a cystine-depleting aminothiol agent approved in the United States and Europe in immediate-release and delayed-release (DR) formulations for the treatment of nephropathic cystinosis in children and adults. It is recommended that CBDR be administered with fruit juice (except grapefruit juice) for maximum absorption. Omeprazole is a proton pump inhibitor that inhibits gastric acid secretion and, theoretically, may cause the premature release of cysteamine by increasing intragastric pH, thereby affecting the PK of CBDR. Methods This open-label, three-period, randomized study in healthy adult subjects was designed primarily to compare the pharmacokinetics of CBDR capsules after a single oral dose administered with orange juice, water, or multiple oral doses of omeprazole with water at steady state. A total of 32 subjects were randomly assigned to receive study agents in one of two treatment sequences. Results All subjects completed the study and baseline characteristics of the overall population and the two treatment sequence populations were similar. Peak mean plasma cysteamine concentrations following co-administration of CBDR capsules with orange juice (1892 ng/mL) were higher compared with co-administration with water (1663 ng/mL) or omeprazole 20 mg and water (1712 ng/mL). Mean time to peak plasma concentration was shorter with omeprazole co-administration (2.5 h) compared with orange juice (3.5 h) or water (3.0 h). Statistical comparisons between treatment groups indicated that exposure as assessed by AUC0–t, AUC0–∞, and Cmax were all within the 80–125% bioequivalence ranges for all comparisons. All treatments were generally well tolerated. Conclusion Overall, the pharmacokinetics of cysteamine bitartrate DR capsules are not significantly impacted by co-administration with orange juice, water only, or omeprazole (with water). Funding Horizon Pharma, Inc.

Supplemental Digital Content is Available in the Text. A novel and potent protein kinase G-1α (PKG-1α) inhibitor is used to demonstrate the important roles of PKG in capsaicin-induced acute pain and in persistent inflammatory pain., Activating PKG-1α induces a long-term hyperexcitability (LTH) in nociceptive neurons. Since the LTH correlates directly with chronic pain in many animal models, we tested the hypothesis that inhibiting PKG-1α would attenuate LTH-mediated pain. We first synthesized and characterized compound N46 (N-((3R,4R)-4-(4-(2-fluoro-3-methoxy-6-propoxybenzoyl)benzamido)pyrrolidin-3-yl)-1H-indazole-5-carboxamide). N46 inhibits PKG-1α with an IC50 of 7.5 nmol, was highly selective when tested against a panel of 274 kinases, and tissue distribution studies indicate that it does not enter the CNS. To evaluate its antinociceptive potential, we used 2 animal models in which the pain involves both activated PKG-1α and LTH. Injecting complete Freund's adjuvant (CFA) into the rat hind paw causes a thermal hyperalgesia that was significantly attenuated 24 hours after a single intravenous injection of N46. Next, we used a rat model of osteoarthritic knee joint pain and found that a single intra-articular injection of N46 alleviated the pain 14 days after the pain was established and the relief lasted for 7 days. Thermal hyperalgesia and osteoarthritic pain are also associated with the activation of the capsaicin-activated transient receptor protein vanilloid-1 (TRPV1) channel. We show that capsaicin activates PKG-1α in nerves and that a subcutaneous delivery of N46 attenuated the mechanical and thermal hypersensitivity elicited by exposure to capsaicin. Thus, PKG-1α appears to be downstream of the transient receptor protein vanilloid-1. Our studies provide proof of concept in animal models that a PKG-1α antagonist has a powerful antinociceptive effect on persistent, already existing inflammatory pain. They further suggest that N46 is a valid chemotype for the further development of such antagonists.

Ketorolac tromethamine is a nonsteroidal anti-inflammatory drug that exhibits analgesic activity with no sedative or anxiolytic properties. Twelve healthy male subjects were enrolled in a study to receive either of 2 treatments over 2 periods in an open-label, randomized, 2-way crossover design: (A) 120 mg of ketorolac tromethamine administered as a continuous subcutaneous infusion over a 24-hour period; or (B) an identical total daily dose administered as 4 intramuscular bolus injections of 30 mg each given every 6 hours (current labeled treatment regimen). The pharmacokinetic and safety profiles were evaluated for both treatments. Both modes of administration have similar values for area under the curve (AUC) and half-life (t1/2 ), suggesting that continuous subcutaneous infusion and repeated intramuscular bolus injections have similar bioavailability. The peak plasma concentration (Cmax ) was 40% lower when ketorolac was administered as a continuous subcutaneous infusion compared with repeat intramuscular bolus injections. The concentration at steady-state (Css ) for continuous subcutaneous infusion was between the Cmax and Ctrough values obtained following the 4 intramuscular injections. Both treatment arms were well tolerated.

The objective of this study was to determine whether grain yield and nitrogen (N) use efficiency (NUE) in winter wheat (Triticum aestivum L.) could be improved by alternate furrow irrigation (AFI). During the 2009–2010 and 2010–2011 crop seasons, the effects of AFI on grain yield, NUE (and its components), the recovery of 15N-labeled fertilizer, N uptake and distribution, and soil nitrate-N were evaluated under field conditions. The irrigation regimes were: W0, non-irrigated; W2, every furrow irrigated at jointing and anthesis; W3, every furrow irrigated before wintering and at jointing and grain filling; and AFI, each of two neighboring furrows irrigated either before wintering or at grain filling, and every furrow irrigated at jointing. Our results show that grain yield, above-ground nitrogen uptake (AGN; including N from fertilizers and soil), and NUE were lowest in the W0 treatment and tended to increase with increasing irrigation frequency. Grain yield, AGN (including N from fertilizers and soil), NUE, nitrogen uptake efficiency (UPE), 15N-recovery efficiency, and N accumulation in grains were significantly higher in the AFI treatment than in the W2 treatment. AGN increased due to a higher amount of N derived from both fertilizers and soil. More N was derived from soil than from fertilizers; thus, the proportion of N derived from fertilizers decreased. No significant differences in grain yield and NUE were observed between the AFI and W3 treatments. AGN decreased when soil N luxury uptake and N accumulation in stems and leaves decreased. Greater N utilization efficiency and a higher N harvest index were observed in the AFI treatment compared with the W3 treatment. In addition, AFI increased nitrate-N contents in the upper soil layer (0–60 cm) and reduced nitrate-N movement to deeper soil compared to the W2 and W3 treatments. Therefore, AFI may be an appropriate irrigation method for improving NUE and decreasing water consumption in winter wheat without reducing yield.

As a major driving element of the structure and function of arid and semiarid ecosystems, rainfall is the essential factor limiting plant biological processes. To clarify the characteristics of transpiration and responses to summer rainfall, sap flow density (F d) of Pinus tabulaeformis and Hippophae rhamnoides was monitored using thermal dissipation probes. In addition, midday leaf water potential (ψm) and leaf stomatal conductance (G s) were also analyzed to determine water use strategies. The results indicated that the diurnal variation in the normalized F d values exhibited a single‐peak curve for P. tabulaeformis, while H. rhamnoides showed multiple peaks. The normalized F d for P. tabulaeformis remained relatively stable regardless of rainfall events. However, there was also a significant increase in the normalized F d for H. rhamnoides in response to rainfall in June and August (p

Ketorolac tromethamine is a nonsteroidal anti-inflammatory drug that exhibits analgesic activity with no sedative or anxiolytic properties. Twelve healthy male subjects were enrolled in a study to receive either of 2 treatments over 2 periods in an open-label, randomized, 2-way crossover design: (A) 120 mg of ketorolac tromethamine administered as a continuous subcutaneous infusion over a 24-hour period; or (B) an identical total daily dose administered as 4 intramuscular bolus injections of 30 mg each given every 6 hours (current labeled treatment regimen). The pharmacokinetic and safety profiles were evaluated for both treatments. Both modes of administration have similar values for area under the curve (AUC) and half-life (t

The lymphoid tyrosine phosphatase (LYP), encoded by the PTPN22 gene, has recently been identified as a promising drug target for human autoimmunity diseases. Like the majority of protein-tyrosine phosphatases LYP can adopt two functionally distinct forms determined by the conformation of the WPD-loop. The WPD-loop plays an important role in the catalytic dephosphorylation by protein-tyrosine phosphatases. Here we investigate the binding modes of two chemotypes of small molecule LYP inhibitors with respect to both protein conformations using computational modeling. To evaluate binding in the active form, we built a LYP protein structure model of high quality. Our results suggest that the two different compound classes investigated, bind to different conformations of the LYP phosphatase domain. Binding to the closed form is facilitated by an interaction with Asp195 in the WPD-loop, presumably stabilizing the active conformation. The analysis presented here is relevant for the design of inhibitors that specifically target either the closed or the open conformation of LYP in order to achieve better selectivity over phosphatases with similar binding sites.

Newly developed monofluoromethylation reaction provided access to various bioactive molecules with an interesting monofluoromethyl unit. An iridium-catalyzed asymmetric version was employed for large-scale methyl-monofluorination of widely used nonsteroidal anti-inflammatory drug ibuprofen (the active S isoform). The methyl-monofluorinated ibuprofen was found to selectively inhibit cyclooxygenase-1 over cyclooxygenase-2 and surprisingly, the compound, with almost equal pharmacokinetic profile, was shown to increase analgesic activity and diminish gastric damage in animal models comparing to the parent drug ibuprofen. Therefore, methyl-monofluorination could be a useful strategy for improving efficacy and safety profile of drugs from the 'profen' family.

INTRODUCTION It has been reported that adult smokers (AS) may be considering smokeless tobacco products as an alternative to smoking. The objective of this study was to evaluate the change in exposure in AS using Marlboro snus (MSNUS) (a tobacco pouch product in test market in June 2007). METHODS AS were randomized into the following groups--CS: subjects (n = 30) continue smoking their own brand; DU: subjects (n = 60) reduced their daily cigarette consumption by >or=50% and were allowed to use MSNUS; SN: subjects (n = 15) stopped smoking their cigarettes but were allowed to use MSNUS; NT: subjects (n = 15) were not allowed to use any tobacco products for the entire duration of the 8-day study. Biomarkers of smoke exposure (BOE) measured at baseline and postbaseline were 24-hr urinary excretion of metabolites of N-nitrosamines, nicotine (urine and plasma), aromatic amines, benzene, and polycyclic aromatic hydrocarbon; urine mutagenicity; and carboxyhemoglobin at various timepoints. RESULTS Statistically significant (p < .05) reductions in all the urinary BOE were observed in the DU group compared with the CS group. After correcting for the residual effect, a proportionate reduction (approximately 50%) in most of the biomarkers was observed. Even larger reductions, similar to the NT group, were observed in the SN group. DISCUSSION The proportionate reduction in exposure when reducing the number of cigarettes by 50% and using MSNUS, under the consumption patterns observed, suggest that the AS did not appear to alter their smoking behavior. The added exposure from MSNUS usage in this group was minimal. The AS sustained substantial reductions in exposure when using MSNUS exclusively.

Nuclear factor kappa B (NF-κB) is an important transcription factor. Aberrant regulation of the NF-κB pathway is frequently observed in a number of major ailments such as cancer and inflammatory diseases. Hence NF-κB modulators have been intensely pursued for their potential therapeutic applications. Numerous reviews have described recent progress in the development of these agents. More recently, a variety of structurally and functionally novel small molecules, identified through high-throughput screens conducted within the Molecular Libraries Screening Center Network (MLSCN) of the NIH Roadmap for Medical Research, have been added to the current list of NF-κB regulators. This review will discuss the inhibitors and activators newly discovered by Columbias Molecular Libraries Screening Center (MLSC) using a well-designed and stable cellular assay.

Lesions of ERBB2, PTEN, and PIK3CA activate the phosphatidylinositol 3-kinase (PI3K) pathway during cancer development by increasing levels of phosphatidylinositol-3,4,5-triphosphate (PIP3). 3-Phosphoinositide-dependent kinase 1 (PDK1) is the first node of the PI3K signal output and is required for activation of AKT. PIP3 recruits PDK1 and AKT to the cell membrane through interactions with their pleckstrin homology domains, allowing PDK1 to activate AKT by phosphorylating it at residue threonine-308. We show that total PDK1 protein and mRNA were overexpressed in a majority of human breast cancers and that 21% of tumors had five or more copies of the gene encoding PDK1, PDPK1. We found that increased PDPK1 copy number was associated with upstream pathway lesions (ERBB2 amplification, PTEN loss, or PIK3CA mutation), as well as patient survival. Examination of an independent set of breast cancers and tumor cell lines derived from multiple forms of human cancers also found increased PDK1 protein levels associated with such upstream pathway lesions. In human mammary cells, PDK1 enhanced the ability of upstream lesions to signal to AKT, stimulate cell growth and migration, and rendered cells more resistant to PDK1 and PI3K inhibition. After orthotopic transplantation, PDK1 overexpression was not oncogenic but dramatically enhanced the ability of ERBB2 to form tumors. Our studies argue that PDK1 overexpression and increased PDPK1 copy number are common occurrences in cancer that potentiate the oncogenic effect of upstream lesions on the PI3K pathway. Therefore, we conclude that alteration of PDK1 is a critical component of oncogenic PI3K signaling in breast cancer. [Cancer Res 2009;69(15):6299–306]

An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.

Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a potent hypoglycemic agent with a novel dihydropyridone scaffold. Several analogs were designed and synthesized. A preliminary structure activity relationship (SARs) showed that the dihydropyridone scaffold is required for the activity.

Despite different embryological origins, islet β-cells and neurons share the expression of many genes and display multiple functional similarities. One shared gene product, vesicular monoamine transporter type 2 (VMAT2, also known as SLC18A2), is highly expressed in human β-cells relative to other cells in the endocrine and exocrine pancreas. Recent reports suggest that the monoamine dopamine is an important paracrine and/or autocrine regulator of insulin release by β-cells. Given the important role of VMAT2 in the economy of monoamines such as dopamine, we investigated the possible role of VMAT2 in insulin secretion and glucose metabolism. Using a VMAT2-specific antagonist, tetrabenazine (TBZ), we studied glucose homeostasis, insulin secretion both in vivo and ex vivo in cultures of purified rodent islets. During intraperitoneal glucose tolerance tests, control rats showed increased serum insulin concentrations and smaller glucose excursions relative to controls after a single intravenous dose of TBZ. One hour following TBZ administration we observed a significant depletion of total pancreas dopamine. Correspondingly, exogenous l-3,4-dihydroxyphenylalanine reversed the effects of TBZ on glucose clearance in vivo. In in vitro studies of rat islets, a significantly enhanced glucose-dependent insulin secretion was observed in the presence of dihydrotetrabenazine, the active metabolite of TBZ. Together, these data suggest that VMAT2 regulates in vivo glucose homeostasis and insulin production, most likely via its role in vesicular transport and storage of monoamines in β-cells.

An efficient synthesis of a series of N -8-quinolinyl benzenesultams as novel NF-κB inhibitors was described via diazotization-induced cyclization of easily accessible N -8-quinolinyl-2-aminobenzenesulfonamides.

A quinazoline that decreases polyglutamine aggregate burden in a cell-based assay was identified from a high-throughput screen of a chemical-compound library, provided by the NIH Molecular Libraries Small Molecule Repository (MLSMR). A structure and activity study yielded leads with submicromolar potency.

The title complex Cu(C8H7O2N)2(OH2) crystallized in the orthorhombic space group, Pbca with unit cell parameters: a = 15.242(2), b = 11.782(4), c = 17.946(4) A, and Z = 8. Two nitrogen atoms, two phenolic oxygen atoms of the ligand, and one water molecule are coordinated with copper to form a distorted tetragonal pyramidal polyhedron.

[reaction: see text] A barrelene derivative fragments to afford benzene and trappable 1,2,3-tricyanocyclopropene. The barrelene anion fragments more easily to liberate benzene and the 1,2,3-tricyanocyclopropenyl anion, which is not trappable or stable in solution. However, the major thermal product from the barrelene anion is a rearranged allyl anion that is formed by disrotatory cleavage of the cyclopropyl ring, a formally Woodward-Hoffmann-forbidden process. Several proposals are offered to rationalize this forbidden rearrangement.

A practical route for the asymmetric large-scale synthesis of methyl-monofluorinated ibuprofen (V) is developed.

The lymphoid tyrosine phosphatase (Lyp, PTPN22) is a critical negative regulator of T cell antigen receptor (TCR) signaling. A single-nucleotide polymorphism (SNP) in the ptpn22 gene correlates with the incidence of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus. Since the disease-associated allele is a more potent inhibitor of TCR signaling, specific Lyp inhibitors may become valuable in treating autoimmunity. Using a structure-based approach, we synthesized a library of 34 compounds that inhibited Lyp with IC(50) values between 0.27 and 6.2 μM. A reporter assay was employed to screen for compounds that enhanced TCR signaling in cells, and several inhibitors displayed a dose-dependent, activating effect. Subsequent probing for Lyp's direct physiological targets by immunoblot analysis confirmed the ability of the compounds to inhibit Lyp in T cells. Selectivity profiling against closely related tyrosine phosphatases and in silico docking studies with the crystal structure of Lyp yielded valuable information for the design of Lyp-specific compounds.

The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and AMP) and characterized in vitro their interactions with corresponding RNA aptamers. The conjugates preserved specific binding to aptamers while showing several 100-fold increase in fluorescence of the dye (the 'light-up' property). In the presence of free small molecules, conjugates can be displaced from aptamers serving also as fluorescent sensors. Our in vitro results provide the proof-of-concept that the small-molecule conjugates with light-up properties can serve as a general approach to label RNA sequences containing aptamers.

Vesicular monoamine transporter type 2 (VMAT2) is a newly emerging target for both diagnostic and therapeutic applications in diabetes mellitus. In pursuit of novel VMAT2 antagonists, we identified a potent hypoglycemic agent with a novel dihydropyridone scaffold. Several analogs were designed and synthesized. A preliminary structure activity relationship (SAR) showed that the dihydropyridone scaffold is required for the activity.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor. Aberrant regulation of the NF-kappaB pathway is frequently observed in a number of major ailments such as cancer and inflammatory diseases. Hence NF-kappaB modulators have been intensely pursued for their potential therapeutic applications. Numerous reviews have described recent progress in the development of these agents. More recently, a variety of structurally and functionally novel small molecules, identified through high-throughput screens conducted within the Molecular Libraries Screening Center Network (MLSCN) of the NIH Roadmap for Medical Research, have been added to the current list of NF-kappaB regulators. This review will discuss the inhibitors and activators newly discovered by Columbia's Molecular Libraries Screening Center (MLSC) using a well-designed and stable cellular assay.

Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC(50)s of the most potent compounds range from micromolar to low nanomolar.

We report here a class of thiazolidine-2,4-diones and 2-thioxothiazolidin-4-ones as potent inhibitors of the lymphoid specific tyrosine phosphatase (Lyp) identified from high throughput screens. Chemical modification by incorporating the known phosphotyrosine (pTyr) mimics led to the discovery of a salicylate-based inhibitor with submicromolar potency.

The interaction of amyloid beta peptide (Abeta) and Abeta-binding alcohol dehydrogenase (ABAD) was recently implicated in the pathogenesis of Alzheimer's disease (AD). Using an ELISA-based screening assay, we identified frentizole, an FDA-approved immunosuppressive drug, as a novel inhibitor of the Abeta-ABAD interaction. Analysis of the frentizole structure-activity relationship led to identification of a novel benzothiazole urea with a 30-fold improvement in potency.

In an effort to generate novel anti-osteoporosis agents, new bisphosphonates with various benzoyl groups on side chains have been synthesized and evaluated for activities on both bone resorption and bone formation in vitro. Several candidates showed distinct dual activities: promoting bone formation and inhibiting bone resorption.

Abstract 1526 Poster Board I-549 Introduction GMI-1070 is a pan-selectin inhibitor which targets E-, P-, and L-selectin, and has demonstrated activity in multiple animal models of disease including sickle cell vaso-occlusive crisis, myocardial infarction, delayed-type hypersensitivity, and microcirculation in diabetes. These Phase 1 healthy volunteer studies evaluated the safety, tolerability and pharmacokinetics (PK) of intravenous (IV) GMI-1070 in single and multiple doses, using a dose-ranging design. Methods A total of 72 healthy volunteers were randomized to receive study drug (GMI-1070 or placebo) in two blinded studies. In the first study, subjects were enrolled in groups of 8 (at a 3:1 ratio), administered a single IV dose of the study drug, and evaluated for safety prior to a dose review by the investigator and medical monitor. After the safety data from each group of 8 was reviewed in a blinded fashion, the decision was made to advance to the next dose group. Doses evaluated in the study were 2, 5, 10, 20, and 40 mg/kg. In the second study, subjects were enrolled similarly in groups of 8 (at a 3:1 ratio), administered multiple IV doses of the study drug over the course of 2 to 4 days, and evaluated for safety and dose escalation in a similar fashion. Doses evaluated in the multiple dose study were: 5, 10, and 20 mg/kg q8 hours for 13 doses (4 days); and finally a loading dose regimen of 40 mg/kg followed by 20 mg/kg q8 hours for 6 doses (2 days). For both studies, safety parameters included adverse events (AEs), clinical lab results, vital signs, ECG, and physical exam. Plasma and urine concentrations of GMI-1070 were measured, and PK parameters were calculated using a non-compartmental analysis. Results All 72 randomized healthy volunteers were included in the safety analysis, and all 54 who received GMI-1070 were included in the PK analysis. There were no serious AEs in either study. All AEs in subjects receiving GMI-1070 were grades 1 or 2 (mild or moderate); no grade 3 (severe) AEs were seen in subjects receiving GMI-1070. One subject did exhibit a pruritic rash while receiving the loading dose regimen of the multiple dose study, and had to discontinue study drug after 3 doses. The rash resolved with treatment. All other AEs in all groups were either possibly related, probably not related, or unrelated to study drug. There was no treatment- or dose-related trend in the incidence of AEs. Overall, there also did not appear to be any clinically significant treatment- or dose-related trends in the clinical lab, vital sign, ECG, or physical exam data in either study. Analysis of the plasma and urine concentrations indicated linear PK over the range of doses tested. The elimination half life is 8 hours with the multiple dose regimen, with a minimum of 89% of the dose recovered in the urine. Conclusions GMI-1070, a pan-selectin inhibitor administered in single or multiple doses IV, at the doses used in these studies, appears to be safe and well-tolerated with linear PK. These studies support proceeding with evaluation of GMI-1070 in the treatment of sickle cell patients in vaso-occlusive crisis. Disclosures Xie: MDS Pharma Services: Employment. Seufert:GlycoMimetics, Inc.: Consultancy. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership. Kramer:GlycoMimetics, Inc.: Consultancy. Thackray:GlycoMimetics, Inc: Employment, Equity Ownership.