Search

Benzylpenicillin methyl phosphate (BPMP, 1), hydrolyzes slowly to produce benzylpencillin and methyl phosphate. BPMP is a substrate and inactivator of E. coli RTEM β-lactamase. A competitive inhibitor of β-lactamase protects the enzyme from inactivation by BPMP. It is proposed that BPMP acylates the e-amino group of Lys-234 of the enzyme.

The reaction of human hemoglobin A with trimesoyl tris(3,5-dibromosalicylate) produces the 3,5-dibromosalicyl ester of the bis(amide) of trimesic acid and the e-amino groups of each of the Lys-82 residues of the two β-subunits of hemoglobin (3,5-dibromosalicyl trimesyl-((Lys-β-82)-(Lys-β-82))-hemoglobin; DBST-Hb). This cross-linked protein containing a reactive ester site behaves as an acylating agent toward a variety of biochemically interesting nucleophiles. The products are acyl derivatives, as established by chromatographic and mass spectral analysis. The procedures demonstrate that the multifunctional reagent converts the protein into a reagent which is capable of directly generating bioconjugates of defined structure

Fuzzy C-Means (FCM) algorithm is an important color quantization technology. Though it is widely used, its runtime is long and its quantization result is not good enough. In the paper, a spatial FCM color quantization algorithm which uses pyramid data structure for the hierarchical analysis of a color image is proposed. Experiments show that the algorithm has better quantization result and shorter runtime than the conventional FCM algorithm and the spatial FCM algorithm before.

In this paper, we propose a novel method for extracting handwritten characters from multi-language document images, which may contain various types of characters, e.g. Chinese, English, Japanese or their mixture. Firstly, text patches in document image are segmented based on connected component analysis. Rules for merging connected components are chosen according to the results of language identification. Then features are extracted for each basic analysis unit-text patch. Genetic algorithm is applied for feature fusion and patch type classification. Finally, a Markov Random Field model is utilized as a post-processing step to further correct the misclassification of text patch type by considering the document context. Experimental results show that the proposed algorithm can apparently improve the performance of handwritten character extraction.

In printing industry, Amplitude Modulation (AM) screening is one of the most important halftone methods to create dot screen, and is used to convert continuous tone image into binary image. On the contrary, in order to get a good visual effect and to do further OCR, we need to remove dot screen by employing inverse halftoning algorithm, however, it is difficult to find a tradeoff between screen suppression and edge details preservation. In this paper, a robust inverse halftoning algorithm based on parameter estimation is proposed, which can deal with the above difficulty perfectly. The proposed algorithm comprises three steps, extraction of screening region, estimation of screening parameters, and inverse transform. Experimental results show that the proposed method is independent with parameters and is robust to obtain good visual performance.

The overexpression of B7-H1 in hepatocellular carcinoma (HCC) mediates HCC immune escape and obstructs the immunotherapy based on tumor-specific CD8+ T cells. Tumor-associated macrophages (TAM) are a major component of cancer-related inflammation and play a central role in tumor promotion. To classify the mechanism underlying the overexpression of B7-H1 in HCC, we examined B7-H1 expression and TAM infiltration in 63 cases of human HCC samples using immunohistochemistry method and found that B7-H1 overexpression was associated with TAM infiltration in HCC tissues. Furthermore, B7-H1 expression was upregulated at both mRNA level and protein level in HCC cells (BEL-7402 and SMMC-7721) cocultured with macrophages in a transwell system. The upregulation of B7-H1 expression induced by macrophage was inhibited by blocking NF-κB or STAT3 signal pathways. These results suggest that overexpression of B7-H1 in HCC may be induced by inflammatory microenvironment involving macrophages and imply that anti-inflammation therapy might be preventive for immune escape and assistant for immunotherapy of HCC.

Trimesoyltris(3,5-dibromosalicylate)(TTDS) was prepared and evaluated as a trifunctional site-directed protein cross-linking reagent. It is synthesized by reaction of trimesoyl chloride with tert-butyl 3,5-dibromosalicylate, followed by deprotection with trifluoroacetic acid. TTDS reacts with the deoxy form of human hemoglobin A and with carbonmonoxyhemoglobin to produce amides from the e-amino groups of Lys-82 of each of the β chains of homoglobin. The third 3,5-dibromosalicylate ester group from TTDS reacts much more slowly, principally undergoing hydrolysis

Cryopreservation of avian ova and embryos is challenging because of the yolky structure of the egg. As an alternative, with the development of effective cryopreservation protocols, ovarian tissue cryopreservation could be used for cryobanking for birds. Pieces of ovarian tissue of week-old Japanese quail (Coturnix japonica) were frozen at 0.5 degrees C/min in a programmable freezer or vitrified by immersion in liquid nitrogen. Straws containing slow-frozen samples were thawed in ice water, and vitrified samples were removed from the vials and transferred into sucrose, with the concentration lowered in sequence at room temperature. Cell viability of tissue was estimated by trypan blue assay, and tissue histology was examined by light microscopy. Frozen-thawed or vitrified-warmed tissue from WB (recessive plumage color) chicks was transplanted into week-old ovariectomized QO (wild-type plumage) chicks, with some chicks receiving fresh tissue as a control group. At sexual maturity, QO recipients were mated to WB males, and the production of WB offspring demonstrated successful cryopreservation and transplantation. Donor-derived offspring were obtained from the ovarian tissue that had been cryopreserved by either slow-freezing or vitrification. The vitrification protocol used in this study showed better outcomes at each level of evaluation. This study demonstrated that the function of ovarian tissue in avian species can be successfully preserved at subzero temperatures and recovered by transplantation. The vitrification protocol is recommended because of high efficiency and overall simplicity.

Trimesoyltris(3,5-dibromosalicylate)(TTDS) was prepared and evaluated as a trifunctional site-directed protein cross-linking reagent. It is synthesized by reaction of trimesoyl chloride with tert-butyl 3,5-dibromosalicylate, followed by deprotection with trifluoroacetic acid. TTDS reacts with the deoxy form of human hemoglobin A and with carbonmonoxyhemoglobin to produce amides from the e-amino groups of Lys-82 of each of the β chains of homoglobin. The third 3,5-dibromosalicylate ester group from TTDS reacts much more slowly, principally undergoing hydrolysis

Systematic SAR studies of in vitro factor Xa inhibitory activity around compound 1 were performed by modifying each of the three phenyl rings. A class of highly potent, selective, efficacious and orally bioavailable direct factor Xa inhibitors was discovered. These compounds were screened in hERG binding assays to examine the effects of substitution groups on the hERG channel affinity. From the leading compounds, betrixaban (compound 11, PRT054021) has been selected as the clinical candidate for development.

Defects inspection and correction is an important topic in the fields of scanned documents preprocessing. In this paper, a very fast and robust algorithm is proposed for locating and removing a special kind of circular noises caused by scanning documents with punched holes. Firstly, original image is reduced according to an elaborately selected ratio. Punched holes after reduction will leave some distinctive small regions. By examining such small regions, holes noises can be fast detected and located. To diminish false detections, Hough transformation is applied to the roughly located regions to further confirm the located holes. Finally, circular noise is eliminated by fitting a bi-linear blending Coons surface which interpolates along the four edges of noisy region. Experiments on a variety of scanned documents with punched holes demonstrate the feasibility and efficiency of the proposed algorithm.

Transplantation of testicular tissue onto the back of immunodeficient nude mice provides a tool to examine testicular development and preserve fertility in mammals. There is no immunodeficient model in birds, but we recently transplanted ovarian tissue between newly hatched chicks from two lines of chickens and produced donor-derived offspring, showing that experimental transplantation is possible in newly hatched chicks. In the present study testicular tissue from newly hatched Barred Plymouth Rock (BPR) chicks was transplanted under the skin of the back, under the skin of the abdomen, or in the abdomen of White Leghorn chicks that had been surgically castrated and immunocompromised. Recipient birds were killed at 10 mo of age. Transplanted tissue was observed in one of five hosts receiving tissue under the skin of the back, two of five hosts receiving tissue under the skin of the abdomen, and three of five chicks with grafts inside the abdominal cavity. In recipients with no regeneration of host testes, testicular transplants grew to the size of normal testes, and histologic analysis showed active spermatogenesis. Subsequent collection of sperm from two successful transplants and surgical insemination of the sperm into the magna of the oviducts of BPR hens resulted in the production of 24 donor-derived chicks. These results demonstrate that the combination of testicular tissue transplantation with intramagnal insemination can produce viable, normal chicks, which could provide a simple approach for the recuperation of live offspring in avian species.

Lines restoration is a specific research issue needed attentions in the image restoration and representation field. Imitating the mechanism of human perception, this article proposes a novel regeneration based method for eliminating degradations of lines in binary document images and engineering drawings. The reformed chain codes expression is used to detect degraded lines in images. Then combining with the information of each original line, we complete lines restoration by regenerating them. Compared with the common methods, the experimental results tested on actual degraded images show the effectiveness and efficiency of our method.

Genetic hypertrophic cardiomyopathy (HCM) results from mutations in the cardiac sarcomere, including β-cardiac myosin, with HCM afflicting about 1 out of every 500 people in the United States. HCM is characterized by hyper-contractility and myocyte hypertrophy. Current agents used to treat HCM include β-blockers and Ca2+ channel blockers to decrease the hyper-contractility and improve cardiac relaxation. A novel and more direct approach to decreasing hyper-contractility and improving diastolic relaxation in HCM patients is by modulating β-cardiac myosin to produce less force. We believe modulation at the sarcomere level provides a focused strategy while lowering the potential for adverse drug events. In this study we examined the effects of MYK0000461, a cardiac myosin selective inhibitor, on adult rat cardiomyocytes to fully understand the mechanism of action on excitation-contraction (E-C) coupling. Cellular contractility was assessed using edge detection and the calcium transient was measured using fura-2 loaded myocytes. MYK0000461 decreased contractility in a dose dependent manner with an IC50 of 250nM without altering the calcium transient. This inhibitory effect can be reversed by stimulation of the β-adrenergic pathway with the known agonist isoproterenol. We hypothesize that modulation of mutant β-cardiac myosin activity with a small molecule agent such as MYK0000461 could potentially treat disorders resulting from hyper-contractility such as HCM.

As microprocessor speeds increase, a large fraction of the execution time is often lost to cache miss penalties. This loss can be particularly severe in processors such as the UltraSPARC-IIICu which have in-order execution and block on cache misses. Such processors rely greatly on the compiler to reduce stalls and achieve high performance. This paper describes a compiler technique for software prefetching that is aware of the specific prefetch behaviors of the target processor. The implementation targets loops containing control-flow and strided or irregular memory access patterns. A two phase locality analysis, capable of handling complex subscript expressions, is used for enhanced identification of prefetch candidates. Prefetch instructions are scheduled with careful consideration of the prefetch behaviors in the target system. Compared to a previous implementation, our technique produced performance improvements of 9% on the geometric mean, and up to 44% on individual tests, in Sun’s first UltraSPARC-IIICu based SPEC CPU2000 submission [5] and has been used in all later submissions to date.

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.

Helper threading is a technique that utilizes a second core or logical processor in a multi-threaded system to improve the performance of the main thread. A helper thread executes in parallel with the main thread that it attempts to accelerate. In this paper, the helper thread merely prefetches data into a shared cache and does not incur any other programmer visible effects. Helper thread prefetching has been proposed as a viable solution in various scenarios where it is difficult to prefetch efficiently within the main thread itself. This paper presents our helper threading experience on SUN's second dual-core SPARC microprocessor, the UltraSPARC IV+. The two cores on this processor share an on-chip L2 and an off-chip L3 cache. We present a compiler framework to automatically construct helper threads and evaluate our scheme on the UltraSPARC IV+ processor. Our preliminary results using helper threads on the SPEC CPU2000 suite show gains of up to 22% on programs that suffer substantial L2 cache misses while at the same time incurring negligible losses on programs that do not suffer L2 cache misses.

A class of N,N-dialkylated 4-(4-arylsulfonylpiperazine-1-carbonyl)-benzamidines and 4-((4-arylsulfonyl)-2-oxo-piperazin-1-ylmethyl)-benzamidines has been discovered as potent factor Xa inhibitors with desirable in vitro and in vivo anticoagulant activity, but with low oral bioavailability. The 5-chloroindole and 6-chlorobenzo[b]thiophene groups are optimal as the factor Xa S1 binding elements. The strategy of incorporating a side chain on the piperazine nucleus to enhance binding affinity has been examined.

A series of benzoxazinone derivatives was designed and synthesized as factor Xa inhibitors. We demonstrated that the naphthyl moiety in the aniline-based compounds 1 and 2 can be replaced with benzene-fused heterobicycles and biaryls to give factor Xa inhibitors with improved trypsin selectivity. The P4 modifications lead to monoamidines which are moderately active. The benzoxazinones 41-45 are potent against factor Xa, retain the improved trypsin selectivity of the corresponding aniline-based compounds, and show strong antithrombotic effect dose responsively.

In this paper, we study how array contraction can enhance locality and improve performance. In our previous work, we have developed a memory minimization scheme, SFC, which is a combination of loop shifting, loop fusion and array contraction. SFC focuses on reducing the memory requirement, and as a by-product, it may enhance cache locality. In this paper, we study how array contraction can contribute to cache locality and performance enhancement. We develop a memory cost model for SFC. We also present a fusion algorithm so that the predicted locality enhancement can be realized. Experimental results on both a real machine and a simulator demonstrate the effectiveness of array contraction on cache locality enhancement and performance improvement.

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3 , a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency ( 14 , IC 50 =0.028 μM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.

We propose a new universal High-Level Information (HLI) format to effectively integrate front-end and back-end compilers by passing front-end information to the back-end compiler. Importing this information into an existing back-end leverages the state-of-the-art analysis and transformation capabilities of existing front-end compilers to allow the back-end greater optimization potential than it has when relying on only locally-extracted information. A version of the HLI has been implemented in the SUIF parallelizing compiler and the GCC back-end compiler. Experimental results with the SPEC benchmarks show that HLI can provide GCC with substantially more accurate data dependence information than it can obtain on its own. Our results show that the number of dependence edges in GCC can be reduced by an average of 48% for the integer benchmark programs and an average of 54% for the floating-point benchmark programs studied, which provides greater flexibility to GCC's code scheduling pass. Even with the scheduling optimization limited to basic blocks, the use of HLI produces moderate speedups compared to using only GCC's dependence tests when the optimized programs are executed on MIPS R4600 and R10000 processors.

Fertilized eggs of chicken and quail were incubated under the simulated microgravity condition provided by a clinostat. The number of Primordial Germ Cells (PGCs) was counted in early embryogenesis, and the reproductive capacity of quail hatched following the simulated microgravity was investigated. Simulated microgravity caused significant decline of PGCs in the blood of early chicken embryos and in the gonads. The numbers of spermatogonia in the hatchling testis were also fewer than those in the control groups. Therefore, simulated microgravity may retard gonadial development and reduce the reproductive capacity.

Substituted acrylamides were used as templates that bridge P1 and P4 binding elements, resulting in a series of potent (sub-nanomolar) and selective factor Xa inhibitors. In this template, cis-geometry of P1 and P4 ligands is highly preferred. SAR on the substituting groups, as well as on modification of P1 and P4 moieties is described. Compounds in this series show good in vivo efficacy in animal models.

Tile-size selection is known to be a complex problem. This paper develops a new selection algorithm targeting relaxation codes. Unlike previous algorithms, this new algorithm considers the effect of loop skewing, which is necessary to tile such codes. It also estimates loop overhead and incorporates them into the execution cost model, which turns out to be critical to the decision between tiling a single loop level vs. tiling two loop levels. Our preliminary experimental results show a significant impact of these previously ignored issues on the execution time of tiled loops in relaxation codes. In our experiments, we measured the cache miss rate and the execution time of five benchmark programs on a single processor and we compared our algorithm with previous algorithms. Our algorithm achieves an average speedup of 1.27 to 1.63 over all the other algorithms.

Red blood cells have been labeled with an anti-renin pharmacophore using the activated labeling agent Boc-Phe-His-ACHPA-Ile-6-NH(CH2)5CO-NHS (4) and the corresponding sulfo-NHS-ester (5). Renin inhibition by labeled cells varies according to the concentrations of 4 or 5 used in the labeling protocols, and with the densities of the red cells employed. Flow cytometry measurements using specific polyclonal antibodies toward the anti-renin pharmacophore confirm that red cells are labeled on their outer surfaces with anti-renin pharmacophores. Inhibitory activity of labeled red cells is clearly associated with the cells themselves, and does not require prior release of an inhibitory entity: renin inhibition increases as a function of the concentration of NHS-ester used to label cells suspended in buffer, and with cell density; on the other hand, the separated supernatant portions of the medium make only minor contributions to the observed inhibitory activities. Renin inhibition also increases with increasing concentrations of ghosts derived from labeled red cells, firmly establishing that activity is intimately associated with cell membranes. Thus, the composite evidence is strongly supportive of inhibitory activity specific to the extracellular surface of red cells, which has been modified by the introduction of anti-renin pharmacophores. This study of inhibitory activity by drug/red blood cell-conjugates represents one of the few examples of a red cell-bound ligand of synthetic origin capable, without prior release, of specifically blocking the activity of its target enzyme. As well, it demonstrates the feasibility of exploiting the activity of covalently bound pharmacophores, free from interference of their carriers, for drug targeting.

We have identified a small molecule inhibitor, MYK0000461, of the cardiac myosin ATPase. This agent was characterized in steady state and transient kinetic assays to understand its mechanism of action. MYK0000461 decreases the steady-state rate of the ATPase activity of purified bovine β-cardiac myosin subfragment-1 (S1) as well as that of bovine cardiac myofibrils, wild type and the mutant R453C of recombinant human β-cardiac myosin S1. We also find that MYK0000461 inhibits cardiac myosin selectively as compared to systems containing rabbit skeletal or chicken smooth muscle myosins Analysis of the individual steps of the chemo-mechanical cycle of cardiac myosin suggests that MYK0000461 exerts its effect by inhibiting the actin-stimulated release of phosphate, presumably by stabilizing the detached state of cardiac myosin prior to the release of phosphate. We find no evidence to suggest that MYK000461 inhibits cardiac myosin in a strongly bound state and no other steps in the chemo-mechanical cycle are affected by MYK0000461. Thus, the enzymatic step governing the weak to strong transition of S1 binding to actin is inhibited without affecting the release from the strongly bound states. This decrease in the rate of transition from the weak to strongly bound state should decrease force production and may underlie its ability to decrease cardiac contractility in cellular and in vivo models of cardiac function. An agent such as MYK0000461 could potentially be used to treat cardiac disorders that stem from hyper contractility such as the genetic hypertrophic cardiomyopathies (HCM). By decreasing the net force of contraction and restoring it back to normal level could potentially be useful in treating patients that suffer from this disease.

Aim Solid phase immunoassays enable us to identify unacceptable donor HLA specific antigens (DSA) which allows for the prediction of compatible donor/recipient combinations. This process is referred to as the virtual crossmatch (vXM). The aim of the study was to evaluate the performance of the vXM against the final flow cytometric crossmatch (FCXM). Methods All FCXM performed in the past three years were included in this study. Flow cytometry antibody screening and Luminex single antigen bead assay (LSA) were performed to determine the presence of HLA antibodies and their specificities. MFI = 1000 was used as cutoff for LSA. A final FCXM either prospective or retrospective for the transplant recipients was performed with potential donors selected upon the vXM. Donor cells used for final crossmatch were treated with pronase. Results Total 2070 FCMX were recorded and analyzed in the study. Among these crossmatches, 1891 were vXM negative, 768 crossmatches were performed for HLA sensitized recipients with 179 positive vXM due to presence of DSA. There were total 297 positive final FCXM, of them 123 were expected and 174 (9%) were unexpected compared with the vXM results. The overall prediction rate of the final negative FCXM was 91%. The overall unexpected positive final FCXM was 8%. The majority of the unexpected final T cell positive and B cell negative FCXM became negative when the FCXM repeated with non-pronase treated donor cells. Conclusions Our vXM practice provided a >90% correct predication of negative FCXM. We could expected that the unexpected positive final FCXM mainly caused by non-HLA reactivity, for example, the pronase treatment and/or presence of antibodies not directed to HLA because the current LSA could finely define the specificities of HLA antibodies. However, it could not be excluded that the presence of anti-DP and/or DQA1 antibodies could trigger a positive B cell FCXM in cases where donors’ DP and DQA1 were not typed. This will be further investigated.

Abstract 2266 Betrixaban is a once daily oral Factor Xa inhibitor being investigated in a Phase 3 clinical trial to prevent venous thromboembolism in acute medically ill patients (APEX Study). Mass balance, metabolite profile and interaction with major CYP enzymes were evaluated in this study. Portola study 06–005 was an open-label, single-dose, mass-balance and metabolic profiling study using 14C-labeled betrixaban in 5 healthy male volunteers. Each subject received a single oral solution containing 40 mg of betrixaban labeled with 100 μCi of 14C. Blood samples were taken serially over a 168-hour interval. Urine samples and fecal samples were collected during the 7–14 day confinement period. Subjects were discharged from the unit when at least one of the following criteria were met: 90% of the radioactivity was recovered in urine and feces, daily excreted radioactivity was 1% or less of administered dose on two consecutive days, or subject reached 336 hours (14 days) post dose. The plasma concentration equivalents of total radioactivity increased rapidly following dosing with a mean peak of 31.69 ng eq/mL occurring at 3.5 hours post-dose. AUC and half-life could not be calculated as radioactivity in plasma could only be detected up to 6 hours post dose. Terminal elimination half life determined in other clinical pharmacology studies was 37 hours. Total radioactivity recovered from urine and feces was approximately 96% (range 92% to 99%), with the majority of 14C recovery in feces (82% to 89% of the dose). The 14C dose recovered in urine, composed of betrixaban and inactive metabolites, ranged from 6% to 13%. The metabolic profile of betrixaban was determined in plasma, urine and feces. Unchanged betrixaban was the predominant component found in human plasma and excreta, accounting for 85.3% of the dose excreted in urine and feces. The major biotransformation pathway for betrixaban was hydrolysis to form PRT062802 and PRT062803, a non-14C labeled metabolite (Figure 1). PRT062803 can be demethylated to form PRT062799 or hydroxylated to form PRT062982. PRT062982 is further conjugated with sulfate to form PRT063069. Both PRT062802 and PRT063069 were major circulating metabolites in human plasma with AUC of 34% and 24% that of betrixaban, respectively. PRT062802 was the only prominent metabolite detected in human urine and feces. In addition to hydrolysis metabolites, two CYP-mediated metabolites, O-desmethyl betrixaban (PRT058326) and N-desmethyl betrixaban (PRT054156), were observed in plasma at trace levels (AUC of each was 10 μM). PRT058326 and PRT054156 have an IC50 for fXa inhibition of approximately 5 nM compared to betrixaban Ki of 0.117 pM. Interaction of betrixaban with CYP enzymes was studied in vitro. CYP inhibition potential was evaluated in human liver microsomes with or without 30 minute pre-incubation of betrixaban. Selective probe substrates were used to monitor CYP activities, i.e. phenacetin for 1A2, tolbutamide for 2C9, S-mephenytoin for 2C19, dextromethorphan for 2D6, and testosterone and midazolam for 3A4. Betrixaban had IC50 > 80 μM for CYP1A2, 2C9, 2D6 and 3A4 for both competitive and time-dependent inhibition. IC50 for 2C19 were 43 and 88 μM for competitive and time-dependent inhibition, respectively. The CYP inhibition IC50's are much higher than the betrixaban therapeutic concentration of 50 nM. CYP induction by betrixaban was also studied using cryopreserved human hepatocytes (n=3). Betrixaban at 1, 10 and 25 μM were incubated in hepatocyte preparation for 48 hours. The activities for CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were determined by measuring the formation of metabolites of the probe substrates similar to those used in the CYP inhibition study. CYP2C19 activities were not quantifiable in all three donors; therefore, induction for this CYP isoform could not be assessed. Betrixaban did not induce the activities of CYP1A2, CYP2C9, and CYP3A4. These results demonstrated that betrixaban was mainly excreted as the unchanged drug most likely via biliary secretion. Renal excretion and metabolism were minor elimination pathways. Betrixaban is unlikely to have drug-drug interactions with CYP-substrate, inducer, or inhibitor drugs. Disclosures: Hutchaleelaha: Portola pharmaceuticals: Employment. Ye:Portola Pharmaceuticals: Employment. Song:Portola Pharmaceuticals: Employment. Lorenz:Portola Pharmaceuticals: Employment. Gretler:Portola Pharmaceuticals: Equity Ownership. Lambing:Portola Pharmaceuticals: Employment.

Abstract 1727 Genetic ablation of Syk in hematopoietic cells blocks various leukocyte immune functions, and protects mice from immune-complex mediated inflammation. These data have helped to identify Syk as an important therapeutic target for immune-mediated diseases. The next step is to test the hypothesis that low level, and specific, pharmacological inhibition of Syk retains the immunomodulatory potential observed with Syk genetic deficiency. With this goal in mind, we provide an update on the development of P505-15, a highly specific and potent small molecule Syk inhibitor which suppresses signaling and activation of primary human and rodent leukocyte immune function. The specificity of P505-15 was tested in a panel of 270 independent purified kinase assays at 300nM. At this concentration, Syk and 8 other kinases were inhibited by ≥ 80%. Subsequent analysis demonstrated a Syk IC50 of 1nM, whereas the next most potently inhibited kinase required an IC50 of 81nM. In a variety of cellular assays we observed potent inhibition of B cell receptor (BCR) induced Syk signaling, but not of Lyn, phorbol 12-myristate 13-acetate (PMA) induced protein kinase C, T cell receptor induced Zap70, or cytokine induced JAK1 (IL6), JAK2 (GM-CSF), or JAK1/3 (IL4) dependent STAT phosphorylation. Consistently, in Ba/F3 cell lines transformed by various kinases, P505-15 only inhibited proliferation of those cells transformed by Syk (IC50 = 0.12μM), and not by Zap70 or JAK family members (IC50 > 6μM). In human whole blood, P505-15 suppressed BCR-induced Syk signaling and cellular activation with IC50's of 0.383μM and 0.362μM, respectively. FceR1-induced basophil degranulation was similarly suppressed with an IC50 of 0.171μM. Importantly, Syk-independent signaling and cellular activation in human whole blood via PMA (B cell assays) or fMLP (basophil degranulation) was unaffected by this compound at 4μM and 1μM, respectively (the highest concentrations tested), again demonstrating its specificity of action. Oral administration of P505-15 in mice led to a reversible inhibition of Syk, with an IC50 of 0.282μM as determined by an ex vivo whole blood BCR stimulation assay. Finally, we tested the immunomodulatory potential of specific Syk inhibition in vivo using rodent models of rheumatoid arthritis. Oral administration of P505-15 resulted in statistically significant and dose-dependent anti-inflammatory activity in both the mouse collagen antibody-induced arthritis and rat collagen induced arthritis models. In each case, anti-inflammatory effects were achieved at sub-micromolar plasma concentrations in which Syk specificity was maintained. These data support the hypothesis that specific Syk inhibition can modulate immune function in vivo, and provide a therapeutic strategy for the treatment of human inflammatory disease by inhibition of this kinase. P505-15 is currently being evaluated in phase I clinical trials. Disclosures: Coffey: Portola Pharmaceuticals: Employment. Francis:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Pak:Portola Pharmaceuticals: Employment. Delaney:Portola Pharmaceuticals: Employment. Betz:Portola Pharmaceuticals: Employment. Jia:Portola Pharmaceuticals: Employment. Xu:Portola Pharmaceuticals: Employment. Bauer:Portola Pharmaceuticals: Employment. Song:Portola Pharmaceuticals: Employment. Pandey:Portola Pharmaceuticals: Employment. Baker:Portola Pharmaceuticals: Employment. Hollenbach:Portola Pharmaceuticals: Employment. Phillips:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals: Employment.

Recent studies indicate that dual inhibition of the spleen tyrosine kinase (Syk) and Janus kinases (JAK) by agents such as R788 (tamatinib fosdium) are clinically efficacious in patients with non-Hodgkin’s lymphoma. Since Syk has been implicated in B cell activation, proliferation, and survival, the present study was designed to determine whether the specific inhibition of Syk alone would suffice to disrupt proliferation and survival of lymphoma cells. To test this, a series of compounds were synthesized and screened to identify those that specifically inhibited Syk in a panel of in vitro multi-kinase assays at 300nM. Of these, three were selected for this study; compounds P459-72, P505-15, and P420-89. All three compounds inhibited purified Syk with IC50’s in the 6-43nM range. P459-72 was highly Syk specific. P505-15 also inhibited purified Lyn with an IC50 of 199nM. P420-89 inhibited multiple kinases in the BCR signaling pathway, in addition to JAK1, 2, and 3; IC50’s of 0.63-6.2nM. Assays using the non-Hodgkin’s lymphoma B cell lines Ramos, SUDHL-4 and -6 showed that each compound also inhibited BCR-induced Syk auto-phosphorylation and BLNK phosphorylation (IC50’s in the 100–400nM range) as well as subsequent Ca2+ flux and ERK phosphorylation (IC50’s in the 50–156nM range). In contrast, the activity of the Src family member Lyn, upstream of Syk in the BCR signaling pathway and responsible for Syk tyrosine phosphorylation at amino acid position 352, was not affected. Cellular proliferation was also attenuated in these lymphomas (IC50’s in the 1–5μM range), and all three compounds induced apoptosis to various extents between 1 and 3μM. Interestingly, while P505-15 did inhibit purified Lyn with an IC50 of 199nM, this did not translate into inhibition of Lyn activity upon BCR cross-linking in cells at concentrations where Syk activity was inhibited. A structurally similar compound with a greater than one hundred fold lower Syk inhibitory activity (P528-85) had no effect on the proliferation and survival of these B cell lines. Finally, the cellular effects of the Syk inhibitors required an active BCR signaling pathway, as the B cell line Toledo which lacks BCR expression was insensitive to these compounds; proliferation was inhibited with IC50’s in the 9-38μM range with no induction of apoptosis below 10μM, the highest concentration tested. Taken together, these data suggest that the specific inhibition of Syk, without concomitant inhibition of JAK, may be sufficient for the treatment of non-Hodgkin’s lymphoma.