Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

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Context:Polycystic ovarian syndrome (PCOS), the most common endocrinopathy of women in the reproductive age group seems to be adversely affected by associated thyroid dysfunction. Both pose independent risks of ovarian failure and pregnancy related complications.Aims:The present study from Eastern India is, therefore, aimed to investigate the prevalence and etiology of different thyroid disorders in PCOS subjects.Settings and Design:Cross-sectional hospital based survey-single centre observational case-control study.Materials and Methods:This prospective single-center study recruited 106 female patients with hypertrichosis and menstrual abnormality among which 80 patients were defined as having PCOS according to the revised 2003 Rotterdam criteria and comprised the study population. Another 80 age-matched female subjects were studied as the control population. Thyroid function and morphology were evaluated by measurement of serum thyroid stimulating hormone (TSH), free thyroxine levels (free T3 and free T4), anti-thyroperoxidase antibody (anti-TPO Ab), clinical examination and ultrasound (USG) of thyroid gland.Statistical Analysis Used:It was done by Student's t-test and Chi-square test using appropriate software (SPSS version 19).Results:This case-control study revealed statistically significant higher prevalence of autoimmune thyroiditis, detected in 18 patients (22.5% vs. 1.25% of control) as evidenced by raised anti-TPO antibody levels (means 28.037 ± 9.138 and 25.72 ± 8.27 respectively; P = 0.035). PCOS patients were found to have higher mean TSH level than that of the control group (4.547 ± 2.66 and 2.67 ± 3.11 respectively; P value < 0.05). There was high prevalence of goiter among PCOS patients (27.5% vs. 7.5% of control, P value > 0.001). On thyroid USG a significantly higher percentage of PCOS patients (12.5%; controls 2.5%) had hypoechoic USG pattern also compatible with the diagnosis of autoimmune thyroiditis.Conclusions:High prevalence of thyroid disorders in PCOS patients thus points towards the importance of early correction of hypothyroidism in the management of infertility associated with PCOS.

Heparin-induced thrombocytopenia (HIT)is a major cause of morbidity and mortality resulting from the associated thrombosis. Extensive studies using our transgenic mouse model of HIT have shown that antibodies reactive with heparinplatelet factor 4 complexes lead to Fc␥RIIA-mediated platelet activation in vitro as well as thrombocytopenia and thrombosis in vivo. We tested PRT-060318 (PRT318), a novel selective inhibitor of the tyrosine kinase Syk, as an approach to HIT treatment. PRT318 completely inhibited HIT immune complex-induced aggregation of both human and transgenic HIT mouse platelets. Transgenic HIT model mice were treated with KKO, a mouse monoclonal HIT-like antibody, and heparin. The experimental group received orally dosed PRT318, whereas the control group received vehicle. Nadir platelet counts of PRT318-treated mice were significantly higher than those of control mice. When examined with a novel thrombosis visualization technique, mice treated with PRT318 had significantly reduced thrombosis. The Syk inhibitor PRT318 thus prevented both HIT immune complex-induced thrombocytopenia and thrombosis in vivo, demonstrating its activity in HIT. (Blood. 2011;117(7): 2241-2246) IntroductionHeparin-induced thrombocytopenia (HIT), characterized by antibodies to macromolecular complexes formed by heparin and platelet factor 4 (PF4), is the most frequent drug-induced immune thrombocytopenia. Patients with HIT are at an increased risk for thrombosis, a major cause of morbidity and mortality in treated patients. Despite this potential side effect, heparins (unfractionated or low molecular weight) remain the drug of choice in clinical situations where high-intensity therapy is needed along with the ability to rapidly modulate the anticoagulant level. 1 The incidence of HIT has therefore not decreased, notwithstanding the introduction of new anticoagulants, primarily because no drug has replaced heparin for the immediate therapy of acute deep vein thrombosis, arterial thrombosis, or extracorporeal circuits during surgery. In addition, indications for its use in the aging population continue to increase.Multiple factors influence the incidence and severity of HIT. The pathogenesis of the disease is well understood, 2-5 although additional progress is being made. Extensive studies in vitro 4,6,7 and in vivo using our transgenic mouse model of HIT 8 show that antibodies reactive with heparin-PF4 complexes lead to Fc receptormediated platelet activation. This activation leads to platelet aggregation, a procoagulant surface, and release of prothrombotic microparticles. In addition, monocytes and other leukocytes bearing Fc␥ receptors can become activated by the HIT immune complex (IC), generating tissue factor and resulting in other prothrombotic and proadhesive changes. [9][10][11] Blocking Fc␥RIIA signaling is an attractive target for therapeutic intervention because Fc␥RIIA-mediated platelet activation (and possibly concurrent monocyte activation) is central to the disease.Fc␥RIIA, like other activating receptors, i...

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a fatal disease with no available disease-modifying therapies. While pathogenic TTR mutations (TTRm) destabilize TTR tetramers, the T119M variant stabilizes TTRm and prevents disease. A comparison of potency for leading TTR stabilizers in clinic and structural features important for effective TTR stabilization is lacking. Here, we found that molecular interactions reflected in better binding enthalpy may be critical for development of TTR stabilizers with improved potency and selectivity. Our studies provide mechanistic insights into the unique binding mode of the TTR stabilizer, AG10, which could be attributed to mimicking the stabilizing T119M variant. Because of the lack of animal models for ATTR-CM, we developed an in vivo system in dogs which proved appropriate for assessing the pharmacokinetics-pharmacodynamics profile of TTR stabilizers. In addition to stabilizing TTR, we hypothesize that optimizing the binding enthalpy could have implications for designing therapeutic agents for other amyloid diseases.

Based on genetic studies that establish the role of spleen tyrosine kinase (Syk) in immune function, inhibitors of this kinase are being investigated as therapeutic agents for inflammatory diseases. Because genetic studies eliminate both adapter functions and kinase activity of Syk, it is difficult to delineate the effect of kinase inhibition alone as would be the goal with small-molecule kinase inhibitors. We tested the hypothesis that specific pharmacological inhibition of Syk activity retains the immunomodulatory potential of Syk genetic deficiency. We report here on the discovery of (4-(3-(2H-1,2,3-triazol-2-yl)phenylamino)-2-((1R,2S)-2-aminocyclohexylamino) pyrimidine-5-carboxamide acetate (P505-15), a highly specific and potent inhibitor of purified Syk (IC 50 1-2 nM). In human whole blood, P505-15 potently inhibited B cell antigen receptor-mediated B cell signaling and activation (IC 50 0.27 and 0.28 M, respectively) and Fc receptor 1-mediated basophil degranulation (IC 50 0.15 M). Similar levels of ex vivo inhibition were measured after dosing in mice (Syk signaling IC 50 0.32 M). Syk-independent signaling and activation were unaffected at much higher concentrations, demonstrating the specificity of kinase inhibition in cellular systems. Oral administration of P505-15 produced dose-dependent anti-inflammatory activity in two rodent models of rheumatoid arthritis. Statistically significant efficacy was observed at concentrations that specifically suppressed Syk activity by ϳ67%. Thus specific Syk inhibition can mimic Syk genetic deficiency to modulate immune function, providing a therapeutic strategy in P505-15 for the treatment of human diseases.

Syk is a protein tyrosine kinase that couples B-cell receptor (BCR) activation with downstream signaling pathways, affecting cell survival and proliferation. Moreover, Syk is involved in BCR-independent functions, such as B cell migration and adhesion. In CLL, Syk becomes activated by external signals from the tissue microenvironment, and was targeted in a first clinical trial with R788 (fostamatinib), a relatively non-specific Syk inhibitor. Here, we characterize the activity of two novel, highly selective Syk inhibitors, PRT318 and P505-15, in assays that model CLL interactions with the microenvironment. PRT318 and P505-15 effectively antagonize CLL cell survival after BCR triggering and in nurselike cell (NLC)-co-cultures. Moreover, they inhibit BCR-dependent secretion of the chemokines CCL3 and CCL4 by CLL cells, and leukemia cell migration towards the tissue homing chemokines CXCL12, CXCL13, and beneath stromal cells. PRT318 and P505-15 furthermore inhibit Syk and ERK phosphorylation after BCR triggering. These findings demonstrate that the selective Syk inhibitors PRT318 and P505-15 are highly effective for inhibition of CLL survival and tissue homing circuits, and support the therapeutic development of these agents in patients with CLL, other B cell malignancies, and autoimmune disorders.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma, and the role of SYK in its pathogenesis is not completely understood. Using tissue microarray, we demonstrated for the first time that SYK protein is activated in 27 of 61 (44%) primary human DLBCL tissues. Among DLBCL cell lines, 7 were sensitive and 3 were resistant to a highly specific SYK inhibitor, PRT060318. In sensitive DLBCL cells, SYK inhibition blocked the G 1 -S transition and caused cell-cycle arrest. This effect was reproduced by genetic reduction of SYK using siRNA. A detailed analysis of the BCR signaling pathways revealed that the consequence of SYK inhibition on PLC␥2 and AKT, as opposed to ERK1/2, was responsible for cell-cycle arrest. Genetic knock-down of these key molecules decelerated the proliferation of lymphoma cells. In addition, BCR signaling can be blocked by PRT060318 in primary lymphoma cells. Together, these findings provide insights into cellular pathways required for lymphoma cell growth and support the rationale for considering SYK inhibition as a potentially useful therapy for DLBCL. The results further suggest the possibility of using PLC␥2 and AKT as biomarkers to predict therapeutic response in prospective clinical trials of specific SYK inhibitors. (Blood. 2011;118(24):6342-6352)