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A 2.5-month-old infant with global developmental delay, initially had generalized tonic spasms followed by appearance of infantile spasms from 4.5 months of age. Thus, he had evolution from early infantile developmental and epileptic encephalopathy (EIDEE) to infantile epileptic spasm syndrome (IESS). Neuroimaging and screening of inborn errors of metabolism were normal, but sleep electroencephalogram showed suppression-burst pattern. Treatment with intramuscular injections of adrenocorticotropic hormone (ACTH) was associated with significant control of infantile spasms, but was followed by development of right hemichoreiform movements 2 days later. Upon continuing ACTH treatment, the dyskinesia generalized, prompting us to stop it and shift to vigabatrin which resulted in partial control of his spasms. Whole-exome sequencing revealed an autosomal dominant heterozygous variation of uncertain significance in the NPRL3 gene. At 6 months of age, he suffered of a probable sudden unexpected death, without any notable illness. The case is unique because both the phenomena—ACTH-induced dyskinesia and probable sudden unexpected death in infancy—are rarely described in the EIDEE-IESS continuum.

BACKGROUND AND AIMS BBP-711 is an investigational small molecule inhibitor of glycolate oxidase (GO), currently in development for the treatment of primary hyperoxaluria type 1 (PH1) and recurrent nephrolithiasis. Selection of BBP-711 as a drug candidate followed structural activity optimization and a pharmacological testing strategy that included functional activity in enzyme assays, target engagement in cellular assays and efficacy studies in a rodent model (Agxt−/− mice) of primary hyperoxaluria type 1. METHOD BBP-711 was developed by iterative synthesis and study of the structure–activity relationship of more than 50 GO inhibitors. Lead molecules including BBP-711 were assessed for functional activity by determining inhibition of the conversion of glycolate to oxalate with purified human, mouse and rat HAOX1 enzymes. The ability of BBP-711 to bind directly to immobilized purified hGO (HAOX1 gene product) was evaluated by surface plasmon resonance. Selectivity of BBP-711 was assessed by determining its inhibition of several physiologically related gene products including that from HAOX2. In vitro efficacy of BBP-711 was tested via inhibition of oxalate production in primary hepatocytes cultured from hyperoxaluric Agxt−/− mice. In vivo efficacy of BBP-711 on urinary oxalate excretion was assessed at various doses given by oral gavage in Agxt −/− mice. Pharmacokinetics of BBP-711 was investigated to assess plasma and liver concentrations associated with efficacy. RESULTS BBP-711 is a potent inhibitor of human, rat and mouse GO (IC50 = 15.4 nM, 22.4 nM and 149 nM, respectively). BBP-711 demonstrated stronger direct binding to hGO1 (KD = 6.31 nM) than to rGO2 (KD = 12.8 nM), indicating its preference for GO1 as a target. BBP-711 specificity was demonstrated with 88%). Higher doses did not result in greater reduction in urinary oxalate. Liver exposures (AUC0-24hr) were higher than plasma exposures at all doses tested. GO engagement by BBP-711 was also assessed in an ex vivo assay. Liver extracts from Agxt−/− and wild-type mice dosed with BBP-711 show a dose-dependent reduction in ex vivo GO activity at 1 and 4-hour post-dose. CONCLUSION Iterative and rational optimization of small molecule GO inhibitors resulted in BBP-711, a potent and specific inhibitor of GO with drug-like properties.

Background Transthyretin (TTR) amyloidosis is an underdiagnosed disease caused by destabilization of TTR due to pathogenic mutations or aging. Both pathogenic and protective mutations illuminate mechanisms of disease and potential interventions. AG10 is a selective, oral TTR stabilizer under development for transthyretin amyloidosis cardiomyopathy (ATTR-CM) that mimics a protective TTR mutation. Objectives This randomized, double-blind, placebo-controlled study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics of AG10 in ATTR-CM patients with symptomatic, chronic heart failure. Methods ATTR-CM, New York Heart Association functional class II to III subjects (n = 49, mutant or wild-type) were randomized 1:1:1 to AG10 400 mg, AG10 800 mg, or placebo twice daily for 28 days. Safety and tolerability were assessed by clinical and laboratory criteria. AG10 plasma levels were measured. TTR stability was assessed by changes in serum TTR, and 2 established ex vivo assays (fluorescent probe exclusion and Western blot). Results AG10 treatment was well-tolerated, achieved target plasma concentrations and demonstrated near-complete stabilization of TTR. TTR stabilization was more complete and less variable at the higher dose with stabilization by fluorescent probe exclusion of 92 ± 10% (mean ± SD) at trough and 96 ± 9% at peak (both p Conclusions AG10 has the potential to be a safe and effective treatment for patients with ATTR-CM. A phase 3 trial is ongoing. (Study of AG10 in Amyloid Cardiomyopathy; NCT03458130)

AG10 is a novel, potent, and selective oral transthyretin (TTR) stabilizer being developed to treat TTR amyloidosis (ATTR). This randomized, double‐blind, placebo‐controlled study evaluated safety, tolerability, pharmacokinetics, and pharmacodynamics (ex vivo stabilization) of orally administered AG10 in healthy adult volunteers. Both mutant and wild‐type ATTR are underdiagnosed diseases with limited therapeutic options. As TTR amyloidogenesis is initiated by dissociation of TTR tetramers destabilized due to inherited mutations or aging, AG10 is designed to treat the disease at its source. Four single and three multiple ascending dose levels of AG10 or matching placebo were orally administered. Safety and tolerability were assessed by vital signs, electrocardiogram, adverse events, and clinical laboratory tests. Pharmacokinetics were measured using a validated bioanalytical assay. Pharmacodynamics were assessed via three pharmacodynamic assays of TTR stabilization. AG10 was uniformly well tolerated, and no safety signals of clinical concern were observed. Pharmacokinetic observations included time to maximum concentration 90%) stabilization of TTR was observed across the entire dosing interval at steady state on the highest dose tested. Serum TTR levels, an in vivo reflection of TTR stabilization by AG10, increased from baseline following 12 days of dosing. AG10 appears to be safe and well tolerated in healthy adult volunteers and can completely stabilize TTR across the dosing interval, establishing clinical proof of concept. Based on these data, AG10 has the potential to be a safe and effective treatment for patients with either mutant or wild‐type ATTR.

BACKGROUND: Infigratinib (BGJ398) is a potent and selective FGFR1-3 inhibitor under evaluation for the treatment of achondroplasia, the most common form of disproportionate short stature. Low doses of infigratinib were shown to be effective in improving skeletal abnormalities in a mouse model of achondroplasia [Demuynck et al. 2019; Komla-Ebri et al. 2016]. At higher doses in adult patients (e.g. 10-100-fold the dose shown to have efficacy in preclinical models of achondroplasia) infigratinib appears to be associated with elevation in phosphorus, an effect known to be associated with FGFR1 inhibition. Specifically, FGFR1 inhibition leads to decreases in FGF23, which, in turn, leads to decreased excretion of phosphate by the kidneys. We sought to understand the relationship between lower doses of infigratinib and changes in phosphorus in preclinical animal models. Methods: Changes in phosphorus were tested at multiple doses in three different species - mouse, rat, and dog - across five different studies. Infigratinib was given orally at doses ranging from 0.03 mg/kg to 30 mg/kg. Both PK and PD (i.e., phosphorus) data was available in all species. Measurement days ranged from day 10 to week 12, although PK/PD measurements occurred within 1 day of each other. All animals were treated in accordance with AVMA guidelines. Results: No significant dose-phosphorus relationship was observed in rats and mice treated with doses of infigratinib ranging from 0.03 mg/kg to 5 mg/kg. A dose-phosphorus and exposure (AUC0-24)-phosphorus relationship was observed at doses of infigratinib ≥10 mg/kg across transgenic mouse, rat, and dog studies. At low doses, the exposure (AUC0-24)-phosphorus relationship showed a shallow slope with linear regression analysis in rats and mice. Conclusions: These findings from five studies in three different species indicate that the exposure-phosphorus relationship is consistent. Importantly, no relationship was observed between dose and phosphorus levels in rats and mice treated with infigratinib at or below 5 mg/kg. Despite the fact that infigratinib is a FGFR1, 2 and 3 inhibitor, low doses of infigratinib shown previously to exert a significant effect in improving skeletal abnormalities in an achondroplasia mouse model, do not seem to result in meaningful changes in phosphorus. These experiments demonstrate that at doses of infigratinib much lower than used in oncology - like those being considered for use in clinical studies of achondroplasia - infigratinib is less likely to cause hyperphosphatemia. Infigratinib will be evaluated in global clinical studies in children with achondroplasia in 2020.

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.

The spleen tyrosine kinase (SYK) regulates immune cell activation in response to engagement of a variety of receptors, making it an intriguing target for the treatment of inflammatory and autoimmune disorders as well as certain B-cell malignancies. We have previously reported on the discovery and preclinical characterization of PRT062607, a potent and highly selective inhibitor of SYK that exhibits robust anti-inflammatory activity in a variety of animal models. Here we present data from our first human studies aimed at characterizing the pharmacokinetics (PK), pharmacodynamics (PD), and safety of PRT062607 in healthy volunteers following single and multiple oral administrations. PRT062607 demonstrated a favorable PK profile and the ability to completely inhibit SYK activity in multiple whole-blood assays. The PD half-life in the more sensitive assays was approximately 24 hours and returned to predose levels by 72 hours. Selectivity for SYK was observed at all dose levels tested. Analysis of the PK/PD relationship indicated an IC50 of 324 nM for inhibition of B-cell antigen receptor-mediated B-cell activation and 205 nM for inhibition of FcεRI-mediated basophil degranulation. PRT062607 was safe and well tolerated across the entire range of doses. Clinical PK/PD was related to in vivo anti-inflammatory activity of PRT062607 in the rat collagen-induced arthritis model, which predicts that therapeutic concentrations may be safely achieved in humans for the treatment of autoimmune disease. PRT062607 has a desirable PK profile and is capable of safely, potently, and selectively suppressing SYK kinase function in humans following once-daily oral dosing.

The heterogeneity and severity of certain autoimmune diseases and B-cell malignancies warrant simultaneous targeting of multiple disease-relevant signaling pathways. Dual inhibition of spleen tyrosine kinase (SYK) and Janus kinase (JAK) represents such a strategy and may elicit several benefits relative to selective kinase inhibition, such as gaining control over a broader array of disease etiologies, reducing probability of selection for bypass disease mechanisms, and the potential that an overall lower level suppression of individual targets may be sufficient to modulate disease activity. To this end, we provide data on the discovery and preclinical development of PRT062070 [4-(cyclopropylamino)-2-({4-[4-(ethylsulfonyl)piperazin-1-yl]phenyl}amino)pyrimidine-5-carboxamide hydrochloride], an orally active kinase inhibitor that demonstrates activity against SYK and JAK. Cellular assays demonstrated specific inhibitory activity against signaling pathways that use SYK and JAK1/3. Limited inhibition of JAK2 was observed, and PRT062070 did not inhibit phorbol 12-myristate 13-acetate-mediated signaling or activation in B and T cells nor T-cell antigen receptor-mediated signaling in T cells, providing evidence for selectivity of action. Potent antitumor activity was observed in a subset of B-cell lymphoma cell lines. After oral dosing, PRT062070 suppressed inflammation and autoantibody generation in a rat collagen-induced arthritis model and blocked B-cell activation and splenomegaly in a mouse model of chronic B-cell antigen receptor stimulation. PRT062070 is currently under evaluation in a phase I dose escalation study in patients with B-cell leukemia and lymphoma (NCT01994382), with proof-of-concept studies in humans planned to assess therapeutic potential in autoimmune and malignant diseases.

Introduction AG10 is a novel, potent and selective oral transthyretin (TTR) stabilizer under clinical development to treat TTR amyloidosis (ATTR). Both mutant and wild-type ATTR are under-diagnosed diseases with no FDA-approved therapies. As ATTR is caused by the destabilization of TTR tetramers due to inherited mutations or aging, AG10 is designed to treat the disease at its source. Hypothesis AG10 is safe and well tolerated at exposures reaching target therapeutic plasma concentrations, resulting in complete stabilization of TTR following repeat dosing in healthy volunteers (HV), and predicting clinical efficacy in treating both mutant and wild-type ATTR. Methods Single and multiple ascending oral doses of AG10 were administered in a double blind manner to 4 single and 3 multiple dose cohorts consisting of 8 HV per cohort randomized 3:1 (AG10: placebo). Safety and tolerability were assessed by vital signs, ECG, adverse events, and safety labs to monitor kidney and liver function, electrolytes, and hematology. Pharmacokinetics were measured using a validated bioanalytical assay. Pharmacodynamics (PD) were assessed via two established ex vivo assays for TTR stabilization, fluorescent probe exclusion and Western blot. Results Single oral doses of 50, 150, 300 and 800 mg, and multiple oral doses to steady state of 100, 300 and 800 mg q12h were well tolerated with a clean safety profile. AG10 was rapidly absorbed with t max max and AUC were dose-dependent, terminal t 1/2 was ∼25 hr, and there was little food effect (slightly increased T max , blunted C max , and 11% increase in AUC following a high fat meal). The PD profile, as assessed by either assay, demonstrated complete stabilization of TTR at C max following single doses of 300 mg or more, and >95% stabilization on average across the entire dosing interval of 800 mg q12h at steady state with low inter-subject variability (Figure). Conclusions AG10, a novel, potent and selective oral TTR stabilizer, is safe and well-tolerated following oral dosing in healthy volunteers and completely stabilizes TTR (>90%) across the dosing interval, establishing clinical proof of concept. Based on these data, AG10 has the potential to be a safe and effective treatment for patients with ATTR.

AG10 is a highly selective and potent oral transthyretin (TTR) stabilizer under development for TTR amyloidosis (ATTR). ATTR cardiomyopathy (ATTR-CM) is under-diagnosed with no FDA-approved therapies indicated for its treatment. Hypothesis: At doses investigated, AG10 achieves complete stabilization

Background: CLEC-2 signals through a cytosolic YXXL downstream of a triacidic amino acid sequence (hemITAM). Results: We show a critical role for the triacidic amino acid in phosphorylation of the cytosolic tyrosine and that CLEC-2 signals via Syk but not Zap-70. Conclusion: CLEC-2 signaling requires a cytosolic triacidic amino acid sequence and Syk. Significance: Proximal signaling events by hemITAM receptors are distinct to those in ITAM receptors., CLEC-2 is a member of new family of C-type lectin receptors characterized by a cytosolic YXXL downstream of three acidic amino acids in a sequence known as a hemITAM (hemi-immunoreceptor tyrosine-based activation motif). Dimerization of two phosphorylated CLEC-2 molecules leads to recruitment of the tyrosine kinase Syk via its tandem SH2 domains and initiation of a downstream signaling cascade. Using Syk-deficient and Zap-70-deficient cell lines we show that hemITAM signaling is restricted to Syk and that the upstream triacidic amino acid sequence is required for signaling. Using surface plasmon resonance and phosphorylation studies, we demonstrate that the triacidic amino acids are required for phosphorylation of the YXXL. These results further emphasize the distinct nature of the proximal events in signaling by hemITAM relative to ITAM receptors.

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 chronic lymphocytic leukemia (CLL), Syk becomes activated by external signals from the tissue microenvironment, and was targeted in a first clinical trial with R788 (fostamatinib), a relatively nonspecific 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 nurse-like cell-co-cultures. Moreover, they inhibit BCR-dependent secretion of the chemokines CCL3 and CCL4 by CLL cells, and leukemia cell migration toward the tissue homing chemokines CXCL12, CXCL13, and beneath stromal cells. PRT318 and P505-15 furthermore inhibit Syk and extracellular signal-regulated kinase 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.

Leptin therapy in human recombinant form has recently been used in HIV-associated lipodystrophy syndrome on experimental basis in some small short-term clinical trials. It has shown its beneficial effects only in hypoleptinemic HIV-infected patients by causing definite improvement in their insulin sensitivity, glucose tolerance, lipid status, and truncal obesity. Leptin prevents lipotoxicity and activates insulin signaling pathways through several postulated mechanisms. Central leptin insufficiency with peripheral hyperleptinemia has come out to be a significant contributor to the development of obesity and metabolic syndrome. In this article, we will review the basis of leptin therapy in HIV patients, with its promises. However, further larger clinical trials are needed to prove its long-term efficacy in the control of metabolic complications related to HIV therapy.

Disease development in human T-cell leukemia virus type 1 (HTLV-1)–infected individuals is positively correlated with the level of integrated viral DNA in T cells. HTLV-1 replication is positively regulated by Tax and Rex and negatively regulated by the p30 and HBZ proteins. In the present study, we demonstrate that HTLV-1 encodes another negative regulator of virus expression, the p13 protein. Expressed separately, p13 localizes to the mitochondria, whereas in the presence of Tax, part of it is ubiquitinated, stabilized, and rerouted to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional coactivator, and, by reducing Tax transcriptional activity, suppresses viral expression. Because Tax stabilizes its own repressor, these findings suggest that HTLV-1 has evolved a complex mechanism to control its own replication. Further, these results highlight the importance of studying the function of the HTLV-1 viral proteins, not only in isolation, but also in the context of full viral replication.

SummaryBetrixaban is an oral direct inhibitor of factor Xa (FXa) being developed for the prevention of venous thromboembolism (VTE). Its antithrombotic effects had not been previously tested in patients. This exploratory clinical trial in the US and Canada randomized 215 patients undergoing elective total knee replacement (TKR) in a 2:2:1 ratio to receive post-operative betrixaban 15 mg or 40 mg p.o. bid or enoxaparin 30 mg s.c. q12h, respectively, for 10–14 days. The betrixaban dosage was blinded, but enoxaparin was not. Primary efficacy outcome was the incidence of VTE, consisting of deep-vein thrombosis (DVT) on mandatory unilateral (operated leg) venography, symptomatic proximal DVT, or pulmonary embolism (PE) through Day 10–14. Safety outcomes included major and clinically significant non-major bleeds through 48 h after treatment. All efficacy and bleeding outcomes were adjudicated by a blinded independent central adjudication committee. Of 214 treated patients, 175 (82%) were evaluable for primary efficacy. VTE incidence was 14/70 (20%; 95% CI: 11, 31) for betrixaban 15 mg, 10/65 (15%; 95% CI: 8, 27) for betrixaban 40 mg, and 4/40 (10%; 95% CI: 3, 24) for enoxaparin. No bleeds were reported for betrixaban 15 mg, 2 (2.4%) clinically significant non-major bleeds with betrixaban 40 mg, and one (2.3%) major and two (4.6%) clinically significant non-major bleeds with enoxaparin. A dose- and concentration-dependent effect of betrixaban on inhibition of thrombin generation and anti-Xa levels was observed. Betrixaban demonstrated antithrombotic activity and appeared well tolerated in knee replacement patients at the doses studied.

Summary A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end-organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N-terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half-life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease-modifying agent in sickle cell patients.

Whereas adaptive immunity has been extensively studied, very little is known about the innate immunity of the host to HTLV-I infection. HTLV-I-infected ATL patients have pronounced immunodeficiency associated with frequent opportunistic infections, and in these patients, concurrent infections with bacteria and/or parasites are known to increase risks of progression to ATL. The Toll-like receptor-4 (TLR4) activation in response to bacterial infection is essential for dendritic cell maturation and links the innate and adaptive immune responses. Recent reports indicate that TLR4 is targeted by viruses such as RSV, HCV, and MMTV. Here we report that HTLV-I has also evolved a protein that interferes with TLR4 signaling; p30 interacts with and inhibits the DNA binding and transcription activity of PU.1 resulting in the down-regulation of the TLR4 expression from the cell surface. Expression of p30 hampers the release of pro-inflammatory cytokines MCP-1, TNF-alpha, and IL-8 and stimulates release of anti-inflammatory IL-10 following stimulation of TLR4 in human macrophage. Finally, we found that p30 increases phosphorylation and inactivation of GSK3-beta a key step for IL-10 production. Our study suggests a novel function of p30, which may instigate immune tolerance by reducing activation of adaptive immunity in ATL patients.

Enzymes of the blood coagulation pathway enhance the inflammatory response leading to endothelial dysfunction, accounting, in part, for the vascular complications occurring in sepsis and cardiovascular disease. The responses of endothelial cell activation include induction of the expression of tissue factor (TF), a membrane glycoprotein that promotes thrombosis, and of E-selectin, a cell adhesion molecule that promotes inflammation. In this report, we demonstrate synergistic interactions between the coagulation factor Xa (fXa) and the proinflammatory cytokines TNF, IL-1beta, and CD40L, leading to enhanced expression of TF and E-selectin in endothelial cells. A detailed analysis of the molecular pathways that could account for this activity of fXa showed that fXa inhibited the cytokine-induced expression of dual specificity phosphatases, MAP kinase phosphatase-L, -4, -5, and -7, blocking a negative regulatory effect on c-Jun N-terminal kinase. The synergistic interaction between fXa and TNF was also involved in the inhibition of A20 and IkappaBalpha expression in the IkappaB kinase-NF-kappaB pathway. The data indicate that inhibition of negative regulatory signaling accounts for the amplification of cytokine-induced endothelial cell activation by fXa.

In immortal cells, the existence of a mechanism for the maintenance of telomere length is critical. In most cases this is achieved by the reactivation of telomerase, a cellular reverse transcriptase that prevents telomere shortening. Here we report that the telomerase gene (hTERT) promoter is up-regulated during transmission of human T-cell lymphotropic virus type-I (HTLV-I) to primary T cells in vitro and in ex vivo adult T-cell leukemia/lymphoma (ATLL) samples, but not asymptomatic carriers. Although Tax impaired induction of human telomerase reverse transcriptase (hTERT) mRNA in response to mitogenic stimulation, transduction of Tax into primary lymphocytes was sufficient to activate and maintain telomerase expression and telomere length when cultured in the absence of any exogenous stimulation. Transient transfection assays revealed that Tax stimulates the hTERT promoter through the nuclear factor κB (NF-κB) pathway. Consistently, Tax mutants inactive for NF-κB activation could not activate the hTERT or sustain telomere length in transduced primary lymphocytes. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays suggested that an increased binding of c-Myc and Sp1 is involved in the NF-κB–mediated activation of the hTERT promoter. This study establishes the role of Tax in regulation of telomerase expression, which may cooperate with other functions of Tax to promote HTLV-I–associated adult T-cell leukemia.

Using N,N-dialkylated benzamidines as the novel P4 motifs, we have designed and synthesized a class of 1-(2-naphthyl)-1H-pyrazole-5-carboxylamides as highly potent and selective fXa inhibitors with significantly improved hydrophilicity and in vitro anticoagulant activity. These benzamidine-P4 fXa inhibitors have displayed excellent oral bioavailability and long half-life.

A variety of P4 motifs have been examined to increase the binding affinity and in vitro anticoagulant potency of our biphenyl 1-(2-naphthyl)-1H-pyrazole-5-carboxylamide-based fXa inhibitors. Highly potent 2-naphthyl-P1 fXa inhibitors (K(i)or =2 nM) with improved in vitro anticoagulant activity (2xTGor =1 microM) and respectable pharmacokinetic properties have been discovered.

Anthranilamides 4 and 5 were designed and synthesized as selective and orally bioavailable factor Xa inhibitors. Structural modifications aimed at lowering their lipophilicity were performed at the central phenyl ring and at the S4 binding biphenyl region by incorporating water solublizing substituents. The resulting compounds (e.g., 7, 8, 14, 30a, and 32b) are highly potent in vitro, and show improved activity in human plasma-based thrombin generation assay.

Hypothyroidism presenting as recurrent hypokalemic paralysis is rare in the literature. This transient and episodic neurological condition is commonly associated with thyrotoxicosis. We report a case of young female admitted with recurrent paralytic attacks since last 1 year. She had no symptom of hypothyroidism. She had weakness of all four limbs, delayed relaxation of ankle jerks, and normal higher mental function. There was no enlargement of thyroid. Serum potassium level ranged from 1.6 to 3.2 meq/L during attack with high serum creatine phosphokinase level. Electromyography was normal. The patient was diagnosed having chronic thyroiditis with high thyroid-stimulating hormone and thyroid-related antibodies. Follow up shows satisfactory result with thyroxine replacement. It is an extremely rare and unusual presentation of hypothyroidism, probably the fourth reported case of hypothyroidism with hypokalemic paralysis, to the best of our knowledge.

The structure-activity relationship of a novel series of substituted piperazinone-based factor Xa inhibitors is described. The most potent compound 34 displays IC(50) of 0.9 nM.

A total of 28 consecutive patients suffering from alcoholic liver disease who were admitted to the Department of Medicine, Nilratan Sircar Medical College and Hospital, Kolkata, from January 2009 to August 2010, were enrolled in this study. Patients in whom liver biopsy was contradicted because of co-morbid conditions like coagulopathy, tense ascites and hepatic space occupying lesions, or those who did not consent for biopsy were excluded from the study.

To overcome the low bioavailability of our substituted acrylamide P1 benzamidine factor Xa inhibitors reported previously, neutral and less basic groups were used to replace the benzamidine. As a result, a series of P1 aminoisoquinoline substituted acrylamide Xa inhibitors was identified to be potent, selective, and orally bioavailable. Modification of P4 moiety of these compounds further improved their pharmacokinetic properties.

Monoamidine FXa inhibitors 3 were designed and synthesized. SAR studies and molecular modeling led to the design of conformationally constrained diaryl ethers 4 and 5, as well as benzopyrrolidinone 7 as potent FXa inhibitors. The monoamidines show high efficacy in a DVT model, but lack desirable oral bioavailability. The benzopyrrolidinone-based aminoisoquinolines 8 do not show significant improvement in oral bioavailability.

The activation of factor X (fX) to factor Xa (fXa) marks the penultimate step in the coagulation cascade and modulating fXa activity may be effective for antithrombotic therapy. Even though fXa inhibitors are screened using in vitro inhibition of human fXa (HfXa) while subsequent evaluation uses in vivo rabbit models, there is limited knowledge of species differences between the coagulation proteins. When comparing amino acid sequences for the human (HfX) and rabbit (RafX) protein, differences are found in the activation peptide and active site regions. In order to study the relative functional characteristics of HfX and RafX, we asked (1) whether fX from the two species is immunologically related, (2) whether the two proteins are activated to fXa in a similar manner, (3) whether HfXa and rabbit factor Xa (RafXa) have similar catalytic activities toward tripeptide substrates. To answer (1), we expressed RafX-glutathione S-transferase (RafX-GST) fusion protein in bacteria and purified the protein for use as an antigen. The resulting monoclonal antibodies were suitable for affinity purification of plasma RafX and for effective anticoagulation in rabbit plasma clotting assays. We found two antibodies (mAb 214 and mAb 290) that anticoagulated rabbit plasma in a dose responsive manner but did not cross-react with human plasma. At a concentration of 500 nM, mAb 214 attained a two-fold extension of rabbit plasma activated partial thromboplastin time (aPTT). To answer (2), we purified plasma RafX and compared the activation of HfX and RafX with Russell's viper venom (RVV-X). Under equivalent reaction conditions, conversion was 30% slower for the rabbit protein. To answer (3), amidolytic activity of HfXa and RafXa were assayed by cleavage of three para-nitroanilide (pNA) substrates (S2222 [Bz-Ile-Glu(gamma-OR)-Gly-Arg-pNA.HCl], S2765 [Z-D-Arg-Gly-Arg-pNA.HCl] and Spectrozyme Xa [MeO-CO-D-CHG-Gly-Arg-pNA.AcOH]). Michaelis constants (K(m)) for the rabbit protein were 187, 72 and 69 microM, respectively, and for the human analog, 255, 63 and 135 microM, respectively. Comparing the extent of substrate turnover (V(max)) for HfXa and RafXa, the latter was shown to cleave all three substrates at a reduced rate. Based on these observations, it can be speculated that the relative antithrombotic potency of active site directed fXa inhibitors might be different between the two species. Predicted human therapeutic doses derived from in vivo results in rabbit models should therefore take species variation into consideration.

A series of potent and selective factor Xa inhibitors was synthesized using various readily available amino acids as central templates. The most potent compound displays IC50 of 3 nM.

Fluorescence and stopped flow methods were used to compare clinically used heparins with regard to their ability to bind to antithrombin and to accelerate the inactivation of factor Xa. Titration of antithrombin with both low molecular weight heparin (LMWH) (enoxaparin, fragmin and ardeparin) and unfractionated heparin (UFH) produced an equivalent fluorescence increase and indicates similar affinity of all heparin preparations to antithrombin. However, relative to UFH enoxaparin, the LMWH with the smallest average molecular mass, contained only 12% material with high affinity for antithrombin. The rate of factor Xa inhibition by antithrombin increased with the concentration of the examined heparins to the same limiting value, but the concentration required for maximal acceleration depended on the preparation. According to these data the high affinity fraction of the heparin preparations increased the intrinsic fluorescence and inhibitory activity equally without additional effects by variations in chain length and chemical composition. In contrast, in the presence of Ca UFH accelerated the inhibition of factor Xa by antithrombin 10-fold more efficiently than comparable concentrations of the high affinity fractions of enoxaparin and fragmin. The bell-shaped dependence of this accelerating effect suggests simultaneous binding of both proteins to heparin. In conclusion, under physiologic conditions the anti-factor Xa activity of heparin results from a composite effect of chain length and the content of material with high affinity to antithrombin. Thus, the reduced antithrombotic activity of LMWH relative to UFH results from a smaller content of high affinity material and the absence of a stimulating effect of calcium.

SummaryIn order to investigate the respective roles of prothrombinase and intrinsic tenase (IXa/VIIIa) in venous thrombosis, we compared the antithrombotic efficacy of inhibitors of these two coagulation complexes. The agents tested were dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated bovine factor IXa (IXai) and Glu-Gly-Arg chloromethyl ketone- inactivated human factor Xa (Xai). In vitro formation of active complexes (prothrombinase or tenase) was inhibited by Xai and IXai resulting in IC50 values of 3 nM and 5 nM, respectively. Antithrombotic activity was measured by inhibition of clot accretion on cotton threads placed in the abdominal vena cava of anesthetized rabbits. Intravenous bolus dosing followed by infusion of Xai during the experimental protocol resulted in a dose dependent reduction of clot weight, a dosage of 16.0 μg/kg + 0.28 μg/kg/min being sufficient to produce a 96% inhibition of thrombosis. A much higher dose of IXai (1.0 mg/kg + 17.3 |ig/kg/min) resulted in a 39% reduction of clot weight. In a rabbit arterio-venous shunt model mimicking arterial thrombosis, the relative efficacy of the two agents was found to be more comparable. The doses required for optimum antithrombotic activity were 128.0 μg/kg + 2.2 μg/kg/min for Xai and 1.0 mg/kg+ 17.3 μg/kg/min for IXai. We conclude that, in this study, prothrombinase rather than tenase inhibition was more effective in reducing venous thrombosis and that these effects can be achieved without disruption of extravascular hemostasis.

Background Selective disruption of the spleen tyrosine kinase (Syk) represents a novel strategy to control B-cell functional responses by inhibition of B-cell antigen receptor (BCR) signaling. PRT062607 (P505-15) is a highly selective small molecule Syk inhibitor that potently suppresses B-cell function in human and rodent blood, and reduces inflammation in rodent models of rheumatoid arthritis (RA). Aims In this study, we sought to determine the potency of Syk inhibition by PRT062607 in whole blood from RA patients, and elucidate covariates that affect the potency of immune-regulation by this compound. Materials and Methods Whole blood was collected from 30 patients diagnosed with RA as part of a single-center outpatient study. Disease severity, serum protein markers of inflammation, and co-medications were related to each other, and to PRT062607 activity in ex vivo Syk-mediated immune function assays. Results We report here that PRT062607 exhibited greater potency in suppressing BCR mediated B-cell functional responses in whole blood from RA patients who received stable methotrexate (MTX) therapy. We demonstrate that the B-cell functional response to BCR ligation is influenced by cytokines and JAK/STAT signaling. Discussion MTX is a known cytokine modulating agent, and this mechanism may act in concert with PRT062607 to control B-cell function. Conclusion These data have important implications for the co-administration of Syk inhibitors and MTX for the treatment of RA.

Anaemia, the major sequelae of chronic renal disease (CRD) needs to be investigated because it acts as an independent risk factor for worsening of cardiovascular survival, cognitive impairment and poor quality of life. In this prospective tertiary care hospital-based study we have followed up 100 randomly assigned CRD patients who were not on dialysis for at least 6 months. Left ventricular mass index (LVMI), ejection fraction (EF), mini-mental status examination (MMSE) and general well-being of these patients were assessed quantitatively on admission and at 3rd and 6th months of follow-up after receiving recombinant human erythropoietin (rHuEPO) and aggressive intravenous iron therapy for anaemia correction. The median study duration was 6 years. Statistical analysis also showed the positive impact of anaemia correction which, even when partial, caused significant improvement in cardiovascular function as evidenced by increase in EF (p = 0.004) and decrease in LVMI (p = 0.016) along with substantial enhancement of general well-being (p0.001). Cognition did not show significant change within a short spell of 6 months. This study thus emphasises on earliest detection and correction of anaemia in CRD population to enhance both short-term and long-term survival as a whole.

Background Direct factor Xa inhibitors [fXaI] have superior anticoagulant efficacy and/or safety relative to warfarin and LMWH but are limited by lack of a specific antidote to reverse anticoagulation during episodes of serious bleeding or before surgery. PRT064445 [PRT] is a modified, human recombinant fXa that is catalytically inactive but retains high-affinity binding to direct fXaI and heparin- antithrombin III complexes. It competes with native fXa for fXaI drugs, thus reversing direct and indirect fXaI-mediated anticoagulation. Methods In this phase I, first-in-man double-blind study, 32 healthy volunteers were randomized (6:2) within dosing cohorts to a single IV bolus of PRT (30 mg, 90 mg, 300 mg, or 600 mg) or placebo and followed for 28 days. Anti-fXa activity was assayed in vitro by adding exogenous rivaroxaban (50 ng/mL) to subject plasma samples. Results The terminal t1/2 was ~6 hours. AUC and Cmax increased disproportionately relative to dose. In the presence of PRT, thrombin generation and anti-fXa activity of rivaroxaban ( Figure ) were reversed in a dose-dependent manner. There were no thrombotic AEs or deaths. There was 1 serious AE (pneumonia) and 3 non-serious infusion-related reactions without anaphylaxis [90 mg (2) and placebo (1)]. One unplanned pregnancy occurred ~10 days post-treatment, followed shortly by a spontaneous abortion. Prothrombin fragment 1 + 2, thrombin-antithrombin complex, and D-dimer transiently increased with dose; other coagulation parameters including PT, aPTT, ACT and platelet activity were unchanged. Conclusions PRT is a promising universal antidote for fXaI. A Phase 2 trial evaluating PRT reversal of several fXaI is ongoing.

Aims Patients with atrial fibrillation (AF) are at increased risk of stroke. Betrixaban is a novel oral factor Xa inhibitor administered once daily, mostly excreted unchanged in the bile and with low (17%) renal excretion. Methods and results Patients with AF and more than one risk factor for stroke were randomized to one of three blinded doses of betrixaban (40, 60, or 80 mg once daily) or unblinded warfarin, adjusted to an international normalized ratio of 2.0–3.0. The primary outcome was major or clinically relevant non-major bleeding. The mean follow-up was 147 days. Among 508 patients randomized, the mean CHADS2 score was 2.2; 87% of patients had previously received vitamin K antagonist therapy. The time in therapeutic range on warfarin was 63.4%. There were one, five, five, and seven patients with a primary outcome on betrixaban 40, 60, 80 mg daily, or warfarin, respectively. The rate of the primary outcome was lowest on betrixaban 40 mg (hazard ratio compared with warfarin = 0.14, exact stratified log-rank P -value 0.04, unadjusted for multiple testing). Rates of the primary outcome with betrixaban 60 or 80 mg were more similar to those of wafarin. Two ischaemic strokes occurred, one each on betrixaban 60 and 80 mg daily. There were two vascular deaths, one each on betrixaban 40 mg and warfarin. Betrixaban was associated with higher rates of diarrhoea than warfarin. Conclusion Betrixaban was well tolerated and had similar or lower rates of bleeding compared with well-controlled warfarin in patients with AF at risk for stroke.

To evaluate the effects of the anticoagulant betrixaban on individual heart rate-corrected QT (QTcI).Ninety-six healthy adults were randomly assigned to single-dose betrixaban 80 and 140 mg (therapeutic and supratherapeutic doses, respectively), placebo, and moxifloxacin 400 mg (positive control) in a four-period crossover study. Electrocardiograms were recorded at pre-dose and post-dose hours 1, 2, 3, 4, 5, 6, 8, 12, 16 and 24.An analysis of covariance determined the placebo-corrected, time-matched mean change from baseline QTcI at the 95% upper confidence interval (UCI; one-sided). The pre-specified clinically significant change for betrixaban-treated groups was10 ms (95% UCI, one-sided). Subjects were monitored for safety and tolerability.Mean QTcI change was10 ms for both betrixaban groups at all time points; expected changes were observed for moxifloxacin, establishing assay sensitivity. Correlation between betrixaban plasma concentration and QTcI duration confirmed the absence of effect on QT.Betrixaban at therapeutic and supratherapeutic doses did not cause clinically relevant changes in QTcI intervals or other electrocardiographic parameters. Betrixaban was well tolerated.

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p′-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the “tenase” complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.

B-cell receptor (BCR) associated kinases including spleen tyrosine kinase (SYK) contribute to the pathogenesis of B-cell malignancies. SYK is persistently phosphorylated in a subset of non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL), and SYK inhibition results in abrogation of downstream kinase activity and apoptosis. P505-15 (also known as PRT062607) is a novel, highly selective, and orally bioavailable small molecule SYK inhibitor (SYK IC(50) = 1 nM) with anti-SYK activity that is at least 80-fold greater than its affinity for other kinases. We evaluated the preclinical characteristics of P505-15 in models of NHL and CLL. P505-15 successfully inhibited SYK-mediated B-cell receptor signaling and decreased cell viability in NHL and CLL. Oral dosing in mice prevented BCR-mediated splenomegaly and significantly inhibited NHL tumor growth in a xenograft model. In addition, combination treatment of primary CLL cells with P505-15 plus fludarabine produced synergistic enhancement of activity at nanomolar concentrations. Our findings support the ongoing development of P505-15 as a therapeutic agent for B-cell malignancies. A dose finding study in healthy volunteers has been completed.