Identification of NVP-CLR457 as an Orally Bioavailable Non-CNS-Penetrant pan-Class IA Phosphoinositol-3-Kinase Inhibitor.

Balanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed.

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Complex and cryptic chromosomal rearrangements involving the MLL gene in acute leukemia: a study of 7 patients and review of the literature.

Chromosomal rearrangements involving the MLL gene have been associated with many different types of hematological malignancies. Most of them are easily recognized by conventional cytogenetics. However, in some cases, complex, unusual or cryptic rearrangements make the MLL involvement difficult or impossible to be detected by conventional cytogenetics. Fluorescent in situ hybridization with a panel of probes coupled with long distance inverse-PCR was used to identify chromosomal rearrangements involving the MLL gene. Seven unusual chromosomal rearrangements were identified, including two complex translocations, three insertions of material of chromosome 11 in another chromosome and one insertion of chromosome material into the MLL gene. Conventional cytogenetics showed three patients to have a deletion of 11q; one had an unexpected t(6;11)(q27;q23) whereas the other two patients had also an insertion of MLL material in another chromosome. Concurrent 3' deletion in the MLL rearrangement was observed in two patients. We recommend a systematic approach to be used in all cases of acute leukemia starting with FISH analyses using a commercially available MLL split signal probe. Should an abnormality be discovered, the analysis has to be completed by further molecular cytogenetic and genomic PCR methods in order to unravel the recombination mechanism.

Leukocyte activation: the link between inflammation and coagulation during heatstroke. A study of patients during the 2003 heat wave in Paris.

OBJECTIVE: The mechanisms linking severe inflammation and coagulation during heatstroke are poorly understood. Here, we examined the roles of the tissue factor pathway, leukocyte activation, and mediators of innate immunity in patients admitted to an intensive care unit for heatstroke during an intense heat wave in Paris. DESIGN: Retrospective observational study. SETTING: Intensive care unit of a university medical center. PATIENTS: Eighteen critically ill severe patients with heatstroke were enrolled in the study and 14 age-matched patients with severe sepsis as controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: High circulating levels of some inflammation and stress mediators (interleukin-6, -8, C5a, interleukin-1 receptor antagonist, heat shock protein 60 and 70) were observed. Blood leukocyte activation was shown by beta2 integrin up-regulation, L-selectin down-regulation, and strong production of reactive oxygen species and interleukin-8 ex vivo. High levels of circulating promatrix metalloproteinase-9 were detected in all the patients studied, and its active form was present in two patients. Overt disseminated intravascular coagulation according to the International Society of Thrombosis and Hemostasis score was present in five patients. Whole-blood tissue factor was present in all the patients and part of this activity was associated with microparticles in five patients. The degrees of inflammation and disseminated intravascular coagulation are correlated with clinical severity. CONCLUSIONS: These results suggest that neutrophil activation plays a key role in the acute activation of coagulation observed during severe heatstroke, despite a rapid and sustained antiinflammatory response. The comparison with a group of patients with severe sepsis suggests some common mechanisms, but more intense responses during heatstroke.

Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma.

The thrombin generation test is used to study coagulation in patients with haemorrhagic diseases or with high thrombotic risk. To our knowledge, this is the first study investigating the relative influence of coagulation factors on thrombin generation in plasma. The aim was to investigate the influence of coagulant factors, anticoagulant factors, and tissue factor (TF) on three parameters: endogenous thrombin potential (ETP), peak thrombin concentration, and lag time for the appearance of thrombin. At a low TF concentration, all factors except factor XI influenced thrombin generation. At a high TF concentration, only the factors of the extrinsic pathway exerted an influence. ETP and peak thrombin were linearly correlated to factor II concentration. Factor V and factor VII effects increased hyperbolically with factor concentration. The influence of factor X on thrombin generation depended on TF concentration. In the absence of factor VIII and factor IX, ETP fell to 60-70% of the normal when peak thrombin fell to 25-30% of the normal. Fibrinogen concentration influenced ETP and peak thrombin and decreasing fibrinogen levels shortened the lag time. As expected, decreasing antithrombin concentration caused dramatic increases in thrombin generation. Protein S prolonged the lag time, especially at a low TF concentration. No effect of protein C was observed, likely due to the absence of thrombomodulin. The thrombin generation test was more sensitive to factor deficiencies at low than at high TF concentration. ETP was not the most critical parameter for studying coagulation factor deficiencies. Instead, peak thrombin was the most sensitive parameter.

Influence of omeprazole on the antiplatelet action of clopidogrel associated with aspirin: the randomized, double-blind OCLA (Omeprazole CLopidogrel Aspirin) study.

OBJECTIVES: This trial sought to assess the influence of omeprazole on clopidogrel efficacy. BACKGROUND: Clopidogrel has proved its benefit in the treatment of atherothrombotic diseases. In a previous observational study, we found clopidogrel activity on platelets, tested by vasodilator-stimulated phosphoprotein (VASP) phosphorylation, to be diminished in patients receiving proton pump inhibitor (PPI) treatment. METHODS: In this double-blind placebo-controlled trial, all consecutive patients undergoing coronary artery stent implantation received aspirin (75 mg/day) and clopidogrel (loading dose, followed by 75 mg/day) and were randomized to receive either associated omeprazole (20 mg/day) or placebo for 7 days. Clopidogrel effect was tested on days 1 and 7 in both groups by measuring platelet phosphorylated-VASP expressed as a platelet reactivity index (PRI). Our main end point compared PRI value at the 7-day treatment period in the 2 groups. RESULTS: Data for 124 patients were analyzed. On day 1, mean PRI was 83.2% (standard deviation [SD] 5.6) and 83.9% (SD 4.6), respectively, in the placebo and omeprazole groups (p = NS), and on day 7, 39.8% (SD 15.4) and 51.4% (SD 16.4), respectively (p < 0.0001). RESULTS: Omeprazole significantly decreased clopidogrel inhibitory effect on platelet P2Y12 as assessed by VASP phosphorylation test. Aspirin-clopidogrel antiplatelet dual therapy is widely prescribed worldwide, with PPIs frequently associated to prevent gastrointestinal bleeding. The clinical impact of these results remains uncertain but merits further investigation.

Correlations between carotid IMT, factor VIII activity level and metabolic disturbances: a cardio-vascular risk factor in the HIV positive persons.

In the HIV infection, the short time-scale between the HIV-induced cardiovascular events and the onset of antiretroviral therapy elicits a thrombophilic co-factor that worsens the induced atherosclerosis. We compared the factor VIII plasma activity, previously implicated in arterial and venous thrombosis, with a surrogate marker of atherosclerosis, the carotid intima-media thickness, and with the usual atherosclerosis risk factors in 154 HIV infected outpatients. The FVIII plasma activity is significantly associated with the carotid intima-media thickness and, strongly, with blood glucose and triglycerides levels. A raised FVIII plasma activity is an important feature of the metabolic syndrome and a putative co-factor of HAART induced cardiovascular events. Thus the prevention of the HAART-induced cardio-vascular events should probably not be exclusively focused on atherosclerosis but likewise on the thrombus formation process.

Do sewage treatment plant discharges substantially impair fish reproduction in polluted rivers?

Sewage treatment plants are frequently associated with the release of xenobiotics and, consequently, with alterations of the reproductive function induced by many of these substances in aquatic organisms. In order to assess the impacts of sewage treatment plant (STP) discharges in polluted rivers, two sentinel species (gudgeon Gobio gobio and stoneloach Barbatula barbatula) were caught during their reproductive cycle upstream and downstream two STPs (STP1--Goffontaine, STP2--Wegnez). Gonadosomatic index, histological (testicular and ovarian stages, atretic follicles, intersexuality) and endocrine (sex steroids, aromatase activity, alkali-labile phosphorus) parameters were assayed. In brief, the results revealed no systematic significant differences (p<0.05) between upstream and downstream sites, whatever the STP, species or sampling period. However, stoneloach females displayed some signs of reproductive impairment and endocrine disruption downstream STP1 (reduced GSI, oocyte diameter and ALP concentrations, increased proportion of atretic follicles) and STP2 (changes in gonadal aromatase activity and plasma levels of 11-KT and T). Few significant changes were observed for gudgeon males and females while there were no significant differences between upstream and downstream sites for stoneloach males. Moreover, plasma E(2) concentrations recorded in gudgeon males sampled in all sites were as high as in females and this was confirmed by high ALP levels. Besides, spermatogenesis of gudgeon males was delayed in STP1 upstream and downstream sites compared to the corresponding sites in STP2. These observations for gudgeon males do not seem related to STP discharge but to a probable estrogenicity of the river. Therefore, as shown by the results, stoneloach seemed more sensitive than gudgeon to STP discharges. In the present study, sewage treatment plant discharges do not substantially impair fish reproduction. In this respect, caution is required when generalising negative impacts of STP discharges.

Screening by fluorescence in situ hybridization for MLL status at diagnosis in 239 unselected patients with acute myeloblastic leukemia.

A large number of abnormalities involving the MLL gene have been associated with hematological malignancies, including acute myeloblastic leukemias (AML). Given the overall unfavorable prognosis of AML with an MLL abnormality, its reliable and accurate detection is needed for informed treatment decision. We therefore investigated the occurrence of MLL abnormalities in 239 unselected consecutive AML patients, using conventional cytogenetic and fluorescent in situ hybridization (FISH) analyses. FISH analysis for MLL was performed using a commercial dual-color probe. Of the 239 patients, 30 (12.6%) showed MLL abnormalities under FISH analysis, 10 (4.2%) showed a split signal indicating the disruption of the MLL gene by translocation or insertion, and 20 (8.4%) showed overrepresentation of the MLL gene without evidence of rearrangement. MLL abnormalities were more frequently found in AML-M5 and M4, mainly as rearrangements, and in AML-M2, mainly as overrepresentation. Our results are in agreement with those reported in other studies. All M2, M4, and M5 AML patients without known recurrent translocations, such as t(8;21) and inv(16), should be investigated by FISH with an MLL probe because it allows the detection of MLL rearrangement that would go undetected by conventional cytogenetics and because it has the ability of detecting multiple copies of the MLL gene in, for example, marker chromosomes and double minutes.

Cryptic 5' MLL gene insertion in an X-chromosome in acute myeloblastic leukemia.

Band 11q23 is known to be involved in translocations and insertions with a variety of partner chromosomes. They lead to MLL rearrangement, resulting in fusion with numerous genes. We report here on a 43-year-old man presenting with asthenia and pancytopenia who was diagnosed with acute myeloblastic leukemia FAB-M5. Conventional cytogenetic techniques showed a del(11)(q21). Using a specific probe for fluorescent in situ hybridization, the MLL gene was found to be disrupted, with the 5' region being inserted into the X-chromosome (around bands q24 approximately q25), as confirmed by whole X-chromosome painting. The accumulating data on acute myeloblastic leukemia demonstrate that the 5'-MLL insertion in an X-chromosome is a rare but recurrent abnormality associated with leukemia, not only in infants, but also in adults.

X chromosome insertion in the MLL gene in a case of childhood acute myeloblastic leukemia.

Band 11q23 is known to be involved in translocations and insertions with a variety of partner chromosomes. These lead to MLL rearrangement, resulting in a fusion with numerous genes. We report here the case of a 5-month-old boy presenting with hemianopsia and severe diffuse intravascular coagulopathy in whom a diagnosis of acute myeloblastic leukemia (AML) French-American-British M4 classification was made. Conventional cytogenetic techniques showed an ins(11;X) (q23;q28q12). Fluorescent in situ hybridization (FISH) with whole chromosome paints confirmed this finding. Using a specific probe, the MLL gene was found to be disrupted, a portion of the X chromosome being inserted between the 5' and 3' regions of the MLL gene. Although some cases of insertion involving chromosomes X and 11 have been reported in AML, this appears to be the first case involving band Xq28. We postulate that this chromosomal rearrangement led to the fusion of the 5' region of the MLL gene with a yet unidentified gene located in band Xq28.