Substituted 1-methyl-4-phenylpyrrolidin-2-ones - Fragment-based design of N-methylpyrrolidone-derived bromodomain inhibitors.

N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.

Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition.

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.

The SMAC mimetic, LCL-161, reduces survival in aggressive MYC-driven lymphoma while promoting susceptibility to endotoxic shock.

Inhibitor of apoptosis proteins (IAPs) antagonize caspase activation and regulate death receptor signaling cascades. LCL-161 is a small molecule second mitochondrial activator of caspase (SMAC) mimetic, which both disengages IAPs from caspases and induces proteasomal degradation of cIAP-1 and -2, resulting in altered signaling through the NFκB pathway, enhanced TNF production and sensitization to apoptosis mediated by the extrinsic pathway. SMAC mimetics are undergoing clinical evaluation in a range of hematological malignancies. Burkitt-like lymphomas are hallmarked by a low apoptotic threshold, conveying sensitivity to a range of apoptosis-inducing stimuli. While evaluating LCL-161 in the Eµ-Myc model of aggressive Burkitt-like lymphoma, we noted unexpected resistance to apoptosis induction despite 'on-target' IAP degradation and NFκB activation. Moreover, LCL-161 treatment of lymphoma-bearing mice resulted in apparent disease acceleration concurrent to augmented inflammatory cytokine-release in the same animals. Indiscriminate exposure of lymphoma patients to SMAC mimetics may therefore be detrimental due to both unanticipated prolymphoma effects and increased susceptibility to endotoxic shock.

Preclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.

Thalidomide-analogue biology: immunological, molecular and epigenetic targets in cancer therapy.

Thalidomide and its analogues (lenalidomide and pomalidomide) are small molecule glutamic acid derivatives of the immunomodulatory drug (IMiD) class. In addition to the immuno-adjuvant and anti-inflammatory properties that define an IMiD, the thalidomide analogues demonstrate an overlapping and diverse range of biological activities, including anti-angiogenic, teratogenic and epigenetic effects. Importantly, the IMiDs possess anti-cancer activity with selectivity for molecularly defined subgroups of hematological malignancies, specifically mature B-cell neoplasms and myelodysplasia with deletion of chromosome 5q. Emerging insight into the pathophysiological drivers of these IMiD-responsive disease states can now be synthesized using previously disclosed IMiD activities and recently discovered thalidomide targets to build unifying models of IMiD mechanism of action. Attention to mechanisms of IMiD-induced clinical toxicities, in particular the recently identified association of lenalidomide with second primary malignancies, provides an additional tool for determination of drug mechanism. This review seeks to define the molecular IMiD targets and biological outputs that underpin their anti-neoplastic activity. It is anticipated that elucidation of important IMiD targets will allow the rational development of new-generation therapeutics with the potential to separate thalidomide-analogue efficacy from clinical toxicity.

Plasmodium species co-infection as a cause of treatment failure.

We report a case of Plasmodium falciparum and Plasmodium malariae coinfection with associated failure of clinical response to artemether + lumefantrine therapy. This case highlights the need to consider co-infection in the setting of apparent treatment failure and the impact of mixed species infection upon host dynamics and clinical presentation. Recognition of malarial co-infection is clinically important for determining appropriate therapy and preventing disease sequelae.

A 'dangerous' group O donor: severe hemolysis in all recipients of organs from a donor with multiple red cell alloantibodies.

Alloimmune hemolysis is a recognized but infrequent complication of solid organ transplantation, particularly where there is incompatibility within the ABO blood group system. We describe severe hemolysis due to passenger lymphocyte syndrome (PLS) in all three recipients of organs from a single donor with multiple red cell (RC) alloantibodies. The first patient, a liver transplant recipient, required augmentation of immunosuppression to treat immune hemolysis due to anti-B, -D, -C and -Cellano (k). This is the first description of PLS caused by alloantibody to the high incidence RC antigen, k. The two single lung transplant recipients developed hemolysis due to anti-D. Both required escalation of immunosuppression and early transfusion support. Three months posttransplant, all three patients have ongoing evidence of compensated hemolysis. This series highlights the potential for severe non-ABO-mediated immune hemolysis following solid organ transplantation. A positive donor RC antibody screen should prompt careful monitoring of organ recipients for hemolysis.

Laboratory tumor lysis syndrome complicating LBH589 therapy in a patient with acute myeloid leukaemia.

LBH589 is a novel cinnamic hydroxamic acid analog (HAA) pan-histone deacetylase inhibitor (HDACi) currently in early phase clinical development.

Efficacy and safety of a high purity, double virus inactivated factor VIII/von Willebrand factor concentrate (Biostate) in patients with von Willebrand disorder requiring invasive or surgical procedures.

Biostate is a double virally inactivated, plasma derived coagulation factor VIII (FVIII)/von Willebrand factor (VWF) concentrate registered and used in Australia, New Zealand and Asia for the treatment of patients with haemophilia A. Although Biostate has been well characterized for FVIII and VWF (ratio 1:2 respectively) and shows a similar VWF multimeric pattern to normal plasma, limited published data is available on its clinical efficacy and safety in patients with von Willebrand disorder (VWD) who require surgical procedures. We retrospectively assessed the efficacy and safety of Biostate in all VWD patients treated at three Australian haemophilia treatment centres undergoing invasive procedures or surgery over a 29-month period between April 2003 and September 2005. A chart review of 43 VWD patients (26 VWD type 1, 12 VWD type 2, 5 VWD type 3; 21 male, 22 female; mean age 52 years, range 19-80 years) undergoing 58 surgical procedures (24 major, 34 minor) was performed. For each procedure, data were collected on Biostate dosage and administration, adverse reactions, haemostatic efficacy and bleeding events. Haemostatic efficacy of Biostate was assessed as excellent in 78% or good in 22% of procedures. There were no bleeding events attributable to lack of efficacy in any patients. No adverse reactions related to the administration of Biostate were observed. These results suggest that Biostate is both safe and efficacious for the prevention of excessive bleeding in VWD patients undergoing surgery or invasive procedures.

Adjuvant rituximab causes prolonged hypogammaglobulinaemia following autologous stem cell transplant for non-Hodgkin's lymphoma.

Rituximab is an anti-CD20 monoclonal antibody that has efficacy in B-cell non-Hodgkin's lymphoma (NHL). Adjuvant immunotherapy with rituximab may reduce relapse rates for high-risk B-cell NHL following high-dose chemotherapy with autologous stem cell transplantation (SCT). However, the potential adverse effects of rituximab on immune reconstitution following SCT are not fully characterized. We performed a retrospective analysis of immunoglobulin (Ig) levels and peripheral blood neutrophil counts in 11 patients who received adjuvant rituximab following autologous SCT for B-cell NHL. Results were compared to a contemporaneous group of 24 patients who received an identical conditioning regimen and autologous SCT for lymphoma, but no adjuvant rituximab. Adjuvant rituximab was associated with a significantly increased incidence of hypogammaglobulinaemia between 12 and 24 months post-SCT, but not neutropenia. Despite suppression of Igs, there were no late or atypical infective complications attributable to rituximab.

Blockade of B7-2, not B7-1, inhibits purified protein derivative-primed T-lymphocyte responses but fails to influence the proportion of Th1 versus Th2 subsets.

The ability to select for a cell-mediated response rather than antibody production following infection with intracellular mycobacteria, would be an advantage in preventing the occurrence of disease. Recent work suggests that the two members of the B7 family of costimulatory molecules, B7-1 and B7-2, may differentially influence the nature of primary immune responses but little is known of their role in this capacity in secondary responses. We have used an in vitro model to investigate whether blocking B7-1 and B7-2 affects changes in the cytokine profiles of Th lymphocytes previously primed to purified protein derivative (PPD) from Mycobacterium bovis. In C57BL/6 and BALB/c mice we found that the proliferative responses of a component of recently activated T lymphocytes, and those returning to the resting state, were inhibited by B7-2 blockade. B7-1 blockade had no distinguishable effect. However, in cultures containing anti-B7-2 antibody, the production of both interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), indicative of cell-mediated and antibody responses, respectively, were reduced. This suggests that intervention in a recall response to mycobacterial antigen by blocking B7-1 or B7-2 molecules, is unlikely to alter the nature of the immune response.

Psychological factors in the longitudinal course of battering: when do the couples split up? When does the abuse decrease?

The longitudinal course of battering was investigated over a 2-year time span. Forty-five batterers and their spouses were assessed with self-report, psychophysiological, and marital interaction measures. Both the stability of the relationship and of the battering were assessed. At the two-year follow-up, 62% of the couples were still married and living together, while 38% had separated or divorced. A combination of six variables, reflecting severity of husband emotional abuse, wife dissatisfaction, husband physiological arousal, and wife defending herself assertively, was 90.2% accurate in predicting separation or divorce 2 years later. Of the couples still living together at follow-up, 46% of the batterers did not reduce their levels of severe violence, while 54% did significantly decrease levels of violence. Husbands who continued to be severely violent at 2-year follow-up were more domineering, globally negative and emotionally abusive toward their wives at Time 1 than husbands who reduced their levels of violence. Even though 54% of the batterers decreased the frequency of violent acts over the 2-year period, only 7% achieved complete desistance. Moreover, husband emotional abuse did not decrease over the 2-year period, even when physical abuse did.

Simvastatin in severe hypercholesterolaemia: a placebo controlled trial.

The effect of simvastatin in 27 patients with severe primary hypercholesterolaemia was assessed by a double-blind placebo controlled parallel group trial. Total serum cholesterol, LDL-cholesterol and apoprotein B (ApoB) were significantly reduced by simvastatin 40 mg daily. Reductions in triglyceride and VLDL-cholesterol and an increase in HDL-cholesterol levels were only significant when calculated as a percentage of baseline, because of wide inter-individual variability. No changes in apoprotein A1, lipoprotein (a), fibrinogen, viscosity or blood pressure were observed. Leucocyte HMG-CoA reductase activity was unchanged after 4 weeks of active treatment but increased by 87% after 3 months (n = 21, P less than 0.05). No severe adverse effects or changes in CK or AST levels were noted. We conclude that simvastatin is effective in the treatment of severe and resistant hypercholesterolaemia, and well tolerated in the short term.

The suppression of exciting thoughts.

We examined how the suppression of an exciting thought influences sympathetic arousal as indexed by skin conductance level (SCL). Subjects were asked to think aloud as they followed instructions to think about or not to think about various topics. Experiment 1 showed that trying not to think about sex, like thinking about sex, elevates SCL in comparison to thinking about or not thinking about less exciting topics (e.g., dancing). Experiment 2 revealed that the suppression of the thought of sex yielded SCL elevation whether or not subjects believed their think-aloud reports would be private or public, and it also revealed that the effect dissipated over the course of a few minutes. Experiment 3 found such dissipation again but showed that subsequent intrusions of the suppressed exciting thought are associated with further elevations in SCL over 30 min. Because such an association was not found when subjects were trying to think about the exciting thought, it was suggested that the suppression of exciting thoughts might be involved in the production of chronic emotional responses such as phobias and obsessive preoccupations.