Smac-derived Aza-peptide As an Aminopeptidase-resistant XIAP BIR3 Antagonist

The peptidic nature of anti-IAPs N-terminus Smac-derived peptides precludes their utilization as potential therapeutic anticancer agents. Recent advances in the development of novel Smac-derived peptidomimetics and non-peptidic molecules with improved anti-IAPs activity and resistance to proteolytic cleavage have been reported and led to a number of candidates that are currently in clinical trials including LCL-161, SM-406/AT-406, GDC-0512/GDC-0917, and birinapant. As an attempt to improve the proteolytic stability of Smac peptides, we developed the Aza-peptide AzaAla-Val-Pro-Phe-Tyr-NH2 (2). Unlike unmodified peptide Ala-Val-Pro-Phe-Tyr-NH2 (1), analogue (2) exhibited resistance towards proteolytic cleavage by two aminopeptidases; LAP and DPP-IV, while retaining its IAP inhibitory activity. This was due to the altered planar geometry of the P1 residue side chain. Our findings showed that using aza-isosteres of bioactive peptide sequences imbue the residue with imperviousness to proteolysis; underscoring a potential approach for developing a new generation of Smac-derived Aza-peptidomimetics.

A selective antagonist of histamine H₄ receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs:involvement of lipocortin-1

BACKGROUND AND PURPOSE: Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

EXPERIMENTAL APPROACH: Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

KEY RESULTS: OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid.

CONCLUSIONS AND IMPLICATIONS: Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

The CXCR2 antagonist, SCH-527123, shows antitumor activity and sensitizes cells to oxaliplatin in preclinical colon cancer models

Colorectal cancer is the second most common cause of cancer-related death in the United States. Recent studies showed that interleukin-8 (IL-8) and its receptors (CXCR1 and CXCR2) are significantly upregulated in both the tumor and its microenvironment, and act as key regulators of proliferation, angiogenesis, and metastasis. Our previous study showed that IL-8 overexpression in colorectal cancer cells triggers the upregulation of the CXCR2-mediated proliferative pathway. The aim of this study was to investigate whether the CXCR2 antagonist, SCH-527123, inhibits colorectal cancer proliferation and if it can sensitize colorectal cancer cells to oxaliplatin both in vitro and in vivo. SCH-527123 showed concentration-dependent antiproliferative effects in HCT116, Caco2, and their respective IL-8-overexpressing variants colorectal cancer cell lines. Moreover, SCH-527123 was able to suppress CXCR2-mediated signal transduction as shown through decreased phosphorylation of the NF-κB/mitogen-activated protein kinase (MAPK)/AKT pathway. These findings corresponded with decreased cell migration and invasion, while increased apoptosis in colorectal cancer cell lines. In vivo results verified that SCH-527123 treatment decreased tumor growth and microvessel density when compared with vehicle-treated tumors. Importantly, these preclinical studies showed that the combination of SCH-527123 and oxaliplatin resulted in a greater decrease in cell proliferation, tumor growth, apoptosis, and angiogenesis that was superior to single-agent treatment. Taken together, these findings suggest that targeting CXCR2 may block tumor proliferation, migration, invasion, and angiogenesis. In addition, CXCR2 blockade may further sensitize colorectal cancer to oxaliplatin treatment.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

Higher plasma levels of complement C3a, C4a and C5a increase the risk of subretinal fibrosis in neovascular age-related macular degeneration.

Background: The aim of this study was to investigate the plasma levels of complement C3a, C4a, and C5a in different types of neovascular age-related macular degeneration (nAMD) and whether the levels were related to patients’ responsiveness to anti-VEGF therapy.

Results: Ninety-six nAMD patients (including 61 with choroidal neovascularisation (CNV), 17 with retinal angiomatous proliferation (RAP), 14 with polypoidal choroidal vasculopathy (PCV) and 4 unclassified patients) and 43 controls were recruited to this case–control study. Subretinal fibrosis was observed in 45 nAMD patients and was absent in 51 nAMD patients. In addition, the responsiveness to anti-VEGF (Lucentis) therapy was also evaluated in nAMD patients. Forty-four patients were complete responders, 48 were partially responders, and only 4 patients did not respond to the therapy. The plasma levels of C3a, C4a and C5a were significantly higher in nAMD patients compared to
controls. Further analysis of nAMD subgroups showed that the levels of C3a, C4a and C5a were significantly increased in patients with CNV but not RAP and PCV. Significantly increased levels of C3a, C4a and C5a were also observed in nAMD patients with subretinal fibrosis but not in those without subretinal fibrosis. Higher levels of C3a were observed in nAMD patients who responded partially to anti-VEGF therapy.

Conclusions: Our results suggest increased systemic complement activation in nAMD patients with CNV but not RAP and PCV. Our results also suggest that higher levels of systemic complement activation may increase the risk of subretinal fibrosis in nAMD patients

The effect of a selective CXCR2 antagonist (AZD5069) on human blood neutrophil count and innate immune functions.

Abstract AIMS: The aim of the present study was to investigate whether selective antagonism of the cysteine-X-cysteine chemokine receptor-2 (CXCR2) receptor has any adverse effects on the key innate effector functions of human neutrophils for defence against microbial pathogens. METHODS: In a double-blind, crossover study, 30 healthy volunteers were randomized to treatment with the CXCR2 antagonist AZD5069 (100 mg) or placebo, twice daily orally for 6 days. The peripheral blood neutrophil count was assessed at baseline, daily during treatment and in response to exercise challenge and subcutaneous injection of granulocyte-colony stimulating factor (G-CSF). Neutrophil function was evaluated by phagocytosis of Escherichia coli and by the oxidative burst response to E. coli. RESULTS: AZD5069 treatment reversibly reduced circulating neutrophil count from baseline by a mean [standard deviation (SD)] of -1.67 (0.67) ×10(9) l(-1) vs. 0.19 (0.78) ×10(9) l(-1) for placebo on day 2, returning to baseline by day 7 after the last dose. Despite low counts on day 4, a 10-min exercise challenge increased absolute blood neutrophil count, but the effect with AZD5069 was smaller and not sustained, compared with placebo treatment. Subcutaneous G-CSF on day 5 caused a substantial increase in blood neutrophil count in both placebo- and AZD5069-treated subjects. Superoxide anion production in E. coli-stimulated neutrophils and phagocytosis of E. coli were unaffected by AZD5069 (P = 0.375, P = 0.721, respectively vs. baseline, Day 4). AZD5069 was well tolerated. CONCLUSIONS: CXCR2 antagonism did not appear adversely to affect the mobilization of neutrophils from bone marrow into the peripheral circulation, phagocytosis or the oxidative burst response to bacterial pathogens. This supports the potential of CXCR2 antagonists as a treatment option for diseases in which neutrophils play a pathological role.

Cost-Effectiveness of Non-Vitamin K Antagonist Oral Anticoagulants for Atrial Fibrillation in Portugal

INTRODUCTION AND OBJECTIVES:Recently, three novel non-vitamin K antagonist oral anticoagulants received approval for reimbursement in Portugal for patients with non-valvular atrial fibrillation (AF). It is therefore important to evaluate the relative cost-effectiveness of these new oral anticoagulants in Portuguese AF patients. METHODS: A Markov model was used to analyze disease progression over a lifetime horizon. Relative efficacy data for stroke (ischemic and hemorrhagic), bleeding (intracranial, other major bleeding and clinically relevant non-major bleeding), myocardial infarction and treatment discontinuation were obtained by pairwise indirect comparisons between apixaban, dabigatran and rivaroxaban using warfarin as a common comparator. Data on resource use were obtained from the database of diagnosis-related groups and an expert panel. Model outputs included life years gained, quality-adjusted life years (QALYs), direct healthcare costs and incremental cost-effectiveness ratios (ICERs). RESULTS:Apixaban provided the most life years gained and QALYs. The ICERs of apixaban compared to warfarin and dabigatran were €5529/QALY and €9163/QALY, respectively. Apixaban was dominant over rivaroxaban (greater health gains and lower costs). The results were robust over a wide range of inputs in sensitivity analyses. Apixaban had a 70% probability of being cost-effective (at a threshold of €20 000/QALY) compared to all the other therapeutic options. CONCLUSIONS:Apixaban is a cost-effective alternative to warfarin and dabigatran and is dominant over rivaroxaban in AF patients from the perspective of the Portuguese national healthcare system. These conclusions are based on indirect comparisons, but despite this limitation, the information is useful for healthcare decision-makers.

Renal response to the angiotensin II receptor subtype 1 antagonist irbesartan versus enalapril in hypertensive patients.

OBJECTIVE: To compare the acute and sustained renal hemodynamic effects on hypertensive patients of 100 mg irbesartan and 20 mg enalapril each once daily. PATIENTS: Twenty patients (aged 35-70 years) with uncomplicated, mild-to-moderate essential hypertension and normal serum creatinine levels completed this study. STUDY DESIGN: After random allocation to treatment (n=10 per group), administration schedule (morning or evening) was determined by further random allocation, with crossover of schedules after 6 weeks' therapy. Treatment and administration assignments were double-blind. Twenty-four-hour ambulatory blood pressure was monitored before and after 6 and 12 weeks of therapy. Renal hemodynamics were determined on the first day of drug administration and 12 and 24 h after the last dose during chronic treatment. RESULTS: Administration of each antihypertensive agent induced a renal vasodilatation with no significant change in glomerular filtration rate. However, the time course appeared to differ: irbesartan had no significant acute effect 4 h after the first dose, but during chronic administration a renal vasodilatory response was found 12 and 24 h after the dose; enalapril was effective acutely and 12 h after administration, but no residual effect was found 24 h after the dose. Both antihypertensive agents lowered mean ambulatory blood pressure effectively, with no significant difference between treatments or between administration schedules (morning versus evening). CONCLUSIONS: Irbesartan and enalapril have comparable effects on blood pressure and renal hemodynamics in hypertensive patients with normal renal functioning. However, the time profiles of the renal effects appear to differ, which might be important for long-term renoprotective effects.

Enforced covalent trimerisation of soluble feline CD134 (OX40)-ligand generates a functional antagonist of feline immunodeficiency virus.

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumour necrosis factor receptor superfamily (TNFRSF) and all primary viral strains tested to date use CD134 for infection. To investigate the effect of the natural ligand for CD134 on FIV infection, feline CD134L was cloned and expressed in soluble forms. However, in contrast to murine or human CD134L, soluble feline CD134L (sCD134L) did not bind to CD134. Receptor-binding activity was restored by enforced covalent trimerisation following the introduction of a synthetic trimerisation domain from tenascin (TNC). Feline and human TNC-CD134Ls retained the species-specificity of the membrane-bound forms of the ligand while murine TNC-CD134L displayed promiscuous binding to feline, human or murine CD134. Feline and murine TNC-CD134Ls were antagonists of FIV infection; however, potency was both strain-specific and substrate-dependent, indicating that the modulatory effects of endogenous sCD134L, or exogenous CD134Lbased therapeutics, may vary depending on the viral strain.

A new selective peroxisome proliferator-activated receptor gamma antagonist with antiobesity and antidiabetic activity.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) plays a key role in adipocyte differentiation and insulin sensitivity. Its synthetic ligands, the thiazolidinediones (TZD), are used as insulin sensitizers in the treatment of type 2 diabetes. These compounds induce both adipocyte differentiation in cell culture models and promote weight gain in rodents and humans. Here, we report on the identification of a new synthetic PPARgamma antagonist, the phosphonophosphate SR-202, which inhibits both TZD-stimulated recruitment of the coactivator steroid receptor coactivator-1 and TZD-induced transcriptional activity of the receptor. In cell culture, SR-202 efficiently antagonizes hormone- and TZD-induced adipocyte differentiation. In vivo, decreasing PPARgamma activity, either by treatment with SR-202 or by invalidation of one allele of the PPARgamma gene, leads to a reduction of both high fat diet-induced adipocyte hypertrophy and insulin resistance. These effects are accompanied by a smaller size of the adipocytes and a reduction of TNFalpha and leptin secretion. Treatment with SR-202 also dramatically improves insulin sensitivity in the diabetic ob/ob mice. Thus, although we cannot exclude that its actions involve additional signaling mechanisms, SR-202 represents a new selective PPARgamma antagonist that is effective both in vitro and in vivo. Because it yields both antiobesity and antidiabetic effects, SR-202 may be a lead for new compounds to be used in the treatment of obesity and type 2 diabetes.

The analysis of Metarhizium robertsii potential as endophytic plant root coloniser, plant growth enhancer and antagonist to bean root pathogen Fusarium solani f. sp. phaseolis.

The soil-inhabiting insect-pathogenic fungus Metarhizium robertsii also colonizes plant roots endophytically, thus showing potential as a plant symbiont. M robertsii is not randomly distributed in soils but preferentially associates with the plant rhizosphere when applied in agricultural settings. Root surface and endophytic colonization of switchgrass (Panicum virgatum) and haricot beans (Phaseolus vulgaris) by M robertsii were examined after inoculation with fungal conidia. Light and confocal microscopies were used to ascertain this rhizosphere association. Root lengths, root hair density and emergence of lateral roots were also measured. Initially, M robertsii conidia adhered to, germinated on, and colonized, roots. Furthermore, plant roots treated with Metarhizium grew faster and the density of plant root hairs increased when compared with control plants. The onset of plant root hair proliferation was initiated before germination of M robertsii on the root (within 1-2 days). Plants inoculated with M robertsii AMAD2 (plant adhesin gene) took significantly longer to show root hair proliferation than the wild type. Cell free extracts of M robertsii did not stimulate root hair proliferation. Longer term (60 days) associations showed that M robertsii endophytically colonized individual cortical cells within bean roots. Metarhizium appeared as an amorphous mycelial aggregate within root cortical cells as well as between the intercellular spaces with no apparent damage to the plant. These results suggested that not only is M robertsii rhizosphere competent but displays a beneficial endophytic association with plant roots that results in the proliferation of root hairs. The biocontrol of bean (Phaseolis vulgaris) root rot fungus Fusarium solani f. sp. phaseolis by Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M robertsii against F. solani. A relative inhibition of ca. 60% of F. solani growth was observed in these assays. Cell free culture filtrates of M robertsii inhibited the germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M robertsii then exposed to F. solani showed healthier plant profiles and lower disease indices compared to plants not colonized by M robertsii. These results suggested that the insect pathogenic/endophytic fungus M robertsii could also be utilized as a biocontrol agent against certain plant pathogens occurring in the rhizosphere.