Rationale and design of an independent randomised controlled trial evaluating the effectiveness of aripiprazole or haloperidol in combination with clozapine for treatment-resistant schizophrenia

<p>Abstract</p> <p>Background</p> <p>One third to two thirds of people with schizophrenia have persistent psychotic symptoms despite clozapine treatment. Under real-world circumstances, the need to provide effective therapeutic interventions to patients who do not have an optimal response to clozapine has been cited as the most common reason for simultaneously prescribing a second antipsychotic drug in combination treatment strategies. In a clinical area where the pressing need of providing therapeutic answers has progressively increased the occurrence of antipsychotic polypharmacy, despite the lack of robust evidence of its efficacy, we sought to implement a pre-planned protocol where two alternative therapeutic answers are systematically provided and evaluated within the context of a pragmatic, multicentre, independent randomised study.</p> <p>Methods/Design</p> <p>The principal clinical question to be answered by the present project is the relative efficacy and tolerability of combination treatment with clozapine plus aripiprazole compared with combination treatment with clozapine plus haloperidol in patients with an incomplete response to treatment with clozapine over an appropriate period of time. This project is a prospective, multicentre, randomized, parallel-group, superiority trial that follow patients over a period of 12 months. Withdrawal from allocated treatment within 3 months is the primary outcome.</p> <p>Discussion</p> <p>The implementation of the protocol presented here shows that it is possible to create a network of community psychiatric services that accept the idea of using their everyday clinical practice to produce randomised knowledge. The employed pragmatic attitude allowed to randomly allocate more than 100 individuals, which means that this study is the largest antipsychotic combination trial conducted so far in Western countries. We expect that the current project, by generating evidence on whether it is clinically useful to combine clozapine with aripiprazole rather than with haloperidol, provides physicians with a solid evidence base to be directly applied in the routine care of patients with schizophrenia.</p> <p>Trial Registration</p> <p><b>Clincaltrials.gov Identifier</b>: NCT00395915</p

Hypoxia/Reoxygenation Cardiac Injury and Regeneration in Zebrafish Adult Heart

<div><p>Aims</p><p>the adult zebrafish heart regenerates spontaneously after injury and has been used to study the mechanisms of cardiac repair. However, no zebrafish model is available that mimics ischemic injury in mammalian heart. We developed and characterized zebrafish cardiac injury induced by hypoxia/reoxygenation (H/R) and the regeneration that followed it.</p><p>Methods and Results</p><p>adult zebrafish were kept either in hypoxic (H) or normoxic control (C) water for 15 min; thereafter fishes were returned to C water. Within 2–6 hours (h) after reoxygenation there was evidence of cardiac oxidative stress by dihydroethidium fluorescence and protein nitrosylation, as well as of inflammation. We used Tg(cmlc2:nucDsRed) transgenic zebrafish to identify myocardial cell nuclei. Cardiomyocyte apoptosis and necrosis were evidenced by TUNEL and Acridine Orange (AO) staining, respectively; 18 h after H/R, 9.9±2.6% of myocardial cell nuclei were TUNEL⁺ and 15.0±2.5% were AO⁺. At the 30-day (d) time point myocardial cell death was back to baseline (n = 3 at each time point). We evaluated cardiomyocyte proliferation by Phospho Histone H3 (pHH3) or Proliferating Cell Nuclear Antigen (PCNA) expression. Cardiomyocyte proliferation was apparent 18–24 h after H/R, it achieved its peak 3–7d later, and was back to baseline at 30d. 7d after H/R 17.4±2.3% of all cardiomyocytes were pHH3⁺ and 7.4±0.6% were PCNA⁺ (n = 3 at each time point). Cardiac function was assessed by 2D-echocardiography and Ventricular Diastolic and Systolic Areas were used to compute Fractional Area Change (FAC). FAC decreased from 29.3±2.0% in normoxia to 16.4±1.8% at 18 h after H/R; one month later ventricular function was back to baseline (n = 12 at each time point).</p><p>Conclusions</p><p>zebrafish exposed to H/R exhibit evidence of cardiac oxidative stress and inflammation, myocardial cell death and proliferation. The initial decrease in ventricular function is followed by full recovery. This model more closely mimics reperfusion injury in mammals than other cardiac injury models.</p></div

Evaluation of Left Ventricle Function by Regional Fractional Area Change (RFAC) in a Mouse Model of Myocardial Infarction Secondary to Valsartan Treatment - Fig 5

<p>(<b>A</b>) Myocyte cross-sectional area (MCSA) in the remote left ventricle (LV) in sham and infarcted groups (n = 6/group). (<b>B</b>) Cell proliferation measured as KI-67 positivity and proliferating fibroblasts detected by double labeling with KI-67 and vimentin. Cells proliferation was expressed as percentage of KI-67 positive cells over total cell number (nuclei), whereas fibroblasts proliferation was expressed as percentage of vimentin/KI-67 double-positive cells over vimentin positive cells. (<b>C</b>) Representative immunofluorescence and quantification of collagen type I staining of the non-infarcted LV (upper panels) and of appendage (lower panels) in sham, MI and MI+Val mice at 4 weeks post-surgery. Interstitial collagen fraction was measured as the percentage of area occupied by collagen on total tissue area. Values are mean ± SEM. * <i>p</i><0.05, *** <i>p</i><0.001 vs. MI. (n = 6/group).</p

Necrotic myocyte cell death induced by H/R <i>in vivo.</i>

<p>Necrotic myocyte cell death was assessed under baseline conditions, and 18 h and 30d after H/R in the Tg(cmlc2:nucDsRed) zebrafish line. At 18 h after H/R it was found a marked increase in necrotic myocardial cell number, which was back to control value at the 30d time point. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R showing colocalization of DAPI, DsRED and AO stainings. Arrows indicate cardiomyocyte AO⁺ nuclei. (b) AO⁺ cardiomyocytes nuclei in control (C) animals, and 18 h and 30d after H/R (n = 3 at each time point; ** <i>p</i><0.01 <i>vs.</i> C).</p

Apoptotic myocyte cell death induced by H/R <i>in vivo.</i>

<p>Apoptotic myocyte cell death was assessed under baseline conditions, and 18 h and 30d after H/R in the Tg(cmlc2:nucDsRed) zebrafish line. At 18 h after H/R it was found a marked increase in apoptotic myocardial cell number, which was back to control value at the 30d time point. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R, showing colocalization of DAPI, DsRED and TUNEL stainings. Arrows indicate cardiomyocyte TUNEL⁺ nuclei, whereas arrow-head indicates non-cardiomyocyte TUNEL⁺ nuclei. (b) TUNEL⁺ cardiomyocytes nuclei in control (C) animals, and 18 h and 30d after H/R (n = 3 at each time point; * <i>p</i><0.05 <i>vs.</i> C).</p

Oxidative stress detection after H/R <i>in vivo.</i>

<p>DHE staining (a-d) and N-Tyr immunofluorescence (e-f) of hearts under control conditions (C) and exposed to H/R. (a) Representative confocal microscopy images of DHE staining in C and 2 h after H/R. (b) Merge of DHE and Hoechst nuclear staining. Calibration bar = 20 µm. White arrow-heads indicate DHE⁺ nuclei. (c) 3D representation of DHE fluorescence intensity distribution in the analyzed area: the <i>z-axis</i> shows the fluorescence intensity in cardiac nuclei, the <i>y-axis</i> and <i>x-axis</i> show the spatial distribution of nuclei on a plane. (d) Graph shows Mean Fluorescence Intensity (MFI) in C and 2 h to 14 h after H/R (n = 4 at each time point; ** <i>p</i><0.01 <i>vs.</i> C). Time course analysis revealed a peak of oxidative stress at 2 h in zebrafish adult heart sections, detected by DHE staining. (e) Representative confocal microscopy images of N-Tyr immunofluorescence, where green fluorescence indicates anti-N-Tyr and Hoechst nuclei staining: control (C, left panel) and 2 h after H/R (right panel). Calibration bar = 10µm. (f) Graph shows Mean Fluorescence Intensity (MFI) in C and 2 h to 14 h after H/R (n = 4 at each time point; ** <i>p</i><0.01 <i>vs.</i> C). H/R induced protein nitrosylation with a peak effect at the 2 h time point.</p

Detection of HIF-1α-dependent genes expression in whole hearts after H/R <i>in vivo.</i>

<p>Graphs show selected HIF-1α-dependent genes expression in whole hearts in control (C) and at different time points (3 h, 6 h, and 9 h) after H/R. (a) <i>Hmox1</i> mRNA expression exhibited a progressive increase and, at the 9 h time point, <i>hmox1</i> was ∼8-fold higher than in C. (b) <i>Vegfaa</i> mRNA increased and achieved its peak 6 h after H/R. (c) <i>Epo</i> mRNA exhibited a peak increase at 3 h which was ∼1.7-fold higher than in C but failed to achieve statistical significance. (n = 6; * <i>p</i><0.05 and *** <i>p</i><0.001 <i>vs.</i> C).</p

Inflammatory response induced by H/R <i>in vivo.</i>

<p>Representative confocal microscopy images (a-h) showing neutrophils (yellow fluorescence or green fluorescence) and macrophages (red fluorescence) infiltration in double transgenic line Tg(MPO:EGFP)×Tg(LysC:DsRed) in control (C) and at different time points (4 h, 6 h, and 14 h) after H/R. Neutrophils are either yellow (LysC⁺/MPO⁺) or green (predominantly, MPO⁺) cells (arrows in the 6 h image); red macrophages are LysC⁺ cells (arrow in the 4 h image). Hoechst stains cell nuclei; (a-d) calibration bar = 100 µm, (e-h) calibration bar = 20 µm.The peak inflammatory response occurred at the 6 h time point after H/R This experiment was performed three times with similar results.</p

Bull’s eye representations of mean regional fractional area change (RFAC) in sham, MI and MI+Val groups at 24 hours, 1 and 4 weeks after MI (n = 12/group).

<p>Slices from LV apex to the base are shown from the inner to the outer circle, whereas red and green tones indicate lower and higher RFAC values (the color bar on the right shows the corresponding scale). • for 0.05</p

Myocardial cells positive for PCNA induced by H/R <i>in vivo.</i>

<p>Cardiomyocytes proliferation was assessed under baseline conditions and 18 h to 30d after H/R in Tg(cmlc2:nucDsRed) zebrafish line. (a) Representative image of a zebrafish heart ventricular section 18 h after H/R showing colocalization of DAPI, DsRed and PCNA stainings. Arrows indicate cardiomyocyte PCNA⁺ nuclei. (b) Following H/R, there was a progressive increase in PCNA⁺ cardiomyocytes nuclei; the peak increase was achieved at the 3d time point, and at 30d the number of PCNA⁺ myocardial cells was back to control value (n = 3 at each time point; ** <i>p</i><0.01 and *** <i>p</i><0.001 <i>vs.</i> C).</p