Efficacy and safety of alirocumab in reducing lipids and cardiovascular events.

BACKGROUND: Alirocumab, a monoclonal antibody that inhibits proprotein convertase subtilisin-kexin type 9 (PCSK9), has been shown to reduce low-density lipoprotein (LDL) cholesterol levels in patients who are receiving statin therapy. Larger and longer-term studies are needed to establish safety and efficacy. METHODS: We conducted a randomized trial involving 2341 patients at high risk for cardiovascular events who had LDL cholesterol levels of 70 mg per deciliter (1.8 mmol per liter) or more and were receiving treatment with statins at the maximum tolerated dose (the highest dose associated with an acceptable side-effect profile), with or without other lipid-lowering therapy. Patients were randomly assigned in a 2:1 ratio to receive alirocumab (150 mg) or placebo as a 1-ml subcutaneous injection every 2 weeks for 78 weeks. The primary efficacy end point was the percentage change in calculated LDL cholesterol level from baseline to week 24. RESULTS: At week 24, the difference between the alirocumab and placebo groups in the mean percentage change from baseline in calculated LDL cholesterol level was -62 percentage points (P<0.001); the treatment effect remained consistent over a period of 78 weeks. The alirocumab group, as compared with the placebo group, had higher rates of injection-site reactions (5.9% vs. 4.2%), myalgia (5.4% vs. 2.9%), neurocognitive events (1.2% vs. 0.5%), and ophthalmologic events (2.9% vs. 1.9%). In a post hoc analysis, the rate of major adverse cardiovascular events (death from coronary heart disease, nonfatal myocardial infarction, fatal or nonfatal ischemic stroke, or unstable angina requiring hospitalization) was lower with alirocumab than with placebo (1.7% vs. 3.3%; hazard ratio, 0.52; 95% confidence interval, 0.31 to 0.90; nominal P=0.02). CONCLUSIONS: Over a period of 78 weeks, alirocumab, when added to statin therapy at the maximum tolerated dose, significantly reduced LDL cholesterol levels. In a post hoc analysis, there was evidence of a reduction in the rate of cardiovascular events with alirocumab. (Funded by Sanofi and Regeneron Pharmaceuticals; ODYSSEY LONG TERM ClinicalTrials.gov number, NCT01507831.).

Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells.

Etoposide (ETP) and anthracyclines are applied for wide anti-cancer treatments. However, the ETP-induced cardiotoxicity remains to be a major safety issue and the underlying cardiotoxic mechanisms are not well understood. This study is aiming to unravel the cardiotoxicity profile of ETP in comparison to anthracyclines using physiologically relevant human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using xCELLigence real-time cell analyser (RTCA), we found that single high dose of ETP induces irreversible increase in hPSC-CMs beating rate and decrease in beating amplitude. We also identified 58 deregulated genes consisting of 33 upregulated and 25 downregulated genes in hPSC-CMs after ETP treatment. Gene ontology (GO) and pathway analysis showed that most upregulated genes are enriched in GO categories like positive regulation of apoptotic process, regulation of cell death, and mitochondria organization, whereas most downregulated genes were enriched in GO categories like cytoskeletal organization, muscle contraction, and Ca2+ ion homeostasis. Moreover, we also found upregulation in 5 miRNAs (has-miR-486-3p, has-miR-34c-5p, has-miR-4423-3p, has-miR-182-5p, and has-miR-139-5p) which play role in muscle contraction, arginine and proline metabolism, and hypertrophic cardiomyopathy (HCM). Immunostaining and transmission electron microscopy also confirmed the cytoskeletal and mitochondrial damage in hPSC-CMs treated with ETP, as well as noticeable alterations in intracellular calcium handling and mitochondrial membrane potential were also observed. The apoptosis inhibitor, Pifithrin-α, found to protect hPSC-CMs from ETP-induced cardiotoxicity, whereas hPSC-CMs treated with ferroptosis inhibitor, Liproxstatin-1, showed significant recovery in hPSC-CMs functional properties like beating rate and amplitude after ETP treatment. We suggest that the damage to mitochondria is a major contributing factor involved in ETP-induced cardiotoxicity and the activation of the p53-mediated ferroptosis pathway by ETP is likely the critical pathway in ETP-induced cardiotoxicity. We also conclude that the genomic biomarkers identified in this study will significantly contribute to develop and predict potential cardiotoxic effects of novel anti-cancer drugs in vitro.

Omics-based responses induced by bosentan in human hepatoma HepaRG cell cultures.

Bosentan is well known to induce cholestatic liver toxicity in humans. The present study was set up to characterize the hepatotoxic effects of this drug at the transcriptomic, proteomic, and metabolomic levels. For this purpose, human hepatoma-derived HepaRG cells were exposed to a number of concentrations of bosentan during different periods of time. Bosentan was found to functionally and transcriptionally suppress the bile salt export pump as well as to alter bile acid levels. Pathway analysis of both transcriptomics and proteomics data identified cholestasis as a major toxicological event. Transcriptomics results further showed several gene changes related to the activation of the nuclear farnesoid X receptor. Induction of oxidative stress and inflammation were also observed. Metabolomics analysis indicated changes in the abundance of specific endogenous metabolites related to mitochondrial impairment. The outcome of this study may assist in the further optimization of adverse outcome pathway constructs that mechanistically describe the processes involved in cholestatic liver injury.

Metabolite signatures of doxorubicin induced toxicity in human induced pluripotent stem cell-derived cardiomyocytes.

Drug-induced off-target cardiotoxicity, particularly following anti-cancer therapy, is a major concern in new drug discovery and development. To ensure patient safety and efficient pharmaceutical drug development, there is an urgent need to develop more predictive cell model systems and distinct toxicity signatures. In this study, we applied our previously proposed repeated exposure toxicity methodology and performed 1H NMR spectroscopy-based extracellular metabolic profiling in culture medium of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to doxorubicin (DOX), an anti-cancer agent. Single exposure to DOX did not show alteration in the basal level of extracellular metabolites while repeated exposure to DOX caused reduction in the utilization of pyruvate and acetate, and accumulation of formate compared to control culture medium. During drug washout, only pyruvate showed reversible effect and restored its utilization by hiPSC-CMs. On the other hand, formate and acetate showed irreversible effect in response to DOX exposure. DOX repeated exposure increased release of lactate dehydrogenase (LDH) in culture medium suggesting cytotoxicity events, while declined ATP levels in hiPSC-CMs. Our data suggests DOX perturbed mitochondrial metabolism in hiPSC-CMs. Pyruvate, acetate and formate can be used as metabolite signatures of DOX induced cardiotoxicity. Moreover, the hiPSC-CMs model system coupled with metabolomics technology offers a novel and powerful approach to strengthen cardiac safety assessment during new drug discovery and development.

MicroRNAs as early toxicity signatures of doxorubicin in human-induced pluripotent stem cell-derived cardiomyocytes.

An in depth investigation at the genomic level is needed to identify early human-relevant cardiotoxicity biomarkers that are induced by drugs and environmental toxicants. The main objective of this study was to investigate the role of microRNAs (miRNAs) as cardiotoxicity biomarkers using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) that were exposed to doxorubicin (DOX) as a "gold standard" cardiotoxicant. hiPSC-CMs were exposed to 156 nM DOX for 2 days or for 6 days of repeated exposure, followed by drug washout and incubation in drug-free culture medium up to day 14 after the onset of exposure. The induced miRNAs were profiled using miRNA microarrays, and the analysis of the data was performed using the miRWalk 2.0 and DAVID bioinformatics tools. DOX induced early deregulation of 14 miRNAs (10 up-regulated and 4 down-regulated) and persistent up-regulation of 5 miRNAs during drug washout. Computational miRNA gene target predictions suggested that several DOX-responsive miRNAs might regulate the mRNA expression of genes involved in cardiac contractile function. The hiPSC-CMs exposed to DOX in a range from 39 to 156 nM did not show a significant release of the cytotoxicity marker lactate dehydrogenase (LDH) compared to controls. Quantitative real-time PCR analyses confirmed the early deregulation of miR-187-3p, miR-182-5p, miR-486-3p, miR-486-5p, miR-34a-3p, miR-4423-3p, miR-34c-3p, miR-34c-5p and miR-1303, and also the prolonged up-regulation of miR-182-5p, miR-4423-3p and miR-34c-5p. Thus, we identified and validated miRNAs showing differential DOX-responsive expression before the occurrence of cytotoxicity markers such as LDH, and these miRNAs also demonstrated the significant involvement in heart failure in patients and animal models. These results suggest that the DOX-induced deregulated miRNAs in human CMs may be used as early sensitive cardiotoxicity biomarkers for screening potential drugs and environmental cardiotoxicants with a similar mechanism of action.

Identification of genomic biomarkers for anthracycline-induced cardiotoxicity in human iPSC-derived cardiomyocytes: an in vitro repeated exposure toxicity approach for safety assessment.

The currently available techniques for the safety evaluation of candidate drugs are usually cost-intensive and time-consuming and are often insufficient to predict human relevant cardiotoxicity. The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The hiPSC-CMs were incubated with 156 nM doxorubicin, which is a well-characterized cardiotoxicant, for 2 or 6 days followed by washout of the test compound and further incubation in compound-free culture medium until day 14 after the onset of exposure. An xCELLigence Real-Time Cell Analyser was used to monitor doxorubicin-induced cytotoxicity while also monitoring functional alterations of cardiomyocytes by counting of the beating frequency of cardiomyocytes. Unlike single exposure, repeated doxorubicin exposure resulted in long-term arrhythmic beating in hiPSC-CMs accompanied by significant cytotoxicity. Global gene expression changes were studied using microarrays and bioinformatics tools. Analysis of the transcriptomic data revealed early expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. The expression of the 84 genes was further studied by real-time PCR in hiPSC-CMs incubated with daunorubicin and mitoxantrone, further anthracycline family members that are also known to induce cardiotoxicity. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by similar mechanisms as doxorubicin, daunorubicin or mitoxantrone. The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.

Gene network activity in cultivated primary hepatocytes is highly similar to diseased mammalian liver tissue.

It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

"Watching the Detectives" report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November 2015.

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.

Efficacy and safety of alirocumab in high cardiovascular risk patients with inadequately controlled hypercholesterolaemia on maximally tolerated doses of statins: the ODYSSEY COMBO II randomized controlled trial.

AIMS: To compare the efficacy [low-density lipoprotein cholesterol (LDL-C) lowering] and safety of alirocumab, a fully human monoclonal antibody to proprotein convertase subtilisin/kexin 9, compared with ezetimibe, as add-on therapy to maximally tolerated statin therapy in high cardiovascular risk patients with inadequately controlled hypercholesterolaemia. METHODS AND RESULTS: COMBO II is a double-blind, double-dummy, active-controlled, parallel-group, 104-week study of alirocumab vs. ezetimibe. Patients (n = 720) with high cardiovascular risk and elevated LDL-C despite maximal doses of statins were enrolled (August 2012-May 2013). This pre-specified analysis was conducted after the last patient completed 52 weeks. Patients were randomized to subcutaneous alirocumab 75 mg every 2 weeks (plus oral placebo) or oral ezetimibe 10 mg daily (plus subcutaneous placebo) on a background of statin therapy. At Week 24, mean ± SE reductions in LDL-C from baseline were 50.6 ± 1.4% for alirocumab vs. 20.7 ± 1.9% for ezetimibe (difference 29.8 ± 2.3%; P < 0.0001); 77.0% of alirocumab and 45.6% of ezetimibe patients achieved LDL-C <1.8 mmol/L (P < 0.0001). Mean achieved LDL-C at Week 24 was 1.3 ± 0.04 mmol/L with alirocumab and 2.1 ± 0.05 mmol/L with ezetimibe, and were maintained to Week 52. Alirocumab was generally well tolerated, with no evidence of an excess of treatment-emergent adverse events. CONCLUSION: In patients at high cardiovascular risk with inadequately controlled LDL-C, alirocumab achieved significantly greater reductions in LDL-C compared with ezetimibe, with a similar safety profile. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01644188.

Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells.

BACKGROUND & AIMS: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories. METHODS: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified. RESULTS: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC. CONCLUSIONS: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation.

Semuloparin for thromboprophylaxis in patients receiving chemotherapy for cancer.

BACKGROUND: Patients receiving chemotherapy for cancer are at increased risk for venous thromboembolism. Limited data support the clinical benefit of antithrombotic prophylaxis. METHODS: In this double-blind, multicenter trial, we evaluated the efficacy and safety of the ultra-low-molecular-weight heparin semuloparin for prevention of venous thromboembolism in patients receiving chemotherapy for cancer. Patients with metastatic or locally advanced solid tumors who were beginning to receive a course of chemotherapy were randomly assigned to receive subcutaneous semuloparin, 20 mg once daily, or placebo until there was a change of chemotherapy regimen. The primary efficacy outcome was the composite of any symptomatic deep-vein thrombosis, any nonfatal pulmonary embolism, and death related to venous thromboembolism. Clinically relevant bleeding (major and nonmajor) was the main safety outcome. RESULTS: The median treatment duration was 3.5 months. Venous thromboembolism occurred in 20 of 1608 patients (1.2%) receiving semuloparin, as compared with 55 of 1604 (3.4%) receiving placebo (hazard ratio, 0.36; 95% confidence interval [CI], 0.21 to 0.60; P<0.001), with consistent efficacy among subgroups defined according to the origin and stage of cancer and the baseline risk of venous thromboembolism. The incidence of clinically relevant bleeding was 2.8% and 2.0% in the semuloparin and placebo groups, respectively (hazard ratio, 1.40; 95% CI, 0.89 to 2.21). Major bleeding occurred in 19 of 1589 patients (1.2%) receiving semuloparin and 18 of 1583 (1.1%) receiving placebo (hazard ratio, 1.05; 95% CI, 0.55 to 1.99). Incidences of all other adverse events were similar in the two study groups. CONCLUSIONS: Semuloparin reduces the incidence of thromboembolic events in patients receiving chemotherapy for cancer, with no apparent increase in major bleeding. (Funded by Sanofi; ClinicalTrials.gov number, NCT00694382.).

Ascorbic Acid-Induced Cardiac Differentiation of Murine Pluripotent Stem Cells: Transcriptional Profiling and Effect of a Small Molecule Synergist of Wnt/ß-Catenin Signaling Pathway.

BACKGROUND: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. METHODS: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. RESULTS: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/ß-catenin signaling pathway, was added to cultures after AA-treatment. CONCLUSION: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.

Efficacy and safety of the proprotein convertase subtilisin/kexin type 9 inhibitor alirocumab among high cardiovascular risk patients on maximally tolerated statin therapy: The ODYSSEY COMBO I study.

BACKGROUND: The ODYSSEY COMBO I study (http://clinicaltrials.gov/show/NCT01644175) evaluated efficacy and safety of alirocumab as add-on therapy to stable maximally tolerated daily statin with or without other lipid-lowering therapy in high cardiovascular risk patients with suboptimally controlled hypercholesterolemia. METHODS: This multicenter, phase 3, randomized (2:1 alirocumab vs placebo), double-blind, 52-week trial enrolled 316 patients with established coronary heart disease or coronary heart disease risk equivalents and hypercholesterolemia. Alirocumab (75 mg every 2 weeks [Q2W]) or placebo Q2W was self-administered subcutaneously via 1 mL prefilled pen. The alirocumab dose was increased to 150 mg Q2W (also 1 mL) at week 12 if week 8 low-density lipoprotein cholesterol (LDL-C) was ≥70 mg/dL. The primary efficacy end point was percent change in LDL-C from baseline to week 24 (intention-to-treat analysis). RESULTS: At week 24, estimated mean (95% CI) changes in LDL-C from baseline were -48.2% (-52.0% to -44.4%) and -2.3% (-7.6% to 3.1%) for alirocumab and placebo, respectively, an estimated mean (95% CI) difference of -45.9% (-52.5% to -39.3%) (P < .0001). Low-density lipoprotein cholesterol <70 mg/dL was achieved by 75% alirocumab versus 9% placebo patients at week 24. At week 12, 83.2% of evaluable alirocumab-treated patients remained on 75-mg Q2W. Treatment-emergent adverse events were comparable between groups. CONCLUSIONS: Alirocumab treatment achieved a significantly greater reduction in LDL-C and allowed a greater proportion of patients to achieve LDL-C goals, versus placebo after 24 weeks in high cardiovascular risk patients with suboptimally controlled hypercholesterolemia at baseline despite receiving maximally tolerated statin with or without other lipid-lowering therapy. The frequency of treatment-emergent adverse events and study medication discontinuations were generally comparable between treatment groups.

Steric considerations on improving the diastereomeric ratio of (+)- and (-)-neomenthyl phenyl sulfoxides using bulky-headed oxidant hexadecyltrimethylammonium periodate and assignment of their configuration.

The bulky-headed oxidant hexadecyltrimethylammonium periodate affords the diastereomeric pairs, (Ss)-(+)/(Rs)-(+) and (Ss)-(-)/(Rs)-(-)-neomenthyl phenyl sulfoxides in stereochemically pure states with improved diastereomeric excess (48% diastereomeric excess [de]) as compared to its nonbulky counterpart, sodium metaperiodate (28% de) from respective (+)/(-)-neomenthyl phenyl sulfides. Steric effects involving the head group volume of hexadecyltrimethylammonium periodate is found to play a role in improving the diastereomeric ratio of the products. The two diastereomers can be readily separated by column chromatography. Absolute configuration at the sulfur center in (+)-neomenthyl phenyl sulfoxide was determined by single-crystal X-ray crystallography and found to be Ss. Relative configurations of other sulfoxides were assigned based on the configuration of (+)-neomenthyl phenyl sulfoxide.

Efficacy and safety of alirocumab, a fully human PCSK9 monoclonal antibody, in high cardiovascular risk patients with poorly controlled hypercholesterolemia on maximally tolerated doses of statins: rationale and design of the ODYSSEY COMBO I and II trials.

BACKGROUND: Alirocumab is a fully human monoclonal antibody to proprotein convertase subtilisin kexin type 9 (PCSK9) under investigation for treatment of hypercholesterolemia and reduction of cardiovascular events. METHODS/DESIGN: The COMBO studies, part of the Phase 3 ODYSSEY clinical trial program, are designed to evaluate the efficacy and safety of alirocumab as add-on therapy to stable, maximally tolerated daily statin, with or without other lipid-lowering therapy (LLT), in a planned 966 patients with hypercholesterolemia at high cardiovascular risk. COMBO I ( http://clinicaltrials.gov/show/NCT01644175) is placebo-controlled, with a double-blind treatment period of 52 weeks, and 306 planned patients who may receive other LLTs in addition to statin therapy. COMBO II ( http://clinicaltrials.gov/show/NCT01644188) has a double-blind treatment period of 104 weeks, comparing alirocumab with ezetimibe in 660 planned patients receiving statin therapy (but no other LLTs). The primary efficacy endpoint is the difference between treatment arms in percent change in low-density lipoprotein cholesterol (LDL-C) from baseline to week 24. Both studies utilized a starting dose of alirocumab 75 mg every 2 weeks (Q2W; administered as 1 mL solution via auto-injector). Patients with LDL-C levels ≥70 mg/dL after 8 weeks of treatment were up-titrated in a blinded manner at week 12 to alirocumab 150 mg Q2W (also 1 mL auto-injector). DISCUSSION: In conclusion, the COMBO studies will provide information on the long-term efficacy and safety of alirocumab in high-risk patients when administered in addition to maximally tolerated statin therapy, with a flexible dosing strategy which allows for individualized therapy based on the degree of LDL-C lowering needed to achieve the desired treatment response. TRIAL REGISTRATIONS COMBO I: NCT01644175 ( NCT01644175). COMBO II: NCT01644188 ( NCT01644188).

Genome-wide profiling of miRNA-gene regulatory networks in mouse postnatal heart development-implications for cardiac regeneration.

Background: After birth, mammalian cardiomyocytes substantially lose proliferative capacity with a concomitant switch from glycolytic to oxidative mitochondrial energy metabolism. Micro-RNAs (miRNAs) regulate gene expression and thus control various cellular processes. Their roles in the postnatal loss of cardiac regeneration are however still largely unclear. Here, we aimed to identify miRNA-gene regulatory networks in the neonatal heart to uncover role of miRNAs in regulation of cell cycle and metabolism. Methods and results: We performed global miRNA expression profiling using total RNA extracted from mouse ventricular tissue samples collected on postnatal day 1 (P01), P04, P09, and P23. We used the miRWalk database to predict the potential target genes of differentially expressed miRNAs and our previously published mRNA transcriptomics data to identify verified target genes that showed a concomitant differential expression in the neonatal heart. We then analyzed the biological functions of the identified miRNA-gene regulatory networks using enriched Gene Ontology (GO) and KEGG pathway analyses. Altogether 46 miRNAs were differentially expressed in the distinct stages of neonatal heart development. For twenty miRNAs, up- or downregulation took place within the first 9 postnatal days thus correlating temporally with the loss of cardiac regeneration. Importantly, for several miRNAs, including miR-150-5p, miR-484, and miR-210-3p there are no previous reports about their role in cardiac development or disease. The miRNA-gene regulatory networks of upregulated miRNAs negatively regulated biological processes and KEGG pathways related to cell proliferation, while downregulated miRNAs positively regulated biological processes and KEGG pathways associated with activation of mitochondrial metabolism and developmental hypertrophic growth. Conclusion: This study reports miRNAs and miRNA-gene regulatory networks with no previously described role in cardiac development or disease. These findings may help in elucidating regulatory mechanism of cardiac regeneration and in the development of regenerative therapies.

Human iPSC-derived preclinical models to identify toxicity of tumor-specific T cells with clinical potential.

The balance between safety and efficacy of T cell therapies remains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful selection of tumor-specific T cells using T cell toxicity screenings are essential. In vitro screening options against vital organs or specialized cell subsets would be preferably included in preclinical pipelines, but options remain limited. Here, we set up preclinical models with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, epicardial cells, and kidney organoids to investigate toxicity risks of tumor-specific T cells more thoroughly. CD8+T cells reactive against PRAME, HA-1H, CD20, or WT1, currently used or planned to be used in phase I/II clinical studies, were included. Using these hiPSC-derived preclinical models, we demonstrated that WT1-specific T cells caused on-target toxicity that correlated with target gene expression. Multiple measures of T cell reactivity demonstrated this toxicity on the level of T cells and hiPSC-derived target cells. In addition, phenotypic analysis illustrated interaction and crosstalk between infiltrated T cells and kidney organoids. In summary, we demonstrated the benefit of hiPSC-derived models in determining toxicity risks of tumor-specific T cells. Furthermore, our data emphasizes the additional value of other measures of T cell reactivity on top of the commonly used cytokine levels.

Correction: Nemade, H.; et al. Cyclooxygenases Inhibitors Efficiently Induce Cardiomyogenesis in Human Pluripotent Stem Cells. Cells 2020, 9, 554.

The authors wish to make the following corrections to this paper [...].