Is the forming of neuronal network activity in human-induced pluripotent stem cells important for the detection of drug-induced seizure risks?

BACKGROUND: Functional network activity is a characteristic for neuronal cells, and the complexity of the network activity represents the necessary substrate to support complex brain functions. Drugs that drastically increase the neuronal network activity may have a potential higher risk for seizures in human. Although there has been some recent considerable progress made using cultures from different types of human-induced pluripotent stem cell (hiPSC) derived neurons, one of the primary limitations is the lack of - or very low - network activity. METHOD: In the present study, we investigated whether the limited neuronal network activity in commercial hiPSC-neurons (CNS.4U®) is capable of detecting drug-induced potential seizure risks. Therefore, we compared the hiPSC-results to those in rat primary neurons with known high neuronal network activity in vitro. RESULTS: Gene expression and electrical activity from in vitro developing neuronal networks were assessed at multiple time-points. Transcriptomes of 7, 28, and 50 days in vitro were analyzed and compared to those from human brain tissues. Data from measurements of electrical activity using multielectrode arrays (MEAs) indicate that neuronal networks matured gradually over time, albeit in hiPSC this developed slower than rat primary cultures. The response of neuronal networks to neuronal active reference drugs modulating glutamatergic, acetylcholinergic and GABAergic pathways could be detected in both hiPSC-neurons and rat primary neurons. However, in comparison, GABAergic responses were limited in hiPSC-neurons. CONCLUSION: Overall, despite a slower network development and lower network activity, CNS.4U® hiPSC-neurons can be used to detect drug induced changes in neuronal network activity, as shown by well-known seizurogenic drugs (affecting e.g., the Glycine receptor and Na+ channel). However, lower sensitivity to GABA antagonists has been observed.

Identification of a series of substituted 2-piperazinyl-5-pyrimidylhydroxamic acids as potent histone deacetylase inhibitors.

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Real-time gene expression analysis in human xenografts for evaluation of histone deacetylase inhibitors.

Real-time analysis of gene expression in experimental tumor models represents a major tool to document disease biology and evaluate disease treatment. However, monitoring gene regulation in vivo still is an emerging field, and thus far it has not been linked to long-term tumor growth and disease outcome. In this report, we describe the development and validation of a fluorescence-based gene expression model driven by the promoter of the cyclin-dependent kinase inhibitor p21waf1,cip1. The latter is a key regulator of tumor cell proliferation and a major determinant in the response to many anticancer agents such as histone deacetylase inhibitors. In response to histone deacetylase inhibitors, induction of fluorescence in A2780 ovarian tumors could be monitored in living mice in a noninvasive real-time manner using whole-body imaging. Single p.o. administration of the histone deacetylase inhibitor MS-275 significantly induces tumor fluorescence in a time- and dose-dependent manner, which accurately predicted long-term antitumoral efficacy in individual mice following extended treatment. These findings illustrate that this technology allows monitoring of the biological response induced by treatment with histone deacetylase inhibitors. In addition to providing experimental pharmacokinetic/pharmacodynamic markers for investigational drugs, this model provides insight into the kinetics of in vivo regulation of transcription, which plays a key role in causing and maintaining the uncontrolled proliferation of tumor tissue.

Discovery of pyrimidyl-5-hydroxamic acids as new potent histone deacetylase inhibitors.

A series of pyrimidyl-5-hydroxamic acids was prepared for evaluation as inhibitors of histone deacetylase (HDAC). Amino-2-pyrimidinyl can be used as a linker to provide HDAC inhibitors of good enzymatic potency.

Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles.

Short nucleic acid sequences specific to oncogene targets such as bcl-2, bcr-abl, and c-myc have been shown to exhibit specific anti-cancer activity in vitro through antigene or antisense activity. Efficient in vivo delivery of oligonucleotides remains a major limitation for the therapeutic application of these molecules. We report herein on the preparation of transferrin-modified nanoparticles containing DNAzymes (short catalytic single-stranded DNA molecules) for tumor targeting as well as their biodistribution using various methods of administration in the mouse. Linear, beta-cyclodextrin-based polymers are complexed with DNAyzme molecules to form sub-50 nm particles termed "polyplexes". The surface properties of the cyclodextrin-containing polyplexes are modified by exploiting the ability of the beta-cyclodextrin substructure and adamantane to form inclusion complexes. Accordingly, conjugates of adamantane with poly(ethylene glycol) (PEG) are prepared and combined with the polyplexes. The adamantane form inclusion complexes with the surface cyclodextrins of the polyplexes to provide a sterically stabilizing layer of PEG. The stabilized polyplexes are also modified with transferrin for increasing targeting to tumor cells expressing transferrin receptors. The preparation, characterization, and in vitro application of these nanoparticles are discussed. The transferrin-polyplexes containing fluorescently-labeled DNAzyme molecules are administered to tumor-bearing nude mice and their biodistribution and clearance kinetics are monitored using a fluorescence imaging system. Four methods of administration are studied: intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. DNAzymes packaged in polyplex formulations are concentrated and retained in tumor tissue and other organs, whereas unformulated DNAzyme is eliminated from the body within 24 hours post-injection. Intravenous and intraperitoneal bolus injections result in the highest fluorescent signal (DNAzyme) at the tumor site. Tumor cell uptake is observed with intravenous bolus injection only, and intracellular delivery requires transferrin targeting.

JNJ-26481585, a novel "second-generation" oral histone deacetylase inhibitor, shows broad-spectrum preclinical antitumoral activity.

PURPOSE: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a "second-generation" class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy. EXPERIMENTAL DESIGN: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors. RESULTS: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC(50), 0.16 nmol/L). CONCLUSIONS: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising "second-generation" HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies.