Abelcunningham7436

In contrast to the European Union and the USA, no laws or regulations mandating pediatric drug development have been established in Japan. Based on the information on drugs approved for pediatric indications in Europe and Japan, we evaluated the recent status of pediatric drug approvals and their characteristics in Japan in comparison with those of Europe.

Drugs approved for pediatric indications between 2007 and 2015 in both regions were included in the study. The proportion of drugs with pediatric indications was calculated by the Anatomical Therapeutic Chemical (ATC) classification, and the status of pediatric formulation development was examined. The time from adult to pediatric indication approval was determined.

A total of 135 drugs were approved for pediatric indications in Europe, with 208 approved in Japan. The proportion of drugs with pediatric indications in Japan among those approved for pediatric indications in Europe was lower among those with ATC classifications of N (Nervous system) and J (Antiinfectives for systemic use) and those with the development of pediatric formulations than among others. Excepting drugs for which adult and pediatric indications were simultaneously approved, the most commonly observed period from the adult indication approval to the pediatric indication approval was more than 12years in Japan and 3-6years in Europe.

The present findings suggested that pediatric development is indeed being promoted in Japan. However, the period from adult to pediatric indication approval was longer in Japan than in Europe, and the development of pediatric drugs for certain diseases has been sluggish, indicating room for further improvement.

The present findings suggested that pediatric development is indeed being promoted in Japan. However, the period from adult to pediatric indication approval was longer in Japan than in Europe, and the development of pediatric drugs for certain diseases has been sluggish, indicating room for further improvement.Antisense oligonucleotide (ASO)-mediated therapy is promising for the treatment of a variety of genetic disorders, such as Duchenne muscular dystrophy. As more ASOs advance in therapeutic development and enter clinical trials, it becomes necessary to have a means of quantifying their amounts in biological samples post-treatment. This information will be valuable for evaluating the safety and pharmacokinetic profiles of ASOs, and in deciding how the efficacy of these drugs can be improved. Gapmers are a class of ASOs characterized by having a central DNA portion that is surrounded by chemically modified nucleotides on both ends. While relatively simple and accessible methods to quantify other ASOs such as phosphorodiamidate morpholino oligomers (PMOs) using enzyme-linked immunosorbent assay (ELISA)-based techniques are available and have been used for in vivo studies, no such method is available for gapmers to our knowledge. Here, we describe a sensitive ELISA protocol that can be used to quantify the levels of locked nucleic acid (LNA) gapmers in mouse muscle tissue.Allele-specific gene silencing by antisense oligonucleotide (ASO) or small interference RNA (siRNA) has been used as a therapeutic approach for conditions caused by dominant gain-of-function mutations. We here present an antisense approach using gapmer ASO to diminish the dominant-negative effect in Ullrich congenital muscular dystrophy (UCMD) caused by dominant mutation in one of the COL6A genes. We provide the details of methods that our lab has used. The methods comprise the design of gapmer ASOs and the in vitro evaluation of gapmer ASOs on the specific silencing of the mutant allele at mRNA levels, and functional assessment at protein levels. A fibroblast cell line cultured from a UCMD patient carrying a dominant mutation in one of the COL6A genes is used as a cellular model.Delivery of conventional antisense oligonucleotides or small interfering RNA (siRNA) molecules into hematolymphoid cells for targeted gene silencing has been proven to be difficult. Here, we describe a simple protocol to knockdown specific gene(s) in malignant hematolymphoid cells using "GapmeR." This protocol could be applicable to a wide range of cell-types and thus solves an important problem for researchers working with cell lines or primary cells derived from patients with hematolymphoid malignancies.Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), have a complex genetic background, in addition to cases where the disease appears to manifest sporadically. The recent discovery of the hexanucleotide repeat expansion in the C9orf72 gene as the causative agent of ALS (C9ALS) gives rise to the opportunity to develop new therapies directed at this mutation , which is responsible for a large proportion of ALS and/or frontotemporal dementia cases. Mammalian models conscientiously replicating the late-onset motor defects and cellular pathologies seen in human patients do not exist. In this context, patient-derived cells give us a platform to test potential antisense oligonucleotide therapies, which could be the key to treat this subtype of motor neuron disease. Recently, we described that locked nucleic acid gapmer oligonucleotide-based treatment targeting C9orf72 repeat expanded transcripts resulted in recovery from the disease-related phenotypes in patient-derived fibroblasts. Our findings highlight the therapeutic potential of C9ALS using this gapmer oligonucleotide-based approach.This chapter describes the use of locked nucleic acid (LNA) GapmeRs for the in vivo knockdown of specific mRNAs in the mouse liver and phenotype analysis. LNA GapmeRs may be tested for efficacy by transfection in cultured cells. read more They are delivered into mice in vivo by intravenous tail injection .Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.Antisense oligonucleotides (ASOs) are widely used for the identification of gene functions and regulation of genes involved in different diseases for therapeutic purposes. For in vitro evaluation of the knockdown activity of gapmer ASOs, we often use lipofection or electroporation to deliver gapmer ASOs into the cells. Here, we describe a method for evaluating the knockdown activity of gapmer ASOs by a cell-free uptake mechanism, termed as gymnosis, using MALAT1 gapmer ASOs modified with 2'-O-methoxyethyl RNA (2'-MOE) or 2'-O,4'-C-ethylene-bridged nucleic acid (ENA). This method is robust because it does not involve the use of any transfection reagent and has minimal effects on cell growth. Further, we describe a convenient technique for performing one-step reverse transcription and real-time qPCR using cell lysates without RNA extraction. Data for up to 96 samples can be obtained following these methods.Oligonucleotide drugs (ODs) have gained increasing attention owing to their promising therapeutic potential. One major obstacle that ODs have been facing is the lack of appropriate in vitro validation systems that can predict in vivo activity and toxicity. We have devised a transfection method called CEM (Ca2+-enrichment method), where the simple enrichment of calcium ion with calcium chloride in culture medium potentiates the activity of various types of naked oligonucleotides including gapmers, siRNA, and phosphorodiamidate morpholino antisense oligonucleotides (PMO) in many cultured cell lines with limited cytotoxicity. We here describe a precise procedure of the method. Besides the benefit of the CEM's predictive power to accurately estimate in vivo activity of ODs of your interest in drug discovery and development settings, this cost-efficient, easy-to-access method can be a robust laboratory technique to modulate gene expressions with ODs with a variety of mechanisms of action.Long noncoding RNAs (lncRNAs) are a recently discovered class of RNA that have diverse intracellular regulatory and structural roles. Because of their wide assortment of functions, lncRNAs can have varied distributions in the nucleus and/or cytoplasm of a cell. However, even though tens of thousands of human lncRNAs have been identified, currently less than 3% have empirically validated functions. RNA knockdown is now a relatively commonplace laboratory technique used to functionally characterize an RNA. These techniques (most commonly antisense therapy and RNA interference) can even have therapeutic benefit to treat a wide variety of genetic or infectious diseases as evidenced by the several RNA knockdown reagents currently in clinical trials. This protocol describes the use of validated gapmer antisense oligonucleotides (ASOs) to knockdown human MALAT1, a nuclear-retained lncRNA that is upregulated in multiple cancer cells. Methods used include cationic lipid transfection into HeLa cells, RNA isolation, and RT-qPCR analysis of the RNA knockdown levels.Heteroduplex oligonucleotides (HDOs) were a novel type of nucleic acid drugs based on an antisense oligonucleotide (ASO) strand and its complementary RNA (cRNA ) strand. HDOs were originally designed to improve the properties of RNase H-dependent ASOs and we reported in our first paper that HDOs conjugated with an α-tocopherol ligand (Toc-HDO ) based on a gapmer ASO showed 20 times higher silencing effect to liver apolipoprotein B (apoB) mRNA in vivo than the parent ASO. Thereafter the HDO strategy was found to be also effective for improving the properties of ASOs modulating blood-brain barrier function and ASO antimiRs which are RNase H-independent ASOs. Therefore, the HDO strategy has been shown to be versatile technology platform to develop effective nucleic acid drugs.Myotonic dystrophy (DM) types 1 (DM1) and 2 (DM2) are caused by autosomal dominant gain-of-function RNA which are, in turn, created by the expansion of repeat sequences in the DMPK and ZNF9 genes, respectively. The expansions are highly unstable and biased for further expansion in somatic cells and across generations. Despite the different genes involved, DM1 and DM2 share several clinical features due to having the similar underlying mechanism of repetitive RNA-mediated toxicity. Both disorders manifest as multisystemic conditions with features including myotonia, cataract development, and abnormalities in cardiac conduction. At present, there is no cure for DM and treatments mostly aim at symptom management. Among the therapeutics being developed, antisense therapy using gapmers is one of the most promising. Compared to other antisense oligonucleotides, gapmers maintain the ability to induce RNase H cleavage while having enhanced target binding affinity and nuclease resistance. This chapter will consolidate the different strategies studied thus far to develop a treatment for DM1 through the targeting of toxic repetitive RNA using gapmers.