Liver biomarker and in vitro assessment confirm the hepatic origin of aminotransferase elevations lacking histopathological correlate in beagle dogs treated with GABAA receptor antagonist NP260

NP260 was designed as a first-in-class selective antagonist of α4-subtype GABA_A receptors that had promising efficacy in animal models of pain, epilepsy, psychosis, and anxiety. However, development of NP260 was complicated following a 28-day safety study in dogs in which pronounced elevations of serum aminotransferase levels were observed, although there was no accompanying histopathological indication of hepatocellular injury. To further investigate the liver effects of NP260, we assayed stored serum samples from the 28-day dog study for liver specific miRNA (miR-122) as well as enzymatic biomarkers glutamate dehydrogenase and sorbitol dehydrogenase, which indicate liver necrosis. Cytotoxicity assessments were conducted in hepatocytes derived from dog, rat, and human liver samples to address the species specificity of the liver response to NP260. All biomarkers, except ALT, returned toward baseline by Day 29 despite continued drug treatment, suggesting adaptation to the initial injury. In vitro analysis of the toxicity potential of NP260 to primary hepatocytes indicated a relative sensitivity of dog > human > rat, which may explain, in part, why the liver effects were not evident in the rodent safety studies. Taken together, the data indicate that a diagnostic biomarker approach, coupled with sensitive in vitro screening strategies, may facilitate interpretation of toxicity potential when an adaptive event masks the underlying toxicity.     

Circulating microRNA 122 in the methionine and choline‐deficient mouse model of non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive form of non‐alcoholic fatty liver disease (NAFLD) and is a major cause of liver cirrhosis and hepatic failure. The methionine choline‐deficient diet (MCD) is a frequently used hepatotoxicity animal model of NASH that induces hepatic transaminase (ALT, AST) elevations and hepatobiliary histological changes similar to those observed in human NASH. Liver‐specific microRNA‐122 (miR‐122) has been shown as a key regulator of cholesterol and fatty acid metabolism in adult liver, and has recently been proposed as a sensitive and specific circulating biomarker of hepatic injury. The purpose of this study was to assess miR‐122 serum levels in mice receiving an MCD diet for 0, 3, 7, 14, 28 and 56 days and compare the performance vs. routine clinical chemistry when benchmarked against the histopathological liver findings. MiR‐122 levels were quantified in serum using RT‐qPCR. Both miR‐122 and ALT/AST levels were significantly elevated in serum at all timepoints. MiR‐122 levels increased on average by 40‐fold after 3 days of initiating the MCD diet, whereas ALT and AST changes were 4.8‐ and 3.3‐fold, respectively. In general, miR‐122 levels remained elevated across all time points, whereas the ALT/AST increases were less robust but correlated with the progressive severity of NASH as assessed by histopathology. In conclusion, serum levels of miR‐122 can potentially be used as a sensitive biomarker for the early detection of hepatotoxicity and can aid in monitoring the extent of NAFLD‐associated liver injury in mouse efficacy models. Copyright © 2013 John Wiley & Sons, Ltd.     

miR-122 Inhibits Hepatocarcinoma Cell Progression by Targeting LMNB2

In the present study, we investigated the role of miR-122 in hepatocarcinoma progression and explored the mechanism. In hepatocarcinoma tissues and cells, we used qRT-PCR to validate the miR-122 expression level. Next, we used colony formation by crystal violet staining assay to compare cell proliferation ability, and we used scratch test or Transwell assay to compare cell migration or invasion ability. We then conducted bioinformatics or luciferase reporter gene assay to prove the regulation effect of miR-122 on lamin B2 (LMNB2), and the biological function of LMNB2 was analyzed. We used nude mouse tumorigenicity assay to test the inhibition effect of miR-122 ASO therapy against hepatocarcinoma. miR-122 was reduced in hepatocarcinoma tissues compared to the paracarcinoma tissues, which was relatively low or high in hepatocarcinoma cell line SMMC7721 or Hep3B, and overexpressed miR-122 inhibited proliferation, migration, and invasion in hepatocarcinoma cells. Additionally, some reports showed that LMNB2 was regulated by miR-122, which inhibited the expression of LMNB2. Moreover, LMNB2 functioned to promote cell proliferation, migration, and invasion. We could achieve the inhibition of hepatocarcinoma using miR-122 therapy through decreasing LMNB2 expression in vivo. Our data indicated that miR-122 could inhibit hepatocellular carcinoma cell progression by targeting LMNB2 and as a therapeutic target for hepatocarcinoma treatment.     

Long Noncoding RNA UCA1 Targets miR-122 to Promote Proliferation,Migration, and Invasion of Glioma Cells

Glioma is the most common and lethal malignant intracranial tumor. Long noncoding RNAs (lncRNAs) have been identified as pivotal regulators in the tumorigenesis of glioma. However, the role of lncRNA urothelial carcinoma-associated 1 (UCA1) in glioma genesis is still unknown. The purpose of this study was to investigate the underlying function of UCA1 on glioma genesis. The results demonstrated that UCA1 was upregulated in glioma tissue and indicated a poor prognosis. UCA1 knockdown induced by si-UCA1 significantly suppressed the proliferative, migrative, and invasive activities of glioma cell lines (U87 and U251). Bioinformatics analysis and luciferase reporter assay verified the complementary binding within UCA1 and miR-122 at the 3¢-UTR. Functional experiments revealed that UCA1 acted as an miR-122 “sponge” to modulate glioma cell proliferation, migration, and invasion via downregulation of miR-122. Overall, the present study demonstrated that lncRNA UCA1 acts as an endogenous sponge of miR-122 to promote glioma cell proliferation, migration, and invasion, which provides a novel insight and therapeutic target in the tumorigenesis of glioma.     

Differentiation of antiinflammatory and antitumorigenic properties of stabilized enantiomers of thalidomide analogs

Therapeutics developed and sold as racemates can exhibit a limited therapeutic index because of side effects resulting from the undesired enantiomer (distomer) and/or its metabolites, which at times, forces researchers to abandon valuable scaffolds. Therefore, most chiral drugs are developed as single enantiomers. Unfortunately, the development of some chirally pure drug molecules is hampered by rapid in vivo racemization. The class of compounds known as immunomodulatory drugs derived from thalidomide is developed and sold as racemates because of racemization at the chiral center of the 3-aminoglutarimide moiety. Herein, we show that replacement of the exchangeable hydrogen at the chiral center with deuterium allows the stabilization and testing of individual enantiomers for two thalidomide analogs, including CC-122, a compound currently in human clinical trials for hematological cancers and solid tumors. Using “deuterium-enabled chiral switching” (DECS), in vitro antiinflammatory differences of up to 20-fold are observed between the deuterium-stabilized enantiomers. In vivo, the exposure is dramatically increased for each enantiomer while they retain similar pharmacokinetics. Furthermore, the single deuterated enantiomers related to CC-122 exhibit profoundly different in vivo responses in an NCI-H929 myeloma xenograft model. The (−)-deuterated enantiomer is antitumorigenic, whereas the (+)-deuterated enantiomer has little to no effect on tumor growth. The ability to stabilize and differentiate enantiomers by DECS opens up a vast window of opportunity to characterize the class effects of thalidomide analogs and improve on the therapeutic promise of other racemic compounds, including the development of safer therapeutics and the discovery of new mechanisms and clinical applications for existing therapeutics.Chirality plays an important role in a variety of disciplines, including pharmaceuticals, foods and flavorings, materials science, and agricultural chemicals. In pharmaceuticals, changing just one chiral center can affect critical compound properties, including potency, off-target side effects, and pharmacokinetics (1–5), thus impacting efficacy and therapeutic index. Since the 1990s, drug molecules originally developed as racemates (a racemate is a 1:1 mixture of two mirror-image compounds or enantiomers) have been separated and developed as single preferred enantiomers (eutomers) because of improved synthesis, purification, and analytical methods. This approach, known as chiral switching, resulted in several new therapeutics based on existing drugs, including esomeprazole (Nexium), escitalopram (Lexapro), levalbuterol (Xopenex), eszopiclone (Lunesta), and levomilnacipran (Fetzima). It also led to new Food and Drug Administration guidance for the characterization and development of stereoisomers (6).There are numerous racemic compounds where chiral switching is impossible, because the chiral center has an exchangeable hydrogen that interconverts on a timescale that is incompatible with storage or dosing of a single pure enantiomer. Some examples of drugs that are still marketed as a mixture of two enantiomers include thalidomide, pioglitazone, bupropion, prasugrel, donepezil, and lorazepam. For each of these molecules, the rate of interconversion of enantiomers under physiological conditions is fast compared with the elimination rate of each molecule.Immunomodulatory drugs derived from thalidomide are an important class of antiinflammatory and antitumorigenic drugs, of which thalidomide is the prototype (7, 8). These compounds (Fig. 1) are all characterized by a nitrogen-substituted 3-aminoglutarimide moiety essential for their therapeutic activity with an exchangeable hydrogen at the chiral center. In addition to thalidomide, a number of analogs, including lenalidomide (Revlimid), pomalidomide (Pomalyst and Imnovid), CC-11006, CC-122, and CC-220, have been or are being developed for the treatment of blood cancers and hematological conditions (e.g., multiple myeloma, myelodysplastic syndrome, lymphoma, and chronic lymphocytic leukemia), solid tumors, and inflammatory diseases (e.g., sarcoidosis, systemic sclerosis, and systemic lupus erythematosus). [We tentatively assign N-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-yl]methyl}cyclopropanecarboxamide (compound 1) as CC-11006, a former development compound for hematological cancers. The chemical structure is disclosed in patents and patent applications related explicitly to this 3-aminoglutarimide (9–11). Experimental data are explicitly disclosed for CC-11006 in the pharmacology review sections for new drug applications (NDAs) 021880 and 204026 corresponding to Revlimid and Pomalyst, respectively. The chemical structure of CC-122 is disclosed in figure 5b in International Patent Application No. WO 2012/125459 (12). The experimental data disclosed in this patent and other patent applications related to the explicit chemical structure (12–16) are also identical to the experimental data in published poster abstracts related to CC-122 (17, 18).]Fig. 1.Structures of thalidomide, pomalidomide, lenalidomide, and protonated and deuterated enantiomers of compound 1 [CC-11006; i.e., (S)-H-1, (R)-H-1, (S)-D-1, and (R)-D-1] as well as protonated and deuterated enantiomers of compound 2 [CC-122; (S)-H-2, ( ...The challenges of dosing and maintaining levels of a single preferred enantiomer of thalidomide analogs are apparent comparing the generally short racemization half-life (rac t_1/2) in human plasma or blood with the longer elimination half-life (elim t_1/2) of thalidomide (19–24) (rac t_1/2 = 2–6 h; elim t_1/2 = 3–8 h), lenalidomide (25–27) (rac t_1/2 < 3 h; elim t_1/2 = 3–8 h), and pomalidomide (28–30) (rac t_1/2 < 1 h; elim t_1/2 = 7.5–9.5 h). The propensity to racemize prior to elimination makes it extremely difficult to assess the properties of the individual enantiomers and impedes mechanistic studies. In fact, the broad spectrum of activity for thalidomide analogs has been extensively studied and suggests multiple target sites of action (7, 31–35). Recently, CC-122 has been defined as a pleiotropic pathway modulator (Celgene Corp) because of its broad range of activity (17, 18). In actuality, each enantiomer may have unique pharmacological and safety profiles. Although there is limited data available and the enantiomers interconvert during the time course of the studies, a few groups have shown that the teratogenicity, in vitro antiinflammatory activity, and in vivo efficacy of protonated thalidomide analogs are caused, in large part, by the (S)-enantiomer (22, 36–38). (S)-pomalidomide was originally advanced into clinical trials as ENMD 0995 (39) but soon abandoned because of the rapid racemization of the exchangeable chiral center (28, 29). Finally, it has recently been shown by X-ray crystallography that the (S)-enantiomers of thalidomide, lenalidomide, and pomalidomide preferentially bind a newly identified target, cereblon (CRBN), believed to be responsible for their efficacy and teratogenicity in a cocrystal with the DDB1–CRBN complex, where DDB1 stands for DNA damage-binding protein 1 (40).Attempts to stabilize the (S)-enantiomer of thalidomide analogs have included replacement of the exchangeable hydrogen with methyl (20, 37, 41, 42) or fluorine groups (43, 44). If and when the stable enantiomer of these analogs was studied, none were superior to the racemic, protonated thalidomide analog. The effects observed included similar or decreased potency, increased degradation, increased toxicity, and/or increased teratogenicity. In vitro stabilization of enantiomers has also been achieved by replacement of the carbonyl group adjacent to the chiral center with an oxetane (45). The impact of this change on in vivo dosing or efficacy has not been reported.Recently, deuterium has been explored to stabilize interconverting enantiomers. Deuterium is a stable isotope of hydrogen with a natural abundance of 0.015%, and it is known for its potential to stabilize chemical bonds. Therefore, deuterium is predicted to not affect the pharmacological properties of a compound, contrary to what can be anticipated with methyl, fluoro, or oxetane functional groups. Furthermore, given the natural abundance of deuterium and its ubiquitous use in past human pharmacokinetic studies, the use of deuterium in therapeutics does not present a safety concern.The use of deuterium to stabilize drugs against undesirable metabolism, known as metabolic switching, began in the 1960s (46–51) and is the predominant approach to deuterated drugs today. Metabolic switching can be a challenging strategy, because it is often difficult to translate from in vitro to in vivo (52), is limited to defined metabolic pathways, and requires the synthesis and testing of numerous analogs.The approach that we describe in this paper, deuterium-enabled chiral switching (DECS), is uniquely differentiated from metabolic switching, in that it is based on chemical stability, is generally independent of metabolism, thus resulting in little or no change in pharmacokinetics, translates from in vitro to in vivo, and requires the synthesis and testing of just two analogs.We are the only group, to our knowledge, to previously and herein report the stabilization and differentiated in vitro and in vivo properties of monodeuterated enantiomers of several thalidomide analogs, including reduced degradation, improved pharmacokinetics, and separation of in vitro antiinflammatory effects (53). Yamamoto et al. (54) previously reported the stabilization of monodeuterated thalidomide enantiomers in aqueous solution but did not differentiate their biochemical properties or their pharmacokinetic and pharmacodynamic properties. Another group has shown improved stability, pharmacokinetics, and separation of in vitro pharmacological effects with pentadeuterated lenalidomide enantiomers (55, 56). However, the latter publications do not differentiate the properties in vivo or allow discrimination of the effects of the additional four deuteria on the 3-aminoglutarimide ring and their pharmacodynamic contributions (55, 56).Herein, we report the synthesis, in vitro characterization, and differentiation of stabilized enantiomers of two unique thalidomide analogs, compounds 1 (57) and 2 (CC-122) (58), and for the first time to our knowledge, we differentiate stabilized enantiomers of thalidomide analogs in in vitro and in vivo efficacy models.     

hsa-miR-100的生物信息学分析

目的 对hsa-miR-100进行靶基因、功能预测等生物信息学分析,为深入研究hsa-miR-100功能提供理论和试验基础.方法 利用Pubmed检索miRNA-100相关文章,通过miRBase、NCBI、UCSC Browser等在线工具分析hsa-miR-100序列,应用miRanda、TargetScan及PicTar预测hsa-miR-100靶基因,结合已证实的靶基因进行功能富集分析和信号通路富集分析.结果 hsa-miR-100与多种肿瘤发生、发展有关,其序列在各物种间具有高度保守性.hsa-miR-100靶基因功能富集于基因沉默、染色质沉默、细胞生物合成负性调节等(P<0.01),涉及肿瘤信号通路、溶酶体信号通路、凋亡信号通路等信号转导通路(P<0.05).结论 hsa-miR-100可能参与肿瘤发生相关的生物学过程.