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MicroRNAs (miRNAs) are short (~ 22 nts) RNAs that regulate gene expression via binding to mRNA. MiRNAs promoting cancer are known as oncomiRs. Targeting oncomiRs is an emerging area of cancer therapy. OncomiR-21 and oncomiR-155 are highly upregulated in lymphoma cells, which are dependent on these oncomiRs for survival. Targeting specific miRNAs and determining their effect on cancer cell progression and metastasis have been the focus of various studies. Inhibiting a single miRNA can have a limited effect, as there may be other overexpressed miRNAs present that may promote tumor proliferation. Herein, we target miR-21 and miR-155 simultaneously using nanoparticles delivered two different classes of antimiRs: phosphorothioates (PS) and peptide nucleic acids (PNAs) and compared their efficacy in lymphoma cell lines.Poly-Lactic-co-Glycolic acid (PLGA) nanoparticles (NPs) containing PS and PNA-based antimiR-21 and -155 were formulated, and comprehensive NP characterizations: morphology (scanning electron microscopy), size (differential light scattering), and surface charge (zeta potential) were performed. Cellular uptake analysis was performed using a confocal microscope and flow cytometry analysis. The oncomiR knockdown and the effect on downstream targets were confirmed by gene expression (real time-polymerase chain reaction) assay.We demonstrated that simultaneous targeting with NP delivered PS and PNA-based antimiRs resulted in significant knockdown of miR-21 and miR-155, as well as their downstream target genes followed by reduced cell viability ex vivo.This project demonstrated that targeting miRNA-155 and miR-21 simultaneously using nanotechnology and a diverse class of antisense oligomers can be used as an effective approach for lymphoma therapy.

Absorption, distribution, metabolism, and excretion (ADME) are the key biologic processes for determination of a drug's pharmacokinetic parameters, which have direct impacts on efficacy and adverse drug reactions (ADRs). The chemical structures, dosage forms, and sites and routes of administration are the principal determinants of ADME profiles and consequent impacts on their efficacy and ADRs. Newly developed large molecule biologic antisense oligonucleotide (ASO) drugs have completely unique ADME that is not fully defined. ASO-based drugs are single-stranded synthetic antisense nucleic acids with diverse modes of drug actions from induction of mRNA degradation, exon skipping and restoration, and interactions with proteins. ASO drugs have a great potential to treat certain human diseases that have remained untreatable with small molecule-based drugs. The ADME of ASO drugs contributes to their unique set of ADRs and toxicity. In this review, to better understand their ADME, the 10 US Food and Drug Administration (FDA)-approved ASO drugs were selected: fomivirsen, pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. A meta-analysis was conducted on their formulation, dosage, sites of administration, local and systematic distribution, metabolism, degradation, and excretion. Membrane permeabilization through endocytosis and nucleolytic degradation by endonucleases and exonucleases are major ADME features of the ASO drugs that differ from small-molecule drugs. The information summarized here provides comprehensive ADME characteristics of FDA-approved ASO drugs, leading to a better understanding of their therapeutic efficacy and their potential ADRs and toxicity. Numerous knowledge gaps, particularly on cellular uptake and subcellular trafficking and distribution, are identified, and future perspectives and directions are discussed. SIGNIFICANCE STATEMENT: Through a systematic analysis of the existing information of absorption, distribution, metabolism, and excretion (ADME) parameters for 10 US Food and Drug Administration (FDA)-approved antisense oligonucleotide (ASO) drugs, this review provides an overall view of the unique ADME characteristics of ASO drugs, which are distinct from small chemical drug ADME. This knowledge is useful for discovery and development of new ASO drugs as well as clinical use of current FDA-approved ASO drugs.

There is large interindividual variability in the efficacy of pre-exposure prophylaxis (PrEP) with tenofovir (TFV) in preventing HIV infection. Naturally occurring mutations in the creatine kinase M-type (CKM) gene examined by Mosher et al. could provide answers to why some individuals who have active serum creatine kinase in clinical assessments may not respond to TFV PrEP.

The maintenance of homeostasis of cytochromes P450 enzymes (P450s) under both physiologic and xenobiotic exposure conditions is ensured by the action of positive and negative regulators. In the current study, the hepatocyte nuclear factor 4α (HNF4A) antisense RNA 1 (HNF4A-AS1), an antisense long noncoding RNA of HNF4A, was found to be a negative regulator of the basal and rifampicin (RIF)-induced expression of nuclear receptors and downstream P450s. In Huh7 cells, knockdown of HNF4A-AS1 resulted in elevated expression of HNF4A, pregnane X receptor (PXR), and P450s (including CYP3A4) under both basal and RIF-induced conditions. Conversely, overexpression of HNF4A-AS1 led to decreased basal expression of constitutive androstane receptor, aryl hydrocarbon receptor, PXR, and all studied P450s. Of note, significantly diminished induction levels of PXR and CYP1A2, 2C8, 2C19, and 3A4 by RIF were also observed in HNF4A-AS1 plasmid-transfected Huh7 cells. Moreover, the negative feedback of HNF4A on HNF4A-AS1-mediated gene expression was validated using a loss-of-function experiment in this study. Strikingly, our data showed that increased enrichment levels of histone 3 lysine 4 trimethylation and HNF4A in the CYP3A4 promoter contribute to the elevated CYP3A4 expression after HNF4A-AS1 knockdown. Overall, the current study reveals that histone modifications contribute to the negative regulation of nuclear receptors and P450s by HNF4A-AS1 in basal and drug-induced levels. SIGNIFICANCE STATEMENT: Utilizing loss-of-function and gain-of-function experiments, the current study systematically investigated the negative regulation of HNF4A-AS1 on the expression of nuclear receptors (including HNF4A, constitutive androstane receptor, aryl hydrocarbon receptor, and pregnane X receptor) and P450s (including CYP1A2, 2E1, 2B6, 2D6, 2C8, 2C9, 2C19, and 3A4) in both basal and rifampicin-induced levels in Huh7 cells. Notably, this study is the first to reveal the contribution of histone modification to the HNF4A-AS1-mediated expression of CYP3A4 in Huh7 cells.

Due to the lack of treatment options for the genetic disease primary hyperoxaluria (PH), including three subtypes PH1, PH2, and PH3, caused by accumulation of oxalate forming kidney stones, there is an urgent need for the development of a drug therapy aside from siRNA drug lumasiran for patients with PH1. After the recent success of drug therapies based on small interfering RNA (siRNA), nedosiran is currently being developed for the treatment of three types of PH as a siRNA-based modality. Through specific inhibition of lactate dehydrogenase enzyme, the key enzyme in biosynthesis of oxalate in liver, phase 1, 2, and 3 clinical trials of nedosiran have achieved the desired primary end point of reduction of urinary oxalate levels in patients with PH1. More PH2 and PH3 patients need to be tested for efficacy. It has also produced a favorable secondary end point on safety and toxicity in PH patients. In addition to common injection site reactions that resolved spontaneously, no severe nedosiran treatment-associated adverse events were reported. Based on the positive results in the clinical studies, nedosiran is a candidate siRNA drug to treat PH patients.

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography–tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography–quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males. SIGNIFICANCE STATEMENT: The current study in an animal model demonstrates that acetaminophen-induced liver injury results in decreased expression and enzyme activities of several examined drug-metabolizing cytochrome P450s (P450s). The extent of such decreases is correlated to the degree of liver injury severity. The generated data may be translated to human health for patients who have drug-induced liver injury with decreased capability to metabolize drugs by certain P450s.

The prevalence of obesity-associated conditions raises new challenges in clinical medication. Although altered expression of drug-metabolizing enzymes (DMEs) has been shown in obesity, the impacts of obese levels (overweight, obesity, and severe obesity) on the expression of DMEs have not been elucidated. Especially, limited information is available on whether parental obese levels affect ontogenic expression of DMEs in children. Here, a high-fat diet (HFD) and three feeding durations were used to mimic different obese levels in C57BL/6 mice. The hepatic expression of five nuclear receptors (NRs) and nine DMEs was examined. In general, a trend of induced expression of NRs and DMEs (except for Cyp2c29 and 3a11) was observed in HFD groups compared to low-fat diet (LFD) groups. Differential effects of HFD on the hepatic expression of DMEs were found in adult mice at different obese levels. Family-based dietary style of an HFD altered the ontogenic expression of DMEs in the offspring older than 15 days. Furthermore, obese levels of parental mice affected the hepatic expression of DMEs in offspring. Overall, the results indicate that obese levels affected expression of the DMEs in adult individuals and that of their children. Drug dosage might need to be optimized based on the obese levels., Graphical abstract A high-fat diet and three feeding durations were used to mimic different obese levels in mice. The hepatic expression of five nuclear receptors and nine drug-metabolizing enzymes (DMEs) was examined. The results indicate that obese levels affected expression of the DMEs in adult individuals and that of their children. Drug dosage might need to be optimized based on the obese levels.Image 1

Precision medicine is a rapidly-developing modality of medicine in human healthcare. Based on each patient׳s unique characteristics, more accurate dosages and drug selection can be made to achieve better therapeutic efficacy and less adverse reactions in precision medicine. A patient׳s individual parameters that affect drug transporter action can be used to develop a precision medicine guidance, due to the fact that therapeutic efficacy and adverse reactions of drugs can both be affected by expression and function of drug transporters on the cell membrane surface. The purpose of this review is to summarize unique characteristics of human breast cancer resistant protein (BCRP) and the genetic variability in the BCRP encoded gene ABCG2 in the development of precision medicine. Inter-individual variability of BCRP/ABCG2 can impact choices and outcomes of drug treatment for several diseases, including cancer chemotherapy. Several factors have been implicated in expression and function of BCRP, including genetic, epigenetic, physiologic, pathologic, and environmental factors. Understanding the roles of these factors in controlling expression and function of BCRP is critical for the development of precision medicine based on BCRP-mediated drug transport. Keywords: BCRP, Epigenetics, Gene polymorphisms, Physiologic factors, Precision medicine

Mammals have developed a variety of antioxidant systems to protect them from the oxygen environment and toxic stimuli. Little is known about the mRNA abundance of antioxidant components during postnatal development of the liver. Therefore, the purpose of this study was to compare the mRNA abundance of antioxidant components during liver development. Livers from male C57BL/6J mice were collected at 12 ages from prenatal to adulthood. The transcriptome was determined by RNA-Seq with transcript abundance estimated by Cufflinks. RNA-Seq provided a complete, more accurate, and unbiased quantification of the transcriptome. Among 33 known antioxidant components examined, three ontogeny patterns of liver antioxidant components were observed: (1) Prenatal-enriched, in which the mRNAs decreased from fetal livers to adulthood, such as metallothionein and heme oxygenase-1; (2) adolescent-rich and relatively stable expression, such as peroxiredoxins; and (3) adult-rich, in which the mRNA increased with age, such as catalase and superoxide dismutase. alternative splicing of several antioxidant genes, such as Keap1, Glrx2, Gpx3, and Txnrd1, were also detected by RNA-Seq. In summary, RNA-Seq revealed the relative abundance of hepatic antioxidant enzymes, which are important in protecting against the deleterious effects of oxidative stress.

The market for large molecule biologic drugs has grown rapidly, including antisense oligonucleotide (ASO) drugs. ASO drugs work as single-stranded synthetic oligonucleotides that reduce production or alter functions of disease-causing proteins through various mechanisms, such as mRNA degradation, exon skipping, and ASO-protein interactions. Since the first ASO drug, fomivirsen, was approved in 1998, the U.S. Food and Drug Administration (FDA) has approved 10 ASO drugs to date. Although ASO drugs are efficacious in treating some diseases that are untargetable by small-molecule chemical drugs, concerns on adverse drug reactions (ADRs) and toxicity cannot be ignored. Illustrative of this, mipomersen was recently taken off the market due to its hepatotoxicity risk. This paper reviews ADRs and toxicity from FDA drug labeling, preclinical studies, clinical trials, and postmarketing real-world studies on the 10 FDA-approved ASO drugs, including fomivirsen and pegaptanib, mipomersen, nusinersen, inotersen, defibrotide, eteplirsen, golodirsen, viltolarsen, and casimersen. Unique and common ADRs and toxicity for each ASO drug are summarized here. The risk of developing hepatotoxicity, kidney toxicity, and hypersensitivity reactions co-exists for multiple ASO drugs. Special precautions need to be in place when certain ASO drugs are administrated. Further discussion is extended on studying the mechanisms of ADRs and toxicity of these drugs, evaluating the existing physiologic and pathologic states of patients, optimizing the dose and route of administration, and formulating personalized treatment plans to improve the clinical utility of FDA-approved ASO drugs and discovery and development of new ASO drugs with reduced ADRs. SIGNIFICANCE STATEMENT: The current review provides a comprehensive analysis of unique and common ADRs and the toxicity of FDA-approved ASO drugs. The information can help better manage the risk of severe hepatotoxicity, kidney toxicity, and hypersensitivity reactions in the usage of currently approved ASO drugs and the discovery and development of new and safer ASO drugs.

More than two decades after the natural gene-silencing mechanism of RNA interference was elucidated, small interfering RNA (siRNA)-based therapeutics have finally broken into the pharmaceutical market. With three agents already approved and many others in advanced stages of the drug development pipeline, siRNA drugs are on their way to becoming a standard modality of pharmacotherapy. The majority of late-stage candidates are indicated for rare or orphan diseases, whose patients have an urgent need for novel and effective therapies. Additionally, there are agents that have the potential to meet the need of a broader population. Inclisiran, for instance, is being developed for hypercholesterolemia and has shown benefit in patients who are uncontrolled even after maximal statin therapy. This review provides a brief overview of mechanisms of siRNA action, physiological barriers to its delivery and activity, and the most common chemical modifications and delivery platforms used to overcome these barriers. Furthermore, this review presents comprehensive profiles of the three approved siRNA drugs (patisiran, givosiran, and lumasiran) and the seven other siRNA candidates in Phase 3 clinical trials (vutrisiran, nedosiran, inclisiran, fitusiran, teprasiran, cosdosiran, and tivanisiran), summarizing their modifications and delivery strategies, disease-specific mechanisms of action, updated clinical trial status, and future outlooks.

The maintenance of homeostasis of cytochromes P450 enzymes (P450s) under both physiologic and xenobiotic exposure conditions is ensured by the action of positive and negative regulators. In the current study, the hepatocyte nuclear factor 4

Obesity and nonalcoholic fatty liver disease (NAFLD) affect expression and function of cytochrome P450 genes (P450s). The increased expression of inflammatory cytokines is a major driver of the downregulation of P450 expression in NAFLD. Decrease in P450 expression could potentially lead to drug-drug interaction, inefficient pharmacological effect of a drug, or hepatotoxicity. An epigenetic modifier, histone 3 lysine 9 methyl transferase enzyme (G9a), known to increase histone 3 lysine 9 methylation, is downregulated in diet-induced obesity animal models. In a liver-specific G9a knockout animal model, expression of P450s was downregulated. Currently, the role of G9a in regulation of P450s in steatosis is unknown. Our hypothesis is that in steatosis G9a plays a role in downregulation of P450 expression. In this study, we used HepaRG cells to induce steatosis using a combination of free fatty acids oleic acid and palmitic acid. The G9a was knocked down and overexpressed using small interfering RNA and adenovirus mediated approaches, respectively. Knockdown and overexpression of G9a in the absence of steatosis decreased and increased expression of nuclear receptors constitutive androstane receptor (CAR), pregnane X receptor, small heterodimer partner, and CYP2B6, 2E1, 2C8, 2C9, and 3A4, respectively. In steatotic conditions, overexpression of G9a prevented fatty acid mediated decreased expression of CAR, CYP2C19, 2C8, 7A1, and 3A4. Our current study suggests that G9a might serve as a key regulator of P450 expression at both the basal level and in early steatotic conditions. Single nucleotide polymorphism of G9a leading to loss/gain of function could lead to the poor metabolizer or ultrarapid metabolizer phenotypes. SIGNIFICANCE STATEMENT: The current study demonstrates that histone modification enzyme G9a is involved in the regulation of expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and small heterodimer partner as well as drug-metabolizing cytochrome P450s (P450s) at basal conditions and in fatty acid induced cellular model of steatosis. Histone 3 lysine 9 methylation should be considered together with histone 3 lysine 4 and histone 3 lysine 27 methylation as the epigenetic mechanisms controlling gene expression of P450s.

Long noncoding RNAs (lncRNAs) are RNAs with a length of over 200 nucleotides that do not have protein-coding abilities. Recent studies suggest that lncRNAs are highly involved in physiological functions and diseases. lncRNAs HNF1α-AS1 and HNF4α-AS1 are transcripts of lncRNA genes HNF1α-AS1 and HNF4α-AS1, which are antisense lncRNA genes located in the neighborhood regions of the transcription factor (TF) genes HNF1α and HNF4α, respectively. HNF1α-AS1 and HNF4α-AS1 have been reported to be involved in several important functions in human physiological activities and diseases. In the liver, HNF1α-AS1 and HNF4α-AS1 regulate the expression and function of several drug-metabolizing cytochrome P450 (P450) enzymes, which also further impact P450-mediated drug metabolism and drug toxicity. In addition, HNF1α-AS1 and HNF4α-AS1 also play important roles in the tumorigenesis, progression, invasion, and treatment outcome of several cancers. Through interacting with different molecules, including miRNAs and proteins, HNF1α-AS1 and HNF4α-AS1 can regulate their target genes in several different mechanisms including miRNA sponge, decoy, or scaffold. The purpose of the current review is to summarize the identified functions and mechanisms of HNF1α-AS1 and HNF4α-AS1 and to discuss the future directions of research of these two lncRNAs.

Expression of cytochrome P450s (P450s) is regulated by epigenetic factors, such as DNA methylation, histone modifications, and noncoding RNAs through different mechanisms. Among these factors, long noncoding RNAs (lncRNAs) have been shown to play important roles in the regulation of gene expression; however, little is known about the effects of lncRNAs on the regulation of P450 expression. The aim of this study was to explore the role of lncRNAs in the regulation of P450 expression by using human liver tissues and hepatoma Huh7 cells. Through lncRNA microarray analysis and quantitative polymerase chain reaction in human liver tissues, we found that the lncRNA hepatocyte nuclear factor 1 alpha antisense 1 (HNF1α-AS1), an antisense RNA of HNF1α, is positively correlated with the mRNA expression of CYP2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 as well as pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Gain- and loss-of-function studies in Huh7 cells transfected with small interfering RNAs or overexpression plasmids showed that HNF1α not only regulated the expression of HNF1α-AS1 and P450s, but also regulated the expression of CAR, PXR, and aryl hydrocarbon receptor (AhR). In turn, HNF1α-AS1 regulated the expression of PXR and most P450s without affecting the expression of HNF1α, AhR, and CAR. Moreover, the rifampicin-induced expression of P450s was also affected by HNF1α and HNF1α-AS1. In summary, the results of this study suggested that HNF1α-AS1 is involved in the HNF1α-mediated regulation of P450s in the liver at both basal and drug-induced levels.

Acetaminophen (APAP) is a commonly used over-the-counter drug for its analgesic and antipyretic effects. However, APAP overdose leads to severe APAP-induced liver injury (AILI) and even death as a result of the accumulation of N-acetyl-p-benzoquinone imine, the toxic metabolite of APAP generated by cytochrome P450s (P450s). Long noncoding RNAs HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1) are regulatory RNAs involved in the regulation of P450 expression in both mRNA and protein levels. This study aims to determine the impact of HNF1α-AS1 and HNF4α-AS1 on AILI. Small hairpin RNAs were used to knock down HNF1α-AS1 and HNF4α-AS1 in HepaRG cells. Knockdown of these lncRNAs altered APAP-induced cytotoxicity, indicated by MTT and LDH assays. Specifically, HNF1α-AS1 knockdown decreased APAP toxicity with increased cell viability and decreased LDH release, whereas HNF4α-AS1 knockdown exacerbated APAP toxicity, with opposite effects in the MTT and LDH assays. Alterations on gene expression by knockdown of HNF1α-AS1 and HNF4α-AS1 were examined in several APAP metabolic pathways, including CYP1A2, CYP2E1, CYP3A4, UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Knockdown of HNF1α-AS1 decreased mRNA expression of CYP1A2, 2E1, and 3A4 by 0.71-fold, 0.35-fold, and 0.31-fold, respectively, whereas knockdown of HNF4α-AS1 induced mRNAs of CYP1A2, 2E1, and 3A4 by 1.3-fold, 1.95-fold, and 1.9-fold, respectively. These changes were also observed in protein levels. Knockdown of HNF1α-AS1 and HNF4α-AS1 had limited effects on the mRNA expression of UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Altogether, our study suggests that HNF1α-AS1 and HNF4α-AS1 affected AILI mainly through alterations of P450-mediated APAP biotransformation in HepaRG cells, indicating an important role of the lncRNAs in AILI. SIGNIFICANCE STATEMENT: The current research identified two lncRNAs, hepatocyte nuclear factor 1α antisense RNA 1 and hepatocyte nuclear factor 4α antisense RNA 1, which were able to affect susceptibility of acetaminophen (APAP)-induced liver injury in HepaRG cells, possibly through regulating the expression of APAP-metabolizing cytochrome P450 enzymes. This discovery added new factors, lncRNAs, which can be used to predict cytochrome P450-mediated drug metabolism and drug-induced toxicity.

The role of intestine-derived factors in promoting liver regeneration after partial hepatectomy (PHx) are not entirely known, but bile acids (BAs) and fibroblast growth factor 15 (Fgf15) that is highly expressed in the mouse ileum could promote hepatocyte proliferation. Fgf15 strongly suppresses the synthesis of BAs, and emerging evidence indicates that Fgf15 is important for liver regeneration. The mechanisms by which Fgf15 promotes liver regeneration are unclear, but Fgf15 may do so indirectly by reducing BA levels and/or directly by promoting cell proliferation. However, it remains undetermined whether these two mechanisms are independent or integrated. In this study, we aimed to clarify these relationships by generating Fgf15 Tet-Off, transgenic mice (Fgf15 Tg) that had very low BA levels as a result from overexpressed Fgf15-mediated suppression of BA synthesis. Compared with wild-type mice, the Fgf15 Tg mice showed increased hepatocyte proliferation even without surgery, and a further induction of the genes in cell-cycle progression after PHx. Moreover, overexpression of Fgf15 by adeno-associated virus (AAV)-Fgf15 transduction or treatment with the recombinant Fgf15 protein led to increased cell proliferation in vivo. Furthermore, Fgf15 Tg mice exhibited an earlier and greater activation of mitogen-activated protein kinase, signal transducer and activator of transcription 3, and NF-κB signaling pathways in the priming stage, and a disruption of the hippo signaling pathway in the termination stage of liver regeneration. Conclusion: Direct in vivo evidence demonstrates that Fgf15 is critical in stimulating the phases of priming and termination of liver regeneration that are critical for cell survival and liver-size determination, independent of BA levels. (Hepatology 2018; 00:000-000).