Improvement of therapeutic efficacy of siRNA drugs by altering ADME

The aim of the study is to generate strong preliminary data to support an external application to enhance therapeutic efficacy and reduce toxicity of small interfering RNA (siRNA) drugs. siRNA offers a unique therapeutic platform to target disease-associated genes for the treatment of various rare life-threatening diseases as well as some common diseases. Accompanying with success of five FDA-approved siRNA drugs in the market, lack of therapeutic efficacy and intolerance of toxicity have made numerous tested siRNA drugs to be terminated in the development pipelines in last decades. Significant knowledge gaps still exist, which prevents us from better understanding how to improve efficacy and to reduce toxicity of siRNA drugs. One of the knowledge gaps is absorption, distribution, metabolism, and excretion (ADME) of siRNA drugs. As same as small chemical drugs, the features of ADME have direct impact on therapeutic efficacy and toxicity of siRNA drugs. Differencing from the ADME of small chemical drugs mediated by phase I and II metabolizing enzymes and transporters, siRNA drugs use completely different sets of proteins for cellular uptake, subcellular distribution, metabolism, and excretion. Understanding how siRNA drugs are uptake into cells and how they are distributed intracellularly by subcellular trafficking from endosomes to lysosomes, and how they are excreted out from cells via exocytosis is critical for improving ADME features of siRNA drugs for better efficacy. Our central hypothesis is that “Altering the key proteins involved in subcellular trafficking and exocytosis has capability to increase proportion of siRNA drugs reaching to their target mRNAs for an increased therapeutic efficacy”. We will test our hypothesis in the following specific aims: (1) to better understand subcellular trafficking of siRNA drugs from early endosomes to late endosomes, to lysosomes, and to exosomes using a high-resolution stimulated emission depletion (STED) image system; and (2) to determine the role of several endosomal compartment proteins in transfer siRNA drugs from late endosomes to lysosomes, including GOLGA8N. The proposed work is significant as it will determine proteins should be therapeutic targets and can be hence given as a combination therapy with patients who are prescribed siRNA drugs. It is also significant as it will create a platform that can be used to investigate other key targets, potentially opening new avenues in the field of therapeutics. This work will create foundational resources for siRNA therapy that will also be used by other researchers.

Funded by the UConn Research Excellence Program (REP)

 

The role of lncRNAs in P450-mediated drug metabolism and drug-induced liver injury

The aim of the project is to determine the roles of long non-coding RNAs (lncRNAs) of HNF1a-AS1 and HNF4a-AS1 in the regulation of cytochrome P450 (CYP) gene expression. Drug-metabolizing CYP enzymes are responsible for therapeutic outcomes of 60-70% drugs. Understanding all key factors in the regulation of expression of the CYP enzymes is critical to predict outcomes of drug therapy. Transcription factors (TFs) of hepatocyte nuclear factor 1a (HNF1a), 4a (HNF4a), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) are the key factors. After binding to their response elements, the TFs facilitate transcriptional regulation through alterations of histone modification status of the CYP genes, the major epigenetic mechanism. A remaining question is how the TFs trigger alterations of histone modifications to facilitate the regulation of CYP expression. lncRNAs may be the key components. Increasing evidence has shown that antisense lncRNAs next to TFs on chromosomal locations are involved in the functions of the TFs in regulation of gene expression. We identified two lncRNAs next to the TFs of HNF1a and HNF4a, which are HNF1a-AS1 and HNF4a-AS1. Several fundamental challenge questions need to be addressed. (1) Whether HNF1a-AS1 and HNF4a-AS1 are the two key factors in maintaining homeostasis of P450 enzymes in liver in a dynamic process, such as drug induction, through enhancing by HNF1a-AS1 or repressing by HNF4a-AS1 in the transcriptional regulation of P450 gene expression? (2) Whether HNF1a-AS1 and HNF4a-AS1 are the two key factors in sensitizing susceptibility of drug induced liver injury (DILI) and following process of liver regeneration? (3) How HNF1a-AS1 and HNF4a-AS1 participate in the regulation of P450 gene expression, directly or indirectly through the regulation of PXR or CAR by alteration of histone modifications? These fundamental questions are so critical for deeply understanding molecular mechanisms in the regulation of CYP-mediated drug metabolism and DILI. In the past five years, we have determined the role of lncRNAs in drug metabolism. Our recent progress has placed us into an appropriate position to provide answers to these fundamental questions. We have developed a comprehensive plan to address these challenge questions with both in vitro and in vivo models in next five years. After completion of the proposed studies, we expect to provide answers to address the challenge questions. The proposed work will elucidate the epigenetic regulatory mechanisms by lncRNAs that control homeostasis of drug-metabolizing CYP expression in a positive and negative feedback loop after exposure to drugs. The proposed work will determine the roles of lncRNAs in P450-mediated drug metabolism and susceptibility to DILI. The generated knowledge will help to identify novel key factors for predicting therapeutic efficacy and DILI.

Funded by NIH/NIGMS 1R35GM140862-01

 

Short- or long-term impacts of drug exposure at early life on drug metabolism, therapeutic efficacy, and drug-induced toxicity

The NIH Funding Opportunity “PAR-13-306: Developmental Pharmacology and Toxicology: Role of Ontogeny” emphasizes the urgency of addressing the issue of “short- or long-term alterations of genes involved in pharmacokinetics in response to drug exposure in early development”. Physicians use drugs to treat various diseases in neonates and infants. However, the current clinical practices have not considered the potential short- or long-term consequences on the patient’s ability to metabolize drugs during adulthood who have received drug treatment early in life. A major reason is the lack of basic research to understand the consequences (short- or long-term) that drug exposure in early life has on drug metabolism, therapeutic efficacy, and potential for drug-induced toxicity. The goal in this study is to understand the short- or long-term impacts of drug exposure at early life on drug metabolism, therapeutic efficacy, and drug-induced toxicity. Based on the previous publications and our preliminary data, we have formed a central hypothesis that “early life exposure to drugs that have the ability to activate nuclear receptors can result in either immediate (short-term) or persistent (long-term) alterations of expression and functions of certain drug metabolizing enzymes, further leading to alterations in therapeutic efficacy and sensitivity to toxicity of other drugs. This effect is dependent on the ability of the drug exposure with a certain dose at a specific age to alter epigenetic memory in liver.” We will use phenobarbital for drug exposure at early life, omeprazole and midazolam for therapeutic efficacy, and acetaminophen for hepatotoxicity. We will focus on P450s (such as Cyp2b, Cyp2c, and Cyp3a) as our target drug metabolizing enzymes and the mouse as our animal model to examine the central hypothesis in the following specific aims. Aim 1 will determine the short- or long-term impacts of drug exposure at early life on drug metabolism. Aim 2 will determine the short- or long-term impacts of drug exposure at early life on therapeutic efficacy. Aim 3 will determine the short- or long-term impacts of drug exposure at early life on drug-induced hepatotoxicity. After completion of the specific aims, we will fill the knowledge gaps on the short- or long-term impacts of drug exposure at early life on drug metabolism. Such knowledge is critical for developmental pharmacology and toxicology to better predict outcomes of drug exposure at early ages on drug responses and sensitivity to drug-induced toxicity throughout the lifetime.

Funded by NIH/NIGMS 1R01GM118367

 

Developmental regulation of drug processing genes

Despite recent rapid progress in understanding the expression patterns and regulatory mechanisms of drug processing genes, namely drug metabolizing enzymes and transporters in adults, little is known about these in the pediatric period. The long-term goal is to understand mechanisms of ontogenic regulation of drug processing genes, so that efficacious and safe drug treatments can be achieved in children. Several factors are known to be essential for normal development, including hepatocyte nuclear factor 1α (HNF1α), farnesoid X receptor (FXR), growth hormone (GH) signaling, and epigenetic influences. HNF1α, a master regulator of early liver development, regulates hepatic expression of a large battery of drug processing genes. Initiation of bile-acid signaling pathways, mediated largely via the FXR, is a hallmark of perinatal liver development. GH is essential for postnatal hepatic gene expression and maturation. The accessibility of transcription factors to the target genes is largely determined by the methylation/acetylation status of histones and DNA sequences. Preliminary studies illustrate that in developing mouse livers, drug processing genes and transcription factors are expressed in distinct dynamic patterns and correlate with epigenetic signatures. The objective of this proposal is to elucidate the regulatory mechanisms of ontogenic expression of drug processing genes in mice. The rationale of this proposal is that its successful completion will generate basic knowledge that will serve as the foundation for further understanding pediatric pharmacology in humans. The central hypothesis is: developmental regulation of drug processing genes is a sequential event regulated by hormones, which activate transcription factors to modify epigenetic signatures and regulate gene expression. This hypothesis will be tested in 2 aims. Aim 1 will determine the ontogenic expression patterns of drug processing genes and the correlation with transcription factors and epigenetic signatures. The relative mRNA expression of major phase I/II enzymes and drug transporters in male mouse livers versus intestine and kidney will be examined, and correlated with expression of transcription factors and chromosome modifications (genome-wide DNA methylation and histone modifications). Aim 2 will elucidate roles of transcription factors and GH in determining ontogenic hepatic expression of drug processing genes in HNF1α-null, FXR-null, and GH deficiency (lit/lit) mice using the same working strategy. This study is novel, because it will use a genome-wide approach to elucidate how alterations of hormones and transcription factors modulate epigenetic signatures and hepatic ontogenic expression of drug processing genes. This study is significant, because little is known about the regulation of hepatic drug processing genes in pediatric stages. Results from this study will: 1) provide basic knowledge on the ontogenic expression patterns of drug processing genes and nuclear receptors in liver, kidney, and intestine; and 2) help to understand how perinatal alterations in hormones and nuclear receptors, via modulating epigenetic signatures, affect stage-specific and long-term expression of drug processing genes.

Funded by NIH/NIEHS 1R01ES019487

 

Control of developmental switch of Cyp3a gene expression in mouse liver

The cytochrome P450 3A subfamily (CYP3A) members, such CYP3A4, CYP3A5, and CYP3A7, are the most abundant P450 enzymes expressed in human liver and intestine and are responsible for metabolizing >50% of drugs. Significant variations in CYP3A activity and gene expression have been found in liver during development, with infants and young children having different ability to metabolize many drugs than adults. Particularly, CYP3A4 and CYP3A7 exhibit profound reciprocal patterns of gene expression with a developmental switch after birth. However, the mechanisms governing the ontogenic expression of the CYP3A genes during development are unknown. Our long-term research goal is to unravel the mechanisms that control drug metabolism during development. The objective of this proposal is to identify the mechanisms controlling the ontogenic expression of the Cyp3a genes in livers during postnatal development by using mouse as a model. Our central hypothesis is that the ontogenic expression patterns of the Cyp3a genes in mouse liver are controlled by transcription factors which change epigenetic modifications at the target chromatin, and place the Cyp3a genes in distinct nuclear positions, allowing the Cyp3a genes to be turned on or off at different developmental stages. To test this hypothesis, we propose to pursue the following specific aims: (1) to establish profiles of histone modifications around the Cyp3a genes in liver cells at different ages; (2) to define chromatin condensation, nuclear positions, and associated histone modifications of the Cyp3a genes in hepatocyte nuclei at different ages; and (3) to examine the roles of the transcription factors in controlling ontogenic expression of the Cyp3a genes during liver maturation. If we prove our hypothesis is correct, the proposed study will identify transcription factors which control the ontogenic expression of the Cyp3a genes through epigenetic mechanisms. It would add greatly to our fundamental knowledge of developmental regulation of gene expression in liver development. This knowledge is particularly novel for drug metabolizing enzymes, because the areas of epigenetics and gene positioning have not received much attention with respect to the ontogeny of genes involved in drug biotransformation. Establishment of this fundamental knowledge is essential for understanding difference of drug responses between pediatric and adult patients and is important for establishing pharmaco-epigenomics based on inter-individual variation of epigenomes in personalized medicine.

Funded by NIH/NIGM-1R01GM087376

 

Chromatin interactions, epigenomics, and transcriptional response to drug induction

Drug-drug interactions (DDIs) are a significant clinical concern, especially for people at age above 50, because ~78% of medicated people at this age group take more than one drug per day, and 4% of them are at risk for either decreasing therapeutic efficacy or increasing adverse drug reactions (ADRs). A possible reason is that some drugs have the ability to induce expression of some proteins involved absorption, distribution, metabolism, and elimination (ADME) of other co-administrated drugs. It is still unknown at molecular levels how the ADME genes alter their expression in response to drug induction. The lack of this knowledge is a barrier to understanding the mechanisms involved in DDIs and results in difficulty predicting drug responses. Our research goal is to better understand the molecular mechanisms involved in drug induction. Rifampicin is selected as a model drug for this study. Based on the literature and our preliminary data, we have formed a central hypothesis that rifampicin activates pregnane X receptor (PXR), a transcription factor that binds to its DNA response elements at multiple genomic locations and participates in long range chromatin interactions in three dimensional (3D) conformation, thus helping bring relevant genomic elements, such as enhancers and promoters into close spatial proximity for efficient and coordinated transcription regulation in this process. The changes in the ADME gene expression further alter therapeutic efficacy or adverse reactions of drugs co-administrated with rifampicin. We will use human primary hepatocytes and established cell cultural lines (HepaRG and HeG2) as in vitro models, and humanized PXR mice as an in vivo model to test our hypothesis with the following specific aims: (1) define PXR-mediated chromatin interaction architecture, epigenomic features, and transcriptional outcome in response to rifampicin induction in human liver cells; (2) characterize PXR-specific transcriptional regulatory programs in humanized PXR mice; and (3) integratively analyze PXR-mediated changes of chromatin interactions, transcription, and histone modifications by computational and network approaches. The completion of this study will provide a comprehensive repertoire of regulatory elements, such as promoters and enhancers, and specific interaction networks of the regulatory functions in response to rifampicin-induction. Through integrated analyses of multiplex datasets generated in this study, we will advance our understanding on how PXR activated by rifampicin participates in maintaining higher-order chromatin structures and transcriptional responses to rifampicin induction. This knowledge will be essential for us to understand molecular mechanisms involved in drug-drug interactions and will help us to predict therapeutic efficacy and adverse drug reactions when patients are administered multiple drugs concomitantly.

Funded by Affinity Research Collaboratives of Institute for System Genomics of the University of Connecticut

 

Review of FDA-approved antisense oligonucleotide (ASO) drugs

FDA has approved 6 ASO drugs to treat various diseases based on antisense inhibitory mechanism. Pharm.D. and undergraduate students have formed a collaborative team working together with Drs. Theodore Rasmussen, Xiaobo Zhong, Jose Manautou, and Raman Bahal to study features of pharmacodynamics, pharmacokinetics, preclinical safety and toxicity, and formulation and delivery system of the drugs.