Pharmacological Traits Research Articles

Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy

Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.

The impact of microRNA expression on cellular proliferation.

As an important class of non-coding regulatory RNAs, microRNAs (miRNAs) play a key role in a range of biological processes. These molecules serve as post-transcriptional regulators of gene expression and their regulatory activity has been implicated in disease pathophysiology and pharmacological traits. We sought to investigate the impact of miRNAs on cellular proliferation to gain insight into the molecular basis of complex traits that depend on cellular growth, including, most prominently, cancer. We examined the relationship between miRNA expression and intrinsic cellular growth (iGrowth) in the HapMap lymphoblastoid cell lines derived from individuals of different ethnic backgrounds. We found a substantial enrichment for miRNAs (53 miRNAs, FDR<0.05) correlated with cellular proliferation in pooled CEU (Caucasian of northern and western European descent) and YRI (individuals from Ibadan, Nigeria) samples. Specifically, 119 miRNAs (59%) were significantly correlated with iGrowth in YRI; of these miRNAs, 18 were correlated with iGrowth in CEU. To gain further insight into the effect of miRNAs on cellular proliferation in cancer, we showed that over-expression of miR-22, one of the top iGrowth-associated miRNAs, leads to growth inhibition in an ovarian cancer cell line (SKOV3). Furthermore, over-expression of miR-22 down-regulates the expression of its target genes (MXI1 and SLC25A37) in this ovarian cancer cell line, highlighting an miRNA-mediated regulatory network potentially important for cellular proliferation. Importantly, our study identified miRNAs that can be used as molecular targets in cancer therapy.

Honey in dermatology and skin care: a review

Honey is a bee-derived, supersaturated solution composed mainly of fructose and glucose, and containing proteins and amino acids, vitamins, enzymes, minerals, and other minor components. Historical records of honey skin uses date back to the earliest civilizations, showing that honey has been frequently used as a binder or vehicle, but also for its therapeutic virtues. Antimicrobial properties are pivotal in dermatological applications, owing to enzymatic H2 O2 release or the presence of active components, like methylglyoxal in manuka, while medical-grade honey is also available. Honey is particularly suitable as a dressing for wounds and burns and has also been included in treatments against pityriasis, tinea, seborrhea, dandruff, diaper dermatitis, psoriasis, hemorrhoids, and anal fissure. In cosmetic formulations, it exerts emollient, humectant, soothing, and hair conditioning effects, keeps the skin juvenile and retards wrinkle formation, regulates pH and prevents pathogen infections. Honey-based cosmetic products include lip ointments, cleansing milks, hydrating creams, after sun, tonic lotions, shampoos, and conditioners. The used amounts range between 1 and 10%, but concentrations up to 70% can be reached by mixing with oils, gel, and emulsifiers, or polymer entrapment. Intermediate-moisture, dried, and chemically modified honeys are also used. Mechanisms of action on skin cells are deeply conditioned by the botanical sources and include antioxidant activity, the induction of cytokines and matrix metalloproteinase expression, as well as epithelial-mesenchymal transition in wounded epidermis. Future achievements, throwing light on honey chemistry and pharmacological traits, will open the way to new therapeutic approaches and add considerable market value to the product.

Pharmacological traits of delta opioid receptors: pitfalls or opportunities?

Delta opioid receptors (DORs) have been considered as a potential target to relieve pain as well as treat depression and anxiety disorders and are known to modulate other physiological responses, including ethanol and food consumption. A small number of DOR-selective drugs are in clinical trials, but no DOR-selective drugs have been approved by the Federal Drug Administration and some candidates have failed in phase II clinical trials, highlighting current difficulties producing effective delta opioid-based therapies. Recent studies have provided new insights into the pharmacology of the DOR, which is often complex and at times paradoxical. This review will discuss the existing literature focusing on four aspects: (1) Two DOR subtypes have been postulated based on differences in pharmacological effects of existing DOR-selective ligands. (2) DORs are expressed ubiquitously throughout the body and central nervous system and are, thus, positioned to play a role in a multitude of diseases. (3) DOR expression is often dynamic, with many reports of increased expression during exposure to chronic stimuli, such as stress, inflammation, neuropathy, morphine, or changes in endogenous opioid tone. (4) A large structural variety in DOR ligands implies potential different mechanisms of activating the receptor. The reviewed features of DOR pharmacology illustrate the potential benefit of designing tailored or biased DOR ligands.

Abstract 2216: The role of cytosine modifications in cellular sensitivity to cytarabine.

Abstract Introduction: Inter-individual differences in cytosine modifications are a result of genetic and environmental influences. 5-methylcytosine and 5-hydroxymethylcytosine are covalent cytosine modifications that occur in CpG dinucleotides throughout the human genome. Objective: To characterize inter-individual variation in cytosine modifications in HapMap samples and investigate the contribution this may have on downstream phenotypes, such as cellular sensitivity to chemotherapeutics, such as cytarabine. Model: Cytosine modification levels at &amp;gt;485,000 CpGs were measured in the International HapMap lymphoblastoid cell lines (LCLs). We utilized the Illumina Infinium 450K HumanMethylation Beadchip array to measure cytosine modification levels in 73 unrelated African (YRI - Yoruba people from Ibadan, Nigeria) and 60 European ancestry (CEU - Caucasians from Utah, US) LCLs. In addition, we evaluated LCL sensitivity to cytarabine in these HapMap cell lines. Results: We performed a linear regression model in order to identify novel associations between cytosine modifications and cytarabine-induced cytotoxicity. From these analyses, we found that promoter methylation of CDC6 and CCDC37 was associated with cytarabine IC50 (p= 3.75 x 10−6 and p= 5.39x 10−6, respectively). Furthermore, it has been shown previously that SNPs associated with pharmacological traits in LCLs are enriched in expression quantitative trait loci (eQTLs). We interrogated SNPs associated with cytosine modification (mSNPs) to help us understand the role of mSNPs in pharmacologic trait-associated genetic variants. We found that SNPs associated with cytarabine are enriched in cis-acting mSNPs in LCLs derived from individuals of European descent. Next, we integrated genome-wide cytosine modification data with genotype and gene expression data available for HapMap LCLs to allow for a more comprehensive understanding of inter-individual genetic variation. We identified a SNP in SLFN5 that was significantly associated with cytarabine sensitivity that is a cis-acting eQTL and mSNP for SLFN5 gene expression and cytosine modification. Conclusions: Analyzing cytosine modification-drug phenotype data may identify novel candidate genes associated with sensitivity to cytarabine. In addition, integrating genotype and gene expression data allow for a better understanding of the biological function underlying pharmacologic trait-associated SNPs. Citation Format: Erika L. Moen, Wenbo Mu, Lucy A. Godley, M. Eileen Dolan, Wei Zhang. The role of cytosine modifications in cellular sensitivity to cytarabine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2216. doi:10.1158/1538-7445.AM2013-2216

Individualized Therapy for Type 2 Diabetes

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, abnormally elevated hepatic glucose production, and reduced glucose-stimulated insulin secretion. Treatment with antihyperglycemic agents is initially successful in type 2 diabetes, but it is often associated with a high secondary failure rate, and the addition of insulin is eventually necessary for many patients, in order to restore acceptable glycemic control and to reduce the risk of development and progression of disease complications. Notably, even patients who appear to have similar requirements of antidiabetic regimens show great variability in drug disposition, glycemic response, tolerability, and incidence of adverse effects during treatment. Pharmacogenomics is a promising area of investigation and involves the search for genetic polymorphisms that may explain the interindividual variability in antidiabetic therapy response. The initial positive results portend that genomic efforts will be able to shed important light on variability in pharmacologic traits. In this review, we summarize the current understanding of genetic polymorphisms that may affect the responses of subjects with T2DM to antidiabetic treatment. These genes belong to three major classes: genes involved in drug metabolism and transporters that influence pharmacokinetics (including the cytochrome P450 [CYP] superfamily, the organic anion transporting polypeptide [OATP] family, and the polyspecific organic cation transporter [OCT] family); genes encoding drug targets and receptors (including peroxisome proliferator-activated receptor gamma [PPARG], the adenosine triphosphate [ATP]-sensitive potassium channel [K(ATP)], and incretin receptors); and genes involved in the causal pathway of T2DM that are able to modify the effects of drugs (including adipokines, transcription factor 7-like 2 (T cell specific, HMG-box) [TCF7L2], insulin receptor substrate 1 [IRS1], nitric oxide synthase 1 (neuronal) adaptor protein [NOS1AP], and solute carrier family 30 (zinc transporter), member 8 [SLC30A8]). In addition to these three major classes, we also review the available evidence on novel genes (CDK5 regulatory subunit associated protein 1-like 1 [CDKAL1], insulin-like growth factor 2 mRNA binding protein 2 [IGF2BP2], potassium voltage-gated channel, KQT-like subfamily, member 1 [KCNQ1], paired box 4 [PAX4] and neuronal differentiation 1 [NEUROD1] transcription factors, ataxia telangiectasia mutated [ATM], and serine racemase [SRR]) that have recently been proposed as possible modulators of therapeutic response in subjects with T2DM.

Genome-Wide Approaches in Pharmacogenomics: Heritability Estimation and Pharmacoethnicity as Primary challenges

The ultimate aim of pharmacogenomics is the identification of genetic variants that underlie interindividual variation in drug response. The furious pace of technological advances [1], such as DNA sequencing and genotyping technologies [2], has begun to enhance our understanding of human diversity in pharmacologic traits. Meanwhile, methodological advances, such as the genome-wide association study (GWAS) approach, have facilitated the discovery of genetic variation with considerable clinical relevance [3]. The sine qua non of the rapidly developing field of pharmacogenomics is the translation of genomic information from these convergent developments into individualized patient care. Exciting promises of the new science abound, including the possibilities of optimized drug therapy, adverse-effect risk prediction, and improved drug discovery and development. The purpose of this article is to explore two of the major challenges to pharmacogenomic progress despite the substantial advances in genome-wide approaches that have already been made.

Abstract PL07-02: Whole genome approaches to identify pharmacogenomic markers of anticancer agents

Abstract Preclinical and clinical genome-wide association studies (GWAS) in oncology provide an unprecedented opportunity for a comprehensive and unbiased assessment of the heritable factors associated with drug response. For chemotherapeutic drugs, there are many challenges with performing a clinical GWAS, including staggering costs because a homogenous population of patients treated with the same dosage regimen and minimal confounding variables is required. To get around these issues, we have developed preclinical cell-based models as a pharmacogenomic discovery tool but also a means to replicate or validate findings from clinical GWAS. Our model utilizes HapMap lymphoblastoid cell lines (LCLs) derived from individuals within different world populations (Caucasian, African, Asian, African American) that are evaluated for drug sensitivity to chemotherapeutic agents (platinating agents, antimetabolites, cyclophosphamide, paclitaxel). Evaluation of inter-ethnic differences revealed that LCLs from Africans tend to be more sensitive to antimetabolites and less sensitive to platinating agents than Caucasian derived LCLs. These LCLs are rich in genetic data as many are part of the International HapMap and the more recent 1000 Genome project. We utilized this data to identify disproportionately more expression quantitative trait loci (eQTLs, indicating that a SNP genotype is associated with the transcript abundance level of a gene) associated with pharmacologic traits than expected by chance based on minor allele frequency. We are attempting to unravel the functional consequence of top pharmacologic GWAS SNPs with local and distant regulatory roles in cellular resistance to chemotherapeutic agents. For example, we discovered a SNP (master regulator) that is local to PRPF39 and distant to more than 100 genes that is associated with cisplatin-induced cytotoxicity in LCLs derived from African populations. Our cell-based model provides potential targets for therapeutic intervention and provides us with a greater understanding of the genetic contribution to interethnic variation in chemotherapeutic sensitivity. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):PL07-02.

Multigenic Control of Drug Response and Regulatory Decision-Making in Pharmacogenomics: The Need for an Upper-Bound Estimate of Genetic Contributions

Nature or nurture? To what extent genetics play a role in drug efficacy and safety? These questions are not new. They are however gaining increasing prominence with the implementation of pharmacogenomics in various facets of medicine ranging from therapeutics, drug development and regulatory science to research funding decisions. For predisposition to common complex diseases, twin and family studies have been the mainstay for estimating genetic components of the attendant risk. On the other hand, the rapid pace of drug development in the pharmaceutical industry and the need for faster regulatory decisions call for an approach of higher throughput to identify the compounds for which heritability is likely to play a significant role in their pharmacokinetics and/or pharmacodynamics. A second predicament related to multifactorial nature of drug effects is that one typically observes a considerable overlap in the distribution of drug response phenotypes among subpopulations identified by each pharmacogenomic biomarker. This is in sharp contrast to monogenic pharmacological traits wherein it is feasible to partition the patient populations into discrete subgroups by analysis of a single gene. Hence, as pharmacogenomic investigations progress from monogenic to increasingly multigenic or multifactorial drug response phenotypes, the regulatory decision-makers are faced with a dilemma: How can a reviewer or a clinician determine if a given separation of a drug response profile by a pharmacogenomic biomarker is worthwhile for clinical implementation? The present manuscript makes an attempt to address these broad and emerging issues in pharmacogenomics and regulatory science. We propose that a comparison of inter- versus intra-subject variability in drug response under minimal environmental exposure may provide an upperbound estimate of heritability of drug efficacy and safety. It is also argued that seemingly modest changes in population averages may underestimate the dramatic impact of a genetic biomarker at the tails of a population. To this end, a conceptual framework for graded risk assessment among subpopulations with overlapping quantitative phenotypes is presented. We conclude with a broader discussion of the evolution of genetic biomarkers from monogenic to multigenic traits in pharmacology, the associated ethical, social and therapeutic policy corollaries and the challenges lying ahead.

A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding.

BMS-378806 is a recently discovered small molecule HIV-1 inhibitor that blocks viral entrance to cells. The compound exhibits potent inhibitory activity against a panel of R5-(virus using the CCR5 coreceptor), X4-(virus using the CXCR4 coreceptor), and R5/X4 HIV-1 laboratory and clinical isolates of the B subtype (median EC50 of 0.04 microM) in culture assays. BMS-378806 is selective for HIV-1 and inactive against HIV-2, SIV and a panel of other viruses, and exhibits no significant cytotoxicity in the 14 cell types tested (concentration for 50% reduction of cell growth, >225 microM). Mechanism of action studies demonstrated that BMS-378806 binds to gp120 and inhibits the interactions of the HIV-1 envelope protein to cellular CD4 receptors. Further confirmation that BMS-378806 targets the envelope in infected cells was obtained through the isolation of resistant variants and the mapping of resistance substitutions to the HIV-1 envelope. In particular, two substitutions, M426L and M475I, are situated in the CD4 binding pocket of gp120. Recombinant HIV-1 carrying these two substitutions demonstrated significantly reduced susceptibility to compound inhibition. BMS-378806 displays many favorable pharmacological traits, such as low protein binding, minimal human serum effect on anti-HIV-1 potency, good oral bioavailability in animal species, and a clean safety profile in initial animal toxicology studies. Together, the data show that BMS-378806 is a representative of a new class of HIV inhibitors that has the potential to become a valued addition to our current armamentarium of antiretroviral drugs.

Quantitative genetics and mouse behavior.

Quantitative differences are observed for most complex behavioral and pharmacological traits within any population. Both environmental and genetic influences regulate such individual differences. The mouse has proven to be a superb model in which to investigate the genetic basis for quantitative differences in complex behaviors. Genetically defined populations of mice, including inbred strains, heterogeneous stocks, and selected lines, have been used effectively to document these genetic differences. Recently, quantitative trait loci methods have been applied to map the chromosomal regions that regulate variation with the goal of eventually identifying the gene polymorphisms that reside in these regions.

Different Levels of Fos Immunoreactivity After Repeated Handling and Injection Stress in Two Inbred Strains of Mice

Expression of Fos and Fos-related antigens was immunohistochemically analyzed in DBA/2J and C57BL/6J inbred mice in response to acute or repeated handling and injection stress. Both strains showed a strong induction of Fos and Fos-related antigens in discrete areas of hypothalamus, amygdala, neocortex, septum, and thalamus 2 h after an acute intraperitoneal injection of normal saline. To habituate animals to this procedure, mice were subjected to repeated handling and injections during 2 weeks preceding the experiment. This procedure led to complete habituation of the immediate early gene response to injection stress in stress-responsive brain areas of C57BL/6J mice, such that no significant difference was found between expression of these proteins in brains of saline-injected animals after repeated stress vs. control animals. In contrast, many brain areas of saline-injected DBA/2J mice still showed elevated Fos and Fos-related antigen expression after repeated injections. These results indicate that identical habituation procedures do not necessarily lead to identical levels of gene expression in brains of inbred strains of mice. In turn, they suggest that genetic components for some behavioral and pharmacological traits identified using inbred strains could be related to different rates of habituation to experimental procedures.

Taxane-mediated gene induction is independent of microtubule stabilization: induction of transcription regulators and enzymes that modulate inflammation and apoptosis.

Pharmacological traits of the antineoplastic agent taxol may originate in part from its effects on gene expression and not simply from its effects on microtubule assembly. This prompts three questions. First, how extensive is gene induction by taxol? Second, is gene induction confined to taxol itself, or does it occur with other taxane analogs? Third, do the functions of any induced genes correspond with known attributes of taxol or taxane analogs? We report that taxol induces numerous early-response genes, not just cytokine genes. Previously unidentified taxol-induced genes include genes coding transcription factors with tumor suppressor effects (krox-24) and enzymes that govern proliferation, apoptosis, and inflammation (2'5'-oligoadenylate synthase, cyclooxygenase-2, and an IkappaB kinase termed chuk). Taxotere, a potent analog of taxol, did not induce any of these genes, implying that taxol modulates gene expression by a mechanism that is distinct from microtubule stabilization and cell cycle arrest. Other taxane analogs induce some of the same genes as taxol, indicating that this process is not unique to taxol. Functional changes coincided with changes in gene expression. For instance, induction of tumor necrosis factor alpha (TNFalpha) accentuated apoptosis in cells treated with taxol compared with corresponding cells treated with taxotere. The functions of several induced genes (e.g., krox-24 and cyclooxygenase-2) are self-consistent with beneficial and adverse effects encountered during taxol administration. These results may be relevant to the safe and effective use of taxol or its analogs in oncology and other areas of medicine.

Interrelationships of behavioral, pharmacological, and biochemical traits pertinent to alcohol

Interrelationships of behavioral, pharmacological, and biochemical traits pertinent to alcohol