Pharmacogenomics in the era of next generation sequencing - from byte to bedside.

Pharmacogenetic research has resulted in the identification of a multitude of genetic variants that impact drug response or toxicity. These polymorphisms are mostly common and have been included as actionable information in the labels of numerous drugs. In addition to common variants, recent advances in Next Generation Sequencing (NGS) technologies have resulted in the identification of a plethora of rare and population-specific pharmacogenetic variations with unclear functional consequences that are not accessible by conventional forward genetics strategies. In this review, we discuss how comprehensive sequencing information can be translated into personalized pharmacogenomic advice in the age of NGS. Specifically, we provide an update of the functional impacts of rare pharmacogenetic variability and how this information can be leveraged to improve pharmacogenetic guidance. Furthermore, we critically discuss the current status of implementation of pharmacogenetic testing across drug development and layers of care. We identify major gaps and provide perspectives on how these can be minimized to optimize the utilization of NGS data for personalized clinical decision-support.

An automated approach to identify scientific publications reporting pharmacokinetic parameters.

Pharmacokinetic (PK) predictions of new chemical entities are aided by prior knowledge from other compounds. The development of robust algorithms that improve preclinical and clinical phases of drug development remains constrained by the need to search, curate and standardise PK information across the constantly-growing scientific literature. The lack of centralised, up-to-date and comprehensive repositories of PK data represents a significant limitation in the drug development pipeline.In this work, we propose a machine learning approach to automatically identify and characterise scientific publications reporting PK parameters from in vivo data, providing a centralised repository of PK literature. A dataset of 4,792 PubMed publications was labelled by field experts depending on whether in vivo PK parameters were estimated in the study. Different classification pipelines were compared using a bootstrap approach and the best-performing architecture was used to develop a comprehensive and automatically-updated repository of PK publications. The best-performing architecture encoded documents using unigram features and mean pooling of BioBERT embeddings obtaining an F1 score of 83.8% on the test set. The pipeline retrieved over 121K PubMed publications in which in vivo PK parameters were estimated and it was scheduled to perform weekly updates on newly published articles. All the relevant documents were released through a publicly available web interface (https://app.pkpdai.com) and characterised by the drugs, species and conditions mentioned in the abstract, to facilitate the subsequent search of relevant PK data. This automated, open-access repository can be used to accelerate the search and comparison of PK results, curate ADME datasets, and facilitate subsequent text mining tasks in the PK domain.

Genetic variation in the farnesoid X-receptor predicts Crohn's disease severity in female patients.

The farnesoid X receptor (FXR) is implicated in Crohn's disease (CD) pathogenesis. It is unclear how genetic variation in FXR impacts CD severity versus genetic variation in nuclear receptors such as pregnane X receptor (PXR) and the multi-drug resistance protein 1 (MDR1, ABCB1). To evaluate FXR-1G > T as a genomic biomarker of severity in CD and propose a plausible molecular mechanism. A retrospective study (n = 542) was conducted in a Canadian cohort of CD patients. Genotypic analysis (FXR-1G > T, MDR1 3435C > T and PXR -25385C > T) as well as determination of the FXR downstream product, fibroblast growth factor (FGF) 19 was performed. Primary outcomes included risk and time to first CD-related surgery. The effect of estrogen on wild type and variant FXR activity was assessed in HepG2 cells. The FXR-1GT genotype was associated with the risk of (odds ratio, OR = 3.34, 95% CI = 1.58-7.05, p = 0.002) and earlier progression to surgery (hazard ratio, HR = 3.00, 95% CI = 1.86-4.83, p < 0.0001) in CD. Female carriers of the FXR-1GT genotype had the greatest risk of surgery (OR = 14.87 95% CI = 4.22-52.38, p < 0.0001) and early progression to surgery (HR = 6.28, 95% CI = 3.62-10.90, p < 0.0001). Women carriers of FXR-1GT polymorphism had a three-fold lower FGF19 plasma concentration versus women with FXR-1GG genotype (p < 0.0001). In HepG2 cells cotransfected with estrogen receptor (ER) and FXR, presence of estradiol further attenuated variant FXR activity. MDR1 and PXR genotypes were not associated with surgical risk. Unlike MDR1 and PXR, FXR-1GT genetic variation is associated with earlier and more frequent surgery in women with CD. This may be through ER-mediated attenuation of FXR activation.

Elevation of serum pyruvate kinase M2 (PKM2) in IBD and its relationship to IBD indices.

OBJECTIVES: Endoscopy remains the gold standard to diagnose and evaluate inflammatory bowel disease (IBD) activity. Current biomarkers or their combinations cannot adequately predict IBD risk, diagnosis, progression or relapse, and response to therapy. Pyruvate kinase M2 (PKM2) is emerging as a significant mediator of the inflammatory process. We aimed to assess levels of serum PKM2 in healthy and newly diagnosed IBD patients and its relationship with IBD indices and microbiota changes. DESIGN AND METHODS: IBD serum samples from newly diagnosed patients were collected and analyzed using a PKM2-ELISA and correlated with disease activity scores, IBD disease type, and intestinal microbiota. Furthermore, we tested the genetic and protein expression of PKM2 in an in vitro intestinal cell model of inflammation. RESULTS: Serum PKM2 levels were 6-fold higher in IBD patients compared to healthy controls, with no sensitivity to disease phenotype (Crohn's Disease or Ulcerative Colitis) or localization of inflammation. Serum PKM2 had considerably less interindividual variability than established IBD fecal biomarkers. A positive Pearson correlation (r=0.6121) existed between serum PKM2 and Bacteroidetes fecal levels in Crohn's disease (CD), while a negative (r=-0.6128) correlation was observed with Actinobacteria fecal levels. Furthermore, LPS (500ng/mL) significantly increased PKM2 expression in vitro, which was significantly suppressed by an anti-inflammatory flaxseed bioactive agent. CONCLUSION: Our data suggests PKM2 as a putative biomarker for IBD and the dysbiosis of microflora in CD. Investigations involving larger number of clinical patients are necessary to validate its use as a serum biomarker of IBD.

Linoorbitides and enterolactone mitigate inflammation-induced oxidative stress and loss of intestinal epithelial barrier integrity.

Barrier integrity dysfunction and oxidative stress are considered hallmarks of inflammatory bowel disease (IBD) pathogenesis. Their mitigation continues to be a drug discovery target in IBD. Natural products may aid treatment of chronic inflammatory diseases, but their use in IBD requires a better understanding of whether individual bioactives may positively modulate disease course. This study investigated the ability of flax linoorbitides (LOBs) and enterolactone (ENL), to mitigate inflammation-induced loss of intestinal epithelial barrier integrity and oxidative stress in vitro. TNF-α with INF-γ and lipopolysaccharide (LPS) induced an inflammatory response in HCT-8 monoculture and Caco-2/RAW-264.7 coculture, respectively. Trans-Epithelial Electrical Resistance (TEER) and Lucifer Yellow rejection for barrier permeability were assessed in differentiated monolayers in the presence and absence of LOBs and ENL. Additionally, RAW 264.7 cells were used to assess protective effects upon induction of oxidative stress. In HCT-8 model, 200 nM of LOB-J, LOB-A, and ENL mitigated the inflammation-induced reduction in TEER with relative TEER values of 108.6%, 63.2%, and 64.2%, respectively, at 24 h relative to time zero. Similarly, at 24 h Caco-2/RAW-264.7 coculture TEER values ranged from ~200% - 243.4% for LOB-A, LOB-J, LOB-ACEJ, and ENL relative to TEER values of untreated cells. ENL and LOBs reduced malondialdehyde (MDA) lipid peroxidation in RAW 264.7 cells upon induction with lipopolysaccharide (LPS). ENL, but not LOBs, caused an increase in zona occludins 1 (ZO-1) protein expression in HCT-8 cells exposed to an inflammatory stimulus to levels comparable to negative control. Our results demonstrate after an inflammatory insult that ENL and the tested LOBs protect intestinal barrier integrity and reduce oxidative stress damage. In conclusion, use of different flax bioactives in the treatment of IBD warrants further investigation.

HPLC-UV method development for fentanyl determination in rat plasma and its application to elucidate pharmacokinetic behavior after i.p. administration to rats.

A simple, rapid and validated high performance liquid chromatography method with UV detection for the quantification of an opioid agonist, fentanyl (FEN), in rat plasma was developed. The assay procedure involved chromatographic separation using a ZIC-HILIC SeQUANT column (250 mm × 4.6 mm, i.d., 5 µm) and a mobile phase of acetonitrile and acetate buffer (pH 3.4, 20mM) of ratio (=65:35, v/v) at a flow rate of 1.2 mL/min and detection wavelength of 201 nm. Plasma sample (100 µL) pretreatment was based on simple deprotienization by acetonitrile spiked with clonidine as an internal standard (I.S.) of 20 ng/mL followed by extraction with tert-butyl methyl ether and centrifugation. The organic layer was evaporated under N(2) gas and reconstituted with 100 µL of acetate buffer (pH 3.4, 20mM), and 50-µL portions of reconstituted sample were injected onto the column. Sample analysis including sample pretreatment was achieved within 35 min. Calibration curve was linear (r ≥ 0.998) from 5 to 100 ng/mL. Both intra- and inter-day assay precisions that are presented through RSD were lower than 12.6% for intra-day and lower than 12.0% for inter-day assessment. Limit of detection was 0.8 ng/mL at S/N of 3. This method was omitting the use of expensive solid phase extraction and time consuming liquid extraction procedures. Moreover, the present method was successfully applied to study pharmacokinetic parameters of FEN after intraperitoneal administration to male Wistar rat. Pharmacokinetic parameters estimated by using moment analysis were T(1/2) 198.3 ± 44.7 min, T(max) 28.3 ± 2.9 min and AUC(0-180) 15.6 ± 2.9(× 10(2))ngmin/mL.