Mass spectral analysis of tryptic digests of cross-linked proteins offers considerable promise as a simple technique to probe protein structure and study protein-protein interactions. We describe the use of a 1:1 mixture of isotopically labeled and unlabeled cross-linkers, disuccinimidyladipate (DSA) and dimethyladipimidate (DMA), to enhance visualization of cross-linked peptides in a tryptic digest. Optimized intramolecular reactions of cytochrome c and ribonuclease A (RNase A) with DSA yielded an average of two cross-links per protein molecule. After digestion of the cross-linked cytochrome c with trypsin and analysis by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization (MALDI), eight modified peptides, five cross-linked and two end-capped, were detected by virtue of their doublet character. An eighth modified peptide's identity remained ambiguous because of its inability to fragment. The lysine-lysine distance constraints obtained are discussed in the context of the known NMR and X-ray structures of cytochrome c. Analysis of cross-linked RNase A by LC/MS and MALDI yielded nine modified peptides, four of which were modified twice, as indicated by the isotopic triplets. Although seven of these peptides contained cross-links, few distance constraints were gained due to the fact that the cross-linked products were variations of modification of the same three lysine residues.

A double-blind crossover study was conducted in four CYP2C19 genotype-defined metabolizer groups to assess whether increase in clopidogrel dosing can overcome reduced pharmacodynamic response in CYP2C19 poor metabolizers (PMs). Ten healthy subjects in each of four metabolizer groups were randomized to a clopidogrel regimen of a 300-mg loading dose (LD) and a 75-mg/day maintenance dose (MD) for 4 days followed by 600-mg LD and 150 mg/day MD, or vice versa. The exposure levels of clopidogrel's active metabolite H4 (clopi-H4) in PMs were 71% lower on the 75-mg/day regimen and 64% lower on the 150-mg/day regimen than the corresponding exposure levels in extensive metabolizers (EMs). In PMs, the maximal platelet aggregation (MPA) induced by adenosine diphosphate (ADP) 5 µmol/l was 10.5% lower on the 75-mg/day regimen and 7.9% lower on the 150-mg/day regimen than the corresponding values in EMs. PMs who were on the clopidogrel regimen of 600-mg LD/150 mg/day MD showed clopi-H4 exposure and MPA levels similar to those in EMs who were on the regimen of 300-mg LD/75 mg/day MD. In a pooled analysis evaluating CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP3A5, CYP2D6, ABCB1, and P2RY12 polymorphisms (N = 396 healthy subjects), only CYP2C19 had a significant impact on antiplatelet response. In healthy CYP2C19 PMs, a clopidogrel regimen of 600-mg LD/150 mg/day MD largely overcomes diminished clopi-H4 exposure and antiplatelet response, as assessed by MPA levels.

The robust transcriptional plasticity of liver mediated through xenobiotic receptors underlies its ability to respond rapidly and effectively to diverse chemical stressors. Thus, drug-induced gene expression changes in liver serve not only as biomarkers of liver injury, but also as mechanistic sentinels of adaptation in metabolism, detoxification, and tissue protection from chemicals. Modern RNA sequencing methods offer an unmatched opportunity to quantitatively monitor these processes in parallel and to contextualize the spectrum of dose-dependent stress, adaptation, protection, and injury responses induced in liver by drug treatments. Using this approach, we profiled the transcriptional changes in rat liver following daily oral administration of 120 different compounds, many of which are known to be associated with clinical risk for drug-induced liver injury by diverse mechanisms. Clustering, correlation, and linear modeling analyses were used to identify and optimize coexpressed gene signatures modulated by drug treatment. Here, we specifically focused on prioritizing 9 key signatures for their pragmatic utility for routine monitoring in initial rat tolerability studies just prior to entering drug development. These signatures are associated with 5 canonical xenobiotic nuclear receptors (AHR, CAR, PXR, PPARα, ER), 3 mediators of reactive metabolite-mediated stress responses (NRF2, NRF1, P53), and 1 liver response following activation of the innate immune response. Comparing paradigm chemical inducers of each receptor to the other compounds surveyed enabled us to identify sets of optimized gene expression panels and associated scoring algorithms proposed as quantitative mechanistic biomarkers with high sensitivity, specificity, and quantitative accuracy. These findings were further qualified using public datasets, Open TG-GATEs and DrugMatrix, and internal development compounds. With broader collaboration and additional qualification, the quantitative toxicogenomic framework described here could inform candidate selection prior to committing to drug development, as well as complement and provide a deeper understanding of the conventional toxicology study endpoints used later in drug development.

Drug-induced liver injury (DILI) is a frequent and dangerous adverse effect faced during preclinical and clinical drug therapy. DILI is a leading cause of candidate drug attrition, withdrawal and in clinic, is the primary cause of acute liver failure. Traditional diagnostic markers for DILI include alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP). Yet, these routinely used diagnostic markers have several noteworthy limitations, restricting their sensitivity, specificity and accuracy in diagnosing DILI. Consequently, new biomarkers for DILI need to be identified.A potential biomarker for DILI is cytokeratin-18 (CK18), an intermediate filament protein highly abundant in hepatocytes and cholangiocytes. Extensively researched in a variety of clinical settings, both full length and cleaved forms of CK18 can diagnose early-stage DILI and provide insight into the mechanism of hepatocellular injury compared to traditionally used diagnostic markers. However, relatively little research has been conducted on CK18 in preclinical models of DILI. In particular, CK18 and its relationship with DILI is yet to be characterised in an in vivo rat model. Such characterization of CK18 and ccCK18 responses may enable their use as translational biomarkers for hepatotoxicity and facilitate management of clinical DILI risk in drug development. The aim of this review is to discuss the application of CK18 as a biomarker for DILI. Specifically, this review will highlight the properties of CK18, summarise clinical research that utilised CK18 to diagnose DILI and examine the current challenges preventing the characterisation of CK18 in an in vivo rat model of DILI.

Human hepatocellular carcinoma cells, HepG2, are often used for drug mediated mitochondrial toxicity assessments. Glucose in HepG2 culture media is replaced by galactose to reveal drug-induced mitochondrial toxicity as a marked shift of drug IC50 values for the reduction of cellular ATP. It has been postulated that galactose sensitizes HepG2 mitochondria by the additional ATP consumption demand in the Leloir pathway. However, our NMR metabolomics analysis of HepG2 cells and culture media showed very limited galactose metabolism. To clarify the role of galactose in HepG2 cellular metabolism, U-13 C 6 -galactose or U-13 C 6glucose was added to HepG2 culture media to help specifically track the metabolism of those two sugars. Conversion to U-13 C 3 -lactate was hardly detected when HepG2 cells were incubated with U-13 C 6 -galactose, while an abundance of U-13 C 3 -lactate was produced when HepG2 cells were incubated with U-13 C 6 -glucose. In the absence of glucose, HepG2 cells increased glutamine consumption as a bioenergetics source. The requirement of additional glutamine almost matched the amount of glucose needed to maintain a similar level of cellular ATP in HepG2 cells. This improved understanding of galactose and glutamine metabolism in HepG2 cells helped optimize the ATP-based mitochondrial toxicity assay. The modified assay showed 96% sensitivity and 97% specificity in correctly discriminating compounds known to cause mitochondrial toxicity from those with prior evidence of not being mitochondrial toxicants. The greatest significance of the modified assay was its improved sensitivity in detecting the inhibition of mitochondrial fatty acid β-oxidation (FAO) when glutamine was withheld. Use of this improved assay for an empirical prediction of the likely contribution of mitochondrial toxicity to human DILI (drug induced liver injury) was attempted. According to testing of 65 DILI positive compounds representing numerous mechanisms of DILI together with 55 DILI negative compounds, the overall prediction of mitochondrial mechanism-related DILI showed 25% sensitivity and 95% specificity.