Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model.

We report for the first time label-free quantification of xenobiotic metabolizing enzymes (XME), transporters, redox enzymes, proteases, and nucleases in six human skin explants and a three-dimensional living skin equivalent model from LabSkin. We aimed to evaluate the suitability of LabSkin as an alternative to animal testing for the development of topical formulations. More than 2000 proteins were identified and quantified from total cellular protein. Alcohol dehydrogenase 1C, the most abundant phase I XME in human skin, and glutathione S-transferase pi 1, the most abundant phase II XME in human skin, were present in similar abundance in LabSkin. Several esterases were quantified and esterase activity was confirmed in LabSkin using substrate-based mass spectrometry imaging. No cytochrome P450 (P450) activity was observed for the substrates tested, in agreement with the proteomics data, where the cognate P450s were absent in both human skin and LabSkin. Label-free protein quantification allowed insights into other related processes such as redox homeostasis and proteolysis. For example, the most abundant antioxidant enzymes were thioredoxin and peroxiredoxin-1. This systematic determination of functional equivalence between human skin and LabSkin is a key step toward the construction of a representative human in vitro skin model, which can be used as an alternative to current animal-based tests for chemical safety and for predicting dosage of topically administered drugs. SIGNIFICANCE STATEMENT: The use of label-free quantitative mass spectrometry to elucidate the abundance of xenobiotic metabolizing enzymes, transporters, redox enzymes, proteases, and nucleases in human skin enhance our understanding of the skin physiology and biotransformation of topical drugs and cosmetics. This will help to develop mathematical models to predict drug metabolism in human skin and to develop more robust in vitro engineered human skin tissue as alternatives to animal testing.

Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells.

The MEKK3/MEK5/ERK5 signaling axis is required for cardiovascular development in vivo. We analyzed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co-A reductase inhibitor drugs. We utilized human cardiac microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co-localization with the tight junction protein ZO-1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co-A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provide the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin-induced cardiotoxicity.

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

Systemic gut microbial modulation of bile acid metabolism in host tissue compartments.

We elucidate the detailed effects of gut microbial depletion on the bile acid sub-metabolome of multiple body compartments (liver, kidney, heart, and blood plasma) in rats. We use a targeted ultra-performance liquid chromatography with time of flight mass-spectrometry assay to characterize the differential primary and secondary bile acid profiles in each tissue and show a major increase in the proportion of taurine-conjugated bile acids in germ-free (GF) and antibiotic (streptomycin/penicillin)-treated rats. Although conjugated bile acids dominate the hepatic profile (97.0 ± 1.5%) of conventional animals, unconjugated bile acids comprise the largest proportion of the total measured bile acid profile in kidney (60.0 ± 10.4%) and heart (53.0 ± 18.5%) tissues. In contrast, in the GF animal, taurine-conjugated bile acids (especially taurocholic acid and tauro-ß-muricholic acid) dominated the bile acid profiles (liver: 96.0 ± 14.5%; kidney: 96 ± 1%; heart: 93 ± 1%; plasma: 93.0 ± 2.3%), with unconjugated and glycine-conjugated species representing a small proportion of the profile. Higher free taurine levels were found in GF livers compared with the conventional liver (5.1-fold; P < 0.001). Bile acid diversity was also lower in GF and antibiotic-treated tissues compared with conventional animals. Because bile acids perform important signaling functions, it is clear that these chemical communication networks are strongly influenced by microbial activities or modulation, as evidenced by farnesoid X receptor-regulated pathway transcripts. The presence of specific microbial bile acid co-metabolite patterns in peripheral tissues (including heart and kidney) implies a broader signaling role for these compounds and emphasizes the extent of symbiotic microbial influences in mammalian homeostasis.

Safety pharmacology--current and emerging concepts.

Safety pharmacology (SP) is an essential part of the drug development process that aims to identify and predict adverse effects prior to clinical trials. SP studies are described in the International Conference on Harmonisation (ICH) S7A and S7B guidelines. The core battery and supplemental SP studies evaluate effects of a new chemical entity (NCE) at both anticipated therapeutic and supra-therapeutic exposures on major organ systems, including cardiovascular, central nervous, respiratory, renal and gastrointestinal. This review outlines the current practices and emerging concepts in SP studies including frontloading, parallel assessment of core battery studies, use of non-standard species, biomarkers, and combining toxicology and SP assessments. Integration of the newer approaches to routine SP studies may significantly enhance the scope of SP by refining and providing mechanistic insight to potential adverse effects associated with test compounds.

Data science in drug discovery safety: Challenges and opportunities.

Early de-risking of drug targets and chemistry is essential to provide drug projects with the best chance of success. Target safety assessments (TSAs) use target biology, gene and protein expression data, genetic information from humans and animals, and competitor compound intelligence to understand the potential safety risks associated with modulating a drug target. However, there is a vast amount of information, updated daily that must be considered for each TSA. We have developed a data science-based approach that allows acquisition of relevant evidence for an optimal TSA. This is built on expert-led conventional and artificial intelligence-based mining of literature and other bioinformatics databases. Potential safety risks are identified according to an evidence framework, adjusted to the degree of target novelty. Expert knowledge is necessary to interpret the evidence and to take account of the nuances of drug safety, the modality, and the intended patient population for each TSA within each project. Overall, TSAs take full advantage of the most recent developments in data science and can be used within drug projects to identify and mitigate risks, helping with informed decision-making and resource management. These approaches should be used in the earliest stages of a drug project to guide decisions such as target selection, discovery chemistry options, in vitro assay choice, and end points for investigative in vivo studies.

Pharmacological activation of the pyruvate dehydrogenase complex reduces statin-mediated upregulation of FOXO gene targets and protects against statin myopathy in rodents.

We previously reported that statin myopathy is associated with impaired carbohydrate (CHO) oxidation in fast-twitch rodent skeletal muscle, which we hypothesised occurred as a result of forkhead box protein O1 (FOXO1) mediated upregulation of pyruvate dehydrogenase kinase-4 (PDK4) gene transcription. Upregulation of FOXO gene targets known to regulate proteasomal and lysosomal muscle protein breakdown was also evident. We hypothesised that increasing CHO oxidation in vivo, using the pyruvate dehydrogenase complex (PDC) activator, dichloroacetate (DCA), would blunt activation of FOXO gene targets and reduce statin myopathy. Female Wistar Hanover rats were dosed daily for 12 days (oral gavage) with either vehicle (control, 0.5% w/v hydroxypropyl-methylcellulose 0.1% w/v polysorbate-80; n = 9), 88 mg( )kg(-1) day(-1) simvastatin (n = 8), 88 mg( )kg(-1) day(-1) simvastatin + 30 mg kg(-1) day(-1) DCA (n = 9) or 88 mg kg(-1) day(-1) simvastatin + 40 mg kg(-1) day(-1) DCA (n = 9). Compared with control, simvastatin reduced body mass gain and food intake, increased muscle fibre necrosis, plasma creatine kinase levels, muscle PDK4, muscle atrophy F-box (MAFbx) and cathepsin-L mRNA expression, increased PDK4 protein expression, and proteasome and cathepsin-L activity, and reduced muscle PDC activity. Simvastatin with DCA maintained body mass gain and food intake, abrogated the myopathy, decreased muscle PDK4 mRNA and protein, MAFbx and cathepsin-L mRNA, increased activity of PDC and reduced proteasome activity compared with simvastatin. PDC activation abolished statin myopathy in rodent skeletal muscle, which occurred at least in part via inhibition of FOXO-mediated transcription of genes regulating muscle CHO utilisation and protein breakdown.

Variation in antibiotic-induced microbial recolonization impacts on the host metabolic phenotypes of rats.

The interaction between the gut microbiota and their mammalian host is known to have far-reaching consequences with respect to metabolism and health. We investigated the effects of eight days of oral antibiotic exposure (penicillin and streptomycin sulfate) on gut microbial composition and host metabolic phenotype in male Han-Wistar rats (n = 6) compared to matched controls. Early recolonization was assessed in a third group exposed to antibiotics for four days followed by four days recovery (n = 6). Fluorescence in situ hybridization analysis of the intestinal contents collected at eight days showed a significant reduction in all bacterial groups measured (control, 10(10.7) cells/g feces; antibiotic-treated, 10(8.4)). Bacterial suppression reduced the excretion of mammalian-microbial urinary cometabolites including hippurate, phenylpropionic acid, phenylacetylglycine and indoxyl-sulfate whereas taurine, glycine, citrate, 2-oxoglutarate, and fumarate excretion was elevated. While total bacterial counts remained notably lower in the recolonized animals (10(9.1) cells/g faeces) compared to the controls, two cage-dependent subgroups emerged with Lactobacillus/Enterococcus probe counts dominant in one subgroup. This dichotomous profile manifested in the metabolic phenotypes with subgroup differences in tricarboxylic acid cycle metabolites and indoxyl-sulfate excretion. Fecal short chain fatty acids were diminished in all treated animals. Antibiotic treatment induced a profound effect on the microbiome structure, which was reflected in the metabotype. Moreover, the recolonization process was sensitive to the microenvironment, which may impact on understanding downstream consequences of antibiotic consumption in human populations.

Analysis of ß-catenin gene mutations and gene expression in liver tumours of C57BL/10J mice produced by chronic administration of sodium phenobarbital.

In this study liver tumours produced in male and female mice of the low spontaneous liver tumour incidence C57BL/10J strain treated for 99 weeks with 1000 ppm in the diet with the model constitutive androstane receptor (CAR) activator sodium phenobarbital (NaPB) were analysed for ß-catenin mutations by Western immunoblotting and DNA/RNA analysis. Some gene array analysis was also performed to identify genes involved in CAR activation and in ß-catenin and Hras gene mutations. Analysis of 8 male and 2 female NaPB-induced liver tumour samples (comprising 2 adenomas, 6 carcinomas and 2 samples containing separate adenomas and carcinomas) revealed truncated ß-catenin forms in just 4 male liver tumour samples, with the presence of the truncated ß-catenin forms being confirmed by ß-catenin exon 1-3 mutation analysis. Microarray gene expression analysis was performed with three of the NaPB-induced male mouse liver tumour samples where ß-catenin mutations had not been identified by Western immunoblotting and DNA/RNA analysis and with three liver samples from both NaPB-induced non-tumour tissue and control animals. Treatment with NaPB resulted in induction of Cyp2b subfamily gene expression in both NaPB-induced mouse liver tumours and in NaPB-treated non-tumour tissue. In addition, the gene expression analysis demonstrated that the ß-catenin and Hras pathways were not modified in NaPB-induced mouse liver tumours not exhibiting truncated ß-catenin forms. Overall, while chronic administration of the model CAR activator NaPB results in both hepatocellular adenoma and carcinoma in the low spontaneous liver tumour incidence C57BL/10J mouse strain, only 40 % of the liver tumours evaluated in this study had ß-catenin mutations. These results are in agreement with previous studies with the CAR activator oxazepam and demonstrate that mouse liver tumours induced by nongenotoxic CAR activators in the absence of initiation with a genotoxic agent are due to a number of mechanisms, including those largely independent of either the Wnt/ß-catenin signalling pathway or Hras oncogene mutations.

Blunted Akt/FOXO signalling and activation of genes controlling atrophy and fuel use in statin myopathy.

Statins are used clinically for cholesterol reduction, but statin therapy is associated with myopathic changes through a poorly defined mechanism. We used an in vivo model of statin myopathy to determine whether statins up-regulate genes associated with proteasomal- and lysosomal-mediated proteolysis and whether PDK gene expression is simultaneously up-regulated leading to the impairment of muscle carbohydrate oxidation. Animals were dosed daily with 80 mg kg(-1) day(-1) simvastatin for 4 (n = 6) and 12 days (n = 5), 88 mg kg(-1) day(-1) simvastatin for 12 days (n = 4), or vehicle (0.5% w/v hydroxypropyl-methylcellulose and 0.1% w/v polysorbate 80; Control, n = 6) for 12 days by oral gavage. We found, in biceps femoris muscle, decreased Akt(Ser473), FOXO1(Ser253) and FOXO3a(Ser253) phosphorylation in the cytosol (P < 0.05, P < 0.05, P < 0.001, respectively) and decreased phosphorylation of FOXO1 in the nucleus after 12 days simvastatin when compared to Control (P < 0.05). This was paralleled by a marked increase in the transcription of downstream targets of FOXO, i.e. MAFbx (P < 0.001), MuRF-1 (P < 0.001), cathepsin-L (P < 0.05), PDK2 (P < 0.05) and PDK4 (P < 0.05). These changes were accompanied by increased PPARalpha (P < 0.05), TNFalpha (P < 0.01), IL6 (P < 0.01), Mt1A (P < 0.01) mRNA and increased muscle glycogen (P < 0.05) compared to Control. RhoA activity decreased after 4 days simvastatin (P < 0.05); however, activity was no different from Control after 12 days. Simvastatin down-regulated PI3k/Akt signalling, independently of RhoA, and up-regulated FOXO transcription factors and downstream gene targets known to be implicated in proteasomal- and lysosomal-mediated muscle proteolysis, carbohydrate oxidation, oxidative stress and inflammation in an in vivo model of statin-induced myopathy. These changes occurred in the main before evidence of extensive myopathy or a decline in the muscle protein to DNA ratio.

Statins but not thiazolidinediones attenuate albumin-mediated chemokine production by proximal tubular cells independently of endocytosis.

BACKGROUND: Proximal tubular epithelial cells (PTEC) secrete chemokines under proteinuric conditions. Both statins and thiazolidinediones (TZDs) possess pleiotropic anti-inflammatory effects. This study examined the ability of statins and TZDs and the natural peroxisome proliferator activated receptor-gamma (PPARgamma) agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) to attenuate the proteinuria-induced pro-inflammatory phenotype of PTEC. METHODS: Mouse PTEC were treated with statins, TZDs and PGJ(2 )and effects on uptake and binding of FITC-albumin determined. PTEC were incubated with fatty acid free bovine serum albumin with or without statins/TZDs/PGJ(2), and the release of MCP-1 and RANTES measured. RESULTS: Statins and TZDs significantly inhibited PTEC albumin endocytosis. PGJ(2 )had no effect. Incubation of PTEC with albumin significantly stimulated production of MCP-1 and RANTES. Co-treatment with statins and PGJ(2) significantly reduced albumin-stimulated chemokine production, an effect reversed by the addition of mevalonate and geranylgeranyl pyrophosphate. In contrast, TZDs had no effect on albumin-mediated chemokine production. CONCLUSION: Statins and PGJ(2), but not TZDs, prevent the development of a PTEC pro-inflammatory phenotype in response to albumin. Albumin endocytosis is not a prerequisite for PTEC chemokine production, and inhibition of albumin endocytosis alone is insufficient to attenuate chemokine production. These studies suggest a therapeutic role for statins and some PPARgamma ligands in proteinuric renal disease.

In Vitro Modeling of Bile Acid Processing by the Human Fecal Microbiota.

Bile acids, the products of concerted host and gut bacterial metabolism, have important signaling functions within the mammalian metabolic system and a key role in digestion. Given the complexity of the mega-variate bacterial community residing in the gastrointestinal tract, studying associations between individual bacterial genera and bile acid processing remains a challenge. Here, we present a novel in vitro approach to determine the bacterial genera associated with the metabolism of different primary bile acids and their potential to contribute to inter-individual variation in this processing. Anaerobic, pH-controlled batch cultures were inoculated with human fecal microbiota and treated with individual conjugated primary bile acids (500 µg/ml) to serve as the sole substrate for 24 h. Samples were collected throughout the experiment (0, 5, 10, and 24 h) and the bacterial composition was determined by 16S rRNA gene sequencing and the bile acid signatures were characterized using a targeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) approach. Data fusion techniques were used to identify statistical bacterial-metabolic linkages. An increase in gut bacteria associated bile acids was observed over 24 h with variation in the rate of bile acid metabolism across the volunteers (n = 7). Correlation analysis identified a significant association between the Gemmiger genus and the deconjugation of glycine conjugated bile acids while the deconjugation of taurocholic acid was associated with bacteria from the Eubacterium and Ruminococcus genera. A positive correlation between Dorea and deoxycholic acid production suggest a potential role for this genus in cholic acid dehydroxylation. A slower deconjugation of taurocholic acid was observed in individuals with a greater abundance of Parasutterella and Akkermansia. This work demonstrates the utility of integrating compositional (metataxonomics) and functional (metabonomics) systems biology approaches, coupled to in vitro model systems, to study the biochemical capabilities of bacteria within complex ecosystems. Characterizing the dynamic interactions between the gut microbiota and the bile acid pool enables a greater understanding of how variation in the gut microbiota influences host bile acid signatures, their associated functions and their implications for health.

Towards better models and mechanistic biomarkers for drug-induced gastrointestinal injury.

Adverse drug reactions affecting the gastrointestinal (GI) tract are a serious burden on patients, healthcare providers and the pharmaceutical industry. GI toxicity encompasses a range of pathologies in different parts of the GI tract. However, to date no specific mechanistic diagnostic/prognostic biomarkers or translatable pre-clinical models of GI toxicity exist. This review will cover the current knowledge of GI ADRs, existing biomarkers and models with potential application for toxicity screening/monitoring. We focus on the current gaps in our knowledge, the potential opportunities and recommend that a systematic approach is needed to identify mechanism-based GI biomarkers with potential for clinical translation.

Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability.

Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.

Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.

The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca(2+ )transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca(2+ )handling (S100A1), sarcomere assembly (telethonin/TCAP) and ß-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

Characterization of the adenosine pharmacology of ticagrelor reveals therapeutically relevant inhibition of equilibrative nucleoside transporter 1.

INTRODUCTION: Studies have shown that ticagrelor has a further adenosine-mediated mechanism of action in addition to its potent inhibition of the P2Y12 receptor, which may explain some of ticagrelor's clinical characteristics. This study aimed to further characterize the adenosine pharmacology of ticagrelor, its major metabolites, and other P2Y12 receptor antagonists. METHODS: Inhibition of nucleoside transporter-mediated [(3)H]adenosine uptake by ticagrelor, its major metabolites, and alternative P2Y12 antagonists was examined in recombinant Madin-Darby canine kidney (MDCK) cells. The pharmacology of ticagrelor and its major metabolites at adenosine A1, A2A, A2B, and A3 receptor subtypes was examined using in vitro radioligand binding and functional assays and ex vivo C-fiber experiments in rat and guinea pig vagus nerves. RESULTS: Ticagrelor (and less effectively its metabolites) and the main cangrelor metabolite inhibited [(3)H]adenosine uptake in equilibrative nucleoside transporter (ENT) 1-expressing MDCK cells, whereas cangrelor and the active metabolites of prasugrel or clopidogrel had no effect. No significant inhibitory activity was observed in MDCK cells expressing ENT2 or concentrative nucleoside transporters 2/3. Ticagrelor demonstrated high affinity (inhibition constant [Ki] = 41 nmol/L) for ENT1. In adenosine receptor-binding experiments, ticagrelor and its major circulating metabolite, AR-C124910XX, had low affinity (Ki > 6 µmol/L) for each of the adenosine A1, A2A, and A2B receptors, whereas ticagrelor had a submicromolar (Ki = 190 nmol/L) affinity for the adenosine A3 receptor. However, in functional assays, at high concentrations (10 µmol/L) ticagrelor only partially inhibited 3 mmol/L adenosine-induced depolarizations in the guinea pig and rat vagus nerve preparations (by 35% and 49%, respectively). CONCLUSIONS: Ticagrelor inhibits cellular adenosine uptake selectively via ENT1 inhibition at concentrations of clinical relevance. However, the low-binding affinity and functional inhibition of adenosine receptors observed with ticagrelor or its metabolites indicate that they possess a negligible adenosine-like activity at clinically relevant concentrations.

Probiotic administration attenuates myocardial hypertrophy and heart failure after myocardial infarction in the rat.

BACKGROUND: Probiotics are extensively used to promote gastrointestinal health, and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut. Here, we determined whether oral probiotic administration can alter the progression of postinfarction heart failure. METHODS AND RESULTS: Rats were subjected to 6 weeks of sustained coronary artery occlusion and administered the probiotic Lactobacillus rhamnosus GR-1 or placebo in the drinking water ad libitum. Culture and 16s rRNA sequencing showed no evidence of GR-1 colonization or a significant shift in the composition of the cecal microbiome. However, animals administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy based on tissue weight assessment and gene expression of atrial natriuretic peptide. Moreover, these animals demonstrated improved hemodynamic parameters reflecting both improved systolic and diastolic left ventricular function. Serial echocardiography revealed significantly improved left ventricular parameters throughout the 6-week follow-up period including a marked preservation of left ventricular ejection fraction and fractional shortening. Beneficial effects of GR-1 were still evident in those animals in which GR-1 was withdrawn at 4 weeks, suggesting persistence of the GR-1 effects after cessation of therapy. Investigation of mechanisms showed a significant increase in the leptin:adiponectin plasma concentration ratio in rats subjected to coronary ligation, which was abrogated by GR-1. Metabonomic analysis showed differences between sham control and coronary artery ligated hearts particularly with respect to preservation of myocardial taurine levels. CONCLUSIONS: The study suggests that probiotics offer promise as a potential therapy for the attenuation of heart failure.