Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

Comparison of Human Long-Term Liver Models for Clearance Prediction of Slowly Metabolized Compounds.

The accurate prediction of human clearance is an important task during drug development. The proportion of low clearance compounds has increased in drug development pipelines across the industry since such compounds may be dosed in lower amounts and at lower frequency. These type of compounds present new challenges to in vitro systems used for clearance extrapolation. In this study, we compared the accuracy of clearance predictions of suspension culture to four different long-term stable in vitro liver models, including HepaRG sandwich culture, the Hµrel stochastic co-culture, the Hepatopac micropatterned co-culture (MPCC), and a micro-array spheroid culture. Hepatocytes in long-term stable systems remained viable and active over several days of incubation. Although intrinsic clearance values were generally high in suspension culture, clearance of low turnover compounds could frequently not be determined using this method. Metabolic activity and intrinsic clearance values from HepaRG cultures were low and, consequently, many compounds with low turnover did not show significant decline despite long incubation times. Similarly, stochastic co-cultures occasionally failed to show significant turnover for multiple low and medium turnover compounds. Among the different methods, MPCCs and spheroids provided the most consistent measurements. Notably, all culture methods resulted in underprediction of clearance; this could, however, be compensated for by regression correction. Combined, the results indicate that spheroid culture as well as the MPCC system provide adequate in vitro tools for human extrapolation for compounds with low metabolic turnover. SIGNIFICANCE STATEMENT: In this study, we compared suspension cultures, HepaRG sandwich cultures, the Hµrel liver stochastic co-cultures, the Hepatopac micropatterned co-cultures (MPCC), and micro-array spheroid cultures for low clearance determination and prediction. Overall, HepaRG and suspension cultures showed modest value for the low determination and prediction of clearance compounds. The micro-array spheroid culture resulted in the most robust clearance measurements, whereas using the MPCC resulted in the most accurate prediction for low clearance compounds.

Utility of preclinical species for uncertainty assessment and correction of prediction of human volume of distribution using the Rodgers-Lukacova model.

Prediction of rat, dog, monkey, and human volume of distribution (VDss) by Rodgers-Lukacova model was evaluated using a data set of more than 100 compounds.The prediction accuracy was best for humans followed by monkeys and dogs with 59, 52, and 41% of compounds within 2-fold, respectively.The accuracy of predictions in preclinical species was indicative of the human situation. This was particularly true for monkeys, where 87% of the compounds that were predicted within 2-fold in monkeys were also predicted within 2-fold in humans.The model's tendency to underestimate VDss was higher in rats and dogs compared to humans and monkeys for all ion classes but zwitterions. Hence, correction of human predictions using prediction errors in rats and dogs resulted in overestimation of VDss.The model had a similar degree of underestimation in humans and monkeys. Correction using monkeys improved the accuracy of the human estimate, especially for basic and zwitterion compounds.A strategy is proposed based on the accuracy of prediction in monkey and monkey scalars for prediction and prospective assessment of the accuracy of human VDss.

Deconvolution of Cytochrome P450 Induction Mechanisms in HepaRG Nuclear Hormone Receptor Knockout Cells.

Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.

Prediction of volume of distribution in humans: analysis of eight methods and their application in drug discovery.

The performance of eight different methods to predict human volume of distribution (VDss) using a large data set (N > 100) was evaluated.The accuracy was assessed by the end points % within two-fold and absolute average fold error (AAFE). The ability to rank order was accessed by the σ and bias was examined using average fold error. Significance of observed differences was established using statistical permutation testing.The Rodgers-Lukova equation, a tissue composition model, for acids and single species scaling based on rat for other ion classes showed the best results in absence of non-rodent data.The semimechanistic Øie-Tozer model based on all thee preclinical species showed the best performance overall (81% within two-fold, AAFE 1.55, σ 0.62). This was not statistically significantly better at the 95% confidence level than the same model based on two preclinical species or single species scaling from monkey. Thus, the use of primates appears difficult to justify when the sole goal is to extrapolate human volume of distribution.

Sensitization to storage proteins in peanut and hazelnut is associated with higher levels of inflammatory markers in asthma.

BACKGROUND: Sensitization to peanuts and hazelnuts is common among young asthmatics and can be primary or a result of cross-reactivity. Sensitization as a result of cross-reactivity to birch pollen is typically associated to tolerance or mild and local symptoms upon intake of peanut or hazelnut. AIM: The aim of this study was to investigate relationships between IgE antibody responses against peanut and hazelnut components, airway and systemic inflammation markers, lung function parameters and reported food hypersensitivity in a cohort of asthmatic children and young adults. METHODS: A population of 408 asthmatic individuals aged 10-35 years were investigated. Information on hypersensitivity symptoms upon intake of peanut or hazelnut were recorded in a standardized questionnaire. Fraction of exhaled nitric oxide (FeNO), blood eosinophil count (B-Eos), spirometry, methacholine challenge outcome and IgE antibodies to peanut and hazelnut allergens were measured by standard clinical and laboratory methods. RESULTS: Subjects sensitized to any of the peanut (Ara h 1, 2 or 3) or hazelnut (Cor a 9 or 14) storage proteins were significantly younger (17.6 vs 21.2 years), had higher levels of FeNO (23.2 vs 16.7 ppb) and B-Eos (340 vs 170 cells/mcl) than those displaying only pollen-related cross-reactive sensitization. Levels of FeNO correlated with levels of IgE to storage proteins in children, but not in adults. Levels of B-Eos correlated with levels of IgE to all allergen components investigated in children, but only to levels of IgE to storage proteins in adults. Anaphylaxis and skin reactions upon intake of peanuts or hazelnuts were more often reported among subjects sensitized to the respective storage proteins than among those with only pollen-related cross-reactive sensitization. As compared to peanut, hazelnut was more often reported to cause gastrointestinal symptoms and less often oral cavity symptoms. CONCLUSIONS: Sensitization to peanut and hazelnut storage proteins was associated with higher levels of inflammation markers and food hypersensitivity symptoms in this population of subjects with asthma.

Determination of low intrinsic clearance in vitro: the benefit of a novel internal standard in human hepatocyte incubations.

1. A novel method utilizing an internal standard in hepatocytes incubations has been developed and demonstrated to decrease the variability in the determination of intrinsic clearance (CLint) in this system. The reduced variability was shown to allow differentiation of lower elimination rate constants from noise. 2. The suggested method was able to compensate for a small but systematic error (0.5 µL/min/106 cells) caused by an evaporation of approximately 15% of the volume during the incubation time. 3. The approach was validated using six commercial drugs (ketoprofen, tolbutamide, phenacetin, etodolac and quinidine) which were metabolized by different pathways. 4. The suggested internal standard, MSC1815677, was extensively characterized and the acquired data suggest that it fulfills the requirements of an internal standard present during the incubation. The proposed internal standard was stable during the incubation and showed a low potential to inhibit drug metabolizing enzymes and transporters. With MSC1815677 we propose a novel simple, robust and cost-effective method to address the challenges in the estimation of low clearance in hepatocyte incubations.

Comparison of methods for the prediction of human clearance from hepatocyte intrinsic clearance for a set of reference compounds and an external evaluation set.

1. We compared direct scaling, regression model equation and the so-called "Poulin et al." methods to scale clearance (CL) from in vitro intrinsic clearance (CLint) measured in human hepatocytes using two sets of compounds. One reference set comprised of 20 compounds with known elimination pathways and one external evaluation set based on 17 compounds development in Merck (MS). 2. A 90% prospective confidence interval was calculated using the reference set. This interval was found relevant for the regression equation method. The three outliers identified were justified on the basis of their elimination mechanism. 3. The direct scaling method showed a systematic underestimation of clearance in both the reference and evaluation sets. The "Poulin et al." and the regression equation methods showed no obvious bias in either the reference or evaluation sets. 4. The regression model equation was slightly superior to the "Poulin et al." method in the reference set and showed a better absolute average fold error (AAFE) of value 1.3 compared to 1.6. A larger difference was observed in the evaluation set were the regression method and "Poulin et al." resulted in an AAFE of 1.7 and 2.6, respectively (removing the three compounds with known issues mentioned above). A similar pattern was observed for the correlation coefficient. Based on these data we suggest the regression equation method combined with a prospective confidence interval as the first choice for the extrapolation of human in vivo hepatic metabolic clearance from in vitro systems.

Sensitization to secretoglobin and lipocalins in a group of young children with risk of developing respiratory allergy.

BACKGROUND: Multiple sensitizations in early age have been reported to be a risk for development of asthma. This study evaluates the emergence and evolution of IgE to aeroallergens among a cohort of children with physician-diagnosed atopic dermatitis and/or showing food allergy symptoms and to examine the relation to asthma development. METHODS: Three-hundred and four children (median age 13.4 months at entry) with food allergy symptoms and/or atopic dermatitis without asthma at inclusion were analysed for IgE antibodies against food-, indoor- and outdoor-allergens and pet allergen components and correlated to the individuals' outcome on asthma inception. RESULTS: At 2 years of follow-up, physician-diagnosed asthma was 19.7% (n = 49) and asthma diagnosed any time was 24% (n = 67). History of persistent cough and asthma of father, combination of milk- and wheat-allergy symptoms and dual sensitization to house dust mite and Japanese cedar were independent risk factors for asthma. Sensitization to dog was the most prevalent inhalant allergen at entry. Asthma children had a higher proportion of sensitization to dog, cat and horse allergens at entry compared with non-asthma children. Being sensitized to both food, house dust mite and pet allergens was strongly associated with asthma (p = 0.0006). Component resolved diagnosis for dog and cat allergens showed that IgE antibodies to Can f 1 and Fel d 1 was common even at very young age. CONCLUSIONS: Early sensitization to inhalant allergens increases the risk of developing asthma as well as having milk and wheat allergy symptoms. Sensitization to dog, was common at an early age despite dog ownership. Sensitization to secretoglobin and lipocalins and less to serum albumins explained the pet sensitization.

A comparative evaluation of models to predict human intestinal metabolism from nonclinical data.

Extensive gut metabolism is often associated with the risk of low and variable bioavailability. The prediction of the fraction of drug escaping gut wall metabolism as well as transporter-mediated secretion (Fg ) has been challenged by the lack of appropriate preclinical models. The purpose of this study is to compare the performance of models that are widely employed in the pharmaceutical industry today to estimate Fg and, based on the outcome, to provide recommendations for the prediction of human Fg during drug discovery and early drug development. The use of in vitro intrinsic clearance from human liver microsomes (HLM) in three mechanistic models - the ADAM, Qgut and Competing Rates - was evaluated for drugs whose metabolism is dominated by CYP450s, assuming that the effect of transporters is negligible. The utility of rat as a model for human Fg was also explored. The ADAM, Qgut and Competing Rates models had comparable prediction success (70%, 74%, 69%, respectively) and bias (AFE = 1.26, 0.74 and 0.81, respectively). However, the ADAM model showed better accuracy compared with the Qgut and Competing Rates models (RMSE =0.20 vs 0.30 and 0.25, respectively). Rat is not a good model (prediction success =32%, RMSE =0.48 and AFE = 0.44) as it seems systematically to under-predict human Fg . Hence, we would recommend the use of rat to identify the need for Fg assessment, followed by the use of HLM in simple models to predict human Fg . © 2017 Merck KGaA. Biopharmaceutics & Drug Disposition Published by John Wiley & Sons, Ltd.

Metabolic profiling of TRPV1 antagonists of the benzothiazole amide series: implications for in vitro genotoxicity assessment.

In vitro metabolic profiling and in vitro genotoxicity assessment are important aspects of the drug discovery program as they eliminate harmful compounds from further development. In standard in vitro genotoxicity testing, induced rat liver S9 is used as an exogenous bio-activation system for detecting promutagens. In this study we show that rat liver S9 is an insufficient system regarding the conversion of TRPV1 antagonists of the benzothiazole amide series into relevant in vivo metabolites. Human and rat hepatocyte experiments demonstrated generation of an aryl amine metabolite that was subsequently N-acetylated. The hydrolyzed metabolites as well as the parent compound were also metabolized into glutathione (GSH) conjugates. Rat liver S9 exhibited a very low amide hydrolysis capacity and no formation of GSH conjugates when supplemented with NADPH and GSH. The discrepancy in metabolic capability between hepatocytes and rat liver S9 led to confounding results in in vitro genotoxicity assessment for this chemical class as judged by the results of Ames test, mouse lymphoma assay, SOS/umu test and Comet assay in rat hepatocytes. This study highlights the pivotal role that understanding the mechanism of metabolite formation has in interpreting as well as designing reliable and relevant in vitro genotoxicity experiments.

Comparative analysis of YAP/TEAD inhibitors in 2D and 3D cultures of primary human hepatocytes reveals a novel non-canonical mechanism of CYP induction.

Induction of cytochrome P450 (CYP) genes constitutes an important cause of drug-drug interactions and preclinical evaluation of induction liability is mandatory for novel drug candidates. YAP/TEAD signaling has emerged as an attractive target for various oncological indications and multiple chemically distinct YAP/TEAD inhibitors are rapidly progressing towards clinical stages. Here, we tested the liability for CYP induction of a diverse set of YAP/TEAD inhibitors with different modes of action and TEAD isoform selectivity profiles in monolayers and 3D spheroids of primary human hepatocytes (PHH). We found that YAP/TEAD inhibition resulted in broad induction of CYPs in 2D monolayers, whereas, if at all, only marginal induction was seen in spheroid culture. Comprehensive RNA-Seq indicated that YAP/TEAD signaling was increased in 2D culture compared to spheroids, which was paralleled by elevated activities of the interacting transcription factors LXR and ESRRA, likely at least in part due to altered mechanosensing. Inhibition of this YAP/TEAD hyperactivation resulted in an overall reduction of hepatocyte dedifferentiation marked by increased hepatic functionality, including CYPs. These results thus demonstrate that the observed induction is due to on-target effects of the compounds rather than direct activation of xenobiotic sensing nuclear receptors. Combined, the presented data link hepatocyte dedifferentiation to YAP/TEAD dysregulation, reveal a novel non-canonical pathway of CYP induction and highlight the advantage of organotypic 3D cultures to predict clinically relevant pharmacokinetic properties, particularly for atypical induction mechanisms.

Identification of M4205─A Highly Selective Inhibitor of KIT Mutations for Treatment of Unresectable Metastatic or Recurrent Gastrointestinal Stromal Tumors.

The treatment of gastrointestinal stromal tumors (GISTs) driven by activating mutations in the KIT gene is a prime example of targeted therapy for treatment of cancer. The approval of the tyrosine kinase inhibitor imatinib has significantly improved patient survival, but emerging resistance under treatment and relapse is observed. Several additional KIT inhibitors have been approved; still, there is a high unmet need for KIT inhibitors with high selectivity and broad coverage of all clinically relevant KIT mutants. An imidazopyridine hit featuring excellent kinase selectivity was identified in a high-throughput screen (HTS) and optimized to the clinical candidate M4205 (IDRX-42). This molecule has a superior profile compared to approved drugs, suggesting a best-in-class potential for recurrent and metastatic GISTs driven by KIT mutations.

The right compound in the right assay at the right time: an integrated discovery DMPK strategy.

The high rate of attrition during drug development and its associated high research and development (R&D) cost have put pressure on pharmaceutical companies to ensure that candidate drugs going to clinical testing have the appropriate quality such that the biological hypothesis could be evaluated. To help achieve this ambition, drug metabolism and pharmacokinetic (DMPK) science and increasing investment have been deployed earlier in the R&D process. To gain maximum return on investment, it is essential that DMPK concepts are both appropriately integrated into the compound design process and that compound selection is focused on accurate prediction of likely outcomes in patients. This article describes key principles that underpin the contribution of DMPK science for small-molecule research based on 15 years of discovery support in a major pharmaceutical company. It does not aim to describe the breadth and depth of DMPK science, but more the practical application for decision making in real-world situations.

Novel bioactivation mechanism of reactive metabolite formation from phenyl methyl-isoxazoles.

Recently, we described a series of phenyl methyl-isoxazole derivatives as novel, potent, and selective inhibitors of the voltage-gated sodium channel type 1.7 (Bioorg Med Chem Lett 21:3871-3876, 2011). The lead compound, 2-chloro-6-fluorobenzyl [3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbamate, showed unprecedented GSH and cysteine reactivity associated with NADPH-dependent metabolism in trapping studies using human liver microsomes. Additional trapping experiments with close analogs and mass spectra and NMR analyses suggested that the conjugates were attached directly to the 5'-methyl on the isoxazole moiety. We propose a mechanism of bioactivation via an initial oxidation of the 5'-methyl generating a stabilized enimine intermediate and a subsequent GSH attack on the 5'-methylene. Efforts to ameliorate reactive metabolite generation were undertaken to minimize the potential risk of toxicity. Formation of reactive metabolites could be significantly reduced or prevented by removing the 5'-methyl, by N-methylation of the carbamate; by replacing the nitrogen with a carbon or removing the nitrogen to obtain a carboxylate; or by inserting an isomeric 5'-methyl isoxazole. The effectiveness of these various chemical modifications in reducing GSH adduct formation is in line with the proposed mechanism. In conclusion, we have identified a novel mechanism of bioactivation of phenyl 5-methyl-isoxazol-4-yl-amines. The reactivity was attenuated by several modifications aimed to prevent the emergence of an enimine intermediate. Whether 5'-methyl isoxazoles should be considered a structural alert for potential formation of reactive metabolites is dependent on their context, i.e., 4'-nitrogen.

A pragmatic approach using first-principle methods to address site of metabolism with implications for reactive metabolite formation.

A majority of xenobiotics are metabolized by cytochrome P450 (CYP) enzymes. The discovery of drug candidates with low propensity to form reactive metabolites and low clearance can be facilitated by understanding CYP-mediated xenobiotic metabolism. Being able to predict the sites where reactive metabolites form is beneficial in drug design to produce drug candidates free of reactive metabolite issues. Herein, we report a pragmatic protocol using first-principle density functional theory (DFT) calculations for predicting sites of epoxidation and hydroxylation of aromatic substrates mediated by CYP. The method is based on the relative stabilities of the CYP-substrate intermediates or the substrate epoxides. Consequently, it concerns mainly the electronic reactivity of the substrates. Comparing to the experimental findings, the presented protocol gave excellent first-ranked epoxidation site predictions of 83%, and when the test was extended to CYP-mediated sites of aromatic hydroxylation, satisfactory results were also obtained (73%). This indicates that our assumptions are valid and also implies that the intrinsic reactivities of the substrates are in general more important than their binding poses in proteins, although the protocol may benefit from the addition of docking information.

Multi-Well Array Culture of Primary Human Hepatocyte Spheroids for Clearance Extrapolation of Slowly Metabolized Compounds.

Accurate prediction of human pharmacokinetics using in vitro tools is an important task during drug development. Albeit, currently used in vitro systems for clearance extrapolation such as microsomes and primary human hepatocytes in suspension culture show reproducible turnover, the utility of these systems is limited by a rapid decline of activity of drug metabolizing enzymes. In this study, a multi-well array culture of primary human hepatocyte spheroids was compared to suspension and single spheroid cultures from the same donor. Multi-well spheroids remained viable and functional over the incubation time of 3 days, showing physiological excretion of albumin and α-AGP. Their metabolic activity was similar compared to suspension and single spheroid cultures. This physiological activity, the high cell concentration, and the prolonged incubation time resulted in significant turnover of all tested low clearance compounds (n = 8). In stark contrast, only one or none of the compounds showed significant turnover when single spheroid or suspension cultures were used. Using multi-well spheroids and a regression offset approach (log(CLint) = 1.1 × + 0.85), clearance was predicted within 3-fold for 93% (13/14) of the tested compounds. Thus, multi-well spheroids represent a novel and valuable addition to the ADME in vitro tool kit for the determination of low clearance and overall clearance prediction. Graphical Abstract.

Current Approaches for Predicting Human PK for Small Molecule Development Candidates: Findings from the IQ Human PK Prediction Working Group Survey.

Accurate prediction of human clearance (CL) and volume of distribution at steady state (Vd,ss) for small molecule drug candidates is an essential component of assessing likely efficacious dose and clinical safety margins. In 2021, the IQ Consortium Human PK Prediction Working Group undertook a survey of IQ member companies to understand the current PK prediction methods being used to estimate these parameters across the pharmaceutical industry. The survey revealed a heterogeneity in approaches being used across the industry (e.g., the use of allometric approaches, differing incorporation of binding terms, and inconsistent use of empirical correction factors for in vitro-in vivo extrapolation, IVIVE), which could lead to different PK predictions with the same input data. Member companies expressed an interest in improving human PK predictions by identifying the most appropriate compound-class specific methods, as determined by physiochemical properties and knowledge of CL pathways. Furthermore, there was consensus that increased understanding of the uncertainty inherent to the compound class-dependent prediction would be invaluable in aiding communication of human PK and dose uncertainty at the time of candidate nomination for development. The human PK Prediction Working Group is utilizing these survey findings to help interrogate clinical IV datasets from across the IQ consortium member companies to understand PK prediction accuracy and uncertainty from preclinical datasets.

Discovery of 5-{2-[5-Chloro-2-(5-ethoxyquinoline-8-sulfonamido)phenyl]ethynyl}-4-methoxypyridine-2-carboxylic Acid, a Highly Selective in Vivo Useable Chemical Probe to Dissect MCT4 Biology.

Due to increased lactate production during glucose metabolism, tumor cells heavily rely on efficient lactate transport to avoid intracellular lactate accumulation and acidification. Monocarboxylate transporter 4 (MCT4/SLC16A3) is a lactate transporter that plays a central role in tumor pH modulation. The discovery and optimization of a novel class of MCT4 inhibitors (hit 9a), identified by a cellular screening in MDA-MB-231, is described. Direct target interaction of the optimized compound 18n with the cytosolic domain of MCT4 was shown after solubilization of the GFP-tagged transporter by fluorescence cross-correlation spectroscopy and microscopic studies. In vitro treatment with 18n resulted in lactate efflux inhibition and reduction of cellular viability in MCT4 high expressing cells. Moreover, pharmacokinetic properties of 18n allowed assessment of lactate modulation and antitumor activity in a mouse tumor model. Thus, 18n represents a valuable tool for investigating selective MCT4 inhibition and its effect on tumor biology.

Accuracy of a point-of-care testing device in children with suspected respiratory allergy.

Measuring IgE antibodies is useful in the diagnostic workup of allergy and asthma. This study was designed to assess the value of a new point-of-care test (ImmunoCAP Rapid Wheeze-Rhinitis Child [ICR]; Phadia AB, Uppsala, Sweden) in the diagnosis of atopy in children with allergy-like symptoms such as rhinitis, eczema, and recurrent episodes of wheezing. Patients (n = 175; average age, 7.2 years) referred from primary care were consecutively enrolled in two pediatric allergy referral centers in Italy and were assessed during a single visit. The ICR test included egg, milk, house-dust mite, timothy, mugwort, wall pellitory, birch, olive, cat, and dog allergens. ICR results were consistent with 78% of the positive clinical diagnoses. Agreement between negative ICR results and physician's clinical judgment ranged between 92 and 99% for the single allergens and averaged 96% for the complete profile. Overall agreement of ICR versus clinical diagnosis was 93%. A false positive ICR rate of 1% was recorded. ICR was positive for 94% of the patients with at least one positive clinical diagnosis. Based on the agreement between the physician's assessment of the clinical relevance of each allergen and the ICR results, we concluded that ICR could be a useful tool for primary care physicians to rule in or out the clinical relevance of single ICR allergens.