Mechanistic safety assessment via multi-omic characterisation of systemic pathway perturbations following in vivo MAT2A inhibition.

The tumour suppressor p16/CDKN2A and the metabolic gene, methyl-thio-adenosine phosphorylase (MTAP), are frequently co-deleted in some of the most aggressive and currently untreatable cancers. Cells with MTAP deletion are vulnerable to inhibition of the metabolic enzyme, methionine-adenosyl transferase 2A (MAT2A), and the protein arginine methyl transferase (PRMT5). This synthetic lethality has paved the way for the rapid development of drugs targeting the MAT2A/PRMT5 axis. MAT2A and its liver- and pancreas-specific isoform, MAT1A, generate the universal methyl donor S-adenosylmethionine (SAM) from ATP and methionine. Given the pleiotropic role SAM plays in methylation of diverse substrates, characterising the extent of SAM depletion and downstream perturbations following MAT2A/MAT1A inhibition (MATi) is critical for safety assessment. We have assessed in vivo target engagement and the resultant systemic phenotype using multi-omic tools to characterise response to a MAT2A inhibitor (AZ'9567). We observed significant SAM depletion and extensive methionine accumulation in the plasma, liver, brain and heart of treated rats, providing the first assessment of both global SAM depletion and evidence of hepatic MAT1A target engagement. An integrative analysis of multi-omic data from liver tissue identified broad perturbations in pathways covering one-carbon metabolism, trans-sulfuration and lipid metabolism. We infer that these pathway-wide perturbations represent adaptive responses to SAM depletion and confer a risk of oxidative stress, hepatic steatosis and an associated disturbance in plasma and cellular lipid homeostasis. The alterations also explain the dramatic increase in plasma and tissue methionine, which could be used as a safety and PD biomarker going forward to the clinic.

Kosaki overgrowth syndrome: A novel pathogenic variant in PDGFRB and expansion of the phenotype including cerebrovascular complications.

Heterozygous activating variants in platelet-derived growth factor, beta (PDGFRB) are associated with phenotypes including Kosaki overgrowth syndrome (KOGS), Penttinen syndrome and infantile myofibromatosis (IM). Here, we present three new cases of KOGS, including a patient with a novel de novo variant c.1477A > T p.(Ser493Cys), and the oldest known individual age 53 years. The KOGS phenotype includes characteristic facial features, tall stature, scoliosis, hyperelastic thin skin, lipodystrophy, variable intellectual and neurological deterioration, and abnormalities on brain imaging. Long-term outcome is unknown. Our cases confirm the phenotypic spectrum includes progressive flexion contractures, camptodactyly, widely spaced teeth, and constriction rings. We also propose novel occasional features including craniosynostosis, ocular pterygia, anterior chamber cleavage syndrome, early osteoporosis, increased pigmentation, recurrent haematomas, predisposition to cellulitis, nail dystrophy, carpal tunnel syndrome, recurrent hypoglycaemia in infancy, joint dislocation, and splenomegaly. Importantly, we report fusiform aneurysm of the basilar artery in two patients. Complications include thrombosis and stroke in the oldest reported patient and fatal rupture at the age of 21 in the patient with the novel variant. We conclude that cerebrovascular complications are part of the phenotypic spectrum of KOGS and KOGS-like disorders and suggest vascular imaging is indicated in these patients.

Predicting Drug-Induced Liver Injury with Bayesian Machine Learning.

Drug induced liver injury (DILI) can require significant risk management in drug development and on occasion can cause morbidity or mortality, leading to drug attrition. Optimizing candidates preclinically can minimize hepatotoxicity risk, but it is difficult to predict due to multiple etiologies encompassing DILI, often with multifactorial and overlapping mechanisms. In addition to epidemiological risk factors, physicochemical properties, dose, disposition, lipophilicity, and hepatic metabolic function are also relevant for DILI risk. Better human-relevant, predictive models are required to improve hepatotoxicity risk assessment in drug discovery. Our hypothesis is that integrating mechanistically relevant hepatic safety assays with Bayesian machine learning will improve hepatic safety risk prediction. We present a quantitative and mechanistic risk assessment for candidate nomination using data from in vitro assays (hepatic spheroids, BSEP, mitochondrial toxicity, and bioactivation), together with physicochemical (cLogP) and exposure (Cmaxtotal) variables from a chemically diverse compound set (33 no/low-, 40 medium-, and 23 high-severity DILI compounds). The Bayesian model predicts the continuous underlying DILI severity and uses a data-driven prior distribution over the parameters to prevent overfitting. The model quantifies the probability that a compound falls into either no/low-, medium-, or high-severity categories, with a balanced accuracy of 63% on held-out samples, and a continuous prediction of DILI severity along with uncertainty in the prediction. For a binary yes/no DILI prediction, the model has a balanced accuracy of 86%, a sensitivity of 87%, a specificity of 85%, a positive predictive value of 92%, and a negative predictive value of 78%. Combining physiologically relevant assays, improved alignment with FDA recommendations, and optimal statistical integration of assay data leads to improved DILI risk prediction.

Null variants and deletions in BRWD3 cause an X-linked syndrome of mild-moderate intellectual disability, macrocephaly, and obesity: A series of 17 patients.

BRWD3 has been described as a cause of X-linked intellectual disability, but relatively little is known about the specific phenotype. We report the largest BRWD3 patient series to date, comprising 17 males with 12 distinct null variants and 2 partial gene deletions. All patients presented with intellectual disability, which was classified as moderate (65%) or mild (35%). Behavioral issues were present in 75% of patients, including aggressive behavior, attention deficit/hyperactivity and/or autistic spectrum disorders. Mean head circumference was +2.8 SD (2.8 standard deviations above the mean), and mean BMI was +2.0 SD (in the context of a mean height of +1.3 SD), indicating a predominant macrocephaly/obesity phenotype. Shared facial features included a tall chin, prognathism, broad forehead, and prominent supraorbital ridge. Additional features, reported in a minority (<30%) of patients included cryptorchidism, neonatal hypotonia, and small joint hypermobility. This study delineates the clinical features associated with BRWD3 null variants and partial gene deletions, and suggests that BRWD3 should be included in the differential diagnosis of patients with an overgrowth-intellectual disability (OGID) phenotype, particularly in male patients with a mild or moderate intellectual disability associated with macrocephaly and/or obesity.

The phenotype of Sotos syndrome in adulthood: A review of 44 individuals.

Sotos syndrome is an overgrowth-intellectual disability (OGID) syndrome caused by NSD1 pathogenic variants and characterized by a distinctive facial appearance, an intellectual disability, tall stature and/or macrocephaly. Other associated clinical features include scoliosis, seizures, renal anomalies, and cardiac anomalies. However, many of the published Sotos syndrome clinical descriptions are based on studies of children; the phenotype in adults with Sotos syndrome is not yet well described. Given that it is now 17 years since disruption of NSD1 was shown to cause Sotos syndrome, many of the children first reported are now adults. It is therefore timely to investigate the phenotype of 44 adults with Sotos syndrome and NSD1 pathogenic variants. We have shown that adults with Sotos syndrome display a wide spectrum of intellectual ability with functioning ranging from fully independent to fully dependent. Reproductive rates are low. In our cohort, median height in adult women is +1.9 SD and men +0.5 SD. There is a distinctive facial appearance in adults with a tall, square, prominent chin. Reassuringly, adults with Sotos syndrome are generally healthy with few new medical issues; however, lymphedema, poor dentition, hearing loss, contractures and tremor have developed in a small number of individuals.

The CHD8 overgrowth syndrome: A detailed evaluation of an emerging overgrowth phenotype in 27 patients.

CHD8 has been reported as an autism susceptibility/intellectual disability gene but emerging evidence suggests that it additionally causes an overgrowth phenotype. This study reports 27 unrelated patients with pathogenic or likely pathogenic CHD8 variants (25 null variants, two missense variants) and a male:female ratio of 21:6 (3.5:1, p < .01). All patients presented with intellectual disability, with 85% in the mild or moderate range, and 85% had a height and/or head circumference ≥2 standard deviations above the mean, meeting our clinical criteria for overgrowth. Behavioral problems were reported in the majority of patients (78%), with over half (56%) either formally diagnosed with an autistic spectrum disorder or described as having autistic traits. Additional clinical features included neonatal hypotonia (33%), and less frequently seizures, pes planus, scoliosis, fifth finger clinodactyly, umbilical hernia, and glabellar hemangioma (≤15% each). These results suggest that, in addition to its established link with autism and intellectual disability, CHD8 causes an overgrowth phenotype, and should be considered in the differential diagnosis of patients presenting with increased height and/or head circumference in association with intellectual disability.

Refining the Primrose syndrome phenotype: A study of five patients with ZBTB20 de novo variants and a review of the literature.

Primrose syndrome is a rare autosomal dominant condition caused by heterozygous missense variants within ZBTB20. Through an exome sequencing approach (as part of the Deciphering Developmental Disorders [DDD] study) we have identified five unrelated individuals with previously unreported, de novo ZBTB20 pathogenic missense variants. All five missense variants targeted the C2H2 zinc finger domains. This genotype-up approach has allowed further refinement of the Primrose syndrome phenotype. Major characteristics (>90% individuals) include an intellectual disability (most frequently in the moderate range), a recognizable facial appearance and brain MRI abnormalities, particularly abnormalities of the corpus callosum. Other frequent clinical associations (in 50-90% individuals) include sensorineural hearing loss (83%), hypotonia (78%), cryptorchidism in males (75%), macrocephaly (72%), behavioral issues (56%), and dysplastic/hypoplastic nails (57%). Based upon these clinical data we discuss our current management of patients with Primrose syndrome.

EED and EZH2 constitutive variants: A study to expand the Cohen-Gibson syndrome phenotype and contrast it with Weaver syndrome.

Overgrowth-intellectual disability (OGID) syndromes are characterized by increased growth (height and/or head circumference ≥+2 SD) in association with an intellectual disability. Constitutive EED variants have previously been reported in five individuals with an OGID syndrome, eponymously designated Cohen-Gibson syndrome and resembling Weaver syndrome. Here, we report three additional individuals with constitutive EED variants, identified through exome sequencing of an OGID patient series. We compare the EED phenotype with that of Weaver syndrome (56 individuals), caused by constitutive EZH2 variants. We conclude that while there is considerable overlap between the EED and EZH2 phenotypes with both characteristically associated with increased growth and an intellectual disability, individuals with EED variants more frequently have cardiac problems and cervical spine abnormalities, boys have cryptorchidism and the facial gestalts can usually be distinguished.

Integrated in vitro models for hepatic safety and metabolism: evaluation of a human Liver-Chip and liver spheroid.

Drug-induced liver injury remains a frequent reason for drug withdrawal. Accordingly, more predictive and translational models are required to assess human hepatotoxicity risk. This study presents a comprehensive evaluation of two promising models to assess mechanistic hepatotoxicity, microengineered Organ-Chips and 3D hepatic spheroids, which have enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Sensitivity of the models to two hepatotoxins, acetaminophen (APAP) and fialuridine (FIAU), was assessed across a range of cytotoxicity biomarkers (ATP, albumin, miR-122, α-GST) as well as their metabolic functionality by quantifying APAP, FIAU and CYP probe substrate metabolites. APAP and FIAU produced dose- and time-dependent increases in miR-122 and α-GST release as well as decreases in albumin secretion in both Liver-Chips and hepatic spheroids. Metabolic turnover of CYP probe substrates, APAP and FIAU, was maintained over the 10-day exposure period at concentrations where no cytotoxicity was detected and APAP turnover decreased at concentrations where cytotoxicity was detected. With APAP, the most sensitive biomarkers were albumin in the Liver-Chips (EC50 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC50 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC50 126 µM) and miR-122 (15-fold) in the liver spheroids, both on day 7. In conclusion, both models exhibited integrated toxicity and metabolism, and broadly similar sensitivity to the hepatotoxicants at relevant clinical concentrations, demonstrating the utility of these models for improved hepatotoxicity risk assessment.

Further delineation of Malan syndrome.

Malan syndrome is an overgrowth disorder described in a limited number of individuals. We aim to delineate the entity by studying a large group of affected individuals. We gathered data on 45 affected individuals with a molecularly confirmed diagnosis through an international collaboration and compared data to the 35 previously reported individuals. Results indicate that height is > 2 SDS in infancy and childhood but in only half of affected adults. Cardinal facial characteristics include long, triangular face, macrocephaly, prominent forehead, everted lower lip, and prominent chin. Intellectual disability is universally present, behaviorally anxiety is characteristic. Malan syndrome is caused by deletions or point mutations of NFIX clustered mostly in exon 2. There is no genotype-phenotype correlation except for an increased risk for epilepsy with 19p13.2 microdeletions. Variants arose de novo, except in one family in which mother was mosaic. Variants causing Malan and Marshall-Smith syndrome can be discerned by differences in the site of stop codon formation. We conclude that Malan syndrome has a well recognizable phenotype that usually can be discerned easily from Marshall-Smith syndrome but rarely there is some overlap. Differentiation from Sotos and Weaver syndrome can be made by clinical evaluation only.

Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury.

Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.

CREBBP mutations in individuals without Rubinstein-Taybi syndrome phenotype.

Mutations in CREBBP cause Rubinstein-Taybi syndrome. By using exome sequencing, and by using Sanger in one patient, CREBBP mutations were detected in 11 patients who did not, or only in a very limited manner, resemble Rubinstein-Taybi syndrome. The combined facial signs typical for Rubinstein-Taybi syndrome were absent, none had broad thumbs, and three had only somewhat broad halluces. All had apparent developmental delay (being the reason for molecular analysis); five had short stature and seven had microcephaly. The facial characteristics were variable; main characteristics were short palpebral fissures, telecanthi, depressed nasal ridge, short nose, anteverted nares, short columella, and long philtrum. Six patients had autistic behavior, and two had self-injurious behavior. Other symptoms were recurrent upper airway infections (n = 5), feeding problems (n = 7) and impaired hearing (n = 7). Major malformations occurred infrequently. All patients had a de novo missense mutation in the last part of exon 30 or beginning of exon 31 of CREBBP, between base pairs 5,128 and 5,614 (codons 1,710 and 1,872). No missense or truncating mutations in this region have been described to be associated with the classical Rubinstein-Taybi syndrome phenotype. No functional studies have (yet) been performed, but we hypothesize that the mutations disturb protein-protein interactions by altering zinc finger function. We conclude that patients with missense mutations in this specific CREBBP region show a phenotype that differs substantially from that in patients with Rubinstein-Taybi syndrome, and may prove to constitute one (or more) separate entities. © 2016 Wiley Periodicals, Inc.

Hepatic effects of repeated oral administration of diclofenac to hepatic cytochrome P450 reductase null (HRN™) and wild-type mice.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit normal hepatic and extrahepatic biotransformation enzyme activities when compared to wild-type (WT) mice, but express no functional hepatic cytochrome P450 activities. When incubated in vitro with [(14)C]-diclofenac, liver microsomes from WT mice exhibited extensive biotransformation to oxidative and glucuronide metabolites and covalent binding to proteins was also observed. In contrast, whereas glucuronide conjugates and a quinone-imine metabolite were formed when [(14)C]-diclofenac was incubated with HRN™ mouse liver, only small quantities of P450-derived oxidative metabolites were produced in these samples and covalent binding to proteins was not observed. Livers from vehicle-treated HRN™ mice exhibited enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration and necrosis and inflammatory cell infiltration, which were not present in livers from WT mice. Elevated liver-derived alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities were also observed in plasma from HRN™ mice. When treated orally with diclofenac for 7 days, at 30 mg/kg/day, the severities of the abnormal liver histopathology and plasma liver enzyme findings in HRN™ mice were reduced markedly. Oral diclofenac administration did not alter the liver histopathology or elevate plasma enzyme activities of WT mice. These findings indicate that HRN™ mice are valuable for exploration of the role played by hepatic P450s in drug biotransformation, but poorly suited to investigations of drug-induced liver toxicity. Nevertheless, studies in HRN™ mice could provide novel insights into the role played by inflammation in liver injury and may aid the evaluation of new strategies for its treatment.

Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

Glutathione metabolism modeling: a mechanism for liver drug-robustness and a new biomarker strategy.

BACKGROUND: Glutathione metabolism can determine an individual's ability to detoxify drugs. To increase understanding of the dynamics of cellular glutathione homeostasis, we have developed an experiment-based mathematical model of the kinetics of the glutathione network. This model was used to simulate perturbations observed when human liver derived THLE cells, transfected with human cytochrome P452E1 (THLE-2E1 cells), were exposed to paracetamol (acetaminophen). METHODS: Human liver derived cells containing extra human cytochrome P4502E1 were treated with paracetamol at various levels of methionine and in the presence and absence of an inhibitor of glutamyl-cysteine synthetase (GCS). GCS activity was also measured in extracts. Intracellular and extracellular concentrations of substances involved in glutathione metabolism were measured as was damage to mitochondria and proteins. A bottom up mathematical model was made of the metabolic pathways around and including glutathione. RESULTS: Our initial model described some, but not all the metabolite-concentration and flux data obtained when THLE-2E1 cells were exposed to paracetamol at concentrations high enough to affect glutathione metabolism. We hypothesized that the lack of correspondence could be due to upregulation of expression of glutamyl cysteine synthetase, one of the enzymes controlling glutathione synthesis, and confirmed this experimentally. A modified model which incorporated this adaptive response adequately described the observed changes in the glutathione pathway. Use of the adaptive model to analyze the functioning of the glutathione network revealed that a threshold input concentration of methionine may be required for effective detoxification of reactive metabolites by glutathione conjugation. The analysis also provided evidence that 5-oxoproline and ophthalmic acid are more useful biomarkers of glutathione status when analyzed together than when analyzed in isolation, especially in a new, model-assisted integrated biomarker strategy. CONCLUSION: A robust mathematical model of the dynamics of cellular changes in glutathione homeostasis in cells has been developed and tested in vitro. GENERAL SIGNIFICANCE: Mathematical models of the glutathione pathway that help examine mechanisms of cellular protection against xenobiotic toxicity and the monitoring thereof, can now be made.

Genetic Diagnosis of Retinoblastoma Using Aqueous Humour-Findings from an Extended Cohort.

The identification of somatic RB1 variation is crucial to confirm the heritability of retinoblastoma. We and others have previously shown that, when tumour DNA is unavailable, cell-free DNA (cfDNA) derived from aqueous humour (AH) can be used to identify somatic RB1 pathogenic variation. Here we report RB1 pathogenic variant detection, as well as cfDNA concentration in an extended cohort of 75 AH samples from 68 patients. We show cfDNA concentration is highly variable and significantly correlated with the collection point of the AH. Cell-free DNA concentrations above 5 pg/µL enabled the detection of 93% of known or expected RB1 pathogenic variants. In AH samples collected during intravitreal chemotherapy treatment (Tx), the yield of cfDNA above 5 pg/µL and subsequent variant detection was low (≤46%). However, AH collected by an anterior chamber tap after one to three cycles of primary chemotherapy (Dx1+) enabled the detection of 75% of expected pathogenic variants. Further limiting our analysis to Dx1+ samples taken after ≤2 cycles (Dx ≤ 2) provided measurable levels of cfDNA in all cases, and a subsequent variant detection rate of 95%. Early AH sampling is therefore likely to be important in maximising cfDNA concentration and the subsequent detection of somatic RB1 pathogenic variants in retinoblastoma patients undergoing conservative treatment.

Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Bioactivation of the cannabinoid receptor antagonist rimonabant to a cytotoxic iminium ion metabolite.

The cannabinoid type 1 receptor (CB1r) antagonist rimonabant was approved in 2006 for the treatment of obesity but was withdrawn in 2008 due to serious drug-related psychiatric disorders. In vitro metabolism studies with rimonabant have revealed high levels of reactive metabolite formation, which resulted in irreversible time-dependent P450 3A4 inhibition and in covalent binding to microsomal proteins. In the present study, an in vitro approach has been used to explore whether metabolic bioactivation of rimonabant might result in cell toxicity. A panel of SV40-T-antigen-immortalized human liver derived (THLE) cells that had been transfected with vectors encoding various human cytochrome P450 enzymes (THLE-1A2, 2C9, 2C19, 2D6, and 3A4) or with an empty vector (THLE-Null) were exposed to rimonabant. Cell toxicity and covalent binding to cellular proteins were evaluated, as was metabolite formation. Rimonabant exhibited markedly potentiated dose and time dependent cytotoxicity to THLE-3A4 cells, compared to that of all other THLE cell lines. This was accompanied by high levels of covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins (1433 pmol drug equivalents/mg protein) and the formation of several metabolites that were not generated by THLE-Null cells. These included N-aminopiperidine (NAP) and an iminium ion species. However, no toxicity was observed when THLE cells were incubated with NAP. Glutathione depletion did not alter the observed potent cell cytotoxicity of rimonabant to THLE-3A4 cells. Preincubation of THLE-3A4 cells with the cytochrome P450 3A4 inhibitor ritonavir blocked the selective toxicity of rimonabant to these cells. In addition, ritonavir pretreatment blocked the metabolism of the compound in the cells and thereby significantly decreased the covalent binding of [(14)C]-rimonabant to THLE-3A4 cell proteins. We conclude that the potent toxicity of rimonabant in THLE-3A4 cells occurs by a mechanistic sequence, which is initiated by cytochrome P450 3A4 mediated formation of a highly cytotoxic reactive iminium ion metabolite that binds covalently to cellular proteins.

Daughter dearest: Sex-biased calcium in mother's milk among rhesus macaques.

Mother's milk provides building blocks necessary for infant development and growth postnatally. Minerals in milk are particularly important for infant skeletal development and may reflect maternal characteristics that are associated with the capacity to synthesize milk and sex-specific developmental priorities of the infant. Using a large sample of mother-infant dyads assigned to the outdoor breeding colony at the California National Primate Research Center (N=104), we investigated the relationship of milk calcium (Ca) and phosphorus (P) concentrations and the ratio of Ca/P to maternal and infant characteristics and to other milk variables. Ca and P are largely associated with casein micelles, and as expected, both Ca and P were positively correlated with protein concentrations in milk. Neither Ca nor P concentrations were associated with maternal parity. Mothers rearing daughters tended to produce higher mean Ca concentration in milk, and consequently a higher Ca/P ratio, than did mothers rearing sons, even though protein concentration was not elevated. These results suggest that the Ca/P ratio in rhesus milk may have been under separate selective pressure from protein content to facilitate the accelerated rate of skeletal calcification that has been observed in female Macaca mulatta infants.

The effect of improving task representativeness on capturing nurses' risk assessment judgements: a comparison of written case simulations and physical simulations.

BACKGROUND: The validity of studies describing clinicians' judgements based on their responses to paper cases is questionable, because - commonly used - paper case simulations only partly reflect real clinical environments. In this study we test whether paper case simulations evoke similar risk assessment judgements to the more realistic simulated patients used in high fidelity physical simulations. METHODS: 97 nurses (34 experienced nurses and 63 student nurses) made dichotomous assessments of risk of acute deterioration on the same 25 simulated scenarios in both paper case and physical simulation settings. Scenarios were generated from real patient cases. Measures of judgement 'ecology' were derived from the same case records. The relationship between nurses' judgements, actual patient outcomes (i.e. ecological criteria), and patient characteristics were described using the methodology of judgement analysis. Logistic regression models were constructed to calculate Lens Model Equation parameters. Parameters were then compared between the modeled paper-case and physical-simulation judgements. RESULTS: Participants had significantly less achievement (ra) judging physical simulations than when judging paper cases. They used less modelable knowledge (G) with physical simulations than with paper cases, while retaining similar cognitive control and consistency on repeated patients. Respiration rate, the most important cue for predicting patient risk in the ecological model, was weighted most heavily by participants. CONCLUSIONS: To the extent that accuracy in judgement analysis studies is a function of task representativeness, improving task representativeness via high fidelity physical simulations resulted in lower judgement performance in risk assessments amongst nurses when compared to paper case simulations. Lens Model statistics could prove useful when comparing different options for the design of simulations used in clinical judgement analysis. The approach outlined may be of value to those designing and evaluating clinical simulations as part of education and training strategies aimed at improving clinical judgement and reasoning.