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OBJECTIVE: To determine if treatment with a 5-HT3 antagonist (ondansetron) reduces need for opioid therapy in infants at risk for neonatal opioid withdrawal syndrome (NOWS). STUDY DESIGN: A multicenter, randomized, placebo controlled, double blind clinical trial of ninety (90) infants. The intervention arms were intravenous ondansetron or placebo during labor followed by a daily dose of ondansetron or placebo in infants for five days. RESULTS: Twenty-two (49%) ondansetron-treated and 26 (63%) placebo-treated infants required pharmacologic treatment (p > 0.05). The Finnegan score was lower in the ondansetron-treated group (4.6 vs. 5.6, p = 0.02). A non-significant trend was noted for the duration of hospitalization. There was no difference in need for phenobarbital or clonidine therapy, or total dose of morphine in the first 15 days of NOWS treatment. CONCLUSIONS: Ondansetron treatment reduced the severity of NOWS symptoms; and there was an indication that it could reduce the length of stay. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov NCT01965704.
Assuntos
Analgésicos Opioides , Síndrome de Abstinência Neonatal , Recém-Nascido , Humanos , Analgésicos Opioides/uso terapêutico , Ondansetron/uso terapêutico , Morfina/efeitos adversos , Síndrome de Abstinência Neonatal/tratamento farmacológico , Fenobarbital/uso terapêuticoRESUMO
Administration of a widely used 5-hydroxytryptamine receptor (5HT3A R) antagonist (ondansetron) potently inhibited the development of experimentally induced opioid dependence and withdrawal responses in mice and humans. However, in several studies examining withdrawal symptoms in subjects with chronic opioid use disorders (OUDs), ondansetron exhibited reduced or absent efficacy. Because attenuation of opioid withdrawal symptomatology is mediated within the brain, this study examined single-dose ondansetron pharmacokinetics in the blood and brain of mice. We demonstrate that ondansetron concentrations in the brain (Cbrain ng/mg) are 1000-fold lower than the blood concentrations (Cblood ng/ml) and decrease rapidly after ondansetron administration; and that a large percentage of brain ondansetron remains in the ventricular fluid. These results indicate that the ondansetron dose, and the time window between ondansetron and opioid administration, and when withdrawal is assessed are critical considerations for clinical studies involving subjects with chronic OUD. The pharmacokinetic results and the dosing considerations discussed here can be used to improve the design of subsequent clinical trials, which will test whether a more prolonged period of ondansetron administration can provide a desperately needed therapy that can prevent the development of neonatal opioid withdrawal syndrome in babies born to mothers with chronic OUD.
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Alcaloides Opiáceos , Transtornos Relacionados ao Uso de Opioides , Síndrome de Abstinência a Substâncias , Humanos , Camundongos , Animais , Ondansetron , Analgésicos Opioides , Alcaloides Opiáceos/uso terapêutico , Encéfalo , Morfina , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Síndrome de Abstinência a Substâncias/tratamento farmacológicoRESUMO
Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax. SIGNIFICANCE: Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247.
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Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Humanos , Glutaratos/metabolismo , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , MutaçãoRESUMO
Introduction: The routine clinical diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is largely restricted to real-time reverse transcription quantitative PCR (RT-qPCR), and tests that detect SARS-CoV-2 nucleocapsid antigen. Given the diagnostic delay and suboptimal sensitivity associated with these respective methods, alternative diagnostic strategies are needed for acute infection. Methods: We studied the use of a clinically validated liquid chromatography triple quadrupole method (LC/MS-MS) for detection of amino acids from plasma specimens. We applied machine learning models to distinguish between SARS-CoV-2-positive and negative samples and analyzed amino acid feature importance. Results: A total of 200 samples were tested, including 70 from individuals with COVID-19, and 130 from negative controls. The top performing model overall allowed discrimination between SARS-CoV-2-positive and negative control samples with an area under the receiver operating characteristic curve (AUC) of 0.96 (95%CI 0.91, 1.00), overall sensitivity of 0.99 (95%CI 0.92, 1.00), and specificity of 0.92 (95%CI 0.85, 0.95). Discussion: This approach holds potential as an alternative to existing methods for the rapid and accurate diagnosis of acute SARS-CoV-2 infection.
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To investigate the pathogenesis of a congenital form of hepatic fibrosis, human hepatic organoids were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD). Here we show that these hepatic organoids develop the key features of ARPKD liver pathology (abnormal bile ducts and fibrosis) in only 21 days. The ARPKD mutation increases collagen abundance and thick collagen fiber production in hepatic organoids, which mirrors ARPKD liver tissue pathology. Transcriptomic and other analyses indicate that the ARPKD mutation generates cholangiocytes with increased TGFß pathway activation, which are actively involved stimulating myofibroblasts to form collagen fibers. There is also an expansion of collagen-producing myofibroblasts with markedly increased PDGFRB protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resemble those present in commonly occurring forms of liver fibrosis. PDGFRB pathway involvement was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. Besides providing insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.
Assuntos
Cirrose Hepática/patologia , Modelos Biológicos , Organoides/patologia , Doenças dos Ductos Biliares/genética , Doenças dos Ductos Biliares/metabolismo , Doenças dos Ductos Biliares/patologia , Colágeno/metabolismo , Células Epiteliais/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Mutação , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Rim Policístico Autossômico Recessivo/tratamento farmacológico , Rim Policístico Autossômico Recessivo/genética , Rim Policístico Autossômico Recessivo/metabolismo , Rim Policístico Autossômico Recessivo/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Respiratory virus infections are significant causes of morbidity and mortality, and may induce host metabolite alterations by infecting respiratory epithelial cells. We investigated the use of liquid chromatography quadrupole time-of-flight mass spectrometry (LC/Q-TOF) combined with machine learning for the diagnosis of influenza infection. METHODS: We analyzed nasopharyngeal swab samples by LC/Q-TOF to identify distinct metabolic signatures for diagnosis of acute illness. Machine learning models were performed for classification, followed by Shapley additive explanation (SHAP) analysis to analyze feature importance and for biomarker discovery. FINDINGS: A total of 236 samples were tested in the discovery phase by LC/Q-TOF, including 118 positive samples (40 influenza A 2009 H1N1, 39 influenza H3 and 39 influenza B) as well as 118 age and sex-matched negative controls with acute respiratory illness. Analysis showed an area under the receiver operating characteristic curve (AUC) of 1.00 (95% confidence interval [95% CI] 0.99, 1.00), sensitivity of 1.00 (95% CI 0.86, 1.00) and specificity of 0.96 (95% CI 0.81, 0.99). The metabolite most strongly associated with differential classification was pyroglutamic acid. Independent validation of a biomarker signature based on the top 20 differentiating ion features was performed in a prospective cohort of 96 symptomatic individuals including 48 positive samples (24 influenza A 2009 H1N1, 5 influenza H3 and 19 influenza B) and 48 negative samples. Testing performed using a clinically-applicable targeted approach, liquid chromatography triple quadrupole mass spectrometry, showed an AUC of 1.00 (95% CI 0.998, 1.00), sensitivity of 0.94 (95% CI 0.83, 0.98), and specificity of 1.00 (95% CI 0.93, 1.00). Limitations include lack of sample suitability assessment, and need to validate these findings in additional patient populations. INTERPRETATION: This metabolomic approach has potential for diagnostic applications in infectious diseases testing, including other respiratory viruses, and may eventually be adapted for point-of-care testing. FUNDING: None.
Assuntos
Influenza Humana/diagnóstico , Aprendizado de Máquina , Metaboloma , Técnicas de Diagnóstico Molecular/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Influenza Humana/metabolismo , Influenza Humana/virologia , Masculino , Metabolômica/métodos , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Orthomyxoviridae/patogenicidade , Ácido Pirrolidonocarboxílico/análiseRESUMO
BACKGROUND & AIMS: Since human induced pluripotent stem cells (iPSCs) develop into hepatic organoids through stages that resemble human embryonic liver development, they can be used to study developmental processes and disease pathology. Therefore, we examined the early stages of hepatic organoid formation to identify key pathways affecting early liver development. METHODS: Single-cell RNA-sequencing and metabolomic analysis was performed on developing organoid cultures at the iPSC, hepatoblast (day 9) and mature organoid stage. The importance of the phosphatidylethanolamine biosynthesis pathway to early liver development was examined in developing organoid cultures using iPSC with a CRISPR-mediated gene knockout and an over the counter medication (meclizine) that inhibits the rate-limiting enzyme in this pathway. Meclizine's effect on the growth of a human hepatocarcinoma cell line in a xenotransplantation model and on the growth of acute myeloid leukemia cells in vitro was also examined. RESULTS: Transcriptomic and metabolomic analysis of organoid development indicated that the phosphatidylethanolamine biosynthesis pathway is essential for early liver development. Unexpectedly, early hepatoblasts were selectively sensitive to the cytotoxic effect of meclizine. We demonstrate that meclizine could be repurposed for use in a new synergistic combination therapy for primary liver cancer: a glycolysis inhibitor reprograms cancer cell metabolism to make it susceptible to the cytotoxic effect of meclizine. This combination inhibited the growth of a human liver carcinoma cell line in vitro and in a xenotransplantation model, without causing significant side effects. This drug combination was also highly active against acute myeloid leukemia cells. CONCLUSION: Our data indicate that phosphatidylethanolamine biosynthesis is a targetable pathway for cancer; meclizine may have clinical efficacy as a repurposed anti-cancer drug when used as part of a new combination therapy. LAY SUMMARY: The early stages of human liver development were modeled using human hepatic organoids. We identified a pathway that was essential for early liver development. Based upon this finding, a novel combination drug therapy was identified that could be used to treat primary liver cancer and possibly other types of cancer.
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Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Leucemia Mieloide Aguda/sangue , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Meclizina/administração & dosagem , Fosfatidiletanolaminas/antagonistas & inibidores , Fosfatidiletanolaminas/biossíntese , Piridinas/administração & dosagem , Quinolinas/administração & dosagem , Adulto , Idoso , Animais , Carcinoma Hepatocelular/patologia , Sobrevivência Celular/efeitos dos fármacos , Quimioterapia Combinada/métodos , Feminino , Técnicas de Inativação de Genes , Glicólise/efeitos dos fármacos , Células Hep G2 , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Organogênese/efeitos dos fármacos , Organogênese/genética , Organoides/efeitos dos fármacos , Organoides/metabolismo , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Estudos Retrospectivos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.
Assuntos
Síndrome de Alagille/genética , Doenças Genéticas Inatas/patologia , Fígado/metabolismo , Organoides/metabolismo , Tetralogia de Fallot/genética , Diferenciação Celular , Doenças Genéticas Inatas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Proteína Jagged-1/genética , Fígado/patologia , Mutação Puntual , Receptores Notch/metabolismo , Transdução de Sinais/genéticaRESUMO
There is considerable biological and physiological heterogeneity among patients who meet standard clinical criteria for acute respiratory distress syndrome (ARDS). In this study, we tested the hypothesis that there exists a subgroup of ARDS patients who exhibit a metabolically distinct profile. We examined undiluted pulmonary edema fluid obtained at the time of endotracheal intubation from 16 clinically phenotyped ARDS patients and 13 control patients with hydrostatic pulmonary edema. Nontargeted metabolic profiling was carried out on the undiluted edema fluid. Univariate and multivariate statistical analyses including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were conducted to find discriminant metabolites. Seven-hundred and sixty unique metabolites were identified in the pulmonary edema fluid of these 29 patients. We found that a subset of ARDS patients (6/16, 38%) presented a distinct metabolic profile with the overrepresentation of 235 metabolites compared with edema fluid from the other 10 ARDS patients, whose edema fluid metabolic profile was indistinguishable from those of the 13 control patients with hydrostatic edema. This "high metabolite" endotype was characterized by higher concentrations of metabolites belonging to all of the main metabolic classes including lipids, amino acids, and carbohydrates. This distinct group with high metabolite levels in the edema fluid was also associated with a higher mortality rate. Thus metabolic profiling of the edema fluid of ARDS patients supports the hypothesis that there is considerable biological heterogeneity among ARDS patients who meet standard clinical and physiological criteria for ARDS.
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Líquidos Corporais/metabolismo , Metabolômica/métodos , Edema Pulmonar/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Adulto , Análise por Conglomerados , Demografia , Análise Discriminante , Feminino , Humanos , Análise dos Mínimos Quadrados , Masculino , Metaboloma , Pessoa de Meia-Idade , Análise de Componente Principal , Regulação para CimaRESUMO
Haloperidol is an effective antipsychotic agent, but it causes Parkinsonian-like extrapyramidal symptoms in the majority of treated subjects. To address this treatment-limiting toxicity, we analyzed a murine genetic model of haloperidol-induced toxicity (HIT). Analysis of a panel of consomic strains indicated that a genetic factor on chromosome 10 had a significant effect on susceptibility to HIT. We analyzed a whole-genome SNP database to identify allelic variants that were uniquely present on chromosome 10 in the strain that was previously shown to exhibit the highest level of susceptibility to HIT. This analysis implicated allelic variation within pantetheinase genes (Vnn1 and Vnn3), which we propose impaired the biosynthesis of cysteamine, could affect susceptibility to HIT. We demonstrate that administration of cystamine, which is rapidly metabolized to cysteamine, could completely prevent HIT in the murine model. Many of the haloperidol-induced gene expression changes in the striatum of the susceptible strain were reversed by cystamine coadministration. Since cystamine administration has previously been shown to have other neuroprotective actions, we investigated whether cystamine administration could have a broader neuroprotective effect. Cystamine administration caused a 23% reduction in infarct volume after experimentally induced cerebral ischemia. Characterization of this novel pharmacogenetic factor for HIT has identified a new approach for preventing the treatment-limiting toxicity of an antipsychotic agent, which could also be used to reduce the extent of brain damage after stroke.
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Antipsicóticos/efeitos adversos , Isquemia Encefálica/genética , Cistamina/uso terapêutico , Haloperidol/efeitos adversos , Fármacos Neuroprotetores/uso terapêutico , Polimorfismo de Nucleotídeo Único , Amidoidrolases/genética , Animais , Antipsicóticos/toxicidade , Isquemia Encefálica/etiologia , Isquemia Encefálica/prevenção & controle , Moléculas de Adesão Celular/genética , Cistamina/administração & dosagem , Cistamina/metabolismo , Proteínas Ligadas por GPI/genética , Haloperidol/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/administração & dosagem , Farmacogenética/métodosRESUMO
BACKGROUND: Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae. RESULTS: We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. CONCLUSIONS: Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.
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Sistemas CRISPR-Cas/genética , Genoma Fúngico , RNA Guia de Cinetoplastídeos/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Cromatina/genética , Humanos , Oxigenases de Função Mista/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítio de Iniciação de TranscriçãoRESUMO
Interspecies differences have limited the predictive utility of toxicology studies performed using animal species. A drug that could be a safe and effective treatment in humans could cause toxicity in animals, preventing it from being used in humans. We investigated whether the use of thymidine kinase (TK)-NOG mice with humanized livers could prevent this unfortunate outcome (i.e., "rescue" a drug for use in humans). A high dose of furosemide is known to cause severe liver toxicity in mice, but it is a safe and effective treatment in humans. We demonstrate that administration of a high dose of furosemide (200 mg/kg i.p.) causes extensive hepatotoxicity in control mice but not in humanized TK-NOG mice. This interspecies difference results from a higher rate of production of the toxicity-causing metabolite by mouse liver. Comparison of their survival curves indicated that the humanized mice were more resistant than control mice to the hepatotoxicity caused by high doses of furosemide. In this test case, humanized TK-NOG mouse studies indicate that humans could be safely treated with a high dose of furosemide.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Furosemida/toxicidade , Hepatócitos/patologia , Fígado/efeitos dos fármacos , Timidina Quinase/genética , Animais , Relação Dose-Resposta a Droga , Furosemida/administração & dosagem , Furosemida/farmacocinética , Hepatócitos/transplante , Humanos , Fígado/patologia , Masculino , Camundongos , Necrose , Especificidade da Espécie , Distribuição TecidualRESUMO
BACKGROUND: We know very little about the genetic factors affecting susceptibility to drug-induced central nervous system (CNS) toxicities, and this has limited our ability to optimally utilize existing drugs or to develop new drugs for CNS disorders. For example, haloperidol is a potent dopamine antagonist that is used to treat psychotic disorders, but 50% of treated patients develop characteristic extrapyramidal symptoms caused by haloperidol-induced toxicity (HIT), which limits its clinical utility. We do not have any information about the genetic factors affecting this drug-induced toxicity. HIT in humans is directly mirrored in a murine genetic model, where inbred mouse strains are differentially susceptible to HIT. Therefore, we genetically analyzed this murine model and performed a translational human genetic association study. METHODS AND FINDINGS: A whole genome SNP database and computational genetic mapping were used to analyze the murine genetic model of HIT. Guided by the mouse genetic analysis, we demonstrate that genetic variation within an ABC-drug efflux transporter (Abcb5) affected susceptibility to HIT. In situ hybridization results reveal that Abcb5 is expressed in brain capillaries, and by cerebellar Purkinje cells. We also analyzed chromosome substitution strains, imaged haloperidol abundance in brain tissue sections and directly measured haloperidol (and its metabolite) levels in brain, and characterized Abcb5 knockout mice. Our results demonstrate that Abcb5 is part of the blood-brain barrier; it affects susceptibility to HIT by altering the brain concentration of haloperidol. Moreover, a genetic association study in a haloperidol-treated human cohort indicates that human ABCB5 alleles had a time-dependent effect on susceptibility to individual and combined measures of HIT. Abcb5 alleles are pharmacogenetic factors that affect susceptibility to HIT, but it is likely that additional pharmacogenetic susceptibility factors will be discovered. CONCLUSIONS: ABCB5 alleles alter susceptibility to HIT in mouse and humans. This discovery leads to a new model that (at least in part) explains inter-individual differences in susceptibility to a drug-induced CNS toxicity.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Alelos , Encéfalo/metabolismo , Haloperidol/toxicidade , Síndromes Neurotóxicas/genética , Polimorfismo de Nucleotídeo Único , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Animais , Antipsicóticos/toxicidade , Barreira Hematoencefálica/metabolismo , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.
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Cefmetazol/farmacocinética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Colestase/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Timidina Quinase/genética , Animais , Bosentana , Doença Hepática Induzida por Substâncias e Drogas/complicações , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colestase/etiologia , Colestase/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ganciclovir/administração & dosagem , Ganciclovir/farmacologia , Hepatócitos/metabolismo , Hepatócitos/fisiologia , Hepatócitos/transplante , Humanos , Taxa de Depuração Metabólica , Especificidade da Espécie , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologia , Sulfonamidas/toxicidade , Timidina Quinase/metabolismo , Distribuição Tecidual , TransgenesRESUMO
We developed a novel method for differentiating adipocyte-derived stem cells (ASCs) into hepatocyte-like cells (iHeps). ASCs are cultured as spherical cellular aggregates and are then induced by culture in chemically defined media for a short time period to differentiate into spherical culture iHeps (SCi-Heps). SCi-Heps have many of the in vitro functional properties of mature hepatocytes, and they can stably reconstitute functioning human liver in vivo in a murine model system. Implantation studies demonstrate that SCi-Heps have a very low malignant potential. All human liver regenerative procedures, including ultrasound-guided direct liver implantation, are scalable and appropriate for human clinical use. These methods can be used to achieve the major promise of regenerative medicine. It may now be possible to regenerate human liver using autologous stem cells obtained from a readily accessible tissue.
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Adipócitos/citologia , Diferenciação Celular , Regeneração Hepática , Células-Tronco/citologia , Animais , Carcinogênese/patologia , Regulação da Expressão Gênica , Humanos , Hospedeiro Imunocomprometido , Masculino , Camundongos Transgênicos , Transplante AutólogoRESUMO
RATIONALE: Metabolomic profiling is a promising methodology of identifying candidate biomarkers for disease detection and monitoring. Although lung cancer is among the leading causes of cancer-related mortality worldwide, the lung tumor metabolome has not been fully characterized. METHODS: We utilized a targeted metabolomic approach to analyze discrete groups of related metabolites. We adopted a dansyl [5-(dimethylamino)-1-naphthalene sulfonamide] derivatization with liquid chromatography/mass spectrometry (LC/MS) to analyze changes of metabolites from paired tumor and normal lung tissues. Identification of dansylated dipeptides was confirmed with synthetic standards. A systematic analysis of retention times was required to reliably identify isobaric dipeptides. We validated our findings in a separate sample cohort. RESULTS: We produced a database of the LC retention times and MS/MS spectra of 361 dansyl dipeptides. Interpretation of the spectra is presented. Using this standard data, we identified a total of 279 dipeptides in lung tumor tissue. The abundance of 90 dipeptides was selectively increased in lung tumor tissue compared to normal tissue. In a second set of validation tissues, 12 dipeptides were selectively increased. CONCLUSIONS: A systematic evaluation of certain metabolite classes in lung tumors may identify promising disease-specific metabolites. Our database of all possible dipeptides will facilitate ongoing translational applications of metabolomic profiling as it relates to lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Dipeptídeos/química , Neoplasias Pulmonares/metabolismo , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/química , Biomarcadores/metabolismo , Carcinoma Pulmonar de Células não Pequenas/química , Estudos de Coortes , Dipeptídeos/metabolismo , Humanos , Neoplasias Pulmonares/químicaRESUMO
BACKGROUND: Better pancreatic cyst fluid biomarkers are needed. OBJECTIVE: To determine whether metabolomic profiling of pancreatic cyst fluid would yield clinically useful cyst fluid biomarkers. DESIGN: Retrospective study. SETTING: Tertiary-care referral center. PATIENTS: Two independent cohorts of patients (n = 26 and n = 19) with histologically defined pancreatic cysts. INTERVENTION: Exploratory analysis for differentially expressed metabolites between (1) nonmucinous and mucinous cysts and (2) malignant and premalignant cysts was performed in the first cohort. With the second cohort, a validation analysis of promising identified metabolites was performed. MAIN OUTCOME MEASUREMENTS: Identification of differentially expressed metabolites between clinically relevant cyst categories and their diagnostic performance (receiver operating characteristic [ROC] curve). RESULTS: Two metabolites had diagnostic significance-glucose and kynurenine. Metabolomic abundances for both were significantly lower in mucinous cysts compared with nonmucinous cysts in both cohorts (glucose first cohort P = .002, validation P = .006; and kynurenine first cohort P = .002, validation P = .002). The ROC curve for glucose was 0.92 (95% confidence interval [CI], 0.81-1.00) and 0.88 (95% CI, 0.72-1.00) in the first and validation cohorts, respectively. The ROC for kynurenine was 0.94 (95% CI, 0.81-1.00) and 0.92 (95% CI, 0.76-1.00) in the first and validation cohorts, respectively. Neither could differentiate premalignant from malignant cysts. Glucose and kynurenine levels were significantly elevated for serous cystadenomas in both cohorts. LIMITATIONS: Small sample sizes. CONCLUSION: Metabolomic profiling identified glucose and kynurenine to have potential clinical utility for differentiating mucinous from nonmucinous pancreatic cysts. These markers also may diagnose serous cystadenomas.
Assuntos
Biomarcadores Tumorais/metabolismo , Líquido Cístico/metabolismo , Cistadenocarcinoma/metabolismo , Cistadenoma/metabolismo , Glucose/metabolismo , Cinurenina/metabolismo , Cisto Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/metabolismo , Estudos de Coortes , Cistadenocarcinoma/diagnóstico , Cistadenocarcinoma Mucinoso/diagnóstico , Cistadenocarcinoma Mucinoso/metabolismo , Cistadenoma/diagnóstico , Cistadenoma Mucinoso/diagnóstico , Cistadenoma Mucinoso/metabolismo , Cistadenoma Seroso/diagnóstico , Cistadenoma Seroso/metabolismo , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-Idade , Cisto Pancreático/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Pseudocisto Pancreático/diagnóstico , Pseudocisto Pancreático/metabolismo , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: We investigated whether human pharmacogenetic factors could be characterized using chimeric NOG mice expressing a thymidine kinase transgene (TK-NOG) with 'humanized' livers. MATERIALS AND METHODS: The rate of human-specific metabolism of two drugs was measured in chimeric mice reconstituted with human hepatocytes with different CYP2C19 and CYP2C9 genotypes. RESULTS: The rate of generation of human-predominant drug metabolites for S-mephenytoin and diclofenac in the chimeric mice was correlated with the CYP2C19 (n=9 donors, P=0.0005) or CYP2C9 (n=7 donors, P=0.0394) genotype, respectively, of the transplanted human hepatocytes. CONCLUSION: This study suggests that TK-NOG mice reconstituted with hepatocytes obtained from a relatively small number (3-10 per genotype) of human donors may be a promising model to identify human pharmacogenetic factors affecting the metabolism of clinically important drugs. For certain compounds, this innovative model system enables pharmacogenetic analyses to be efficiently performed in vivo within a human context and with control of all confounding environmental variables.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Diclofenaco/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Mefenitoína/metabolismo , Farmacogenética , Animais , Proteínas de Transporte/fisiologia , Células Cultivadas , Quimera/genética , Quimera/metabolismo , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C9 , Hepatócitos/transplante , Humanos , Fígado/citologia , Camundongos , Camundongos Transgênicos , Timidina Quinase/fisiologiaRESUMO
Interspecies differences in drug metabolism have made it difficult to use preclinical animal testing data to predict the drug metabolites or potential drug-drug interactions (DDIs) that will occur in humans. Although chimeric mice with humanized livers can produce known human metabolites for test substrates, we do not know whether chimeric mice can be used to prospectively predict human drug metabolism or a possible DDI. Therefore, we investigated whether they could provide a more predictive assessment for clemizole, a drug in clinical development for the treatment of hepatitis C virus (HCV) infection. Our results demonstrate, for the first time, that analyses performed in chimeric mice can correctly identify the predominant human drug metabolite before human testing. The differences in the rodent and human pathways for clemizole metabolism were of importance, because the predominant human metabolite was found to have synergistic anti-HCV activity. Moreover, studies in chimeric mice also correctly predicted that a DDI would occur in humans when clemizole was coadministered with a CYP3A4 inhibitor. These results demonstrate that using chimeric mice can improve the quality of preclinical drug assessment.