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Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells1-7. Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy7-9; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.
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Antineoplásicos , Células Clonales , Resistencia a Antineoplásicos , Neoplasias , Humanos , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Células Clonales/patología , Código de Barras del ADN Taxonómico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , RNA-Seq , Análisis de Expresión Génica de una Sola Célula , Células Tumorales Cultivadas , Antineoplásicos/farmacologíaRESUMEN
Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.
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Envejecimiento , Pulmón , Melanoma , Metástasis de la Neoplasia , Células del Estroma , Microambiente Tumoral , Anciano , Envejecimiento/patología , Fibroblastos/patología , Humanos , Pulmón/patología , Melanoma/patología , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Neoplasia Residual , Proteínas Proto-Oncogénicas , Proteínas Tirosina Quinasas Receptoras , Piel/patología , Células del Estroma/patología , Proteína Wnt-5a , Tirosina Quinasa c-Mer , Tirosina Quinasa del Receptor AxlRESUMEN
Metastatic melanoma is an aggressive disease, despite recent improvements in therapy. Eradicating all melanoma cells even in drug-sensitive tumors is unsuccessful in patients because a subset of cells can transition to a slow-cycling state, rendering them resistant to most targeted therapy. It is still unclear what pathways define these subpopulations and promote this resistant phenotype. In the current study, we show that Wnt5A, a non-canonical Wnt ligand that drives a metastatic, therapy-resistant phenotype, stabilizes the half-life of p53 and uses p53 to initiate a slow-cycling state following stress (DNA damage, targeted therapy, and aging). Inhibiting p53 blocks the slow-cycling phenotype and sensitizes melanoma cells to BRAF/MEK inhibition. In vivo, this can be accomplished with a single dose of p53 inhibitor at the commencement of BRAF/MEK inhibitor therapy. These data suggest that taking the paradoxical approach of inhibiting rather than activating wild-type p53 may sensitize previously resistant metastatic melanoma cells to therapy.
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Melanoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína Wnt-5a/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Melanoma/genética , Melanoma/patología , Terapia Molecular Dirigida , Mutación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Microambiente Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/fisiologíaRESUMEN
Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.
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Glucuronosiltransferasa , Isoenzimas , Microsomas Hepáticos , Proteínas Recombinantes , Animales , Humanos , Ácidos Grasos/metabolismo , Ácidos Grasos/química , Glucuronosiltransferasa/metabolismo , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/química , Isoenzimas/metabolismo , Isoenzimas/genética , Cinética , Microsomas Hepáticos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Magnetismo , Microsomas/química , Microsomas/metabolismoRESUMEN
RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect many RNA species and exponentially amplify their signals at once, while also reducing the time and cost compared with the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection to detect 10 different RNA species in more than 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect that the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.
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ARN , Transcriptoma , ARN/genéticaRESUMEN
Campylobacter jejuni is a Gram-negative pathogenic bacterium commonly found in chickens and is the leading cause of human diarrheal disease worldwide. The various serotypes of C. jejuni produce structurally distinct capsular polysaccharides (CPSs) on the exterior surfaces of the cell wall. The capsular polysaccharide from C. jejuni serotype HS:5 is composed of a repeating sequence of d-glycero-d-manno-heptose and d-glucitol-6-phosphate. We previously defined the pathway for the production of d-glycero-d-manno-heptose in C. jejuni. Here, we elucidate the biosynthetic pathway for the assembly of cytidine diphosphate (CDP)-6-d-glucitol by the combined action of two previously uncharacterized enzymes. The first enzyme catalyzes the formation of CDP-6-d-fructose from cytidine triphosphate (CTP) and d-fructose-6-phosphate. The second enzyme reduces CDP-6-d-fructose with NADPH to generate CDP-6-d-glucitol. Using sequence similarity network (SSN) and genome neighborhood network (GNN) analyses, we predict that these pairs of proteins are responsible for the biosynthesis of CDP-6-d-glucitol and/or CDP-d-mannitol in the lipopolysaccharides (LPSs) and capsular polysaccharides in more than 200 other organisms. In addition, high resolution X-ray structures of the second enzyme are reported, which provide novel insight into the manner in which an open-chain nucleotide-linked sugar is harbored in an active site cleft.
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Campylobacter jejuni , Animales , Humanos , Sorbitol/metabolismo , Pollos/metabolismo , Polisacáridos/metabolismo , Citidina Difosfato/metabolismo , Fructosa/metabolismo , Polisacáridos Bacterianos/metabolismoRESUMEN
BACKGROUND: There are little data on changes in insulin sensitivity during the first few years of life following in utero human immunodeficiency virus (HIV) and antiretroviral (ARV) exposure. METHODS: The Tshilo Dikotla study enrolled pregnant persons with HIV (PWH) (receiving tenofovir/emtricitabine or lamivudine plus dolutegravir or efavirenz) and pregnant individuals without HIV, as well as their liveborn children. Newborns were randomized to receive either zidovudine (AZT) or nevirapine (NVP) postnatal prophylaxis. Homeostasis Model Assessment for Insulin Resistance (HOMA-IR) was assessed at birth and 1, 18, 24, and 36 months of life. We fit linear mixed-effects models to evaluate the association between in utero HIV/ARV exposure and average HOMA-IR from birth through 36 months of life, adjusting for confounders. RESULTS: A total of 419 children were included (287 with in utero HIV/ARV exposure and uninfected [CHEU] and 132 without in utero HIV/ARV exposure [CHUU]). CHEU were born to older women (29.6 vs 25.3 years of age) with higher gravidity (3 vs 1). HOMA-IR was persistently higher in CHEU versus CHUU in adjusted analyses (mean difference of 0.07 in log10 HOMA-IR, P = .02) from birth through 36 months of life. Among CHEU, no differences in HOMA-IR were observed from birth through 36 months by in utero ARV exposure status or between AZT and NVP infant prophylaxis arms. CONCLUSIONS: In utero HIV/ARV exposure was associated with lower insulin sensitivity throughout the first 36 months of life, indicating persistent early life metabolic disturbances which may raise concern for poorer metabolic health later in life.
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A stable aluminum tris(dithiolene) triradical (3) was experimentally realized through a low-temperature reaction of the sterically demanding lithium dithiolene radical (2) with aluminum iodide. Compound 3 was characterized by single-crystal X-ray diffraction, UV-vis and EPR spectroscopy, SQUID magnetometry, and theoretical computations. The quartet ground state of triradical 3 has been unambiguously confirmed by variable-temperature continuous wave EPR experiments and SQUID magnetometry. Both SQUID magnetometry and broken-symmetry DFT computations reveal a small doublet-quartet energy gap [ΔEDQ = 0.18 kcal mol-1 (SQUID); ΔEDQ = 0.14 kcal mol-1 (DFT)]. The pulsed EPR experiment (electron spin echo envelop modulation) provides further evidence for the interaction of these dithiolene-based radicals with the central aluminum nucleus of 3.
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BACKGROUND: The role of body fat on metabolic complications remains poorly understood in young people living with perinatally acquired HIV (YPHIV). OBJECTIVE: Our objective was to assess the association of changes in adiposity over 2 years with metabolic outcomes in YPHIV. METHODS: The PHACS Adolescent Master Protocol (AMP) study enrolled YPHIV from 2007 to 2009 across 15 US sites, including Puerto Rico. We included YPHIV aged 7-19 years with body composition data assessed by whole-body dual-energy X-ray absorptiometry (DXA) at baseline and 2 years later. Metabolic outcomes included homeostatic model assessment of insulin resistance (HOMA-IR) and non-high-density lipoprotein cholesterol (non-HDL-C). We fitted linear regression models to assess the association of increase in body fat over 2 years with metabolic outcomes at years 2 and 3. RESULTS: In all, 232 participants had a second DXA and either HOMA-IR or non-HDL-C measured at year 2. Participant characteristics at the first DXA were: age 12 years (9-14) [median (Q1-Q3)], 69% Black, and median CD4 count 714 cells/µL; 70% with HIV RNA <400 copies/mL. In adjusted analyses for every 1% increase in body fat from baseline to year 2, HOMA-IR was higher by 1.03-fold at year 3 (95% CI: 1.00, 1.05). We observed that for every 1% increase in body fat from baseline to year 2, non-HDL-C was 0.72 mg/dL higher at year 2 (95% CI: -0.04-1.49) and 0.81 mg/dL higher at year 3 (95% CI: -0.05-1.66). CONCLUSIONS: Increases in adiposity over time may lead to downstream decreased insulin sensitivity and dyslipidaemia in YPHIV.
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Infecciones por VIH , Resistencia a la Insulina , Adolescente , Humanos , Infecciones por VIH/complicaciones , Adiposidad , Obesidad/complicaciones , Colesterol , Tejido Adiposo/diagnóstico por imagen , Absorciometría de FotónRESUMEN
Correction for 'Pericyclic reaction benchmarks: hierarchical computations targeting CCSDT(Q)/CBS and analysis of DFT performance' by Pascal Vermeeren et al., Phys. Chem. Chem. Phys., 2022, 24, 18028-18042, https://doi.org/10.1039/D2CP02234F.
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E2H2 (E = As, Sb, Bi) structures involving multiple bonds have attracted much attention recently. The E2H3+ cations (protonated E2H2) are predicted to be viable with substantial proton affinities (>180 kcal/mol). Herein, the bonding characters and energetics of a number of E2H3+ isomers are explored through CCSD(T) and DFT methods. For the As2H3+ system, the CCSD(T)/cc-pVQZ-PP method predicts that the vinylidene-like structure lies lowest in energy, with the trans and cis isomers higher by 6.7 and 9.3 kcal/mol, respectively. However, for Sb2H3+ and Bi2H3+ systems, the trans isomer is the global minimum, while the energies of the cis and vinylidene-like structures are higher, respectively, by 2.0 and 2.4 kcal/mol for Sb2H3+ and 1.6 and 15.0 kcal/mol for Bi2H3+. Thus, the vinyledene-like structure is the lowest energy for the arsenic system but only a transition state of the bismuth system. With permanent dipole moments, all minima may be observable in microwave experiments. Besides, we have also obtained transition states and planar-cis structures with higher energies. The current results should provide new insights into the various isomers and provide a number of predictions for future experiments.
RESUMEN
High-level potential energy surfaces for three reactions of hypobromous acid with atomic hydrogen were computed at the CCSDTQ/CBS//CCSDT(Q)/complete basis set level of theory. Focal point analysis was utilized to extrapolate energies and gradients for energetics and optimizations, respectively. The H attack at Br and subsequent Br-O cleavage were found to proceed barrierlessly. The slightly submerged transition state lies -0.2 kcal mol-1 lower in energy than the reactants and produces OH and HBr. The two other studied reaction paths are the radical substitution to produce H2O and Br with a 4.0 kcal mol-1 barrier and the abstraction at hydrogen to produce BrO and H2 with an 11.2 kcal mol-1 barrier. The final product energies lie -37.2, -67.9, and -7.3 kcal mol-1 lower in energy than reactants, HOBr + H, for the sets of products OH + HBr, H2O + Br, and H2 + BrO, respectively. Additive corrections computed for the final energetics, particularly the zero-point vibrational energies and spin-orbit corrections, significantly impacted the final stationary point energies, with corrections up to 6.2 kcal mol-1.
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Mitochondrial dysfunction and excessive reactive oxygen species production contributes to the pathophysiology of aging. Coenzyme Q10 is thought to protect mitochondria from oxidative damage; thus, mitoquinone was developed as mitochondria-targeted analogue with similar antioxidant activity. Mitoquinone is the oxidized form of mitoquinol. Mitoquinone/mitoquinol mesylate has been proposed as a food ingredient. As part of the safety analysis, we performed genotoxicity assays and a 39-week toxicity study to determine overall toxicity potential. Mitoquinone mesylate showed no evidence of genotoxic potential in two in vitro assays, bacterial reverse mutation and human lymphocyte chromosome aberration, nor in the in vivo micronucleus test in rats. In the 39-week study in dogs, there were no findings observed, which were considered to represent adverse systemic toxicity; therefore, the high dose level (40 mg/kg/day) was considered the NOAEL. The principal findings in this study were fecal disturbances and vomiting. These findings were considered to be due to a local, possibly irritant effect of the test substance on the gastrointestinal tract and were not considered adverse as there were no impacts on clinical or histopathology. This highest dose exceeds the expected daily human intake more than 100-fold. Data from well-designed clinical trials actively collecting safety endpoints corroborate that 20 mg/day can be safely consumed and is not likely to result in significant gastrointestinal complaints. These results support the conclusion that the use of mitoquinone/mitoquinol mesylate as a food ingredient is safe.
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Aggression refers to a wide range of behaviors with lasting individual and societal consequences. Recurrent, unplanned aggressive behavior is the core diagnostic criterion for intermittent explosive disorder (IED). In this study, we compared two behavioral measures of aggression in the laboratory: the Taylor Aggression Paradigm (TAP) and the Point-Subtraction Aggression Paradigm (PSAP). This sample (n = 528) included community participants who met DSM-5 criteria for IED (n = 156), met DSM-5 criteria for a nonaggressive psychiatric disorder (n = 205), or did not meet DSM-5 criteria for any psychiatric disorder (n = 167). All participants completed the TAP, a single-session PSAP, and relevant self-report measures. MANOVA analyses demonstrated differences between IED participants and nonaggressive participants; however, these group differences were no longer significant for the PSAP after including demographic variables. Correlation analyses found that the TAP and PSAP were positively related to one another and the composite variables associated with aggressive behavior (i.e., history of aggression, impulsivity, and propensity to experience anger) and; dependent correlations revealed that past aggression and trait anger were more strongly related to the TAP. Differences in TAP and PSAP outcomes may be partially attributed to operationalizations of aggression and methods of aggression and provocation. Further, as aggressive and nonaggressive participants differed on the PSAP somewhat mirroring the TAP, our results add to growing evidence of the validity of a single-session PSAP; further research is needed to fully establish single-session PSAP as a laboratory aggression task compared to the multi-session PSAP.
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Agresión , Trastornos Disruptivos, del Control de Impulso y de la Conducta , Humanos , Agresión/psicología , Agresión/fisiología , Masculino , Femenino , Adulto , Trastornos Disruptivos, del Control de Impulso y de la Conducta/psicología , Adulto Joven , Conducta Impulsiva/fisiología , Persona de Mediana Edad , Adolescente , Ira/fisiologíaRESUMEN
Results of 2 parallel phase 2 trials of transplantation of unrelated umbilical cord blood (UCB) or bone marrow (BM) from HLA-haploidentical relatives provided equipoise for direct comparison of these donor sources. Between June 2012 and June 2018, 368 patients aged 18 to 70 years with chemotherapy-sensitive lymphoma or acute leukemia in remission were randomly assigned to undergo UCB (n = 186) or haploidentical (n = 182) transplant. Reduced-intensity conditioning comprised total-body irradiation with cyclophosphamide and fludarabine for both donor types. Graft-versus-host disease prophylaxis for UCB transplantation was cyclosporine and mycophenolate mofetil (MMF) and for haploidentical transplantation, posttransplant cyclophosphamide, tacrolimus, and MMF. The primary end point was 2-year progression-free survival (PFS). Treatment groups had similar age, sex, self-reported ethnic origin, performance status, disease, and disease status at randomization. Two-year PFS was 35% (95% confidence interval [CI], 28% to 42%) compared with 41% (95% CI, 34% to 48%) after UCB and haploidentical transplants, respectively (P = .41). Prespecified analysis of secondary end points recorded higher 2-year nonrelapse mortality after UCB, 18% (95% CI, 13% to 24%), compared with haploidentical transplantation, 11% (95% CI, 6% to 16%), P = .04. This led to lower 2-year overall survival (OS) after UCB compared with haploidentical transplantation, 46% (95% CI, 38-53) and 57% (95% CI 49% to 64%), respectively (P = .04). The trial did not demonstrate a statistically significant difference in the primary end point, 2-year PFS, between the donor sources. Although both donor sources extend access to reduced-intensity transplantation, analyses of secondary end points, including OS, favor haploidentical BM donors. This trial was registered at www.clinicaltrials.gov as #NCT01597778.
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Sangre Fetal/fisiología , Enfermedad Aguda , Adulto , Anciano , Trasplante de Médula Ósea/efectos adversos , Causas de Muerte , Enfermedad Crónica , Femenino , Enfermedad Injerto contra Huésped/epidemiología , Enfermedad Injerto contra Huésped/etiología , Antígenos HLA/inmunología , Hematopoyesis , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Supervivencia sin Progresión , Trasplante Haploidéntico/efectos adversos , Resultado del Tratamiento , Donante no Emparentado , Adulto JovenRESUMEN
Omidubicel is an ex vivo expanded hematopoietic progenitor cell and nonexpanded myeloid and lymphoid cell product derived from a single umbilical cord blood unit. We report results of a phase 3 trial to evaluate the efficacy of omidubicel compared with standard umbilical cord blood transplantation (UCBT). Between January 2017 and January 2020, 125 patients age 13 to 65 years with hematologic malignancies were randomly assigned to omidubicel vs standard UCBT. Patients received myeloablative conditioning and prophylaxis with a calcineurin inhibitor and mycophenolate mofetil for graft-versus-host disease (GVHD). The primary end point was time to neutrophil engraftment. The treatment arms were well balanced and racially diverse. Median time to neutrophil engraftment was 12 days (95% confidence interval [CI], 10-14 days) for the omidubicel arm and 22 days (95% CI, 19-25 days) for the control arm (P < .001). The cumulative incidence of neutrophil engraftment was 96% for patients receiving omidubicel and 89% for patients receiving control transplants. The omidubicel arm had faster platelet recovery (55% vs 35% recovery by 42 days; P = .028), had a lower incidence of first grade 2 to 3 bacterial or invasive fungal infection (37% vs 57%; P = .027), and spent more time out of hospital during the first 100 days after transplant (median, 61 vs 48 days; P = .005) than controls. Differences in GVHD and survival between the 2 arms were not statistically significant. Transplantation with omidubicel results in faster hematopoietic recovery and reduces early transplant-related complications compared with standard UCBT. The results suggest that omidubicel may be considered as a new standard of care for adult patients eligible for UCBT. The trial was registered at www.clinicaltrials.gov as #NCT02730299.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Sangre Fetal/trasplante , Neoplasias Hematológicas/terapia , Adolescente , Adulto , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Femenino , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/etiología , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
OBJECTIVE: Infants and toddlers with classical congenital adrenal hyperplasia (CAH) are at high risk for morbidity/mortality arising from life-threatening adrenal crisis. Management of acute illnesses in CAH requires an understanding of factors leading to emergency department (ED) visits and hospitalizations in the first few years of life. We, therefore, examined adrenal crisis at prehospital and ED stages of illness in young children with CAH as they related to medical outcomes. PATIENTS AND DESIGN: Retrospective cohort study of 39 children with CAH due to 21-hydroxylase deficiency (0-4 years of age) and 27 age-matched controls. MEASUREMENTS: ED visit, acute illness symptoms (fever, vomiting, diarrhoea) and other characteristics (hospitalizations, administration of stress-dose hydrocortisone, electrolyte abnormalities). RESULTS: CAH infants and toddlers had significantly higher rates of ED visits (0.50 [0.25-0.88] per person-year) than controls (0 [0-0] per person-year; p < .001). Moreover, CAH children under 6 months old had significantly higher rates of ED visits compared with older ages. Only 50% (51/102) of illness-related ED visits in CAH children were preceded by the administration of either oral (46/51) or intramuscular (11/51) stress dosing by parents. A total of 10.8% of ED visits resulted in hospital admission. Controlling for age and 17-hydroxyprogesterone at diagnosis, electrolyte abnormalities and administration of parenteral hydrocortisone in the ED significantly predicted hospital admission. Receiving a hydrocortisone injection before the ED was a significant predictor of having electrolyte abnormalities. CONCLUSIONS: Infants and toddlers with classical CAH are at high risk for acute illness and hospitalizations and often do not receive adequate stress dosing before the ED.
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Hiperplasia Suprarrenal Congénita , Humanos , Lactante , Preescolar , Adulto , Adolescente , Hiperplasia Suprarrenal Congénita/diagnóstico , Hidrocortisona , Enfermedad Aguda , Estudios Retrospectivos , Hospitalización , ElectrólitosRESUMEN
The 1 : 2 reaction of the imidazole-based dithiolate (2) with GeCl2 ⢠dioxane in THF/TMEDA gives 3, a TMEDA-complexed dithiolene-based germylene. Compound 3 is converted to monothiolate-complexed (5) and N-heterocyclic carbene-complexed (7) germanium(II) dithiolene complexes via Lewis base ligand exchange. A bis-dithiolene-based germylene (8), involving a 3c-4e S-Ge-S bond, has also been synthesized through controlled hydrolysis of 7. The bonding nature of 3, 5, and 8 was investigated by both experimental and theoretical methods.
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OBJECTIVE: An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. METHODS: In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. RESULTS: The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. CONCLUSIONS: This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.
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Transportadores de Anión Orgánico , Rifampin , Humanos , Atorvastatina/metabolismo , Rifampin/farmacología , Rifampin/metabolismo , Células HEK293 , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Interacciones Farmacológicas , Transportadores de Anión Orgánico/metabolismoRESUMEN
PURPOSE: After single oral dosing of the glycine reuptake transporter (GlyT1) inhibitor, iclepertin (BI 425809), a single major circulating metabolite, M530a, was identified. However, upon multiple dosing, a second major metabolite, M232, was observed with exposure levels ~ twofold higher than M530a. Studies were conducted to characterize the metabolic pathways and enzymes responsible for formation of both major human metabolites. METHODS: In vitro studies were conducted with human and recombinant enzyme sources and enzyme-selective inhibitors. The production of iclepertin metabolites was monitored by LC-MS/MS. RESULTS: Iclepertin undergoes rapid oxidation to a putative carbinolamide that spontaneously opens to an aldehyde, M528, which then undergoes reduction by carbonyl reductase to the primary alcohol, M530a. However, the carbinolamide can also undergo a much slower oxidation by CYP3A to form an unstable imide metabolite, M526, that is subsequently hydrolyzed by a plasma amidase to form M232. This difference in rate of metabolism of the carbinolamine explains why high levels of the M232 metabolite were not observed in vitro and in single dose studies in humans, but were observed in longer-term multiple dose studies. CONCLUSIONS: The long half-life iclepertin metabolite M232 is formed from a common carbinolamine intermediate, that is also a precursor of M530a. However, the formation of M232 occurs much more slowly, likely contributing to its extensive exposure in vivo. These results highlight the need to employ adequate clinical study sampling periods and rigorous characterization of unexpected metabolites, especially when such metabolites are categorized as major, thus requiring safety assessment.