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A spinal epidural abscess is a rare condition characterized by the accumulation of pus between the dura mater and vertebral column, often caused by hematogenous spread from a distant site or local spread from infection in nearby structures. The abscess leads to compression of the spinal cord and can result in neurological damage, including dysfunction or permanent neurological deficits. Treatment of spinal epidural abscesses should not be delayed and requires a combination of decompression by surgical drainage and antibiotic therapy. The authors present a rare case in which a spinal epidural abscess developed from a hospital-acquired pressure ulcer, further complicated by bacteremia.
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Male rhesus monkeys (n = 24) had a biopsy of prefrontal cortical area 46 prior to chronic ethanol self-administration (n = 17) or caloric control (n = 7). Fourteen months of daily self-administration (water vs. 4% alcohol, 22 h access/day termed "open-access") was followed by two cycles of prolonged abstinence (5 weeks) each followed by 3 months of open-access alcohol and a final abstinence followed by necropsy. At necropsy, a biopsy of Area 46, contralateral to the original biopsy, was obtained. Gene expression data (RNA-Seq) were collected comparing biopsy/necropsy samples. Monkeys were categorized by drinking status during the final post-abstinent drinking phase as light (LD), binge (BD), heavy (HD) and very heavy (VHD drinkers). Comparing pre-ethanol to post-abstinent biopsies, four animals that converted from HD to VHD status had significant ontology enrichments in downregulated genes (necropsy minus biopsy n = 286) that included immune response (FDR < 9 × 10-7) and plasma membrane changes (FDR < 1 × 10-7). Genes in the immune response category included IL16 and 18, CCR1, B2M, TLR3, 6 and 7, SP2 and CX3CR1. Upregulated genes (N = 388) were particularly enriched in genes associated with the negative regulation of MAP kinase activity (FDR < 3 × 10-5), including DUSP 1, 4, 5, 6 and 18, SPRY 2, 3, and 4, SPRED2, BMP4 and RGS2. Overall, these data illustrate the power of the NHP model and the within-subject design of genomic changes due to alcohol and suggest new targets for treating severe escalated drinking following repeated alcohol abstinence attempts.
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Circuit manipulation has been a staple technique in neuroscience to identify how the brain functions to control complex behaviors. Chemogenetics, including designer receptors exclusively activated by designer drugs (DREADDs), have proven to be a powerful tool for the reversible modulation of discrete brain circuitry without the need for implantable devices, thereby making them especially useful in awake and unrestrained animals. This study used a DREADD approach to query the role of the nucleus accumbens (NAc) in mediating the interoceptive effects of 1.0 g/kg ethanol (i.g.) in rhesus monkeys (n = 7) using a drug discrimination procedure. After training, stereotaxic surgery was performed to introduce an AAV carrying the human muscarinic 4 receptor DREADD (hM4Di) bilaterally into the NAc. The hypothesis was that decreasing the output of the NAc by activation of hM4Di with the DREADD actuator, clozapine-n-oxide (CNO), would potentiate the discriminative stimulus effect of ethanol (i.e., a leftward shift the ethanol dose discrimination curve). The results showed individual variability shifts of the ethanol dose-response determination under DREADD activation. Characterization of the expression and function of hM4Di with MRI, immunohistochemical, and electrophysiological techniques found the selectivity of NAc transduction was proportional to behavioral effect. Specifically, the proportion of hM4Di expression restricted to the NAc was associated with the potency of the discriminative stimulus effects of ethanol. Together, these experiments highlight the NAc in mediating the interoceptive effects of ethanol, provide a framework for validation of chemogenetic tools in primates, and underscore the importance of robust within-subjects examination of DREADD expression for interpretation of behavioral findings.
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Clozapina , Etanol , Animais , Encéfalo , Clozapina/farmacologia , Etanol/farmacologia , Macaca mulatta , Núcleo AccumbensRESUMO
Hazardous, heavy drinking increases risk for developing alcohol use disorder (AUD), which affects ~7% of adult Americans. Thus, understanding the molecular mechanisms promoting risk for heavy drinking is essential to developing more effective AUD pharmacotherapies than those currently approved by the FDA. Using genome-wide bisulfate sequencing, we identified DNA methylation (DNAm) signals within the nucleus accumbens core (NAcC) that differentiate nonheavy and heavy ethanol-drinking rhesus macaques. One differentially DNAm region (D-DMR) located within the gene neurobeachin (NBEA), which promotes synaptic membrane protein trafficking, was hypermethylated in heavy drinking macaques. A parallel study identified a similar NBEA D-DMR in human NAcC that distinguished alcoholic and nonalcoholic individuals. To investigate the role of NBEA in heavy ethanol drinking, we engineered a viral vector carrying a short hairpin RNA (shRNA) to reduce the expression of NBEA. Using two murine models of ethanol consumption: 4 days of drinking-in-the-dark and 4 weeks of chronic intermittent access, the knockdown of NBEA expression did not alter average ethanol consumption in either model. However, it did lead to a significant increase in the ethanol preference ratio. Following withdrawal, whole-cell patch clamp electrophysiological experiments revealed that Nbea knockdown led to an increase in spontaneous excitatory postsynaptic current amplitude with no alteration in spontaneous inhibitory postsynaptic currents, suggesting a specific role of NBEA in trafficking of glutamatergic receptors. Together, our findings suggest that NBEA could be targeted to modulate the preference for alcohol use.
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Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Proteínas de Transporte/genética , Proteínas do Tecido Nervoso/genética , Adulto , Idoso , Animais , Metilação de DNA/efeitos dos fármacos , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Núcleo Accumbens/efeitos dos fármacosRESUMO
BACKGROUND: The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. Humans who inherit mutant TP53 alleles develop a wide range of early onset cancers, a disorder called Li-Fraumeni Syndrome (LFS). Trp53-deficient mice recapitulate most but not all of the cancer phenotypes observed in TP53-deficient human cancers, indicating that new animal models may complement current mouse models and better inform on human disease development. MATERIALS AND METHODS: The recent application of CRISPR/Cas9 genetic engineering technology has permitted the emergence of golden Syrian hamsters as genetic models for wide range of diseases, including cancer. Here, the first cancer phenotype of TP53 knockout golden Syrian hamsters is described. RESULTS: Hamsters that are homozygous for TP53 mutations become moribund on average ~ 139 days of age, while hamsters that are heterozygous become moribund at ~ 286 days. TP53 homozygous knockout hamsters develop a wide range of cancers, often synchronous and metastatic to multiple tissues, including lymphomas, several sarcomas, especially hemangiosarcomas, myeloid leukemias and several carcinomas. TP53 heterozygous mutants develop a more restricted tumor spectrum, primarily lymphomas. CONCLUSIONS: Overall, hamsters may provide insights into how TP53 deficiency leads to cancer in humans and can become a new model to test novel therapies.
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The COVID-19 pandemic has revealed the pronounced vulnerability of the elderly and chronically ill to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced morbidity and mortality. Cellular senescence contributes to inflammation, multiple chronic diseases, and age-related dysfunction, but effects on responses to viral infection are unclear. Here, we demonstrate that senescent cells (SnCs) become hyper-inflammatory in response to pathogen-associated molecular patterns (PAMPs), including SARS-CoV-2 spike protein-1, increasing expression of viral entry proteins and reducing antiviral gene expression in non-SnCs through a paracrine mechanism. Old mice acutely infected with pathogens that included a SARS-CoV-2-related mouse ß-coronavirus experienced increased senescence and inflammation, with nearly 100% mortality. Targeting SnCs by using senolytic drugs before or after pathogen exposure significantly reduced mortality, cellular senescence, and inflammatory markers and increased antiviral antibodies. Thus, reducing the SnC burden in diseased or aged individuals should enhance resilience and reduce mortality after viral infection, including that of SARS-CoV-2.
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Envelhecimento , Senescência Celular/efeitos dos fármacos , Infecções por Coronavirus/mortalidade , Flavonóis/uso terapêutico , Moléculas com Motivos Associados a Patógenos/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , COVID-19/imunologia , COVID-19/mortalidade , Linhagem Celular , Infecções por Coronavirus/imunologia , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Feminino , Flavonóis/farmacologia , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Hepatite Murina/imunologia , Quercetina/farmacologia , Quercetina/uso terapêutico , Receptores de Coronavírus/genética , Receptores de Coronavírus/metabolismo , Organismos Livres de Patógenos Específicos , Tratamento Farmacológico da COVID-19RESUMO
GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase ß-galactosidase (ß-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing ß-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (ß-gal-/-). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult ß-gal-/- mice showed clearance of enzyme activity in the plasma and an increase in ß-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating ß-gal-/- mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated ß-gal-/- mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed ß-gal-/- mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in ß-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active ß-gal fusion enzyme in vivo that is readily taken up into tissues. Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to ß-gal-/- mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in ß-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).
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Cryopreserved tissues are increasingly needed in biomedical applications. However, successful cryopreservation is generally only reported for thin tissues (≤1 mm). This work presents several innovations to reduce cryoprotectant (CPA) toxicity and improve tissue cryopreservation, including 1) improved tissue warming rates through radiofrequency metal form and field optimization and 2) an experimentally verified predictive model to optimize CPA loading and rewarming to reduce toxicity. CPA loading is studied by microcomputed tomography (µCT) imaging, rewarming by thermal measurements, and modeling, and viability is measured after loading and/or cryopreservation by alamarBlue and histology. Loading conditions for three common CPA cocktails (6, 8.4, and 9.3 m) are designed, and then fast cooling and metal forms rewarming (up to 2000 °C min-1 ) achieve ≥90% viability in cryopreserved 1-2 mm arteries with various CPAs. Despite high viability by alamarBlue, histology shows subtle changes after cryopreservation suggesting some degree of cell damage especially in the central portions of thicker arteries up to 2 mm. While further studies are needed, these results show careful CPA loading and higher metal forms warming rates can help reduce CPA loading toxicity and improve outcomes from cryopreservation in tissues while also offering new protocols to preserve larger tissues ≥1 mm in thickness.
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Criopreservação , Crioprotetores , Crioprotetores/farmacologia , Temperatura Alta , Ondas de Rádio , Microtomografia por Raio-XRESUMO
Bromfenac is a nonsteroidal anti-inflammatory drug that was approved and subsequently withdrawn from the market because of reported cases of acute hepatotoxicity. Recently, in vitro studies have revealed that bromfenac requires UDPGA and alamethicin supplemented human liver microsomes (HLM) to form a major metabolite, bromfenac indolinone (BI). Bromfenac and BI form thioether adducts through a bioactivation pathway in HLM and hepatocytes. [J. P. Driscoll et al., Chem. Res. Toxicol. 2018, 31, 223-230.] Here, Cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) reaction phenotyping experiments using recombinant enzymes were performed on bromfenac and BI to identify the CYP and UGT enzymes responsible for bromfenac's metabolism and bioactivation. It was determined that UGT2B7 converts bromfenac to BI, and that while CYP2C8, CYP2C9, and CYP2C19 catalyze the hydroxylation of bromfenac, only CYP2C9 forms thioether adducts when incubated with NAC or GSH as trapping agents. Although CYP2C9 was shown to form a reactive intermediate, no inhibition of CYP2C9 was observed when an IC50 shift assay was performed. Reaction phenotyping experiments with BI and recombinant CYP enzymes indicated that CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 were responsible for the formation of an aliphatic hydroxylated metabolite. An aromatic hydroxylation on the indolinone moiety was also formed by CYP1A2 and CYP3A4. The aromatic hydroxylated BI is a precursor to the quinone methide and quinone imine intermediates in the proposed bioactivation pathway. Through time-dependent inhibition (TDI) experiments, it was revealed that BI can cause an IC50 shift in CYP1A2 and CYP3A4. However, BI does not inhibit the other isoforms that were also responsible for the formation of the aliphatic hydroxylation, an alternative biotransformation that does not undergo further downstream bioactivation. The results of these metabolism studies with bromfenac and BI add to our understanding of the relationship between biotransformation, reactive intermediate generation, and a potential mechanistic link to the hepatotoxicity of this compound.
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Anti-Inflamatórios não Esteroides/farmacologia , Benzofenonas/farmacologia , Bromobenzenos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/metabolismo , Biotransformação , Humanos , Fenótipo , Proteínas Recombinantes/metabolismoRESUMO
Alcohol use disorder (AUD) is a chronic condition with devastating health and socioeconomic effects. Still, pharmacotherapies to treat AUD are scarce. In a prior study aimed at identifying novel AUD therapeutic targets, we investigated the DNA methylome of the nucleus accumbens core (NAcc) of rhesus macaques after chronic alcohol use. The G-protein coupled receptor 39 (GPR39) gene was hypermethylated and its expression downregulated in heavy alcohol drinking macaques. GPR39 encodes a Zn2+-binding metabotropic receptor known to modulate excitatory and inhibitory neurotransmission, the balance of which is altered in AUD. These prior findings suggest that a GPR39 agonist would reduce alcohol intake. Using a drinking-in-the-dark two bottle choice (DID-2BC) model, we showed that an acute 7.5 mg/kg dose of the GPR39 agonist, TC-G 1008, reduced ethanol intake in mice without affecting total fluid intake, locomotor activity or saccharin preference. Furthermore, repeated doses of the agonist prevented ethanol escalation in an intermittent access 2BC paradigm (IA-2BC). This effect was reversible, as ethanol escalation followed agonist "wash out". As observed during the DID-2BC study, a subsequent acute agonist challenge during the IA-2BC procedure reduced ethanol intake by ~47%. Finally, Gpr39 activation was associated with changes in Gpr39 and Bdnf expression, and in glutamate release in the NAcc. Together, our findings suggest that GPR39 is a promising target for the development of prevention and treatment therapies for AUD.
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Alcoolismo , Comportamento Animal/efeitos dos fármacos , Comportamento de Ingestão de Líquido/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Sulfonamidas/farmacologia , Consumo de Bebidas Alcoólicas , Alcoolismo/tratamento farmacológico , Alcoolismo/prevenção & controle , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Pirimidinas/administração & dosagem , Sulfonamidas/administração & dosagemRESUMO
Translating chemogenetic techniques from nonhuman primates to potential clinical applications has been complicated in part due to in vivo conversion of the chemogenetic actuator, clozapine N-oxide (CNO), to its pharmacologically active parent compound, clozapine, a ligand with known side effects, including five boxed warnings from the Food and Drug Administration. Additionally, the limited solubility of CNO requires high concentrations of potentially toxic detergents such as dimethylsulfoxide (DMSO). To address these concerns, pharmacokinetic profiling of commercially available CNO in DMSO (CNO-DMSO, 10% v/v DMSO in saline) and a water-soluble salt preparation (CNO-HCl, saline) was conducted in rhesus macaques. A time course of blood plasma and cerebrospinal fluid (CSF) concentrations of CNO and clozapine was conducted (30-240 minutes post-administration) following a range of doses (3-10 mg/kg, i.m. and/or i.v.) of CNO-DMSO or CNO-HCl. CNO-HCl resulted in 6- to 7-fold higher plasma concentrations of CNO compared to CNO-DMSO, and relatively less clozapine (3%-5% clozapine/CNO in the CNO-DMSO group and 0.5%-1.5% clozapine/CNO in the CNO-HCl group). Both groups had large between-subjects variability, pointing to the necessity of performing individual CNO pharmacokinetic studies prior to further experimentation. The ratio of CNO measured in the CSF was between 2% and 6% of that measured in the plasma and did not differ across drug preparation, indicating that CSF concentrations may be approximated from plasma samples. In conclusion, CNO-HCl demonstrated improved bioavailability compared with CNO-DMSO with less conversion to clozapine. Further investigation is needed to determine if brain concentrations of clozapine following CNO-HCl administration are pharmacologically active at off-target monoaminergic receptor systems in the primate brain.
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Antipsicóticos/farmacocinética , Clozapina/análogos & derivados , Ácido Clorídrico/farmacocinética , Animais , Antipsicóticos/sangue , Antipsicóticos/líquido cefalorraquidiano , Clozapina/sangue , Clozapina/líquido cefalorraquidiano , Clozapina/farmacocinética , Feminino , Ácido Clorídrico/sangue , Ácido Clorídrico/líquido cefalorraquidiano , Macaca mulatta , MasculinoRESUMO
Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug-drug interaction potential. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug-drug interaction risk. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.
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Benzilaminas/farmacocinética , Uracila/análogos & derivados , Animais , Benzilaminas/química , Benzilaminas/metabolismo , Células CACO-2 , Miosinas Cardíacas/metabolismo , Cardiomiopatia Hipertrófica/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Interações Medicamentosas , Hepatócitos/metabolismo , Humanos , Macaca fascicularis , Masculino , Taxa de Depuração Metabólica , Camundongos Endogâmicos ICR , Microssomos Hepáticos , Ratos Sprague-Dawley , Uracila/química , Uracila/metabolismo , Uracila/farmacocinéticaRESUMO
AIM: Immobilized metal ion affinity chromatography is widely employed for purifying polyhistidine-tagged recombinant proteins from cell lysates. The technique can be applied for quantification of therapeutic proteins in biological matrices by LC-MS/MS. RESULTS: A protein reagent-free workflow was developed for quantifying polyhistidine-tagged proteins by LC-MS/MS. The workflow includes target protein enrichment by immobilized metal ion affinity chromatography, on-bead trypsin digestion and quantification of signature peptides by LC-MS/MS. It was applied to quantify a 6×His-tagged protein in a mouse pharmacokinetic study with assay sensitivity of 10.0 ng/ml and linearity up to 10,000 ng/ml. CONCLUSION: The protein reagent-free workflow developed herein can overcome reagent limitation and serve as a viable approach for quantifying polyhistidine-tagged therapeutic proteins to support discovery pharmacokinetic and pharmacodynamic studies.
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Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Histidina/química , Fragmentos de Peptídeos/análise , Proteínas Recombinantes/farmacocinética , Espectrometria de Massas em Tandem/métodos , Animais , Metais/química , Camundongos , Camundongos Nus , Ratos , Distribuição TecidualRESUMO
OBJECTIVES: The interleukin-6 receptor (IL-6R) blocker tocilizumab (TCZ) reduces inflammatory disease activity in rheumatoid arthritis (RA) but elevates lipid concentrations in some patients. We aimed to characterise the impact of IL-6R inhibition on established and novel risk factors in active RA. METHODS: Randomised, multicentre, two-part, phase III trial (24-week double-blind, 80-week open-label), MEASURE, evaluated lipid and lipoprotein levels, high-density lipoprotein (HDL) particle composition, markers of coagulation, thrombosis and vascular function by pulse wave velocity (PWV) in 132 patients with RA who received TCZ or placebo. RESULTS: Median total-cholesterol, low-density lipoprotein-cholesterol (LDL-C) and triglyceride levels increased in TCZ versus placebo recipients by week 12 (12.6% vs 1.7%, 28.1% vs 2.2%, 10.6% vs -1.9%, respectively; all p<0.01). There were no significant differences in mean small LDL, mean oxidised LDL or total HDL-C concentrations. However, HDL-associated serum amyloid A content decreased in TCZ recipients. TCZ also induced reductions (>30%) in secretory phospholipase A2-IIA, lipoprotein(a), fibrinogen and D-dimers and elevation of paraoxonase (all p<0.0001 vs placebo). The ApoB/ApoA1 ratio remained stable over time in both groups. PWV decreases were greater with placebo than TCZ at 12â weeks (adjusted mean difference 0.79â m/s (95% CI 0.22 to 1.35; p=0.0067)). CONCLUSIONS: These data provide the first detailed evidence for the modulation of lipoprotein particles and other surrogates of vascular risk with IL-6R inhibition. When compared with placebo, TCZ induced elevations in LDL-C but altered HDL particles towards an anti-inflammatory composition and favourably modified most, but not all, measured vascular risk surrogates. The net effect of such changes for cardiovascular risk requires determination.
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Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Dislipidemias/metabolismo , Receptores de Interleucina-6/antagonistas & inibidores , Idoso , Arildialquilfosfatase/metabolismo , Doenças Cardiovasculares/diagnóstico por imagem , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Método Duplo-Cego , Feminino , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/metabolismo , Fosfolipases A2 do Grupo II/metabolismo , Humanos , Lipoproteína(a)/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Onda de Pulso , Proteína Amiloide A Sérica/metabolismo , Triglicerídeos/sangue , UltrassonografiaRESUMO
The N-glycan diversity of human serum glycoproteins, i.e., the human blood serum N-glycome, is both complex and constrained by the range of glycan structures potentially synthesizable by human glycosylation enzymes. The known glycome, however, has been further limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to include polysialylated N-glycans. Sample preparation improvements included acidified, microwave-accelerated, PNGase F N-glycan release to promote lactonization, and sodium borohydride reduction, that were both optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. Online separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient, providing additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) was utilized. When these improved methods are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described (Kronewitter et al. Anal. Chem. 2014, 86, 6268-6276), we are able to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrated the application of these advances in the context of the human serum glycome, and for which our initial observations included the detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.
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Análise Química do Sangue/instrumentação , Análise Química do Sangue/métodos , Polissacarídeos/sangue , Espectrometria de Massas por Ionização por Electrospray , Boroidretos/química , Cromatografia Líquida de Alta Pressão , Glicômica , Humanos , Ácido N-Acetilneuramínico/química , Nanotecnologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/isolamento & purificaçãoRESUMO
Glycomics quintavariate-informed quantification (GlyQ-IQ) is a biologically guided glycomics analysis tool for identifying N-glycans in liquid chromatography-mass spectrometry (LC-MS) data. Glycomics LC-MS data sets have convoluted extracted ion chromatograms that are challenging to deconvolve with existing software tools. LC deconvolution into constituent pieces is critical in glycomics data sets because chromatographic peaks correspond to different intact glycan structural isomers. The biological targeted analysis approach offers several key advantages to traditional LC-MS data processing. A priori glycan information about the individual target's elemental composition allows for improved sensitivity by utilizing the exact isotope profile information to focus chromatogram generation and LC peak fitting on the isotopic species having the highest intensity. Glycan target annotation utilizes glycan family relationships and in source fragmentation in addition to high specificity feature LC-MS detection to improve the specificity of the analysis. The GlyQ-IQ software was developed in this work and evaluated in the context of profiling the N-glycan compositions from human serum LC-MS data sets. A case study is presented to demonstrate how GlyQ-IQ identifies and removes confounding chromatographic peaks from high mannose glycan isomers from human blood serum. In addition, GlyQ-IQ was used to generate a broad human serum N-glycan profile from a high resolution nanoelectrospray-liquid chromatography-tandem mass spectrometry (nESI-LC-MS/MS) data set. A total of 156 glycan compositions and 640 glycan isomers were detected from a single sample. Over 99% of the GlyQ-IQ glycan-feature assignments passed manual validation and are backed with high-resolution mass spectra.
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Glicômica/métodos , Polissacarídeos/análise , Polissacarídeos/sangue , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida/métodos , Humanos , SoftwareRESUMO
We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/síntese química , Naftiridinas/síntese química , Inibidores de Proteínas Quinases/síntese química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Citocromo P-450 CYP3A , Inibidores do Citocromo P-450 CYP3A , Cães , Descoberta de Drogas , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Macaca fascicularis , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Ratos , Relação Estrutura-Atividade , Células U937 , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses.
Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Leucemia Mieloide Aguda/tratamento farmacológico , Naftiridinas/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Compostos Heterocíclicos com 3 Anéis/uso terapêutico , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Nus , Naftiridinas/farmacocinética , Naftiridinas/uso terapêutico , Neoplasias Experimentais , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sorafenibe , Células U937 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of urea based calcimimetics was optimized for potency and oral bioavailability. Crucial to this process was overcoming the poor pharmacokinetic properties of lead thiazole 1. Metabolism-guided modifications, characterized by the use of metabolite identification (ID) and measurement of time dependent inhibition (TDI) of CYP3A4, were essential to finding a compound suitable for oral dosing. Calcimimetic 18 exhibited excellent in vivo potency in a 5/6 nephrectomized rat model and cross-species pharmacokinetics.