RESUMEN
4ß-Hydroxycholesterol (4ß-HC) in plasma has been used as a biomarker to assess CYP3A drug-drug interaction (DDI) potential during drug development. However, due to the long half-life and narrow dynamic range of 4ß-HC, its use has been limited to the identification of CYP3A inducers, but not CYP3A inhibitors. The formation of 1ß-hydroxydeoxycholic acid (1ß-OH DCA) from deoxycholic acid (DCA) is mediated by CYP3A, thus 1ß-OH DCA can potentially serve as an alternative to 4ß-HC for assessment of CYP3A DDI potential. To study this feasibility, we developed a sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantitation of 1ß-OH DCA and its glycine and taurine conjugates in human plasma with the lower limit of quantitation of 50 pg/ml, which enabled the quantitation of basal levels and further reduction. The method was applied to a DDI study to assess how 1ß-OH DCA and its glycine and taurine conjugates would respond to CYP3A induction or inhibition. Rifampin induction resulted in an increase of 1ß-OH DCA and its conjugates in plasma, with 6.8-, 7.8-, 8.3-, and 10.3-fold increases of area under the curve from the time of dosing to the last measurable concentration (AUCLST), area under the curve from the time of dosing to 24 hours (AUC24h), C max, and mean concentrations for total 1ß-OH DCA (total of all three forms), respectively. Importantly, inhibition with itraconazole resulted in notable reduction of these biomarkers, with 84%, 85%, 82%, and 81% reductions of AUCLST, AUC24h, C max, and mean concentrations for total 1ß-OH DCA, respectively. These preliminary data demonstrate for the first time that total 1ß-OH DCA in plasma has the potential to serve as a biomarker for CYP3A DDI assessment in early clinical development and may provide key advantages over 4ß-HC. SIGNIFICANCE STATEMENT: The authors have reported the use of total 1ß-hydroxydeoxycholic acid (1ß-OH DCA) (sum of 1ß-OH DCA and its glycine and taurine conjugates) plasma exposure as a biomarker for CYP3A activity. Itraconazole inhibition led to an 81%-85% decrease of total 1ß-OH DCA plasma exposures, whereas rifampin induction led to a 6.8- to 10.3-fold increase of total 1ß-OH DCA plasma exposures. Using 1ß-OH DCA exposures in plasma also provides the benefit of allowing pharmacokinetic and biomarker assessment using the same matrix.
Asunto(s)
Biomarcadores , Inductores del Citocromo P-450 CYP3A , Citocromo P-450 CYP3A , Ácido Desoxicólico , Interacciones Farmacológicas , Hidroxicolesteroles , Humanos , Citocromo P-450 CYP3A/metabolismo , Biomarcadores/sangre , Ácido Desoxicólico/sangre , Inductores del Citocromo P-450 CYP3A/farmacología , Hidroxicolesteroles/sangre , Espectrometría de Masas en Tándem/métodos , Masculino , Adulto , Rifampin/farmacología , Rifampin/sangre , Inhibidores del Citocromo P-450 CYP3A/farmacología , Inhibidores del Citocromo P-450 CYP3A/farmacocinética , Cromatografía Liquida/métodos , Taurina/sangre , Taurina/análogos & derivadosRESUMEN
The blood-brain barrier (BBB) plays a critical role in preventing harmful endogenous and exogenous substances from penetrating the brain. Optimal brain penetration of small-molecule central nervous system (CNS) drugs is characterized by a high unbound brain/plasma ratio (Kp,uu). While various medicinal chemistry strategies and in silico models have been reported to improve BBB penetration, they have limited application in predicting Kp,uu directly. We describe a physics-based computational approach, a quantum mechanics (QM)-based energy of solvation (E-sol), to predict Kp,uu. Prospective application of this method in internal CNS drug discovery programs highlights the utility and accuracy of this new method, which showed a categorical accuracy of 79% and an R2 of 0.61 from a linear regression model.
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Barrera Hematoencefálica , Encéfalo , Transporte Biológico/fisiología , Fármacos del Sistema Nervioso Central , Simulación por ComputadorRESUMEN
A series of structurally diverse azaspirodecanone and spirooxazolidinone analogues were designed and synthesized as potent and selective somatostatin receptor subtype 5 (SSTR5) antagonists. Four optimized compounds each representing a subseries showed improvement in their metabolic stability and pharmacokinetic profiles compared to those of the original lead compound 1 while maintaining pharmacodynamic efficacy. The optimized cyclopropyl analogue 13 demonstrated efficacy in a mouse oral glucose tolerance test and an improved metabolic profile and pharmacokinetic properties in rhesus monkey studies. In this Communication, we discuss the relationship among structure, in vitro and in vivo activity, metabolic stability, and ultimately the potential of these compounds as therapeutic agents for the treatment of type 2 diabetes. Furthermore, we show how the use of focused libraries significantly expanded the structural class and provided new directions for structure-activity relationship optimization.
RESUMEN
The World Health Organization has identified antimicrobial resistance as a global public health threat since the prevalence and spread of antibiotic resistance among bacterial pathogens worldwide are staggering. Carbapenems, such as imipenem and meropenem, have been used to treat multidrug-resistant bacteria; however, since the development of resistance to carbapenems, ß-lactam antibiotics in combination with ß-lactamase inhibitors (BLI) has been one of the most successful strategies to enhance the activity of ß-lactam antibiotics. Relebactam (REL) is a new BLI which has been found to inhibit class A and class C ß-lactamases in vitro REL has been reported to restore imipenem's activity against both imipenem-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae Reported here are the in vivo efficacy studies of the imipenem-cilastatin (IMI)-REL combination in mouse models of disseminated and pulmonary infection caused by imipenem-resistant clinical isolates of P. aeruginosa and K. pneumoniae The combination was also evaluated in a P. aeruginosa delayed pulmonary model of infection. IMI-REL was found to be effective in the disseminated model of infection with log reduction in P. aeruginosa CFU of 3.73, 3.13, and 1.72 at REL doses of 40, 20, and 10 mg/kg, respectively. For K. pneumoniae, log reductions in CFU of 2.36, 3.06, and 2.29 were reported at REL doses of 80, 40, and 20 mg/kg, respectively. The combination was less effective in the delayed pulmonary model than in the immediate pulmonary model; however, overall REL was found to be effective against these imipenem-resistant strains.
Asunto(s)
Compuestos de Azabiciclo/uso terapéutico , Combinación Cilastatina e Imipenem/uso terapéutico , Animales , Cilastatina/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Femenino , Imipenem/uso terapéutico , Ratones , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Inhibidores de beta-Lactamasas/uso terapéuticoRESUMEN
The paper describes the SAR/SPR studies that led to the discovery of phenoxy cyclopropyl phenyl acetamide derivatives as potent and selective GPR119 agonists. Based on a cis cyclopropane scaffold discovered previously, phenyl acetamides such as compound 17 were found to have excellent GPR119 potency and improved physicochemical properties. Pharmacokinetic data of compound 17 in rat, dog and rhesus will be described. Compound 17 was suitable for QD dosing based on its predicted human half-life, and its projected human dose was much lower than that of the recently reported structurally-related benzyloxy compound 2. Compound 17 was selected as a tool compound candidate for NHP (Non-Human Primate) efficacy studies.
Asunto(s)
Acetamidas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Acetamidas/farmacocinética , Animales , Semivida , Humanos , Puntos Cuánticos , Ratas , Relación Estructura-ActividadRESUMEN
The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human ß3-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established ß3 potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 3/farmacología , Piperazinas/farmacología , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Agonistas de Receptores Adrenérgicos beta 3/química , Agonistas de Receptores Adrenérgicos beta 3/uso terapéutico , Humanos , Piperazinas/química , Piperazinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a carcinogenic heterocyclic aromatic amine formed during the high-temperature cooking of meats. The cytochrome P450-mediated N-hydroxylation of the exocyclic amine group of PhIP produces 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, an electrophilic metabolite that forms adducts with DNA and proteins. Previous studies conducted by our laboratory showed that the reaction of N-oxidized PhIP metabolites with human albumin in vitro primarily occurs at the Cys34 residue, to produce an acid-labile linked sulfinamide adduct. On the basis of these findings, we developed a sensitive ultraperformance liquid chromatography-mass spectrometry method to measure acid-labile albumin-PhIP adducts in human volunteers administered a dietary-relevant dose of 14C-labeled PhIP [Dingley, K. H., et al. (1999) Cancer Epidemiol., Biomarkers Prev. 8, 507-512]. Mild acid treatment of albumin (0.1 N HCl, 37 °C for 1 h) or proteolytic digestion with Pronase [50 mM ammonium bicarbonate buffer (pH 8.5) at 37 °C for 18 h] released similar amounts of covalently bound PhIP, which was characterized by multistage scanning and quantified by Orbitrap mass spectrometry. The amount of [14C]PhIP recovered by acid treatment of albumin 24 h following dosing accounted for 7.2-21.3% of the [14C]PhIP bound to albumin based on accelerator mass spectrometry measurements. 2-Amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine, a hydrolysis product of the Cys34 S-N linked sulfenamide adduct of PhIP, was not detected in either acid-treated or protease-treated samples. These findings suggest that a portion of the PhIP bound to albumin in vivo probably occurs as an acid-labile sulfinamide adduct formed at the Cys34 residue.
Asunto(s)
Albúminas/metabolismo , Imidazoles/metabolismo , Espectrometría de Masas/métodos , Calibración , Cromatografía Liquida/métodos , Humanos , HidrólisisRESUMEN
GPR142 has been identified as a potential glucose-stimulated insulin secretion (GSIS) target for the treatment of type 2 diabetes mellitus (T2DM). A class of triazole GPR142 agonists was discovered through a high throughput screen. The lead compound 4 suffered from poor metabolic stability and poor solubility. Lead optimization strategies to improve potency, efficacy, metabolic stability, and solubility are described. This optimization led to compound 20e, which showed significant reduction of glucose excursion in wild-type but not in GPR142 deficient mice in an oral glucose tolerance test (oGTT) study. These studies provide strong evidence that reduction of glucose excursion through treatment with 20e is GPR142-mediated, and GPR142 agonists could be used as a potential treatment for type 2 diabetes.
RESUMEN
A novel series of benzo-[1,2,4]-triazolo-[1,4]-oxazepine GPR142 agonists are described. The series was designed to address the suboptimal PK (pharmacokinetic) and off-target profile of a class of N-aryl-benzo-[1,4]-oxazepine-4-carboxamides, represented by 1, that were identified from a high-throughput screen of the Merck compound collection for GPR142 agonists. This work led to the discovery of 3-phenoxy-benzo-[1,2,4]-triazolo-[1,4]-oxazepine 47, a potent GPR142 agonist with an off-target and PK profile suitable for in vivo studies. This compound and a related analogue 40 were shown to be active in mouse oral glucose tolerance tests (OGTTs). Furthermore, a GPR142 knock-out mouse OGTT study with compound 40 provides evidence that its glucose-lowering effect is mediated by GPR142.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Descubrimiento de Drogas , Oxazepinas/farmacología , Receptores Acoplados a Proteínas G/agonistas , Triazoles/farmacología , Animales , Relación Dosis-Respuesta a Droga , Prueba de Tolerancia a la Glucosa , Ratones , Ratones Noqueados , Estructura Molecular , Oxazepinas/síntesis química , Oxazepinas/química , Ratas , Receptores Acoplados a Proteínas G/deficiencia , Relación Estructura-Actividad , Triazoles/síntesis química , Triazoles/químicaRESUMEN
The discovery of vibegron, a potent and selective human ß3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical ß3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived ß3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.
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Agonistas de Receptores Adrenérgicos beta 3/uso terapéutico , Pirimidinonas/uso terapéutico , Pirrolidinas/uso terapéutico , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Agonistas de Receptores Adrenérgicos beta 3/farmacocinética , Agonistas de Receptores Adrenérgicos beta 3/toxicidad , Animales , Células CHO , Cricetinae , Cricetulus , Descubrimiento de Drogas , Femenino , Humanos , Lipidosis/inducido químicamente , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Modelos Moleculares , Pirimidinonas/farmacocinética , Pirimidinonas/toxicidad , Pirrolidinas/farmacocinética , Pirrolidinas/toxicidad , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Relación Estructura-Actividad , Vejiga Urinaria/efectos de los fármacos , Micción/efectos de los fármacos , Difracción de Rayos XRESUMEN
The paper will describe the synthesis and SAR studies that led to the discovery of benzamide (reverse amide) as potent and selective human ß3-adrenergic receptor agonist. Based on conformationally restricted pyrrolidine scaffold we discovered earlier, pyrrolidine benzoic acid intermediate 22 was synthesized. From library synthesis and further optimization efforts, several structurally diverse reverse amides such as 24c and 24i were found to have excellent human ß3-adrenergic potency and good selectivity over the ß1 and ß2 receptors. In addition to human ß1, ß2, ß3 and hERG data, PK of selected compounds will be described.
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Agonistas de Receptores Adrenérgicos beta 3/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Receptores Adrenérgicos beta 3/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/síntesis química , Agonistas de Receptores Adrenérgicos beta 3/química , Benzamidas/síntesis química , Benzamidas/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
We report herein the design and synthesis of a series of potent and selective GPR119 agonists. Our objective was to develop a GPR119 agonist with properties that were suitable for fixed-dose combination with a DPP4 inhibitor. Starting from a phenoxy analogue (1), medicinal chemistry efforts directed toward reducing half-life and increasing solubility led to the synthesis of a series of benzyloxy analogues. Compound 28 was chosen for further profiling because of its favorable physicochemical properties and excellent GPR119 potency across species. This compound exhibited a clean off-target profile in counterscreens and good in vivo efficacy in mouse oGTT.
RESUMEN
Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.
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Aductos de ADN/aislamiento & purificación , Animales , Aductos de ADN/química , Humanos , Marcaje Isotópico , Espectrometría de MasasRESUMEN
A series of conformationally restricted acetanilides were synthesized and evaluated as ß3-adrenergic receptor agonists (ß3-AR) for the treatment of overactive bladder (OAB). Optimization studies identified a five-membered ring as the preferred conformational lock of the acetanilide. Further optimization of both the aromatic and thiazole regions led to compounds such as 19 and 29, which have a good balance of potency and selectivity. These compounds have significantly reduced intrinsic clearance compared to our initial series of pyridylethanolamine ß3-AR agonists and thus have improved unbound drug exposures. Both analogues demonstrated dose dependent ß3-AR mediated responses in a rat bladder hyperactivity model.
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Acetanilidas/síntesis química , Acetanilidas/farmacología , Agonistas de Receptores Adrenérgicos beta 3/síntesis química , Agonistas de Receptores Adrenérgicos beta 3/farmacología , Vejiga Urinaria Hiperactiva/tratamiento farmacológico , Acetanilidas/uso terapéutico , Agonistas de Receptores Adrenérgicos beta 3/uso terapéutico , Animales , Células CHO , Cricetinae , Cricetulus , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Conformación MolecularRESUMEN
Epidemiologic evidence indicates that exposure to heterocyclic amines in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of heterocyclic amines, which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of heterocyclic amine bioactivation in humans, the most mass abundant heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers where administered a dietary relevant dose of [(14)C]PhIP 48 to 72 hours before surgery to remove colon tumors. Urine was collected for 24 hours after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All 10 subjects were phenotyped for cytochrome P4501A2 (CYP1A2), N-acetyltransferase 2, and sulfotransferase 1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N(2)-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N(2)-glucuronide had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N(2)-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.
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Carcinógenos/metabolismo , Colon/metabolismo , Aductos de ADN/orina , Imidazoles/metabolismo , Arilamina N-Acetiltransferasa/fisiología , Arilsulfotransferasa/fisiología , Citocromo P-450 CYP1A2/fisiología , Glucuronosiltransferasa/fisiología , HumanosRESUMEN
2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) has been implicated as a major mutagenic heterocyclic amine in the human diet and is carcinogenic in the rat prostate. To validate PhIP-induced rat prostatic neoplasia as a model of human prostate cancer progression, we sought to study the earliest histologic and morphologic changes in the prostate and to follow progressive changes over time. We fed sixty-seven 5-week-old male Fischer F344 rats with PhIP (400 ppm) or control diets for 20 weeks, and then sacrificed animals for histomorphologic examination at the ages of 25, 45, and 65 weeks. Animals treated with PhIP showed significantly more inflammation (P = .002, > .001, and .016 for 25, 45, and 65 weeks, respectively) and atrophy (P = .003, > .001, and .006 for 25, 45, and 65 weeks, respectively) in their prostate glands relative to controls. Prostatic intraepithelial neoplasia (PIN) occurred only in PhIP-treated rats. PIN lesions arose in areas of glandular atrophy, most often in the ventral prostate. Atypical cells in areas of atrophy show loss of glutathione S-transferase pi immunostaining preceding the development of PIN. None of the animals in this study developed invasive carcinomas, differing from those in previous reports. Overall, these findings suggest that the pathogenesis of prostatic neoplasia in the PhIP-treated rat prostate proceeds from inflammation to postinflammatory proliferative atrophy to PIN.
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Imidazoles/toxicidad , Inflamación , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Animales , Atrofia , Carcinógenos/toxicidad , Proliferación Celular , Modelos Animales de Enfermedad , Glutatión Transferasa/metabolismo , Masculino , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/inducido químicamente , Ratas , Ratas Endogámicas F344RESUMEN
Accelerator mass spectrometry (AMS) is currently one of the most sensitive methods available for the trace detection of DNA adducts and is particularly valuable for measuring adducts in humans or animal models. However, the standard approach requires administration of a radiolabeled compound. As an alternative, we have developed a preliminary 14C-postlabeling assay for detection of the highly mutagenic O6-methyldeoxyguanosine (O6-MedG), by AMS. Procedures were developed for derivatising O6-MedG using unlabeled acetic anhydride. Using conventional liquid chromatography/mass spectrometry (LC/MS) analysis, the limit of detection (LOD) for the major product, triacetylated O6-MedG, was 10 fmol. On reaction of O6-MedG with 14C-acetic anhydride, using a specially designed enclosed system, the predominant product was 14C-di-acetyl O6-MedG. This change in reaction profile was due to a modification of the reaction procedure, introduced as a necessary safety precaution. The LOD for 14C-di-acetyl O6-MedG by AMS was determined as 79 amol, approximately 18,000-fold lower than that achievable by liquid scintillation counting (LSC). Although the assay has so far only been carried out with labeled standards, the degree of sensitivity obtained illustrates the potential of this assay for measuring O6-MedG levels in humans.
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Aductos de ADN/análisis , Desoxiguanosina/análogos & derivados , Monitoreo del Ambiente/métodos , Espectrometría de Masas/métodos , Anhídridos Acéticos/química , Radioisótopos de Carbono/análisis , Desoxiguanosina/análisis , Desoxiguanosina/química , Humanos , Espectrometría de Masas/instrumentación , Aceleradores de Partículas/instrumentaciónRESUMEN
Accelerator mass spectrometry (AMS) is the most sensitive method for detecting and quantifying rare long-lived isotopes with high precision. In this chapter, we review the principles underlying AMS-based biomedical studies, focusing on important practical considerations and experimental procedures needed for the detection and quantitation of (14)C- and (3)H-labeled compounds in various experiment types.
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ADN/análisis , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Proteínas/análisis , Animales , Radioisótopos de Carbono/análisis , Humanos , Aceleradores de Partículas/instrumentación , Tritio/análisisRESUMEN
A protocol is described for the isolation of DNA and subsequent preparation of samples for the measurement of adduct levels by accelerator mass spectrometry (AMS). AMS is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. However, special precautions must be taken to avoid cross-contamination of isotope between samples and to produce a sample that is compatible with AMS. The DNA isolation method described is based on digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen DNA isolation columns. DNA is then precipitated with isopropanol, washed repeatedly with 70% ethanol to remove salt, and then dissolved in water. This method has been used to generate reliably good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. For quantification of adduct levels from 14C-labeled compounds, DNA samples are then converted to graphite, and the 14C content is measured by AMS.
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Aductos de ADN/análisis , ADN/aislamiento & purificación , Espectrometría de Masas/métodos , Animales , Radioisótopos de Carbono , Pruebas de Carcinogenicidad , ADN/química , Daño del ADN , Humanos , Pruebas de MutagenicidadRESUMEN
The mycotoxin ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rodents. However, the mechanism of OTA-induced tumor formation is unknown and conflicting results have been obtained regarding the potential of OTA to bind to DNA. OTA is poorly metabolized, and no reactive intermediates capable of interacting with DNA have been detected in vitro or in vivo. Recently, a hydroquinone/quinone redox couple and a carbon-bonded OTA-deoxyguanosine (OTA-dG) adduct formed by electrochemical oxidation and photoreaction of OTA have been reported and suggested to be involved in OTA carcinogenicity. This study was designed to characterize the role of DNA binding and to determine if formation of these derivatives occurs in vivo and in relevant activation systems in vitro using specific and sensitive methods. Horseradish peroxidase activation of OTA and its dechlorinated analogue ochratoxin B (OTB) yielded ochratoxin A-hydroquinone (OTHQ), but the postulated OTA-dG adduct was not detectable using LC-MS/MS. In support of this, no OTA-related DNA adducts were observed by 32P-postlabeling. In vivo, only traces of OTHQ were found in the urine of male F344 rats treated with high doses of OTA (2 mg/kg body wt) for 2 weeks, suggesting that this metabolite is not formed to a relevant extent. In agreement with the in vitro data, OTA-dG was not detected by LC-MS/MS in liver and kidney DNA extracted from treated animals. In addition, DNA binding of OTA and OTB was assessed in male rats given a single dose of 14C-OTA or 14C-OTB using accelerator mass spectrometry, a highly sensitive method for quantifying extremely low concentrations of radiocarbon. The 14C content in liver and kidney DNA from treated animals was not significantly different from controls, indicating that OTA does not form covalent DNA adducts in high yields. In summary, the results presented here demonstrate that DNA binding of OTA is not detectable with sensitive analytical methods and is unlikely to represent a mechanism for OTA-induced tumor formation.