RESUMEN
Nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in the quantitation of small and large molecules. Recently, we demonstrated that (1)H NMR could be used to quantitate drug metabolites isolated in submilligram quantities from biological sources. It was shown that these metabolites, once quantitated by NMR, were suitable to be used as reference standards in quantitative LC/MS-based assays, hence circumventing the need for radiolabeled material or synthetic standards to obtain plasma exposure estimates in humans and preclinical species. The quantitative capabilities of high-field NMR is further demonstrated in the current study by obtaining the mass balance of fluorinated compounds using (19)F-NMR. Two fluorinated compounds which were radio-labeled with carbon-14 on metabolically stable positions were dosed in rats and urine and feces collected. The mass balance of the compounds was obtained initially by counting the radioactivity present in each sample. Subsequently, the same sets of samples were analyzed by (19)F-NMR, and the concentrations determined by this method were compared with data obtained using radioactivity counting. It was shown that the two methods produced comparable values. To demonstrate the value of this analytical technique in drug discovery, a fluorinated compound was dosed intravenously in dogs and feces and urine collected. Initial profiling of samples showed that this compound was excreted mainly unchanged in feces, and hence, an estimate of mass balance was obtained using (19)F-NMR. The data obtained by this method was confirmed by additional quantitative studies using mass spectrometry. Hence cross-validations of the quantitative (19)F-NMR method by radioactivity counting and mass spectrometric analysis were demonstrated in this study. A strategy outlining the use of fluorinated compounds in conjunction with (19)F-NMR to understand their routes of excretion or mass balance in animals is proposed. These studies demonstrate that quantitative (19)F-NMR could be used as an alternate technique to obtain an estimate of the mass balance of fluorinated compounds, especially in early drug development where attrition of the compounds is high, and cost savings could be realized through the use of such a technique rather than employing radioactive compounds. The potential application of qNMR in conducting early human ADME studies with fluorinated compounds is also discussed.
Asunto(s)
Descubrimiento de Drogas/métodos , Compuestos de Flúor/farmacocinética , Espectroscopía de Resonancia Magnética/métodos , Animales , Radioisótopos de Carbono , Perros , Heces/química , Compuestos de Flúor/orina , Radioisótopos de Flúor , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
It is important to gain an understanding of the pharmacological activities of metabolite(s) of compounds in development, especially if they are found in systemic circulation in humans. Pharmacological evaluation of metabolites is normally conducted with synthetic standards, which become available during various stages of drug development. However, the synthesis of metabolite standards may be protracted, taking anywhere from several weeks to months to be completed. This often slows down early pharmacological evaluation of metabolites. Once a metabolite(s) is found to possess comparable (or greater) pharmacological activity than the parent compound, additional studies are performed to better understand the implications of circulating pharmacologically active metabolite(s). To conduct some of these studies as early as possible without slowing the progression of a compound in development is important, especially if critical go or no-go decisions impinge on the outcomes from these studies. Early pharmacological evaluation of significant metabolites is hereby proposed to be conducted in the drug discovery stage so that all pertinent studies and information can be gathered in a timely manner for decision-making. It is suggested that these major metabolites be isolated, either from biological or chemical sources, and quantified appropriately. For biologically generated metabolites, NMR is proposed as the tool of choice to quantitate these metabolites before their evaluation in pharmacological assays. For metabolites that have the same UV characteristics as the parent compound, quantitation can be conducted using UV spectroscopy instead of NMR. In this article, we propose a strategy that could be used to determine the pharmacological activities of metabolites isolated in submilligram quantities.
Asunto(s)
Descubrimiento de Drogas , Microsomas Hepáticos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Evaluación Preclínica de Medicamentos , Humanos , Macaca fascicularis , Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Masculino , Preparaciones Farmacéuticas/química , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [(14)C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182-195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the epsilon-amine of lysine attacking to the beta-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37 degrees C for 18 h.
Asunto(s)
Química Farmacéutica/métodos , Quinolinas/sangre , Albúmina Sérica/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Radioisótopos de Carbono/sangre , Humanos , Mapeo Peptídico/métodos , Péptidos/metabolismo , Ensayo de Unión Radioligante/métodosRESUMEN
Methylnaltrexone (MNTX) is a peripherally acting mu-opioid receptor antagonist and is currently indicated for the treatment of opioid-induced constipation in patients with advanced illness who are receiving palliative care, when response to laxative therapy has not been sufficient. Sulfation to MNTX-3-sulfate (M2) and carbonyl reduction to methyl-6alpha-naltrexol (M4) and methyl-6beta-naltrexol (M5) are the primary metabolic pathways for MNTX in humans. The objectives of this study were to investigate MNTX in vitro metabolism in human and nonclinical species and to identify the human enzymes involved in MNTX metabolism. Of the five commercially available sulfotransferases investigated, only SULT2A1 and SULT1E1 catalyzed M2 formation. Formation of M4 and M5 was catalyzed by NADPH-dependent hepatic cytosolic enzymes, which were identified using selective chemical inhibitors (10 and 100 microM) for aldo-keto reductase (AKR) isoforms, short-chain dehydrogenase/reductase including carbonyl reductase, alcohol dehydrogenase, and quinone oxidoreductase. The results were then compared with the effects of the same inhibitors on 6beta-naltrexol formation from naltrexone, a structural analog of MNTX, which is catalyzed mainly by AKR1C4. The AKR1C inhibitor phenolphthalein inhibited MNTX and naltrexone reduction up to 98%. 5beta-Cholanic acid 3alpha,7alpha-diol, the AKR1C2 inhibitor, and medroxyprogesterone acetate, an inhibitor of AKR1C1, AKR1C2, and AKR1C4, inhibited MNTX reduction up to 67%. Other inhibitors were less potent. In conclusion, the carbonyl reduction of MNTX to M4 and M5 in hepatic cytosol was consistent with previous in vivo observations. AKR1C4 appeared to play a major role in the carbonyl reduction of MNTX, although multiple enzymes in the AKR1C subfamily may be involved. Human SULT2A1 and SULT1E1 were involved in MNTX sulfation.
Asunto(s)
Enzimas/metabolismo , Hígado/enzimología , Naltrexona/análogos & derivados , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa , Aldo-Ceto Reductasas , Animales , Biocatálisis/efectos de los fármacos , Citosol/enzimología , Perros , Inhibidores Enzimáticos/farmacología , Haplorrinos , Humanos , Cinética , Espectrometría de Masas , Ratones , Estructura Molecular , NADP/metabolismo , Naltrexona/metabolismo , Oxidación-Reducción , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Sulfotransferasas/metabolismo , Ésteres del Ácido Sulfúrico/metabolismoRESUMEN
Methylnaltrexone (MNTX), a selective mu-opioid receptor antagonist, functions as a peripherally acting receptor antagonist in tissues of the gastrointestinal tract. This report describes the metabolic fate of [(3)H]MNTX or [(14)C]MNTX bromide in mice, rats, dogs, and humans after intravenous administration. Separation and identification of plasma and urinary MNTX metabolites was achieved by high-performance liquid chromatography-radioactivity detection and liquid chromatography/mass spectrometry. The structures of the most abundant human metabolites were confirmed by chemical synthesis and NMR spectroscopic analysis. Analysis of radioactivity in plasma and urine showed that MNTX underwent two major pathways of metabolism in humans: sulfation of the phenolic group to MNTX-3-sulfate (M2) and reduction of the carbonyl group to two epimeric alcohols, methyl-6alpha-naltrexol (M4) and methyl-6beta-naltrexol (M5). Neither naltrexone nor its metabolite 6beta-naltrexol were detected in human plasma after administration of MNTX, confirming an earlier observation that N-demethylation was not a metabolic pathway of MNTX in humans. The urinary metabolite profiles in humans were consistent with plasma profiles. In mice, the circulating and urinary metabolites included M5, MNTX-3-glucuronide (M9), 2-hydroxy-3-O-methyl MNTX (M6), and its glucuronide (M10). M2, M5, M6, and M9 were observed in rats. Dogs produced only one metabolite, M9. In conclusion, MNTX was not extensively metabolized in humans. Conversion to methyl-6-naltrexol isomers (M4 and M5) and M2 were the primary pathways of metabolism in humans. MNTX was metabolized to a higher extent in mice than in rats, dogs, and humans. Glucuronidation was a major metabolic pathway in mice, rats, and dogs, but not in humans. Overall, the data suggested species differences in the metabolism of MNTX.
Asunto(s)
Naltrexona/análogos & derivados , Antagonistas de Narcóticos/farmacocinética , Adulto , Animales , Biotransformación , Cromatografía Líquida de Alta Presión , Remoción de Radical Alquila , Perros , Glucuronidasa/metabolismo , Humanos , Inyecciones Intravenosas , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Ratones , Naltrexona/administración & dosificación , Naltrexona/farmacocinética , Antagonistas de Narcóticos/administración & dosificación , Compuestos de Amonio Cuaternario/administración & dosificación , Compuestos de Amonio Cuaternario/farmacocinética , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Analyzing brain microdialysate samples by mass spectrometry is challenging due to the high salt content of the artificial cerebral spinal fluid (aCSF), low analyte concentrations and small sample volumes collected. A drug and its major metabolites can be examined in brain microdialysates by targeted approaches such as selected reaction monitoring (SRM) which provides selectivity and high sensitivity. However, this approach is not well suited for metabolite profiling in the brain which aims to determine biotransformation pathways. Identifying minor metabolites, or metabolites that arise from brain metabolism, remains a challenge and, for a drug in early discovery, identification of metabolites present in the brain can provide useful information for understanding the pharmacological activity and potential toxicological liabilities of the drug. A method is described here for rapid metabolite profiling in brain microdialysates that involves sample clean-up using C18 ZipTips to remove salts followed by direct infusion nanoelectrospray with an LTQ/Orbitrap mass spectrometer using real-time internal recalibration. Full scan mass spectra acquired at high resolving power (100 K at m/z 400) were examined manually and with mass defect filtering. Metabolite identification was aided by sub-parts-per-million mass accuracy and structural characterization was accomplished by tandem mass spectrometry (MS/MS) experiments in the Orbitrap or LTQ depending on the abundance of the metabolite. Using this approach, brain microdialysate samples from rats dosed with one of four CNS drugs (imipramine, reboxetine, citalopram or trazodone) were examined for metabolites. For each drug investigated, metabolites, some of which not previously reported in rat brain, were identified and characterized.
Asunto(s)
Química Encefálica , Encéfalo/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Biotransformación , Fármacos del Sistema Nervioso Central/química , Fármacos del Sistema Nervioso Central/metabolismo , Masculino , Microdiálisis/instrumentación , Ratas , Ratas Sprague-Dawley , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
In addition to mass accuracy, the ability of a mass spectrometer to faithfully measure the isotopic distribution of an ion, defined as spectral accuracy, is also important. Although time-of-flight mass spectrometers are reported to possess high spectral accuracy capability compared with other mass spectrometers, the Orbitrap has not yet been investigated. Ten natural products (moxidectin, erythromycin, digoxin, rifampicin, amphotericin B, rapamycin, gramicidin S, cyclosporin A, vancomycin, and thiostrepton) ranging in molecular weight from 639 to 1663 Da were measured on an LTQ/Orbitrap mass spectrometer with resolving power settings of 7.5, 15, 30, 60, and 100 K. The difference in the observed profile isotope pattern compared with the theoretical calculation after peak shape calibration, denoted spectral error, was calculated using the program MassWorks (Cerno Bioscience, Danbury, CT, USA). Spectral errors were least at 7.5 K resolving power (< or = 3%) but exceeded 10% for some compounds at 100 K. The increasing spectral error observed at higher resolving power for compounds with complex fine structure might be explained by the phenomena of isotopic beat patterns as observed in FTICR. Several compounds with prominent doubly charged ions allowed comparison of spectral accuracies of singly- versus doubly-charged ions. When using spectral error to rank elemental compositions with formula constraints (C(0-100)H(0-200)N(0-50)O(0-50)Cl(0-5)S(0-5)) and a mass tolerance < or = 2 parts-per-million, the correct formula was ranked first 35% of the time. However, spectral error considerations eliminated >99% of possible elemental formulas for compounds with molecular weight >900 Da.
Asunto(s)
Elementos Químicos , Iones/análisis , Espectrometría de Masas/métodos , Productos Biológicos/química , Calibración , Cromatografía Líquida de Alta Presión , Calor , Iones/química , Espectrometría de Masas/instrumentación , Estructura Molecular , Peso Molecular , Programas Informáticos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Nuclear magnetic resonance (NMR) spectroscopy has traditionally been considered as an indispensable tool in elucidating structures of metabolites. With the advent of Fourier transform (FT) spectrometers, along with improvements in software and hardware (such as high-field magnets, cryoprobes, versatile pulse sequences, and solvent suppression techniques), NMR is increasingly being considered as a critical quantitative tool, despite its lower sensitivity as compared to mass spectrometry. A specific quantitative application of NMR is in determining the concentrations of biologically isolated metabolites, which could potentially be used as reference standards for further quantitative work by liquid chromatography/mass spectrometry. With the recent demands from regulatory agencies on quantitative information on metabolites, it is proposed that NMR will play a significant role in strategies aimed at addressing metabolite coverage in toxicological species. Traditionally, biologically isolated metabolites have not been considered as a way of generating "reference standards" for further quantitative work. However, because of the recent FDA guidance on safety testing of metabolites, one has to consider means of authenticating and quantitating biologically or nonbiologically generated metabolites. 1H NMR is being proposed as the method of choice, as it is able to be used as both a qualitative and a quantitative tool, hence allowing structure determination, purity check, and quantitative measurement of the isolated metabolite. In this publication, the application of NMR as a powerful and robust analytical technique in determining the concentrations of in vitro or in vivo isolated metabolites is discussed. Furthermore, to demonstrate the reliability and accuracy of metabolite concentrations determined by NMR, validation and cross-validation with gravimetric and mass spectrometric methods were conducted.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Preparaciones Farmacéuticas/análisis , Pruebas de Toxicidad/métodos , Acetaminofén/análisis , Acetaminofén/química , Acetaminofén/metabolismo , Animales , Cromatografía Liquida , Espectrometría de Masas , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Fenacetina/análisis , Fenacetina/química , Fenacetina/metabolismo , RatasRESUMEN
The recent guidance on "Safety Testing of Drug Metabolites" issued by the U.S. Food and Drug Administration, Center for Drug Evaluation and Research (CDER) has highlighted the importance of identifying and characterizing drug metabolites as early as possible in drug discovery and development. Furthermore, upon identifying significant circulating metabolites in human plasma, it has become important to demonstrate that these metabolites are present at an equal or greater exposure level (area under the curve, AUC) in any one of the preclinical species used in safety testing. Frequently, synthetic standards of metabolites are not available, and hence, obtaining their AUC values can be a challenge. In this report, we demonstrate how combinations of nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography/ultraviolet/mass spectrometry (LC/UV/MS), and plasma pooling methods were used to obtain reliable AUC values of metabolites present in the plasma of preclinical species from short-term safety studies. Plasma pooling methods were compared to the traditional approaches of obtaining quantitative information on the levels of circulating metabolites in preclinical species. The exposure values obtained via sample pooling were comparable to those obtained by traditional methods of analyzing samples individually. In the absence of synthetic chemical standards, calculations of AUC values of metabolites, using either sample pooling or traditional approaches, were achieved through the use of UV detectors. In cases where the UV properties of metabolites were significantly different from their parent compounds, NMR was used as a quantitative tool to obtain exposure values. NMR was found to be useful in quantitating biologically produced metabolites, which could subsequently be used as reference compounds for further quantitative studies. The limitations of UV detectors to obtain exposure estimates are discussed. A practical solution is presented that will enable us to obtain a quantitative assessment of metabolite exposure in humans and coverage in toxicology species, hence, circumventing the use of radiolabeled compounds or authentic chemically synthesized standards of metabolites.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Preparaciones Farmacéuticas/sangre , Pruebas de Toxicidad/métodos , Algoritmos , Animales , Área Bajo la Curva , Cromatografía Líquida de Alta Presión , Perros , Evaluación Preclínica de Medicamentos , Femenino , Masculino , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Radioisótopos/química , Ratas , Estándares de Referencia , Espectrofotometría Ultravioleta , Espectrometría de Masas en Tándem , Pruebas de Toxicidad/normasRESUMEN
A robust bioanalytical method capable of measuring acetyl and palmitoyl carnitines was developed and validated. Application of hydrophilic interaction chromatography (HILIC) enabled retention of these highly polar and difficult to analyze compounds on a silica HPLC column. The chromatography was conducted with a high percentage of an organic component in the mobile phase, allowing high sensitivity for the pre-existing positively charged quaternary ammonium ions by electrospray ionization mass spectrometry. Successful application of the method to reliably quantify naturally occurring acyl carnitines in mouse plasma depended on the use of corresponding deuterated analogues. The specificity of the method, achieved through the use of stable isotope labeled compounds in combination with a mass spectral multiple reaction monitoring technique, permitted a non-invasive assessment of the overall change in the levels of these acyl carnitines in the plasma of intact animals administered peroxisome proliferator activated receptor (PPAR) agents. These acyl carnitines, as carriers of the corresponding long-chain fatty acids for transport into mitochondria, can be employed as potential biomarkers for significant alteration in the beta-oxidation process in an intact animal.
Asunto(s)
Carnitina/análogos & derivados , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Palmitoilcarnitina/sangre , Animales , Biomarcadores/sangre , Biomarcadores/química , Calibración , Carnitina/sangre , Marcaje Isotópico , Masculino , Ratones , Ratones Endogámicos , Estructura Molecular , Oxidación-Reducción , Palmitoilcarnitina/química , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Metabolite identification studies remain an integral part of pre-clinical and clinical drug development programs. Analysis of biological matrices, such as plasma, urine, feces and bile, pose challenges due to the large amounts of endogenous components that can mask a drug and its metabolites. Although direct infusion nanoelectrospray using capillaries has been used routinely for proteomic studies, metabolite identification has traditionally employed liquid chromatographic (LC) separation prior to analysis. A method is described here for rapid metabolite profiling in biological fluids that involves initial sample clean-up using pipette tips packed with reversed-phase material (i.e. ZipTips) to remove matrix components followed by direct infusion nanoelectrospray on an LTQ/Orbitrap mass spectrometer using a protonated polydimethylcyclosiloxane cluster ion for internal calibration. We re-examined samples collected from a prazosin metabolism study in the rat. Results are presented that demonstrate that sub parts-per-million accuracies can be achieved on molecular ions, facilitating identification of metabolites, and on product ions, facilitating structural assignments. The data also show that the high-resolution measurements (R = 100,000 at m/z 400) enable metabolites of interest to be resolved from endogenous components. The extended analysis times available with nanospray enables signal averaging for 1 min or more that is valuable when metabolites are present in low concentrations as encountered here in plasma and brain. Using this approach, the metabolic fate of a drug can be quickly obtained. A limitation of this approach is that metabolites that are structural isomers cannot be distinguished, although such information can be collected by LC/MS during follow-on experiments.
Asunto(s)
Líquidos Corporales/metabolismo , Microquímica/métodos , Nanotecnología/métodos , Prazosina/farmacocinética , Manejo de Especímenes/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Antihipertensivos/análisis , Antihipertensivos/farmacocinética , Líquidos Corporales/química , Masculino , Microquímica/instrumentación , Nanotecnología/instrumentación , Prazosina/análisis , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
Tigecycline, a novel, first-in-class glycylcycline antibiotic, has been approved for the treatment of complicated intra-abdominal infections and complicated skin and skin structure infections. The pharmacokinetics, metabolism, and excretion of [(14)C]tigecycline were examined in healthy male volunteers. Tigecycline has been shown to bind to bone; thus, to minimize the amount of radioactivity binding to bone and to maximize the recovery of radioactivity, tigecycline was administered intravenously (30-min infusion) as a single 100-mg dose, followed by six 50-mg doses, every 12 h, with the last dose being [(14)C]tigecycline (50 microCi). After the final dose, the pharmacokinetics of tigecycline in serum showed a long half-life (55.8 h) and a large volume of distribution (21.0 l/kg), whereas radioactivity in serum had a shorter half-life (6.9 h) and a smaller volume of distribution (3.3 l/kg). The major route of elimination was feces, containing 59% of the radioactive dose, whereas urine contained 32%. Unchanged tigecycline was the predominant drug-related compound in serum, urine, and feces. The major metabolic pathways identified were glucuronidation of tigecycline and amide hydrolysis followed by N-acetylation to form N-acetyl-9-aminominocycline. The glucuronide metabolites accounted for 5 to 20% of serum radioactivity, and approximately 9% of the dose was excreted as glucuronide conjugates within 48 h. Concentrations of N-acetyl-9-aminominocycline were approximately 6.5% and 11% of the tigecycline concentrations in serum and urine, respectively. Excretion of unchanged tigecycline into feces was the primary route of elimination, and the secondary elimination pathways were renal excretion of unchanged drug and metabolism to glucuronide conjugates and N-acetyl-9-aminominocycline.
Asunto(s)
Antibacterianos/farmacocinética , Minociclina/análogos & derivados , Acetilación , Adulto , Antibacterianos/administración & dosificación , Área Bajo la Curva , Biotransformación , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Heces/química , Glucurónidos/metabolismo , Semivida , Humanos , Infusiones Intravenosas , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Minociclina/administración & dosificación , Minociclina/farmacocinética , TigeciclinaRESUMEN
Prazosin (2-[4-(2-furanoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline) is an antihypertensive agent that was introduced to the market in 1976. It has since established an excellent safety record. However, in vitro metabolism of prazosin has not been investigated. This study describes the in vitro biotransformation of prazosin in liver microsomes from rats, dogs, and humans, as well as rat and human cryopreserved hepatocytes and characterization of metabolites using liquid chromatography/tandem mass spectrometry. The major in vivo biotransformation pathways reported previously in rats and dogs include demethylation, amide hydrolysis, and O-glucuronidation. These metabolic pathways were also confirmed in our study. In addition, several new metabolites were characterized, including a stable carbinolamine, an iminium species, and an enamine-all formed via oxidation of the piperazine ring. Two ring-opened metabolites generated following oxidative cleavage of the furan ring were also identified. Using semicarbazide hydrochloride as a trapping agent, an intermediate arising from opening of the furan ring was captured as a pyridazine product. In the presence of glutathione, three glutathione conjugates were detected in microsomal incubations, although they were not detected in cryopreserved hepatocytes. These data support ring opening of the furan via a reactive gamma-keto-alpha,beta-unsaturated aldehyde intermediate. In the presence of UDP-glucuronic acid, prazosin underwent conjugation to form an N-glucuronide not reported previously. Our in vitro investigations have revealed additional metabolic transformations of prazosin and have shown the potential of prazosin to undergo bioactivation through metabolism of the furan ring to a reactive intermediate.
Asunto(s)
Antagonistas Adrenérgicos alfa/metabolismo , Antihipertensivos/metabolismo , Prazosina/metabolismo , Vasodilatadores/metabolismo , Animales , Biotransformación , Células Cultivadas , Cromatografía Líquida de Alta Presión , Criopreservación , Perros , Femenino , Glutatión/metabolismo , Hepatocitos/metabolismo , Humanos , Masculino , Microsomas Hepáticos/metabolismo , NADP/metabolismo , Ratas , Ratas Sprague-Dawley , Espectrometría de Masas en Tándem , Uridina Difosfato Ácido Glucurónico/metabolismoRESUMEN
Following the recent withdrawal of several prominent drugs from US and European markets because of detrimental drug-drug interactions, metabolic drug interactions have received considerable attention in the pharmaceutical industry. In turn, the question of drug safety has received significant legal, regulatory and commercial emphasis, bringing this issue to the forefront of both industry and government drug agendas. The value of predicting the drug interactions of compounds as early as possible in the drug discovery process for all therapeutic areas cannot be underestimated. From 1964 to 1999, approximately 8% of the drugs approved by the FDA were later withdrawn from the US market. Pharmaceutical companies are facing increasing pressure to prove the long-term safety of their products, and this is complicated by the fact that animal models are not perfectly predictive of human responses, and may provide contradictory information. The failure to address safety concerns successfully during the drug optimization process may result in companies withdrawing any approved drugs from the market; drug safety issues not only present human health consequences, but also have a negative economic and public relations impact on the pharmaceutical industry. This paper discusses the significance of drug interactions, and addresses strategies to evaluate the potential of a drug candidate for drug interactions.
Asunto(s)
Interacciones Farmacológicas , Animales , Bebidas , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática , Interacciones Alimento-Droga , HumanosRESUMEN
A P450 catalyzed N-para-hydroxy metabolite was suggested to be a prerequisite for N-dephenylation occurrence. Although two mechanisms have been proposed to describe this process as a consequence of either a chemical degradation or P450 lead epoxidation of the hydroxy metabolite, direct evidence has not been demonstrated. In this study, we started with a novel technique using a dipeptide, Lys-Phe, to trap the byproduct of N-dephenylation, a quinone-like compound, forming a peptide adduct to facilitate LC/MS characterization. N-dephenylation via chemical degradation was assessed by LC/MS characterization of the resulting (Lys-Phe)(2)-quinone from 4-hydroxyphenyl-2-naphthylamine following interaction with Lys-Phe in pH 7.4 buffer. N-dephenylation mediated by P450 catalysis proposed was investigated in N-para-hydroxy benzodioxane derivative incubated with mouse liver microsomes in the presence of Lys-Phe in 50/50 H(2)(16)O/H(2)(18)O. LC/MS demonstrated that only one of two hydroxy oxygens in the byproduct was exchanged with water and the MS signal intensity of the (16)O labeled peptide adduct was equal to that of (18)O labeled. These observations suggested us that the origin of the oxygen in the byproduct was from water only, not from O(2). Therefore, it appears that N-dephenylation occurs via a stepwise process, namely the substrate is initially metabolized to a N-para-hydroxy metabolite by P450, which was readily oxidized to a quinone imine/iminium chemically or enzymatically, then hydrolyzed resulting in N-dephenylation. However, in our studies, the proposed P450 mechanism involving epoxidation of a N-para-hydroxy metabolite was disproved.
Asunto(s)
2-Naftilamina/análogos & derivados , Benzoquinonas/metabolismo , Microsomas Hepáticos/metabolismo , 2-Naftilamina/química , 2-Naftilamina/metabolismo , Animales , Benzoquinonas/química , Cromatografía Liquida/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Dioxanos/química , Dioxanos/metabolismo , Dipéptidos/química , Dipéptidos/metabolismo , Espectroscopía de Resonancia por Spin del Electrón/métodos , Indicadores y Reactivos/química , Indicadores y Reactivos/metabolismo , Lisina/química , Lisina/metabolismo , Espectrometría de Masas/métodos , Ratones , Microsomas Hepáticos/química , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Oxígeno , Fenilalanina/química , Fenilalanina/metabolismoRESUMEN
Absorption, distribution, metabolism, excretion and toxicology (ADMET) studies are widely used in drug discovery and development to help obtain the optimal balance of properties necessary to convert lead compounds into drugs that are safe and effective for human use. Drug discovery efforts have been aimed at identifying and addressing metabolism issues at the earliest possible stage, by developing and applying innovative liquid chromatography-mass spectrometry (LC-MS)-based techniques and instrumentation, which are both faster and more accurate than prior techniques. Such new approaches are demonstrating considerable potential to improve the overall safety profile of drug candidates throughout the drug discovery and development process. These emerging techniques streamline and accelerate the process by eliminating potentially harmful candidates earlier and improving the safety of new drugs. In the area of drug metabolism, for example, revolutionary changes have been achieved by the combination of LC-MS with innovative instrumentation such as triple quadrupoles, ion traps and time-of-flight mass spectrometry. In turn, most ADMET studies have come to rely on LC-MS for the analysis of an ever-increasing workload of potential candidates. This article provides a discussion on the importance of LC-MS in supporting drug metabolism studies, and highlights the relative merits of current applications for LC-MS in drug metabolism testing and analysis. These applications include in vitro and in vivo testing, pharmacokinetic profiling, chiral separations, stable isotope labeling, metabolic activation testing, metabolite characterization and radiolabeled-drug testing.
Asunto(s)
Cromatografía Liquida , Espectrometría de Masas , Preparaciones Farmacéuticas/metabolismo , Animales , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Humanos , Preparaciones Farmacéuticas/análisis , Farmacocinética , Trazadores Radiactivos , EstereoisomerismoRESUMEN
A novel technique to study the reactivity of acyl glucuronide metabolites to protein has been developed and is described herein. Considered here are acyl glucuronide metabolites, which have undergone the rearrangement of the glucuronic acid moiety at physiological temperature and pH. The investigation of the reactivity of these electrophilic metabolites was carried out by measuring the rate of reaction of rearranged AG metabolites in forming the corresponding acyl glucuronide-peptide adduct in the presence of Lys-Phe. This differs from the parallel technique used in forming AG adducts of proteins that have been previously reported. In the study described here, the Schiff base adduct, diclofenac acyl glucuronide-Lys-Phe product, was generated and structurally elucidated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. The product structure was proved to be a Schiff base adduct by chemical derivatization by nucleophilic addition of HCN and chemical reduction with NaCNBH(3), followed by LC/MS/MS analysis. It is proposed here that the degree of reactivity of acyl glucuronides as measured by covalent binding to protein is proportional to the amount of its peptide adduct generated with the peptide technique described. The application of this technique to the assessment of the degree of reactivity of acyl glucuronide metabolites was validated by developing a reactivity rank of seven carboxylic acid-containing drugs. Consistency was achieved between the ranking of reactivity in the peptide technique for these seven compounds and the rankings found in the literature. In addition, a correlation (R(2) = 0.95) was revealed between the formation of a peptide adduct and the rearrangement rate of the primary acyl glucuronide of seven tested compounds. A structure effect on the degree of reactivity has demonstrated the rate order: acetic acid > propionic acid > benzoic acid derivatives. A rational explanation of this order was proposed, based on the inherent electronic and steric properties of each specific aglycone. In addition, adaptation of this technique to automation in order to more rapidly assess the ranking of reactivity of acyl glucuronide covalent binding to proteins by new chemical entities is proposed.
Asunto(s)
Ácidos Carboxílicos/química , Cromatografía Liquida/métodos , Glucurónidos/química , Espectrometría de Masas/métodos , Péptidos/química , Bases de Schiff/química , Acilación , Aductos de ADN/química , Lisina/química , Fenilalanina/química , Valor Predictivo de las PruebasRESUMEN
Troglitazone (TGZ), the first glitazone used for the treatment of type II diabetes mellitus and removed from the market for liver toxicity, was shown to bind covalently to microsomal protein and glutathione (GSH) following activation by cytochrome P450 (P450). The covalent binding of (14)C-TGZ in dexamethasone-induced rat liver microsomes was NADPH-dependent and required the active form of P450; it was completely inhibited by ketoconazole (10 microM) and GSH (4 mM). The covalent binding in P450 3A4 Supersomes (9.2 nmol of TGZ Eq/nmol P450) was greater than that with P450 1A2 (0.7), 2C8 (3.7), 2C19 (1.4), 2E1 (0.6), and 2D6 (1.1) and 3A5 (3.0). The covalent binding in liver microsomes from rats pretreated with dexamethasone (5.3 nmol of TGZ Eq bound/nmol P450) was greater than that from rats pretreated with vehicle (3.5), beta-naphthoflavone (0.4), phenobarbital (1.1), or pyridine (2.5). A TGZ-GSH adduct was detected by liquid chromatography-tandem mass spectrometry and radioactivity detection with a deprotonated quasi-molecular ion [M-H](-) at m/z 745, with fragment ions at m/z 438 (deprotonated TGZ moiety), and at m/z 306 (deprotonated GSH moiety). The TGZ-GSH adduct was determined to be 5-glutathionyl-5-[4-(6-hydroxy-2,5,7,8-tetramethylchroman-2-ylmethoxy)benzyl]-thiazolidine-2,4-dione based on collision-induced dissociation fragmentation, and one- and two-dimensional NMR analysis of the isolated adduct. The synthetic 5-hydroxy TGZ and the benzylidene derivative of TGZ did not react with GSH or GSH ethyl ester. The mechanisms for metabolic activation of TGZ may involve an ultimate reactive sulfonium ion which could be formed from an initial sulfoxide followed by a formal Pummerer rearrangement, or a C5 thiazolidinedione radical or a sulfur cation radical.
Asunto(s)
Cromanos/farmacocinética , Hipoglucemiantes/farmacocinética , Microsomas Hepáticos/metabolismo , Tiazolidinedionas/farmacocinética , Animales , Biotransformación , Radioisótopos de Carbono , Células Cultivadas , Cromanos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Dexametasona/farmacología , Glutatión/química , Glutatión/metabolismo , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Hipoglucemiantes/metabolismo , Isoenzimas/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Microsomas Hepáticos/enzimología , Unión Proteica , Ratas , Ratas Endogámicas F344 , Tiazolidinedionas/metabolismo , TroglitazonaRESUMEN
Structural information on metabolites can be a considerable asset for enhancing and streamlining the process of developing new drug candidates. Modern approaches that generate and use metabolite structural information can accelerate the drug discovery and development process by eliminating potentially harmful candidates earlier in the process and improving the safety of new drugs. This review examines the relative merits of current and potential strategies for dealing with metabolite characterization.
Asunto(s)
Diseño de Fármacos , Preparaciones Farmacéuticas/metabolismo , Farmacología Clínica , Animales , Biotransformación , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Humanos , Preparaciones Farmacéuticas/químicaRESUMEN
Troglitazone (TGZ) was the first glitazone used for the treatment of type II diabetes mellitus. TGZ undergoes an oxidative chroman ring-opening reaction to form a quinone product. Recently, cytochrome P450 (P450) was shown to be able to catalyze the formation of TGZ quinone. TGZ quinone was the major metabolite formed by dexamethasone-induced rat liver microsomes or myeloperoxidase (MPO) incubated with TGZ. The ultimate source for the quinone carbonyl oxygen atom of TGZ quinone was investigated using (18)O water in both enzyme reaction systems followed by liquid chromatography/tandem mass spectometry analysis of the TGZ quinone product. The resultant TGZ quinone formed by either liver microsomes or MPO contained a single atom of (18)O. The (18)O atom was determined to be the quinone carbonyl oxygen by collision-induced dissociation fragmentation of the (18)O-labeled TGZ quinone. The formation of TGZ quinone was inhibited approximately 90% by coincubation with ascorbic acid or cysteine in the MPO reaction system but only 10 to 20% in liver microsomes, which might reflect the difference in the mechanism by which TGZ quinone is formed by P450 and peroxidase. These results suggest that P450 catalyze an atypical reaction to form TGZ quinone, involving the incorporation of an oxygen from water into the quinone carbonyl position.