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1.
Arch Insect Biochem Physiol ; : e21643, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31667894

RESUMO

Mass spectrometry imaging (MSI) can visualize the composition, abundance, and spatial distribution of molecules in tissues or cells, which has been widely used in the research of life science. Insects, especially the agricultural pests, have received a great deal of interests from the scientists in biodiversity and food security. This review introduces the major characteristics of MSI, summarizes its application to the investigation of insect endogenous metabolites, exogenous metabolites, and the spatiotemporal changes of metabolites between insects and plants, and discusses its shortfalls and perspectives. The significance of these concerns is beneficial for future insect research such as physiology and metabolism.

2.
Turk J Gastroenterol ; 30(6): 532-540, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31144659

RESUMO

BACKGROUND/AIMS: Critically ill patients with cirrhosis with pneumonia are at an increased risk for mortality. Only a few accurate predictive models are existing specific to these patients. The aim of the present study was to compare the existing prognostic models and to develop an improved mortality risk model for patients with cirrhosis and pneumonia. MATERIALS AND METHODS: A total of 231 patients were enrolled in our study (70% training and 30% validation cohorts). All participants were followed up for at least 21 days. Model for End-stage Liver Disease and Pneumonia (MELD-P) was derived by the Cox proportional hazards model. The performances of prognostic scoring systems were compared by calculation of the area under the receiver operating characteristic (AUROC) curve. RESULTS: MELD-P showed better discriminative capabilities than existing scoring systems. Four clinical variables, including loge bilirubin (hazard ratio (HR) 1.29, 95% confidence interval (CI) 1.01-1.73), loge international normalized ratio (HR 3.57, 95% CI 1.30-9.78), loge pulse oxygen saturation/fraction of inspired oxygen (HR 0.38, 95% CI 0.14-0.99), and vasopressors used (HR 3.72, 95% CI 1.85-7.49), were considered as independent prognostic values associated with 21-day mortality. MELD-P had AUROC curve values of 0.78 (95% CI 0.71-0.84) in predicting in-hospital mortality, 0.78 (95% CI 0.70-0.84) at 21-day, 0.88 (95% CI 0.82-0.93) at 14-day, and 0.87 (95% CI 0.81-0.92) at 7-day. A similar result was obtained in validation cohort. CONCLUSION: MELD-P, as the first model specifically designed to evaluate the risk of mortality in critically ill patients with cirrhosis and pneumonia, performs well on the mortality assessment of short-term mortality.

3.
J Med Chem ; 62(7): 3228-3250, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30893553

RESUMO

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

4.
Eur J Gastroenterol Hepatol ; 31(7): 824-831, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30601338

RESUMO

BACKGROUND AND AIM: Critically ill patients with cirrhosis are at an increased risk of mortality. Our study aimed to externally validate the ability of the prothrombin time-international normalized ratio to albumin ratio (PTAR), an objective and simple scoring system, to predict 90-day mortality in critically ill patients with cirrhosis. PATIENTS AND METHODS: A total of 865 patients were entered into the study, and all the participants were followed up for at least 90 days. Clinical parameters on the first day of intensive care unit admission were included to compare survivors with nonsurvivors. RESULTS: After multivariable adjustment, the association between the risk of 90-day mortality and PTAR remained statistically significant with a hazard ratio of 2.71 (95% confidence interval: 1.99-3.68). The PTAR score showed good discrimination ability for predicting 90-day mortality with an area under receiver operating characteristic curve of 0.72 (95% confidence interval: 0.68-0.75). To improve its feasibility, we regrouped the PTAR scores into three levels of risk (low risk: <0.55, intermediate risk: 0.55-1.00, and high risk: ≥1.00); the 90-day mortality rates were 20.1% (74/368), 41.7% (168/403), and 73.4% (69/94), respectively. CONCLUSION: The PTAR score system is a convenient and practical tool for predicting the prognosis of critically ill patients with cirrhosis.

5.
Sci Rep ; 8(1): 14256, 2018 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250036

RESUMO

DNA barcoding, based on a fragment of cytochrome c oxidase I (COI) mtDNA, is as an effective molecular tool for identification, discovery, and biodiversity assessment for most animals. However, multiple gene markers coupled with more sophisticated analytical approaches may be necessary to clarify species boundaries in cases of cryptic diversity or morphological plasticity. Using 339 moths collected from mountains surrounding Beijing, China, we tested a pipeline consisting of two steps: (1) rapid morphospecies sorting and screening of the investigated fauna with standard COI barcoding approaches; (2) additional analyses with multiple molecular markers for those specimens whose morphospecies and COI barcode grouping were incongruent. In step 1, 124 morphospecies were delimited into 116 barcode units, with 90% of the conflicts being associated with specimens identified to the genus Hypena. In step 2, 55 individuals representing all 12 Hypena morphospecies were analysed using COI, COII, 28S, EF-1a, Wgl sequences or their combinations with the BPP (Bayesian Phylogenetics and Phylogeography) multigene species delimitation method. The multigene analyses supported the delimitation of 5 species, consistent with the COI analysis. We conclude that a two-step barcoding analysis pipeline is able to rapidly characterize insect biodiversity and help to elucidate species boundaries for taxonomic complexes without jeopardizing overall project efficiency by substantially increasing analytical costs.

6.
Acta Pharm Sin B ; 8(2): 252-260, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29719786

RESUMO

In the present study, total membrane proteins from tumor cell lines including HepG2, Hep3B2, H226, Ovcar3 and N87 were extracted and digested with γLysC and trypsin. The resulting peptide lysate were pre-fractionated and subjected to untargeted quantitative proteomics analysis using a high resolution mass spectrometer. The mass spectra were processed by the MaxQuant and the protein abundances were estimated using total peak area (TPA) method. A total of 6037 proteins were identified, and the analysis resulted in the identification of 2647 membrane proteins. Of those, tumor antigens and absorption, metabolism, disposition and elimination (ADME) proteins including UDP-glucuronosyltransferase, cytochrome P450, solute carriers and ATP-binding cassette transporters were detected and disclosed significant variations among the cell lines. The principal component analysis was performed for the cluster of cell lines. The results demonstrated that H226 is closely related with N87, while Hep3B2 aligned with HepG2. The protein cluster of Ovcar3 was apart from that of other cell lines investigated. By providing for the first time quantitative untargeted proteomics analysis, the results delineated the expression profiles of membrane proteins. These findings provided a useful resource for selecting targets of choice for anticancer therapy through advancing data obtained from preclinical tumor cell line models to clinical outcomes.

7.
J Med Chem ; 60(12): 5193-5208, 2017 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-28541707

RESUMO

PI3Kδ plays an important role controlling immune cell function and has therefore been identified as a potential target for the treatment of immunological disorders. This article highlights our work toward the identification of a potent, selective, and efficacious PI3Kδ inhibitor. Through careful SAR, the successful replacement of a polar pyrazole group by a simple chloro or trifluoromethyl group led to improved Caco-2 permeability, reduced Caco-2 efflux, reduced hERG PC activity, and increased selectivity profile while maintaining potency in the CD69 hWB assay. The optimization of the aryl substitution then identified a 4'-CN group that improved the human/rodent correlation in microsomal metabolic stability. Our lead molecule is very potent in PK/PD assays and highly efficacious in a mouse collagen-induced arthritis model.


Assuntos
Artrite Experimental/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Relação Estrutura-Atividade , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Células CACO-2/efeitos dos fármacos , Células CACO-2/imunologia , Cães , Canal de Potássio ERG1/metabolismo , Inibidores Enzimáticos/química , Feminino , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Coelhos
8.
Bioorg Med Chem Lett ; 27(13): 2849-2853, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28209465

RESUMO

Selective PI3Kδ inhibitors have recently been hypothesized to be appropriate immunosuppressive agents for the treatment of immunological disorders such as rheumatoid arthritis. However, few reports have highlighted molecules that are highly selective for PI3Kδ over the other PI3K isoforms. In this letter, isoform and kinome selective PI3Kδ inhibitors are presented. The Structural Activity Relationship leading to such molecules is outlined.


Assuntos
Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
9.
Springerplus ; 5(1): 1180, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27512639

RESUMO

BACKGROUND: Local anesthetic infiltration has been used to manage postoperative pain in various surgeries. The present study was aimed to investigate the effect of skin infiltration with 0.5 % ropivacaine on postoperative pain in patients undergoing craniotomy. METHODS: One hundred and six patients with ASA I/II scheduled to undergo elective craniotomy were enrolled during March to November in 2015 in this prospective, randomized, placebo-controlled, double-blind study. After the anesthesia induction, skin along the incision was infiltrated with 0.5 % ropicavaine (group R, n = 53) or 0.9 % normal saline (group C, n = 53), respectively. Morphine was used as rescue analgesic postoperatively. Morphine consumption during the first 24 postoperative hours was recorded as the primary outcome, and the time to first rescue requirement was also recorded. Pain was assessed at 2, 4, 8, 24 h, 7 days, 3 months after surgery by visual analog scale (VAS). Heart rate and mean arterial pressure were recorded before anesthesia induction (T1), after anesthesia induction (T2), after scalp infiltration (T3), during skull drilling (T4), mater cutting (T5) and skin closure (T6). RESULTS: Morphine consumption during the first 24 postoperative hours was significantly higher in group C than in group R (13.36 [6.5, 20] vs. 6.3 [0, 10] mg, P < 0.05). The first time of patients needed rescue analgesic was prolonged in group R as compared with group C (6.16 [3.4, 8.0] vs. 3.87 [2.3, 4] h, P < 0.05). Postoperative VAS and hemodynamic signs during the first 24 h showed no significant difference in two groups. The incidence of persistent pain on 7 days and 3 months postoperatively had no significant differences between two groups. Besides one patient (2 %) enduring moderate pain (VAS 4-7) in group C, the number of patients suffering from mild pain (VAS 1-3) was 17 (33.3 %) in group R and 17 (34 %) in group C 3 months after surgery. CONCLUSION: The results suggest 0.5 % ropivacaine scalp infiltration before skin incision has favorable analgesic effect in reducing morphine consumption and prolong the time of first rescue analgesic requirement after surgery. Trial registration Chinese Clinical Trial Registry (http://www.chictr.org.cn/) registration number: ChiCTR-IPR-14005717.

10.
Clin Vaccine Immunol ; 22(11): 1166-75, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26376928

RESUMO

Replication-competent adenovirus (rcAd)-based vaccine vectors may theoretically provide immunological advantages over replication-incompetent Ad vectors, but they also raise additional potential clinical and regulatory issues. We produced replication-competent Ad serotype 26 (rcAd26) vectors by adding the E1 region back into a replication-incompetent Ad26 vector backbone with the E3 or E3/E4 regions deleted. We assessed the effect of vectorization on the replicative capacity of the rcAd26 vaccines. Attenuation occurred in a stepwise fashion, with E3 deletion, E4 deletion, and human immunodeficiency virus type 1 (HIV-1) envelope (Env) gene insertion all contributing to reduced replicative capacity compared to that with the wild-type Ad26 vector. The rcAd26 vector with E3 and E4 deleted and containing the Env transgene exhibited 2.7- to 4.4-log-lower replicative capacity than that of the wild-type Ad26 in vitro. This rcAd26 vector is currently being evaluated in a phase 1 clinical trial. Attenuation as a result of vectorization and transgene insertion has implications for the clinical development of replication-competent vaccine vectors.


Assuntos
Vacinas contra Adenovirus/genética , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Vetores Genéticos , Replicação Viral , Vacinas contra Adenovirus/imunologia , Ensaios Clínicos Fase I como Assunto , Expressão Gênica , Humanos , Mutagênese Insercional , Sorogrupo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
11.
Bioorg Med Chem Lett ; 24(9): 2206-11, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24685542

RESUMO

Investigation of various heterocyclic core isosteres of imidazopyrazines 1 & 2 yielded purine derivatives 3 & 8 as potent and selective BTK inhibitors. Subsequent SAR studies of the purine series led to the discovery of 20 as a leading compound. Compound 20 is very selective when screened against a panel of 400 kinases and is a potent inhibitor in cellular assays of human B cell function including B-Cell proliferation and CD86 cell surface expression and exhibited in vivo efficacy in a mouse PCA model. Its X-ray co-crystal structure with BTK shows that the high selectivity is gained from filling a BTK specific lipophilic pocket. However, physical and ADME properties leading to low oral exposure hindered further development.


Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Purinas/química , Purinas/farmacologia , Tirosina Quinase da Agamaglobulinemia , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/enzimologia , Linfócitos B/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Ratos
12.
Int J Clin Exp Med ; 7(12): 5943-6, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25664139

RESUMO

Hyperthermia is relatively common in inpatients, but hyperthermia occurring in the immediate postoperative period after undergoing neurosurgery has some unique characteristics. This case report concerns a patient who developed immediate postoperative hyperthermia up to 39.3°C (the axillary temperature) in the post-anesthesia care unit (PACU) after resection of a seminoma from the thalamus and third ventricle. Having been re-intubated and mechanically ventilated, the elevated temperature was treated on the PACU by cooling the skin with ice and antipyretic drugs. Within 2 hours after the surgery, the patient's body temperature had fallen to 37.8°C and vital signs were stable. The patient was then transferred to the neurology intensive care unit for further management. The patient was discharged 70 days after surgery with normal body temperature. During excision of a space-occupying lesion in the thalamus or hypothalamus, clinicians must be mindful of the possibility of hyperthermia and administer appropriate treatments immediately.

13.
J Mass Spectrom ; 48(3): 413-22, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23494800

RESUMO

Ticlopidine, an antiplatelet drug, undergoes extensive oxidative metabolism to form S-oxide, N-oxide, hydroxylated and dealkylated metabolites. However, metabolism of ticlopidine via conjugation has not been thoroughly investigated. In this study, multiple data acquisition and processing tools were applied to the detection and characterization of ticlopidine conjugates in rat bile. Accurate full-scan mass spectrometry (MS) and collision-induced dissociation (CID) MS/MS data sets were recorded using isotope pattern-dependent acquisition on an LTQ/Orbitrap system. In addition, mass spectral data from online H/D exchanging and high collision energy dissociation (HCD) were recorded. Data processes were carried out using extracted ion chromatography (EIC), mass defect filter (MDF) and isotope pattern filter (IPF). The total ion chromatogram displayed a few major conjugated metabolites and many endogenous components. Profiles from EIC and IPF processes exhibited multiple conjugates with no or minimal false positives. However, ticlopidine conjugates that were not predictable or lost a chorine atom were not found by EIC or IPF, respectively. MDF was able to detect almost all of ticlopidine conjugates although it led to a few more false positives. In addition to CID spectra, data from HCD, H/D exchanging experiments and isotope pattern simulation facilitated structural characterization of unknown conjugates. Consequently, 20 significant ticlopidine conjugates, including glucuronide, glutathione, cysteinylglycine, cysteine and N-acetylcysteine conjugates, were identified in rat bile, a majority of which are associated with bioactivation and not previously reported. This study demonstrates the utility and limitation of various high-resolution MS-based data acquisition and processing techniques in detection and characterization of conjugated metabolites.


Assuntos
Bile/metabolismo , Inibidores da Agregação de Plaquetas/análise , Inibidores da Agregação de Plaquetas/metabolismo , Ticlopidina/análise , Ticlopidina/metabolismo , Animais , Bile/química , Cromatografia Líquida de Alta Pressão , Masculino , Ratos , Ratos Wistar , Espectrometria de Massas em Tandem
14.
Anal Chem ; 83(23): 8937-44, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21970614

RESUMO

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 µg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed.


Assuntos
Muramidase/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Animais , Galinhas , Cromatografia Líquida de Alta Pressão , Humanos , Muramidase/sangue , Peptídeos/análise , Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Extração em Fase Sólida
15.
J Pharm Biomed Anal ; 54(5): 979-86, 2011 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-21168298

RESUMO

Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-µL fractions (within 4.1% CV) and 250-µL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Preparações Farmacêuticas/metabolismo , Radioisótopos/análise , Contagem de Cintilação/métodos , Animais , Bile/metabolismo , Buspirona/metabolismo , Buspirona/urina , Radioisótopos de Carbono/análise , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Cães , Avaliação Pré-Clínica de Medicamentos/instrumentação , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Preparações Farmacêuticas/urina , Reprodutibilidade dos Testes , Contagem de Cintilação/instrumentação , Manejo de Espécimes , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos
16.
Chem Res Toxicol ; 23(5): 909-17, 2010 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-20297803

RESUMO

The bioactivation of ticlopidine, a widely used antiplatelet drug, into reactive metabolites and their subsequent covalent binding to cellular macromolecules are thought to be involved in the occurrence of idiosyncratic hepatotoxicity in patients. In the present study, GSH/stable isotope-labeled GSH was used as the trapping agent to investigate the bioactivation pathways of ticlopidine in rat liver microsomes. The samples were analyzed by high-resolution linear ion trap/Orbitrap followed by multiple mass defect filtering (MDF). In total, 17 GSH adducts were detected, and a comprehensive profile for ticlopidine bioactivation has been proposed. The results show that ticlopidine can be directly bioactivated by rat P450s, forming GSH adducts through two major bioactivation pathways, thiophene-S-oxidation and thiophene epoxidation. These adducts were also formed substantially in human liver microsomes. Moreover, ticlopidine can be metabolized via multiple pathways before giving rise to reactive intermediates. The GSH adducts derived from epoxidation of the chlorophenyl moiety of ticlopide and bioactivation of N-dealkylated metabolites are reported here for the first time. The formation of a number of ticlopidine GSH adducts from diversified metabolic pathways mediated by P450s implies a high potential for protein binding and provides a conceivable link between the high reactivity of ticlopidine after bioactivation and the ticlopidine-induced toxicity. Additionally, the current approach has the following advantages as compared to previous high-resolution LC/MS methodologies. First, novel MDF utilized doubly charged ions as filter templates to detect the GSH adducts, mainly doubly charged in the ion source, resulting in broader detection coverage. Second, multiple mass defect filter templates were for the first time applied to reveal different classes of GSH adducts. Finally, a quick check of isotopic doublets and full examination of isotope fingerprints in the accurate mass were introduced to screen out false positives and enhance the identification of low abundant GSH adducts.


Assuntos
Inibidores da Agregação de Plaquetas/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Ticlopidina/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/química , Humanos , Microssomos Hepáticos/metabolismo , Inibidores da Agregação de Plaquetas/toxicidade , Ratos , Ticlopidina/toxicidade
17.
Chem Res Toxicol ; 22(4): 690-8, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19253935

RESUMO

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.


Assuntos
Glutationa/metabolismo , Medicamentos sob Prescrição/metabolismo , Cromatografia Líquida de Alta Pressão , Compostos de Dansil/química , Glutationa/química , Humanos , Fígado/enzimologia , Fígado/metabolismo , Espectrometria de Massas , Medicamentos sob Prescrição/química , Medicamentos sob Prescrição/toxicidade
18.
Chem Res Toxicol ; 21(7): 1477-83, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18549250

RESUMO

The present study describes a novel integrated approach for rapid analysis of reactive metabolites with a linear ion trap mass spectrometer (LTQ). In this approach, an isotope pattern-dependent scanning method was applied to the data acquisition of glutathione (GSH)-trapped reactive metabolites. Recorded full-scan MS and MS/MS data sets were further processed with neutral loss filtering, product ion filtering, and extracted ion chromatographic analysis to search for protonated molecules and MS/MS spectra of GSH adducts. To evaluate the effectiveness and reliability of the approach, GSH adducts of carbamazepine, diclofenac, 4-ethylphenol, acetaminophen, p-cresol, and omeprazole were analyzed, which were formed in human liver microsome incubations fortified with a mixture of nonlabeled GSH and stable isotope-labeled GSH at a 1:0.8 ratio. Results demonstrate that the combination of the isotope pattern-dependent scanning with the postacquisition data mining was very effective in detecting low levels of GSH adducts, regardless of their fragmentation patterns. As compared to a neutral loss scanning method performed with a triple quadrupole mass spectrometer, the LTQ-based approach had several major advantages, including the superior selectivity and sensitivity in detecting different classes of GSH adducts and the higher throughput capability of the detection and MS/MS spectral acquisition of GSH adducts in a single LC/MS run. Overall, this analytical approach provides a simple and efficient means for screening for reactive metabolites using a linear ion trap LC/MS platform.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Glutationa/metabolismo , Preparações Farmacêuticas/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Acetaminofen/química , Acetaminofen/metabolismo , Carbamazepina/química , Carbamazepina/metabolismo , Cresóis/química , Cresóis/metabolismo , Diclofenaco/química , Diclofenaco/metabolismo , Glutationa/análise , Omeprazol/química , Omeprazol/metabolismo , Preparações Farmacêuticas/química , Fenóis/química , Fenóis/metabolismo , Valor Preditivo dos Testes
19.
J Pharm Biomed Anal ; 47(2): 351-9, 2008 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-18295429

RESUMO

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of indocyanine green (ICG) in dog plasma and bile. An ICG analog (IR-820) was used as an internal standard. A protein precipitation method was used with acetonitrile for plasma sample preparation, whereas bile samples were diluted with water (120-fold) prior to analysis. Using MS/MS in the multiple reaction monitoring mode, ICG and IR-820 were detected in both matrices without interference. The lower limit of quantitation for ICG in dog plasma was 3 ng/mL, with an intra- and inter-day accuracy (%Bias) and precision (%CV) of less than 15%, so it was possible to study the pharmacokinetics of ICG in bile duct-cannulated dogs and assess their liver function after surgery. The method described herein is sensitive, selective and faster than other existing methods (e.g., spectrophotometry, HPLC/UV-vis detection, or HPLC/fluorescence detection).


Assuntos
Ânions/análise , Bile/química , Cromatografia Líquida/métodos , Corantes/análise , Verde de Indocianina/análise , Espectrometria de Massas em Tandem/métodos , Animais , Ânions/sangue , Bile/metabolismo , Corantes/química , Corantes/farmacocinética , Cães , Verde de Indocianina/química , Verde de Indocianina/farmacocinética , Estrutura Molecular , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
20.
J Org Chem ; 73(3): 992-1003, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18181642

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are major environmental carcinogens produced in the combustion of fossil fuels, tobacco, and other organic matter. Current evidence indicates that PAHs are transformed enzymatically to active metabolites that react with DNA to form adducts that result in mutations. Three activation pathways have been proposed: the diol epoxide path, the radical-cation path, and the quinone path. The latter involves aldo-keto reductase mediated oxidation of PAH dihydrodiol metabolites to catechols that enter into redox cycles with quinones. This results in generation of reactive oxygen species (ROS) that attack DNA, and the PAH quinones also react with DNA to form adducts. Several strategies for synthesis of the stable adducts formed by the o-quinone metabolites of carcinogenic PAHs with 2'-deoxyribonucleosides were investigated and compared. The PAH quinones studied were benz[a]anthracene-3,4-dione and its 7-methyl- and 7,12-dimethyl- derivatives. The parent PAHs represent a range of carcinogenicity from inactive to highly potent. Two synthetic methods were devised that differ in the catalyst employed, Pd(OAc)(2) or CuI. The Pd-mediated method involved coupling a protected amino-catechol PAH derivative with a halo-2'-deoxyribonucleoside. The copper-mediated method entailed reaction of a halo-PAH catechol derivative with a 2'-deoxyribonucleoside. Adducts of benz[a]anthracene-3,4-dione (and its 7-methyl- and 7,12-dimethyl- derivatives) with 2'-deoxyadenosine and 2'-deoxyguanosine were prepared by these methods. Availability of adducts of these types through synthesis makes possible for the first time biological studies to determine the role of these adducts in tumorigenesis. The copper-mediated method offers advantages of economy, adaptability to large-scale preparation, utility for synthesis of (13)C- or (15)N-labeled analogues, and nonformation of bis-adducts as secondary products.


Assuntos
Carcinógenos/química , Desoxirribonucleosídeos/química , Hidrocarbonetos Policíclicos Aromáticos/química , Quinonas/química , Quinonas/metabolismo , Aminação , Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Catecóis/química , Cobre/química , Halogenação , Isomerismo , Estrutura Molecular , Paládio/química
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