RESUMO
Dysfunction of copper homeostasis can lead to a host of disorders, which might be toxic sometimes. 4-Methoxy-5-hydroxy-canthin-6-one (CAN) is one of the major constituents from Picrasma quassioides and responsible for its therapeutic effects. In this work, we evaluated the toxic effect of CAN (7.5µM) on zebrafish embryos. CAN treatment decreased survival, delayed hatching time and induced malformations (loss of pigmentation, pericardial edema, as well as hematologic and neurologic abnormalities). Besides, exogenous copper supplementation rescued the pigmentation and cardiovascular defects in CAN-treated embryos. Further spectroscopic studies revealed a copper-chelating activity of CAN. Then its regulation on the expressions of copper homeostasis related genes also be analyzed. In addition, CAN lowered the total activity of SOD, elevated the ROS production and altered the oxidative related genes transcriptions, which led to oxidative stress. In conclusion, we demonstrated that CAN (7.5µM) might exert its toxic effects in zebrafish embryos by causing copper dyshomeostasis and oxidative stress. It will give insight into the risk assessment and prevention of CAN-mediated toxicity.
Assuntos
Cobre/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Indóis/toxicidade , Naftiridinas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Peixe-Zebra/embriologia , Animais , Dose Letal Mediana , Estrutura MolecularRESUMO
Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.
Assuntos
Naftiridinas/metabolismo , Piridazinas/metabolismo , Tiazóis/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Glutationa/química , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Microssomos Hepáticos/metabolismo , Conformação Molecular , Naftiridinas/química , Naftiridinas/farmacocinética , Naftiridinas/toxicidade , Ligação Proteica , Piridazinas/química , Piridazinas/farmacocinética , Piridazinas/toxicidade , Ratos , Fatores de Risco , Espectrometria de Massas por Ionização por Electrospray , Tiazóis/química , Tiazóis/toxicidadeRESUMO
The concentrations of dopamine (DA), serotonin (5HT) and their metabolites were quantified in 5 brain areas of rats exposed to saline, cocaine (15 mg/kg b.i.d.), amitriptyline (10 mg/kg), or amfonelic acid (AFA, 1.5 mg/kg) throughout gestation. Male pups from 3 similarly treated dams were fostered to 2 surrogate dams. The process of breeding and rearing was repeated 4 times with new dams to build the groups to 4-12, since only one pup per litter was used for any one measurement. AFA was used to mimic the dopamine (DA) uptake blockade and stimulant properties of cocaine and amitriptyline was used to mimic the other pharmacological effects of cocaine. At postnatal days (PND) 30, 60, and 180, one pup per litter was removed for HPLC analysis of monoamines. A second pup received 0.3 mg/kg haloperidol, catalepsy assessed after 1 hr, and the brain used for analysis. The cataleptic response to haloperidol was unaffected by any prenatal treatment. The striatum from PND 30 cocaine rats had decreased levels of DA without a decrease in DA metabolites. At PND 60 in cocaine exposed rats, DA and DOPAC concentrations were increased, and 5HT levels were decreased in the striatum. The amitriptyline-exposed group exhibited decreased 5HT and 5-HIAA levels in the striatum. The hypothalamus of the cocaine group had lower levels of 5-HIAA, and other brain areas had a trend for lower levels of 5HT and 5-HIAA. At PND 180, DOPAC was increased in the striatum and prefrontal cortex of the cocaine group. Haloperidol-induced altered monoamine metabolism was unaffected by any prenatal treatment at any age. These data suggest that age-related changes in the DA and 5HT neurotransmission systems occur in rats exposed prenatally to cocaine. However, the ability of the dopaminergic system to respond to a challenge by a DA receptor blocker is unaltered by these in utero treatments.
Assuntos
Envelhecimento/metabolismo , Amitriptilina/toxicidade , Encéfalo/metabolismo , Cocaína/toxicidade , Dopamina/metabolismo , Naftiridinas/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Serotonina/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Catalepsia/fisiopatologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Feminino , Haloperidol/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Ácido Nalidíxico/análogos & derivados , Especificidade de Órgãos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Gravidez , Ratos , Ratos Sprague-Dawley , Valores de ReferênciaRESUMO
Calycanthine, the principal alkaloid of the order Calycanthaceae, has been isolated from a species of the genus Psychotria (Rubiaceae) occurring in some Pacific islands. The drug is considered as a very powerful convulsant poison but very little is known about its mechanism of action. The symptoms observed were similar to those of some neuropoisons such as strychnine. The properties of this alkaloid have been investigated on the genesis, conduction, and transmission of the nerve impulse, using giant axons of the cockroach (Periplaneta americana). Calycanthine hydrochloride (10(-5) M), which did not alter nervous conduction in pre- and post-synaptic fibers, significantly reduced the efficacy of the synaptic transmission.
Assuntos
Alcaloides/toxicidade , Sistema Nervoso Central/efeitos dos fármacos , Convulsivantes/toxicidade , Naftiridinas/toxicidade , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Alcaloides/isolamento & purificação , Animais , Axônios/efeitos dos fármacos , Convulsivantes/isolamento & purificação , Masculino , Periplaneta , Plantas/análise , Sinapses/efeitos dos fármacosAssuntos
Naftiridinas/síntese química , Triazóis/síntese química , Animais , Anti-Infecciosos , Anti-Inflamatórios não Esteroides , Antifúngicos , Antiprotozoários , Comportamento Animal/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Camundongos , Naftiridinas/farmacologia , Naftiridinas/toxicidade , Ratos , Relação Estrutura-Atividade , Triazóis/farmacologia , Triazóis/toxicidadeRESUMO
DL50 for 10-gamma-dimethylaminopropyl-7-chlorobenzo(b)-(1-8)-naphtyridone-5 hydrochloride (IF C-45) determined on several species of animals at different ways of administration, had values similar to those of imipramine and amitryptyline. IF C-45 a-cumulates in 34-4% of A-DL50. The evaluation of chronic toxicity was performed on rats, giving IF C-45 daily for 3 months the doses of 30, 25 and 12-5 mg/kg. The results of hematological, biochemical and histological estimations did not show in these animals any marked toxic effects.