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1.
Biomed Chromatogr ; 33(1): e4377, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30187929

RESUMO

In this study, a sensitive hydrophilic interaction liquid chromatography (HILIC) method was developed to determine pseudojervine (PJV), veratrosine (VTS), jervine (JV), veratramine (VTM), veramarine (VA) and veratroylzygadenine (VTG) in rat plasma. Separations were carried out using LC-MS/MS with a Chrom Matrix HP amide column (5 m, 10 cm × 3.0 mm i.d.). The mobile phases were (A) 0.01 mm formic acid and (B) acetonitrile. Good linearity was found for all analytes (R2  > 0.995) in the concentration range from 5 to 1000 µg/L with LLOQ at 5 µg/L for VTM and VTS; and from 1 to 1000 µg/L with LLOQ at 1 µg/L for PJV, JV, VA and VTG. Accuracy of the assay varied from 90.5 to 108.1%. The extraction recovery and matrix effect of six analytes ranged from 72.2 to 95.5% and from 79.2 to 98.4%. According to the stability test, six analytes in rat plasma were stable during the analysis process. On the basis of validation of the assay, the pharmacokinetics of the six steroid alkaloids were investigated after oral administration of Lilu extracts to rats.


Assuntos
Cromatografia Líquida/métodos , Alcaloides de Veratrum/sangue , Alcaloides de Veratrum/farmacocinética , Animais , Estabilidade de Medicamentos , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray/métodos , Esteroides/sangue , Esteroides/química , Esteroides/farmacocinética , Alcaloides de Veratrum/química
2.
Biopharm Drug Dispos ; 36(5): 308-24, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25765359

RESUMO

Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has rarely been explored. The objective of the current study was to provide a comprehensive investigation of its metabolic pathways. The in vitro metabolic profiles of veratramine were evaluated by incubating it with liver microsomes and cytosols. The in vivo metabolic profiles in plasma, bile, urine and feces were monitored by UPLC-MS/MS after oral (20 mg/kg) and i.v. (50 µg/kg) administration in rats. Meanwhile, related P450s inhibitors and recombinant P450s and SULTs were used to identify the isozymes responsible for its metabolism. Eleven metabolites of veratramine, including seven hydroxylated, two sulfated and two glucuronidated metabolites, were characterized. Unlike most alkaloids, the major reactive sites of veratramine were on ring A and B instead of on the amine moiety. CYP2D6 was the major isozyme mediating hydroxylation, and substrate inhibition was observed with a Vmax , Ki and Clint of 2.05 ± 0.53 nmol/min/mg, 33.08 ± 10.13 µ m and 13.58 ± 1.27 µL/min/mg. SULT2A1, with Km , Vmax and Clint values of 19.37 ± 0.87 µ m, 1.51 ± 0.02 nmol/min/mg and 78.19 ± 8.57 µL/min/mg, was identified as the major isozyme contributing to its sulfation. In conclusion, CYP2D6 and SULT2A1 mediating hydroxylation and sulfation were identified as the major biotransformation for veratramine.


Assuntos
Arilsulfotransferase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Alcaloides de Veratrum/farmacocinética , Animais , Bile/química , Citosol/metabolismo , Fezes/química , Humanos , Isoenzimas/metabolismo , Fígado/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Ratos Sprague-Dawley , Alcaloides de Veratrum/sangue , Alcaloides de Veratrum/farmacologia , Alcaloides de Veratrum/urina
3.
J Pharm Biomed Anal ; 66: 282-6, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22516681

RESUMO

Cyclopamine (1) was isolated from the plant Veratrum californicum Durand (Liliacea) and identified as the teratogen responsible for severe craniofacial birth defects including cyclops in the offspring of sheep grazing on mountain ranges in central Idaho. More recently, cyclopamine (1) was found to inhibit the hedgehog (Hh) signaling pathway which plays a critical role in embryonic development and is implicated in several types of cancer. Thus, cyclopamine (1) and cyclopamine derivatives have been targeted as potential pharmaceutical treatments for certain cancers and other diseases associated with the Hh signaling pathway. A monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay was developed to detect and measure cyclopamine (1) and cyclopamine derivatives in biological samples. The limits of detection of the assay for cyclopamine (1), 3-keto-N-aminoethyl aminocaproyl digyrocinnamoyl-cyclopamine (8), and N-(4-l-rhamnopyranosyl-1H-1,2,3-triazol-1-yl)-methylcyclopamine (11) were 2.9 pg, 0.41 pg and 2.6 pg, respectively. This assay was also found to be useful for the detection and measurement of cyclopamine (1) in sera from mice that had been dosed with cyclopamine (1). The simple ELISA method described herein demonstrates the potential of using these techniques for the rapid screening of biological samples for the presence and levels of cyclopamine (1) and other cyclopamine derivatives that are Hh inhibitors with anticancer potential.


Assuntos
Cinamatos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Alcaloides de Veratrum/sangue , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos Fitogênicos/sangue , Proteínas Hedgehog/antagonistas & inibidores , Limite de Detecção , Camundongos , Camundongos Endogâmicos C57BL , Ovinos , Transdução de Sinais/efeitos dos fármacos
4.
Chin J Cancer ; 30(7): 472-81, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21718593

RESUMO

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Cyclopamine (Cyc), an alkaloid from the Veratrum plant, is a specific natural product inhibitor of the Hh pathway that acts by targeting smoothened (SMO) protein. However, its poor solubility, acid sensitivity, and weak potency relative to other Hh antagonists prevent the clinical development of Cyc as a therapeutic agent. Here, we report properties of cyclopamine tartrate salt (CycT) and its activities in Hh signaling-mediated cancer in vitro and in vivo. Unlike Cyc, CycT is water soluble (5-10 mg/mL). The median lethal dose (LD50) of CycT was 62.5 mg/kg body weight compared to 43.5 mg/kg for Cyc, and the plasma half-life (T1/2) of CycT was not significantly different from that of Cyc. We showed that CycT had a higher inhibitory activity for Hh signaling-dependent motor neuron differentiation than did Cyc (IC50 = 50 nmol/L for CycT vs. 300 nmol/L for Cyc). We also tested the antitumor effectiveness of these Hh inhibitors using two mouse models of basal cell carcinomas (K14cre:Ptch1(neo/neo) and K14cre:SmoM2(YFP)). After topical application of CycT or Cyc daily for 21 days, we found that all CycT-treated mice had tumor shrinkage and decreased expression of Hh target genes. Taken together, we found that CycT is an effective inhibitor of Hh signaling-mediated carcinogenesis.


Assuntos
Carcinoma Basocelular/patologia , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos , Alcaloides de Veratrum/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Proteínas Hedgehog/antagonistas & inibidores , Camundongos , Neurônios Motores/citologia , Plantas Medicinais/química , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened , Solubilidade , Tartaratos/sangue , Tartaratos/farmacologia , Veratrum/química , Alcaloides de Veratrum/sangue , Alcaloides de Veratrum/isolamento & purificação
5.
Toxicol Sci ; 104(1): 189-97, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18411234

RESUMO

The Hedgehog (Hh) signaling pathway is an essential regulator of embryonic development and appears to play important roles in postnatal repair and cancer progression and metastasis. The teratogenic Veratrum alkaloid cyclopamine is a potent Hh antagonist and is used experimentally both in vitro and in vivo to investigate the role of Hh signaling in diverse biological processes. Here, we set out to establish an administration regimen for cyclopamine-induced teratogenicity in the mouse. The dysmorphogenic concentration of cyclopamine was determined in vitro via mouse whole-embryo culture assays to be 2.0 microM. We administered cyclopamine to female C57BL/6J mice at varied doses by oral gavage, ip injection, or osmotic pump infusion and assessed toxicity and pharmacokinetic (PK) models. Bolus administration was limited by toxicity and rapid clearance. In vivo cyclopamine infusion at 160 mg/kg/day yielded a dam serum steady-state concentration of approximately 2 microM with a corresponding amniotic fluid concentration of approximately 1.5 microM. Gross facial defects were induced in 30% of cyclopamine-exposed litters, with affected embryos exhibiting cleft lip and palate. This is the first report describing the PKs and teratogenic potential of cyclopamine in the mouse and demonstrates that transient Hh signaling inhibition induces facial clefting anomalies in the mouse that mimic common human birth defects.


Assuntos
Fenda Labial/induzido quimicamente , Fissura Palatina/induzido quimicamente , Proteínas Hedgehog/antagonistas & inibidores , Teratogênios/toxicidade , Alcaloides de Veratrum/toxicidade , Líquido Amniótico/química , Animais , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Transdução de Sinais , Teratogênios/farmacocinética , Alcaloides de Veratrum/administração & dosagem , Alcaloides de Veratrum/sangue , Alcaloides de Veratrum/farmacocinética
6.
J Agric Food Chem ; 51(3): 582-6, 2003 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-12537426

RESUMO

Veratrum californicum was responsible for large losses of sheep grazing high mountain ranges in central Idaho in the 1950s. Veratrum induces various birth defects including the cyclopic-type craniofacial defect (monkey-faced lambs) that is specifically induced in lambs after pregnant ewes grazed the plant on the 14th day of gestation. The steroidal alkaloids cyclopamine (1) and jervine (2) were isolated from Veratrum and shown to be primarily responsible for the malformations. Cyclopamine (1) and jervine (2) are potent teratogens that inhibit Sonic hedgehog (Shh) signaling during gastrulation-stage embryonic development, producing cyclopia and holoprosencephaly. Although losses to the sheep industry from Veratrum are now relatively infrequent, occasional incidents of toxicoses and craniofacial malformations are still reported in sheep and other species. However, the benefits to biomedical research using cyclopamine (1) as a tool to study human diseases have greatly expanded. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) to detect and measure cyclopamine (1) and jervine (2) was developed using polyclonal antibodies produced in ewes. The limits of detection of the assay were 90.0 and 22.7 pg for cyclopamine (1) and jervine (2), respectively. This assay was used for the detection and measurement of cyclopamine (1) spiked into sheep blood. The simple extraction-ELISA methods developed in this study demonstrate the potential of using these techniques for the rapid screening of biological samples to detect the presence and concentration of cyclopamine (1) and jervine (2) and will be beneficial to pharmacological studies and livestock diagnostics.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Teratogênios/análise , Alcaloides de Veratrum/análise , Veratrum/química , Anormalidades Induzidas por Medicamentos/veterinária , Animais , Especificidade de Anticorpos , Soros Imunes , Ovinos/sangue , Doenças dos Ovinos/induzido quimicamente , Alcaloides de Veratrum/sangue , Alcaloides de Veratrum/envenenamento
7.
Hum Toxicol ; 2(2): 321-5, 1983 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-6862477

RESUMO

1 Nine cases of accidental poisoning of children with sneezing powder are reported. Symptoms, besides sneezing, included gastrointestinal disturbances and syncope, whilst examination demonstrated bradycardia and hypotension. 2 The powder, as supplied, carried no information on its constituents but Veratrum alkaloids were identified on analysis. The signs and symptoms observed were compatible with poisoning from these compounds. 3 As a result of these observations, it was possible to trace the manufacturers and a change was made to a safer formulation. This example emphasises the value of toxic vigilance by Poison Control Centres.


Assuntos
Jogos e Brinquedos , Espirro , Alcaloides de Veratrum/envenenamento , Adolescente , Criança , Cromatografia em Camada Delgada , Feminino , Lavagem Gástrica , Humanos , Masculino , Espectrometria de Massas , Alcaloides de Veratrum/sangue
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