RESUMO
Traditional solid phase extraction (SPE) suffers from a lack of specific adsorption. To overcome this problem, a combination of adsorption method and molecular imprinting technology by polydopamine modification was proposed to realize specific recognition of target compounds in SPE, which is of great significance to improve the separation efficiency of SPE. Cellulose hydrogel beads were prepared by dual cross-linking curing method and modified with polydopamine to make them hydrophilic and biocompatible. Subsequently, cellulose hydrogel-based molecularly imprinted beads (MIBs) were synthesized by surface molecular imprinting technology and used as novel column fillers in SPE to achieve efficient adsorption (34.16 mg·g-1) with specific selectivity towards camptothecin (CPT) in 120 min. The simulation and NMR analysis revealed that recognition mechanism of MIBs involved hydrogen bond interactions and Van der Waals effect. The MIBs were successful used in separating CPT from Camptotheca acuminata fruits, exhibiting impressive adsorption capacity (1.19 mg·g-1) and efficient recovery of CPT (81.54 %). Thus, an environmentally friendly column filler for SPE was developed, offering a promising avenue for utilizing cellulose-based materials in the selective separation of natural products.
Assuntos
Camptotecina , Celulose , Hidrogéis , Impressão Molecular , Extração em Fase Sólida , Camptotecina/química , Camptotecina/isolamento & purificação , Celulose/química , Adsorção , Impressão Molecular/métodos , Hidrogéis/química , Extração em Fase Sólida/métodos , Camptotheca/química , Polímeros/química , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Frutas/químicaRESUMO
BACKGROUND: In the present study, we have explored the utility of QSAR modelling, in silico ADMET, docking, chemical semi-synthesis, and in vitro evaluation studies for the identification of active camptothecin (CPT) derivatives against cancer-targeting human liver (HepG2) and lung (A549) cancer cell lines. METHODS: Two QSAR models were developed as screenings tools using the multiple linear regression (MLR) method followed by ADMET and docking studies. The regression coefficient (r2) and cross-validation regression coefficients (rCV2T) of the QSAR model for the HepG2 cell line was 0.95 and 0.90, respectively, and for the A549 cell line, it was 0.93 and 0.81, respectively. RESULTS: In silico studies show that CPT derivatives (CPT-1 and CPT-6) possess drug-like properties. Docking performed on DNA Topoisomerase-I showed significant binding affinity. Finally, predicted active derivatives were chemically semi synthesized, spectroscopically characterized, and evaluated in-vitro for cytotoxic/anticancer activity against HepG2 and A549 cell lines. CONCLUSION: The experimental results are consistent with the predicted results. These findings may be of immense importance in the anticancer drug development from an inexpensive and widely available natural product, camptothecin.
Assuntos
Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Camptotecina/isolamento & purificação , Magnoliopsida/química , Extratos Vegetais/isolamento & purificação , Células A549 , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/química , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Modelos Lineares , Simulação de Acoplamento Molecular/métodos , Extratos Vegetais/farmacologia , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Transdução de SinaisRESUMO
Camptothecin the third most in demand alkaloid, is commercially extracted in India from the endangered plant, Nothapodytes nimmoniana. Endophytes, the microorganisms that reside within plants, are reported to have the ability to produce host-plant associated metabolites. Hence, our research aims to establish a sustainable and high camptothecin yielding endophyte, as an alternative source for commercial production of camptothecin. A total of 132 endophytic fungal strains were isolated from different plant parts (leaf, petiole, stem and bark) of N. nimmoniana, out of which 94 were found to produce camptothecin in suspension culture. Alternaria alstroemeriae (NCIM1408) and Alternaria burnsii (NCIM1409) demonstrated camptothecin yields up to 426.7 ± 33.6 µg/g DW and 403.3 ± 41.6 µg/g DW, respectively, the highest reported production to date. Unlike the reported product yield attenuation in endophytes with subculture in axenic state, Alternaria burnsii NCIM1409 could retain and sustain the production of camptothecin up to ~ 200 µg/g even after 12 continuous subculture cycles. The camptothecin biosynthesis in Alternaria burnsii NCIM1409 was confirmed using 13C carbon labelling (and cytotoxicity analysis on different cancer cell lines) and this strain can now be used to develop a sustainable bioprocess for in vitro production of camptothecin as an alternative to plant extraction.
Assuntos
Alternaria/metabolismo , Camptotecina/biossíntese , Camptotecina/isolamento & purificação , Alcaloides/metabolismo , Camptotecina/metabolismo , Endófitos/metabolismo , Índia , Magnoliopsida/metabolismo , Folhas de Planta/metabolismoRESUMO
In the study, endophytic fungi isolated from Ophiorrhiza mungos were screened for camptothecin (CPT) biosynthetic potential by high performance liquid chromatography (HPLC). Among the 16 fungi screened, OmF3, OmF4, and OmF6 were identified to synthesize CPT. Further LC-MS analysis also showed the presence of CPT specific m/z of 349 for the extracts from OmF3, OmF4, and OmF6. However, the fragmentation masses with m/z of 320, 305, 277 and 220 specific to the CPT could be identified only for the OmF3 and OmF4. These CPT producing fungi were further identified as Meyerozyma sp. OmF3 and Talaromyces sp. OmF4. The cultures of these two fungi were then supplemented with nanoparticles and analyzed for the quantitative enhancement of CPT production by LC-MS/MS. From the result, Meyerozyma sp. OmF3 was found to produce 947.3 ± 12.66 µg/L CPT, when supplemented with 1 µg/mL zinc oxide nanoparticles and the same for uninduced parental strain OmF3 was only 1.77 ± 0.13 µg/L. At the same time, Talaromyces sp. OmF4 showed the highest production of 28.97 ± 0.37 µg/L of CPT when cultured with 10 µg/mL silver nanoparticles and the same for uninduced strain was 1.19 ± 0.24 µg/L. The observed quantitative enhancement of fungal CPT production is highly interesting as it is a rapid and cost effective method. The study is remarkable due to the identification of novel fungal sources for CPT production and its enhancement by nanoparticle supplementation.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Camptotecina/isolamento & purificação , Fungos/química , Nanopartículas Metálicas/química , Animais , Antineoplásicos Fitogênicos/biossíntese , Antineoplásicos Fitogênicos/química , Camptotecina/biossíntese , Camptotecina/química , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas em TandemRESUMO
Camptothecin (CPT), a potent inhibitor of topoisomerase I and HIF-1α, failed to demonstrate utility as an anti-cancer agent in early clinical trial investigations, primarily due to limited clinical activity and significant toxicity attributable to unfavorable physicochemical properties (e.g. low plasma solubility, pH-labile lactone ring). NLG207 (formerly CRLX101), a nanoparticle-drug conjugate (NDC) of CPT designed to optimize plasma pharmacokinetics and facilitate drug delivery to tumors, is included as part of combination treatment in two Phase II clinical trials ongoing at the National Cancer Institute (NCT02769962 and NCT03531827). To better understand the potential for drug-drug interactions and to correlate drug exposure to clinical outcomes and pharmacodynamic biomarkers, a robust analytical method was developed to measure CPT in human plasma. Two sample processing methods were developed to quantify both NDC-bound CPT and free CPT, primarily via alteration of pH conditions. A solid-phase extraction recovered >79 % of CPT prior to quantitative analysis by ultra HPLC-MS/MS. Dynamic calibration ranges of 10 to 10,000â¯ng/mL and 1 to 1000â¯ng/mL for total and free CPT, respectively were utilized to capture clinical ranges. NLG207 NDCs demonstrated significant rates of CPT release in human plasma at room temperature after 2â¯h but were shown to be stable at 4⯰C for 24â¯h and through 4 freeze/thaw cycles. This assay was used to quantitate CPT plasma concentrations in clinical samples to confirm clinical utility following NLG207 treatment in subjects with advanced prostate cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Camptotecina/sangue , Ciclodextrinas/sangue , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Benzamidas , Camptotecina/isolamento & purificação , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Ciclodextrinas/isolamento & purificação , Ciclodextrinas/farmacocinética , Ciclodextrinas/uso terapêutico , Interações Medicamentosas , Estabilidade de Medicamentos , Humanos , Masculino , Pessoa de Meia-Idade , Nitrilas , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacocinética , Feniltioidantoína/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/sangueRESUMO
We have reported previously that 9-methoxycamptothecin (MCPT) showed significant antitumor activity in vitro. Here, agarose gel electrophoresis experiments were performed to evaluate MCPT's unwinding ability toward plasmid DNA and inhibitory activities against topoisomerases (Topo) I and II. Binding properties of MCPT to calf thymus DNA (CT-DNA) were evaluated by UV-vis, melting temperature, fluorescence, circular dichroism methodologies and molecular docking technique. Results showed that MCPT at 100 µM inhibited Topo I activity, but had no effect on Topo II. Studies on the binding properties indicated that minor groove binding was the most probable binding mode of MCPT to DNA. The abilities of MCPT to act as Topo I inhibitor and minor groove binding agent may be related to its strong antitumor activity.
Assuntos
Camptotecina/análogos & derivados , Magnoliopsida/química , Inibidores da Topoisomerase I/farmacologia , Camptotecina/isolamento & purificação , Camptotecina/farmacologia , Dicroísmo Circular , DNA , Simulação de Acoplamento Molecular , Inibidores da Topoisomerase I/isolamento & purificaçãoRESUMO
Large-volume sample stacking (LVSS) is commonly used as an effective online preconcentration method in capillary zone electrophoresis (CZE). In this paper, the method LVSS combined with CZE has been proposed to analyze camptothecin alkaloids. Optimum separation can be achieved in the following conditions: pH 9.0; 25mm borate buffer containing 20 mm sulfobutylether-ß-cyclodextrin and 20 mm ionic liquid 1-ethyl-3-methyllimidazole l-lactate; applied voltage 20 kV; and capillary temperature 25 °C. The LVSS was optimized as hydrodynamic injection 4 s at 5.0 psi and the polarity switching time was 0.17 min. Under the above conditions, the analytes could be separated completely in <20 min and the detector response was increased compared with conventional hydrodynamic injection. The limits of detection were between 0.20 and 0.78 µg/L. A good linearity was obtained with correlation coefficients from 0.9991 to 0.9997. The recoveries ranged from 97.72 to 103.2% and the results demonstrated excellent accuracy. In terms of the migration time and peak area, the experiment was reproducible. The experimental results indicated that baseline separation can be obtained and this method is suitable for the quantitative determination of camptothecin alkaloids in real samples.
Assuntos
Camptotheca/química , Camptotecina/análise , Camptotecina/isolamento & purificação , Eletroforese Capilar/métodos , Extratos Vegetais/química , Camptotecina/análogos & derivados , Camptotecina/química , Frutas/química , Concentração de Íons de Hidrogênio , Modelos Lineares , Casca de Planta/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura , beta-Ciclodextrinas/químicaRESUMO
Camptothecins, a kind of monoterpene-quinoline alkaloids from Camptotheca acuminata Decne, have long attracted much attention worldwide as an anti-cancer drug. However, there is still a lack of effective methods for the accumulation and discovery of camptothecin analogues from botanic resources for camptothecin-based drug research. This work develops a one-step method for the targeted accumulation, quick detection, and identification of camptothecin analogues from C. acuminata fruit using bilayer solid-phase extraction coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (bilayer-SPE-UHPLC-Q-TOF-MS/MS). The bilayer-SPE cartridge, with polyamide (PA) as the upper layer and octadecyl silane (ODS) as the lower layer, was designed for the removal of flavonoid and ellagic acid impurities and the enrichment of camptothecins for further MS analysis. Subsequently, the mass spectrometry fragmentations, especially multistage retro-Diels-Alder cleavage, were summarized based on the MS/MS data of 10 reference camptothecins. The UHPLC-Q-TOF-MS/MS conditions were optimized, and the MS/MS data of the potential camptothecin analogues in the bilayer-SPE enriched fractions were analyzed. A total of 30 camptothecin analogues, including 15 new compounds, were identified from the fruit according the fragmentation pathways of the reference standards. The proposed structure of peak 20 was confirmed using its NMR data through rapid enrichment and purification. Overall, the bilayer-SPE enrichment and reliable mass spectrometry fragmentation in our work could provide an effective and simple method for the exploration of the biosynthesis pathway and metabolomics of camptothecin analogues.
Assuntos
Camptotheca/química , Camptotecina/análise , Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão , Frutas/química , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Alcaloides/química , Antineoplásicos/análise , Antineoplásicos/isolamento & purificação , Camptotecina/isolamento & purificaçãoRESUMO
BACKGROUND: Camptothecin (CPT) is a potent drug against cancers, originally from plants. The endophytic fungi could produce the secondary metabolite same as the host and is used as medicine. OBJECTIVES: The aim of this paper was to investigate an endophytic fungal CPT with anti-neoplastic activity. METHODS: Endophytic fungi were isolated from Camptotheca acuminata in China. CPT from strain S-019 was characterized by TLC, HPLC and EI-MS analysis. Anti-tumor activity of fungal CPT was detected by MTT and fluorescent dye methods using Vero and PC-3 cells. RESULTS: A total of 94 endophytic fungi strains were isolated from tissues of C. acuminata and 16 fungi strains displayed cytotoxic activity on Vero or PC3 cells. Of which, the fungal strain S-019, classified as Fusarium solani, displayed impressive cytotoxic activity on cancer cells and was found to produce CPT by analysis of TLC, HPLC and EI-MS methods. Bioassay studies confirmed that the fungi CPT had potent cytotoxicity on Vero cells and induced apoptosis of Vero cells. CONCLUSION: The endophytic fungi from camptotheca trees are a reliable source for natural anticancer compounds. The endophytic fungi could produce CPT same as plant. The fungal CPT exhibited effective activity at inhibiting cell growth and inducing apoptosis on Vero cells.
Assuntos
Antineoplásicos/uso terapêutico , Camptotheca/microbiologia , Camptotecina/uso terapêutico , Fusarium/química , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Camptotheca/química , Camptotecina/química , Camptotecina/isolamento & purificação , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatografia Líquida de Alta Pressão , Endófitos/química , Endófitos/isolamento & purificação , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Células Vero/efeitos dos fármacosRESUMO
A novel and efficient ultrasonic assisted-reflux synergistic extraction (UARSE) method for extracting camptothecin (CPT) and betulinic acid (BA) from Camptotheca acuminata Decne. fruits has been developed in this study. The advantages of the ultrasonic and reflux extraction methods have been combined in the UARSE method and used to extract CPT and BA for the first time. The parameters influencing the efficiency of UARSE were optimized using the Box-Behnken design (BBD) to obtain the maximum extraction yield of CPT and BA. The optimal extraction conditions were as follows: 225 W for the ultrasonic power; 24 min for the extraction time; and 32 mL/g for the liquid-solid ratio. The extraction yields obtained by UARSE were 2.386 ± 0.112 mg/g for CPT and 17.192 ± 0.808 mg/g for BA, which were 1.43-fold and 1.33-fold, respectively, higher than by using heating reflux extraction (HRE) and ultrasonic-assisted extraction (UAE). In addition, the 24-min extraction time using UARSE was 80% and 60% less than those provided by HRE and UAE, respectively. Therefore, UARSE can be considered a rapid and efficient method for extracting CPT and BA from the fruits of C. acuminata Decne.
Assuntos
Camptotheca/química , Camptotecina/química , Triterpenos/química , Ultrassom , Camptotecina/isolamento & purificação , Frutas/química , Triterpenos Pentacíclicos , Triterpenos/isolamento & purificação , Ácido BetulínicoRESUMO
Ultrasound-assisted extraction (UAE) of commercially important natural product camptothecin (CPT) from Nothapodytes nimmoniana plant has been investigated. The influences of process factors such as electric acoustic intensity, solid to liquid ratio, duty cycle, temperature and particle size on the maximum extraction yield and kinetic mechanisms of the entire extraction process have been investigated. The kinetics results showed that increasing the intensity, duty cycle, solid to liquid ratio and decreasing the particle size lead to substantial increase in extraction yields compared to classical stirring extraction. Different kinetic models were applied to fit the experimental data. The second order rate model appears to be the best. The extraction rate constant, initial extraction rate and the equilibrium concentration for all experimental conditions have been calculated. SEM analysis of spent plant material clearly showed hollow openings on cell structure, which could be directly correlated to explosive disruption by the action of ultrasound waves. Overall 1.7-fold increase in extraction yields of CPT (0.32% w/w) and decrease in time from 6h to 18min was observed over the stirring method.
Assuntos
Camptotecina/isolamento & purificação , Fracionamento Químico/métodos , Magnoliopsida/química , Modelos Teóricos , Ondas Ultrassônicas , Cinética , Tamanho da Partícula , Temperatura , Fatores de TempoRESUMO
The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a â¼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.
Assuntos
Antineoplásicos Fitogênicos/biossíntese , Camptotheca/metabolismo , Camptotecina/biossíntese , Cinuramina/análogos & derivados , Antineoplásicos Fitogênicos/análise , Antineoplásicos Fitogênicos/isolamento & purificação , Cultura Axênica , Camptotheca/efeitos dos fármacos , Camptotecina/análise , Camptotecina/isolamento & purificação , Técnicas de Cultura de Células , Criopreservação , Meios de Cultura/química , Glucosídeos Iridoides/farmacologia , Cinuramina/química , Cinuramina/metabolismo , Sorbitol/farmacologia , Triptaminas/farmacologiaRESUMO
This work reported that ionic liquid (IL) ([Bmim] [PF6 ]) and sulfobutylether-ß-CD (SBE-ß-CD) were used as electrolyte additives for the separation and determination of camptothecin (CPT) alkaloids by CZE. Separation parameters such as the buffer type, pH, and concentration of the running buffer, the concentration of SBE-ß-CD and IL, temperature, and separation voltage were all investigated in order to achieve the maximum possible resolution. The four analytes were baseline separated within 10 min in capillary at the separation voltage of 15 kV with a running buffer consisting of 20 mM borate buffer, 20 mM IL, and 100 mM SBE-ß-CD at pH 9.0. Under such conditions, good linearity about two orders of magnitudes of peak areas was achieved for the investigated CPT alkaloids with the correlation coefficients ranging from 0.9946 to 0.9985. For all analytes, detection limits (S/N = 3) and quantitation limits (S/N = 10) range from 0.05 to 0.92 µg/mL and 0.17 to 3.06 µg/mL, respectively. The proposed method has not only been successfully applied to the separation and determination of CPT alkaloids but also showed that IL seemed to be a promising additive in CZE separation.
Assuntos
Camptotecina/análise , Camptotecina/isolamento & purificação , Eletroforese Capilar/métodos , Imidazóis/química , Líquidos Iônicos/química , beta-Ciclodextrinas/química , Camptotecina/química , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos TestesRESUMO
Realistic in vitro models are critical in the drug development process. In this study, a novel in vitro platform is employed to assess drug penetration and metabolism. This platform, which utilizes a 3D printed fluidic device, allows for dynamic dosing of three dimensional cell cultures, also known as spheroids. The penetration of the chemotherapeutic irinotecan into HCT 116 colon cancer spheroids was examined with MALDI imaging mass spectrometry (IMS). The active metabolite of irinotecan, SN-38, was also detected. After twenty-four hours of treatment, SN-38 was concentrated to the outside of the spheroid, a region of actively dividing cells. The irinotecan prodrug localization contrasted with SN-38 and was concentrated to the necrotic core of the spheroids, a region containing mostly dead and dying cells. These results demonstrate that this unique in vitro platform is an effective means to assess drug penetration and metabolism in 3D cell cultures. This innovative system can have a transformative impact on the preclinical evaluation of drug candidates due to its cost effectiveness and high throughput.
Assuntos
Camptotecina/análogos & derivados , Neoplasias do Colo/tratamento farmacológico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Esferoides Celulares/efeitos dos fármacos , Camptotecina/administração & dosagem , Camptotecina/isolamento & purificação , Técnicas de Cultura de Células/métodos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Irinotecano , Dispositivos Lab-On-A-Chip , Impressão Tridimensional/instrumentação , Esferoides Celulares/metabolismoRESUMO
Camptothecin (CPT) and its derivative have been revealed to possess special anti-cancer activity, extraction methods are necessary for trace determination of CPTs in complex samples. In this work, we prepared a high efficient boronic acid-based polymer monolithic layer for microextraction of CPTs. A disposable membrane filter-based extraction device was developed, and boronic acid groups were co-polymerized into a polyporous polymer skeleton and served as the monolithic sorbent. The prepared poly(4-VB-MA-TRIM) showed good stability and great extraction efficiency toward four CPTs. After optimization of extraction conditions, poly(4-VB-MA-TRIM)-based solid-phase microextraction was coupled HPLC for determination of CPTs in biological samples. The method exhibited low limits of detection of 0.05-0.2 ng mL(-1), which is significantly more sensitive than reported HPLC methods. The method also showed wide linear range (0.1-100 and 0.5-200 ng mL(-1)), good linearity (R(2)≥0.9981) and good reproducibility (RSD ≤3.76%). The method has been applied in plasma samples, with good selectivity and good recoveries ranging from 85.1 to 104.7%.
Assuntos
Antineoplásicos Fitogênicos/sangue , Ácidos Borônicos/química , Camptotecina/sangue , Cromatografia Líquida de Alta Pressão/métodos , Polímeros/química , Microextração em Fase Sólida/métodos , Antineoplásicos Fitogênicos/isolamento & purificação , Camptotecina/isolamento & purificação , Cromatografia Líquida de Alta Pressão/instrumentação , Desenho de Equipamento , Humanos , Limite de Detecção , Microextração em Fase Sólida/instrumentaçãoRESUMO
A novel conjugate of camptothecin and artesunate (C-Q) was prepared and its cytotoxicity was evaluated using the MTT assay. In addition, the antitumour activity and toxicity of C-Q were investigated in mice, and interaction between transferrin (TF) and C-Q was investigated to evaluate its interaction with biological macromolecules. In the MTT assay, C-Q showed better inhibitory activity against MCF7 breast cancer cells and SMMC-7721 liver cancer cells than camptothecin or artesunate. In vivo, C-Q showed lower toxicity and better antitumour activity compared with camptothecin. Fluorescence spectroscopy showed static quenching of TF in the presence of C-Q, and thermodynamic parameters (ΔH>0 and ΔG<0) indicated that the reaction was spontaneous and endothermic. The main binding force between C-Q and TF was hydrophobic, as indicated by thermodynamic parameters (ΔH>0 and ΔS>0). Thus, synchronous fluorescence spectra showed that C-Q had no influence on the conformation of TF. Our results indicated that C-Q represents a novel potential anticancer therapeutic vector with advantages over current methods of CPT and ART administration. This novel drug delivery system allows the use of these drugs in a manner associated with few side effects for normal tissue, and which facilitates synergistic effects of anti-tumour drugs.
Assuntos
Antineoplásicos/química , Artemisininas/química , Camptotecina/química , Medicamentos de Ervas Chinesas/química , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Artemisininas/isolamento & purificação , Artemisininas/farmacologia , Artesunato , Camptotecina/isolamento & purificação , Camptotecina/farmacologia , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/farmacologia , Feminino , Fígado/efeitos dos fármacos , Fígado/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SN-38 is a highly potent anticancer drug but its poor solubility in aqueous solvent and adverse side effects limit clinical applications. To overcome these limitations, SN-38-loaded-injectable drug delivery depots have been intratumorally administered in xenograft tumor model in nude mice. The extraction and high performance liquid chromatography (HPLC) were performed in order to determine the amount of SN-38 inside tumors. SN-38 was extracted from tumors using DMSO. HPLC analysis was validated and resulted in linearity over the concentration range from 0.03 to 150 µg/mL (r(2) ≥ 0.998). Lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.308 µg/mL and 1.02 µg/mL, respectively. The extraction efficiency (% recovery) of SN-38 in porcine tissues was similar to that of tumors which provided more than 90% recovery in all concentrations. Moreover, the variability of precision and accuracy within and between-day were less than 15%. Therefore, this extraction and HPLC protocol was applied to determine the amount of SN-38 in tumors. Results show higher remaining amount of SN-38 in tumor from SN-38-loaded polymeric depots than that of SN-38 solution. These results reveal that SN-38-loaded polymeric depots can prevent the leakage of free-drug out of tumors and can sustain higher level of SN-38 inside tumor. Thus, the therapeutic efficacy can be elevated by SN-38-loaded polymeric depots.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Neoplasias Encefálicas/tratamento farmacológico , Camptotecina/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Sistemas de Liberação de Medicamentos , Polímeros/administração & dosagem , Animais , Antineoplásicos Fitogênicos/análise , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/uso terapêutico , Camptotecina/análise , Camptotecina/isolamento & purificação , Camptotecina/farmacocinética , Camptotecina/uso terapêutico , Modelos Animais de Doenças , Irinotecano , Camundongos NusRESUMO
New tandem mass spectrometric method coupled with liquid chromatography (LC-MS/MS) has been developed to determine the total concentration of camptothecin derivatives (irinotecan and SN-38) regardless of inter-conversion phenomenon between carboxylate and lactone forms. At first, all sample solutions were acidified for 1h in order to completely convert CPT derivatives into their lactone forms and then CPT derivatives were extracted with organic solution containing diethyl ether and ethyl acetate (2:1, v/v) just after alkalization in the range pH 8.0-8.5 in acid-treated solutions. Analytes were separated on a reverse phase C18 column (150×2.1mm) and eluted isocratically with a mobile phase which consisted of acetonitrile-methanol-buffer (0.1% formic acid, 5mM ammonium formate) (3:4:3, v/v). CPT derivatives were monitored by tandem mass spectrometry in electrospay-positive ionization and multiple reaction mode programmed to the following transitions (m/z): '587.6â167.2' of CPT-11, '393.6â349.3' of SN-38 and '349.4â 305.2' of CPT. The method was validated to have the proper linearity (r(2)>0.99) over the range of 5-1000ng/ml of CPT-11 and 1-250ng/ml of SN-38 with good accuracy (89.8-114.3%) and precision (less than 10%). In all stability tests, concentration of CPT-11 and SN-38 had been left in the acceptable range of 88.8-110.7% when sample solutions were acidified before determination of CPT derivatives. Newly developed LC-MS/MS method was suitable for the determination of CPT derivatives of both rabbit plasma and tumor tissues in the pharmacokinetic study.
Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Camptotecina/análogos & derivados , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Antineoplásicos Fitogênicos/sangue , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/metabolismo , Camptotheca/química , Camptotecina/sangue , Camptotecina/isolamento & purificação , Camptotecina/metabolismo , Camptotecina/farmacocinética , Irinotecano , Limite de Detecção , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Coelhos , Reprodutibilidade dos TestesRESUMO
Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/isolamento & purificação , Camptotecina/farmacologia , Meliaceae/química , Casca de Planta/química , Antineoplásicos Fitogênicos/química , Camptotecina/química , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Espectrometria de Massas , Conformação Molecular , Relação Estrutura-AtividadeRESUMO
Camptothecine, a potent eukaryotic topoisomerase inhibitor, is an important anticancer compound. The global demand for this compound was estimated to be $1 billion in 2003 and is only further expected to increase. Partly to meet the expected increase in demand, in the recent past, several efforts have been made to discover newer and alternative plant and fungal sources of camptothecine. In this study we report a rich source of camptothecine and its natural derivatives, Pyrenacantha volubilis (Icacinaceae) from the eastern coast of peninsular India. Camptothecine and its derivatives were analyzed using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in all plant parts such as twigs, leaves, roots, seedling, ripened whole fruit, fruit coat, seed coat and cotyledons. Cotyledons and ripened whole fruits contained the highest amount of camptothecine (1.35% and 0.60% dry weight respectively). LC-MS and ESI-MS/MS analyses revealed besides camptothecine, other derivatives and precursors such as 10-hydroxycamptothecine, 9-methoxycamptothecine, 20-deoxycamptothecine, deoxypumiloside, strictosidine and strictosamide. Pure camptothecine was isolated from fruits and structurally confirmed using NMR. Seed extracts were found to be effective against breast cancer, ovarian, colon and carcinoma cell lines (with IC50 values of 4.0 µg/mL, 6.5 µg/mL, 25.0 µg/mL and 25.0 µg/mL respectively). We discuss the results in the context of exploring alternative sources of camptothecine.