RESUMO
BACKGROUND: The function of platelets have extended way beyond the horizon of haemostasis and thrombosis, and are recognised as active participants in vascular inflammation, as well as in prothrombotic complications of cardiovascular diseases. We describe and compare platelet function in type II diabetes (with and without cardiovascular manifestation) and healthy individuals using scanning electron microscopy and flow cytometry. METHODS: Thirty subjects were recruited per group and informed consent was obtained from all participants. Diabetic patients were recruited from the diabetic clinic of the Steve Biko Academic Hospital (South Africa). Blood samples were drawn from all participants so that platelet specific antigens were analyzed in citrated whole blood. The platelet parameters used in the study were platelet identifiers (CD41 and CD42) and markers of platelet activation (CD62 and CD63). RESULTS: Results show that, compared to healthy individuals, both diabetic groups showed a significant difference in both platelet identifiers (CD41-PE, CD42b-PE) as well as markers indicating platelet activation (CD62P-PE and CD63-PE). INTERPRETATION: The flow cytometric data shows that the platelet surface receptors and platelet activation are statistically elevated. This is suggestive of enhanced platelet activation and it appears as if platelets are displaying 'angry' behaviour. The lysosomal granules may play a significant role in diabetes with cardiovascular complications. These results were confirmed by ultrastructural analysis.
Assuntos
Plaquetas/citologia , Diabetes Mellitus Tipo 2/sangue , Citometria de Fluxo , Ativação Plaquetária/fisiologia , Trombose/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/análise , Biomarcadores/sangue , Feminino , Citometria de Fluxo/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/imunologia , Adulto JovemRESUMO
Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 µM and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1 fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells. This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias Esofágicas/tratamento farmacológico , Estradiol/administração & dosagem , Estrenos/administração & dosagem , Sulfonamidas/administração & dosagem , Linhagem Celular Tumoral , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Estradiol/análogos & derivados , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.
Assuntos
Antineoplásicos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Simulação por Computador , Eritrócitos/efeitos dos fármacos , Estradiol/análogos & derivados , Estrenos/farmacologia , Sulfonamidas/farmacologia , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Anidrase Carbônica II/efeitos dos fármacos , Inibidores da Anidrase Carbônica/farmacocinética , Proteínas de Transporte/farmacocinética , Proteínas de Transporte/farmacologia , Descoberta de Drogas , Eritrócitos/ultraestrutura , Estradiol/farmacocinética , Estradiol/farmacologia , Estradiol/toxicidade , Estrenos/farmacocinética , Feminino , Hemólise/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Pesquisa Qualitativa , Sulfonamidas/farmacocinética , Sulfonamidas/toxicidade , TemperaturaRESUMO
2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-ß-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 µM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.
Assuntos
Antineoplásicos/farmacologia , Caspase 7/metabolismo , Caspase 8/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacologia , Estrenos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Sulfonamidas/farmacologia , Adenocarcinoma , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias da Mama , Feminino , Humanos , Células MCF-7 , Dobramento de ProteínaRESUMO
Research into potential anticancer agents has shown that 2-methoxyestradiol exerts antiproliferative activity in vitro and in vivo in an estrogen receptor-independent manner. Due to its limited biological accessibility and rapid metabolic degradation, several new analogues have been developed in recent years. This study investigated the in vitro effects of a novel in silicodesigned compound (C16) in an estrogen receptor-positive breast adenocarcinoma epithelial cell line (MCF-7), an estrogen receptor-negative breast adenocarcinoma epithelial cell line (MDA-MB-231) and a nontumorigenic breast cell line (MCF-12A). Light microscopy revealed decreased cell density, cells blocked in metaphase and the presence of apoptotic characteristics in all three cell lines after exposure to C16 for 24 h. Polarizationoptical transmitted light differential interference contrast revealed the presence of several rounded cells and decreased cell density. The xCELLigence real-time label-independent approach revealed that C16 exerted antiproliferative activity. Significant inhibition of cell growth was demonstrated after 24 h of exposure to 0.2 µM C16 in all three cell lines. However, the non-tumorigenic MCF-12A cell line recovered extremely well after 48 h when compared to the tumorigenic cell lines. This indicates that C16 acts as an antiproliferative agent, possesses antimitotic activity and induces apoptosis in vitro. These features warrant further investigation.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estradiol/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Estradiol/análogos & derivados , Feminino , Humanos , Células MCF-7 , Metáfase , Enxofre/químicaRESUMO
2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/patologia , Estriol/análogos & derivados , Autofagia/efeitos dos fármacos , Contagem de Células , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estriol/farmacologia , Humanos , Microscopia de Interferência , Projetos Piloto , Coloração e RotulagemRESUMO
Current chemotherapeutic agents have many side effects and are toxic to normal cells, providing impetus to identify agents that can effectively eliminate tumorigenic cells without damaging healthy cells. The aim of this study was to examine whether combining a novel BRD4 inhibitor, ITH-47, with the antimitotic estradiol analogue, ESE-15-ol, would have a synergistic effect on inhibiting the growth of two different breast cancer cell lines in vitro. Our docking and molecular dynamics studies showed that compared to JQ1, ITH-47 showed a similar binding mode with hydrogen bonds forming between the ligand nitrogens of the pyrazole, ASN99, and water of the BRD4 protein. Data from cell growth studies revealed that the GI50 of ITH-47 and ESE-15-ol after 48 hours of exposure was determined to be 15 µM and 70 nM, respectively, in metastatic MDA-MB-231 breast cancer cells. In tumorigenic MCF-7 breast cancer cells, the GI50 of ITH-47 and ESE-15-ol was 75 µM and 60 nM, respectively, after 48 hours of exposure. Furthermore, the combination of 7.5 µM and 14 nM of ITH-47 and ESE-15-ol, respectively, resulted in 50% growth inhibition of MDA-MB-231 cells resulting in a synergistic combination index (CI) of 0.7. Flow cytometry studies revealed that, compared to the control, combination-treated MDA-MB-231 cells had significantly more cells present in the sub-G1 phase and the combination treatment induced apoptosis in the MDA-MB-231 cells. Compared to vehicle-treated cells, the combination-treated cells showed decreased levels of the BRD4, as well as c-Myc protein after 48 hours of exposure. In combination, the selective BRD4 inhibitor, ITH-47, and ESE-15-ol synergistically inhibited the growth of MDA-MB-231 breast cancer cells, but not of the MCF-7 cell line. This study provides evidence that resistance to BRD4 inhibitors may be overcome by combining inhibitors with other compounds, which may have treatment potential for hormone-independent breast cancers.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Antimitóticos/farmacologia , Antineoplásicos , Apoptose/efeitos dos fármacos , Azepinas/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Estradiol/análogos & derivados , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Mitose/efeitos dos fármacos , Metástase Neoplásica , Proteínas Nucleares/genética , Sulfonamidas/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Triazóis/farmacologiaRESUMO
BACKGROUND: Due to their high proliferative requirements, tumorigenic cells possess altered metabolic systems whereby cells utilize higher quantities of glutamine and glucose. These altered metabolic requirements make it of interest to investigate the effects of physiological non-tumorigenic concentrations of glucose and glutamine on tumorigenic cells since deprivation of either results in a canonical amino acid response in mammalian cell. METHODS: The influence of short-term exposure of tumorigenic cells to correlating decreasing glutamine- and glucose quantities were demonstrated in a highly glycolytic metastatic breast cell line and a cervical carcinoma cell line. Thereafter, cells were propagated in medium containing typical physiological concentrations of 1 mM glutamine and 6 mM glucose for 7 days. The effects on morphology were investigated by means of polarization-optical transmitted light differential interference contrast. Flow cytometry was used to demonstrate the effects of glutamine-and glucose starvation on cell cycle progression and apoptosis induction. Fluorometrics were also conducted to investigate the effects on intrinsic apoptosis induction (mitocapture), reactive oxygen species production (2,7-dichlorofluorescein diacetate) and acidic vesicle formation (acridine orange). RESULTS: Morphological data suggests that glutamine-and glucose deprivation resulted in reduced cell density and rounded cells. Glutamine-and glucose starvation also resulted in an increase in the G2M phase and a sub-G1 peak. Complete starvation of glutamine and glucose resulted in the reduction of the mitochondrial membrane potential in both cell lines with MDA-MB-231 cells more prominently affected when compared to HeLa cells. Further, starved cells could not be rescued sufficiently by propagating since cells possessed an increase in reactive oxygen species, acidic compartments and vacuole formation. CONCLUSION: Starvation from glutamine and glucose for short periods resulted in decreased cell density, rounded cells and apoptosis induction by means of reactive oxygen species generation and mitochondrial dysfunction. In addition, the metastatic cell line reacted more prominently to glutamine-and glucose starvation due to their highly glycolytic nature. Satisfactory cellular rescue was not possible as cells demonstrated oxidative stress and depolarized mitochondrial membrane potential. This study contributes to the knowledge regarding the in vitro effects and signal transduction of glucose and/or l-glutamine deprivation in tumorigenic cell lines.
RESUMO
2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1-25 µM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 µM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Estradiol/análogos & derivados , 2-Metoxiestradiol , Adenocarcinoma , Caspase 3/metabolismo , Forma Celular/efeitos dos fármacos , Citocromos c/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática , Estradiol/farmacologia , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Sulfonamidas/farmacologia , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.
Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Sulfonamidas/farmacologia , Simulação por Computador , Inibidores da Anidrase Carbônica/farmacologia , Eritrócitos/efeitos dos fármacos , Estradiol/análogos & derivados , Estrenos/farmacologia , Antineoplásicos/farmacologia , Sulfonamidas/toxicidade , Sulfonamidas/farmacocinética , Temperatura , Inibidores da Anidrase Carbônica/farmacocinética , Disponibilidade Biológica , Microscopia Eletrônica de Varredura , Proteínas de Transporte/farmacologia , Proteínas de Transporte/farmacocinética , Anidrase Carbônica II/efeitos dos fármacos , Pesquisa Qualitativa , Eritrócitos/ultraestrutura , Estradiol/toxicidade , Estradiol/farmacologia , Estradiol/farmacocinética , Estrenos/farmacocinética , Descoberta de Drogas , Hemólise/efeitos dos fármacos , Antineoplásicos/farmacocinéticaRESUMO
2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.
Assuntos
Humanos , Apoptose , Autofagia , Contagem de Células , Carcinoma de Células Escamosas/patologia , Ciclo Celular , Divisão Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Estriol/análogos & derivados , Estriol/farmacologia , Neoplasias Esofágicas/patologiaRESUMO
2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.(AU)