The application of a haemorrhage assessment tool in evaluating control of bleeding in a pilot trauma haemorrhage trial.

OBJECTIVES: To determine whether it was feasible to use a haemorrhage assessment tool (HAT) within a trauma trial and whether the data obtained could differentiate patients who had achieved haemostasis. BACKGROUND: Major haemorrhage is one of the leading causes of death worldwide, affecting 40% of trauma patients. Clinical trials evaluating haemostatic interventions often use transfusion outcomes as a primary endpoint. Transfusion is highly dependent on local practice, limiting its reliability as a robust, transferable endpoint. METHODS: A five-point HAT questionnaire was applied to participants enrolled into the EFIT-1 trial. This RCT evaluated the feasibility of administering a 6 g fibrinogen concentrate to patients with severe trauma haemorrhage. RESULTS: Of participants, 98% completed a HAT; 75% participants had 'achieved haemostasis' at the time of tool completion, as determined by clinical acumen alone. HAT scores were able to differentiate which participants required transfusion after 3 h. Of participants, 56% were transfused red blood cells when they scored 0-2, compared to 17% with HAT scores between 3 and 5. CONCLUSION: This study has confirmed the feasibility of using a HAT during the emergency care of patients suffering trauma haemorrhage, and future studies should be conducted to determine its value as an endpoint in haemostasis studies.

In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer.

BACKGROUND: STX2484 is a novel non-steroidal compound with potent anti-proliferative activity. These studies aimed to identify STX2484's mechanism of action, in vivo efficacy and activity in taxane-resistant breast cancer models. METHODS: Effects of STX2484 and paclitaxel on proliferation, cell cycle and apoptosis were assessed in vitro in drug-resistant (MCF-7(DOX)) and non-resistant cells (MCF-7(WT)). STX2484 efficacy in ßIII tubulin overexpression in MCF-7 cells was also determined. Anti-angiogenic activity was quantified in vitro by a co-culture model and in vivo using a Matrigel plug assay. An MDA-MB-231 xenograft model was used to determine STX2484 efficacy in vivo. RESULTS: STX2484 is a tubulin disruptor, which induces p53 expression, Bcl2 phosphorylation, caspase-3 cleavage, cell cycle arrest and apoptosis. In addition, STX2484 is a potent anti-angiogenic agent in vitro and in vivo. In breast cancer xenografts, STX2484 (20 mg kg(-1) p.o.) suppressed tumour growth by 84% after 35 days of daily dosing, with limited toxicity. In contrast to paclitaxel, STX2484 efficacy was unchanged in two clinically relevant drug-resistant models. CONCLUSIONS: STX2484 is an orally bioavailable microtubule-disrupting agent with in vivo anti-angiogenic activity and excellent in vivo efficacy with no apparent toxicity. Crucially, STX2484 has superior efficacy to paclitaxel in models of clinical drug resistance.

Effect of tamoxifen or anastrozole on steroid sulfatase activity and serum androgen concentrations in postmenopausal women with breast cancer.

BACKGROUND: In postmenopausal women estrogens can be formed by the aromatase pathway, which gives rise to estrone, and the steroid sulfatase (STS) route which can result in the formation of estrogens and androstenediol, a steroid with potent estrogenic properties. Aromatase inhibitors, such as anastrozole, are now in clinical use whereas STS inhibitors, such as STX64, are still undergoing clinical evaluation. STX64 was recently shown to block STS activity and reduce serum androstenediol concentrations in postmenopausal women with breast cancer. In contrast, little is known about the effects of aromatase inhibitors or anti-estrogens on STS activity or serum androgen levels. PATIENTS AND METHODS: Study 1: Blood was collected from ten postmenopausal women with breast cancer before and after two-week treatment with anastrozole and serum concentrations of androstenediol and other androgens and estrogens were assessed. Study 2: Blood samples were collected from 15 breast cancer patients before and after four-week treatment with anastrozole and 10 patients before and after four-week treatment with tamoxifen. Blood was used to assess STS activity in peripheral blood lymphocytes (PBLs) and serum dehydroepiandrosterone sulfate and dehydroepiandrosterone levels. RESULTS: Neither anastrozole nor tamoxifen had any significant effect on STS activity as measured in PBLs. Anastrozole did not affect serum androstenediol concentrations. CONCLUSION: Anastrozole and tamoxifen did not inhibit STS activity and serum androstenediol concentrations were not reduced by aromatase inhibition. As androstenediol has estrogenic properties, it is possible that the combination of an aromatase inhibitor and STS inhibitor may give a therapeutic advantage over the use of either agent alone.

Class III beta-tubulin expression and in vitro resistance to microtubule targeting agents.

BACKGROUND: Class III beta-tubulin overexpression is a marker of resistance to microtubule disruptors in vitro, in vivo and in the clinic for many cancers, including breast cancer. The aims of this study were to develop a new model of class III beta-tubulin expression, avoiding the toxicity associated with chronic overexpression of class III beta-tubulin, and study the efficacy of a panel of clinical and pre-clinical drugs in this model. METHODS: MCF-7 (ER+ve) and MDA-MB-231 (ER-ve) were either transfected with pALTER-TUBB3 or siRNA-tubb3 and 24 h later exposed to test compounds for a further 96 h for proliferation studies. RT-PCR and immunoblotting were used to monitor the changes in class III beta-tubulin mRNA and protein expression. RESULTS: The model allowed for subtle changes in class III beta-tubulin expression to be achieved, which had no direct effect on the viability of the cells. Class III beta-tubulin overexpression conferred resistance to paclitaxel and vinorelbine, whereas downregulation of class III beta-tubulin rendered cells more sensitive to these two drugs. The efficacy of the colchicine-site binding agents, 2-MeOE2, colchicine, STX140, ENMD1198 and STX243 was unaffected by the changes in class III beta-tubulin expression. CONCLUSION: These data indicate that the effect of class III beta-tubulin overexpression may depend on where the drug's binding site is located on the tubulin. Therefore, this study highlights for the first time the potential key role of targeting the colchicine-binding site, to develop new treatment modalities for taxane-refractory breast cancer.

A retrospective review of patients with head injury with coexistent anticoagulant and antiplatelet use admitted from a UK emergency department.

BACKGROUND AND AIM: Conflicting evidence exists surrounding the increased risk of adverse outcome conferred by preinjury anticoagulant and antiplatelet treatment in patients with head injury. The aim of this study was to determine the epidemiology of patients with head injury on anticoagulant and antiplatelet treatment admitted to a hospital from an emergency department (ED). METHODS: This was a retrospective analysis of all patients with head injury admitted to a hospital from a major UK ED between 1 January 2005 and 31 December 2007. RESULTS: 399 patients met the inclusion criteria. 110 patients underwent CT, with 24 having traumatic haemorrhage. Of 271 patients on aspirin, 75 (28%) underwent CT, with 19 of these (25%) having traumatic haemorrhage. Of 89 patients on warfarin, 27 (30%) underwent CT, with 4 of these (15%) having traumatic haemorrhage. Seven of the 24 (29%) patients with traumatic haemorrhage on CT did not undergo urgent ED scanning. All these patients were on aspirin. CONCLUSIONS: This study confirms the need for caution in the early discharge of patients with head injury taking anticoagulant medication. This study also raises concerns that patients taking antiplatelet medication prior to injury may also be at high risk of developing covert serious intracranial haemorrhage and suggests the need for a well-designed cohort study looking at antiplatelet risk in head injury.

Management of syncope in the Emergency Department.

Syncope is a common presenting complaint to the emergency department (ED). Its assessment is difficult. Some serious causes of syncope are transient and patients with a potentially life threatening condition may appear well by the time they reach the ED. Accurate history taking is vital and is often diagnostic whilst identification of a cardiac cause is associated with an increased mortality. This is related to underlying cardiac disease; patients presenting with syncope who have significant cardiac disease should be investigated thoroughly to determine the nature of the underlying heart disease and the cause of syncope. Early work suggested that as many as 30% of patients with cardiac syncope died within one year of presentation. This led to physicians admitting many patients with unexplained syncope however presently there is little evidence that focussed investigation, or even admission leads to an improved prognosis. Studies looking at syncope clinical decision units have though shown these to be of some benefit. Risk stratification studies on syncope in the ED have attempted to help emergency physicians target high-risk patients once those with clearly identifiable conditions have been identified and managed. These clinical decision rules have suffered from poor external validation and in the USA where many of these tools were developed, a universal consensus approach remains lacking. Although no individual tool has yet been successfully implemented into standard practice, as a whole they have probably enabled emergency physicians to become more aware of the risk factors that are likely to lead to poor outcome. It is likely that serious outcome in syncope although significant, is not quite as common as previously thought. Presently the American College of Emergency Physician (ACEP) guidelines are the most useful guidelines written for the emergency physician. With biochemical markers showing some promise, further work may lead to incorporation of these into existing clinical decision rules and guidelines to improve their sensitivity and specificity.

Syncope management in the UK and Republic of Ireland.

AIMS: To establish the current practice of emergency department (ED) management of syncope in the UK and Republic of Ireland. METHODS: A survey of "major" or "intermediate" size ED in the UK and Republic of Ireland conducted by postal and telephone questionnaire. RESULTS: 177 (70%) ED responded. 32 (18%) ED have syncope guidelines, which are based on a range of existing guidelines. 97 ED (55%) have an observation ward or clinical decision unit and 48 (49%) of these admit syncope patients to these units. 32 ED (18%) have access to a specialist syncope outpatient clinic. This is most likely to be run by general practitioner specialists (43%) or general physicians (24%). 81% of ED felt that improved research-based guidelines would be useful when managing syncope patients. CONCLUSION: The ED management of syncope patients in the UK and Republic of Ireland is varied. Only 18% of ED have specific guidelines for managing this difficult condition and only 18% have access to a specialist syncope clinic. A robust consensus UK syncope guideline is clearly required.

CCL5 secreted by senescent aged fibroblasts induces proliferation of prostate epithelial cells and expression of genes that modulate angiogenesis.

There is increased interest in the effects of secretory products from aged cells on promoting both benign and malignant cell growth. We identified a human fibroblast line, AG04382, from an aged donor that naturally demonstrated senescence-associated features and whose conditioned media significantly induced proliferation of benign prostatic hyperplasia (BPH1) cells. Candidate cytokines mediating this effect were identified with protein arrays and validated by ELISA. We found that the AG04382 fibroblast line secreted high levels of CXCL5, CCL5, and CCL2, but relative to the other lines, its conditioned media was unique in its increased expression of CCL5. Blocking studies using specific antibodies against CXCL5, CCL5, and CCL2 in the conditioned media of AG04382 showed that only CCL5 contributed significantly to BPH1 proliferation. Stimulation of BPH1 cells with rhuCCL5 resulted in increased proliferation and migration, as well as significant changes in the expression of genes that influence angiogenesis. These data suggest that CCL5 is a candidate chemokine secreted by aged cells that promotes prostate growth and regulates angiogenesis.

BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure.

The anti-proliferative and anti-angiogenic properties of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2), are enhanced in a series of sulphamoylated derivatives of 2-MeOE2. To investigate possible mechanisms of resistance to these compounds, a cell line, A2780.140, eightfold less sensitive to the 3,17-O,O-bis-sulphamoylated derivative, STX140, was derived from the A2780 ovarian cancer cell line by dose escalation. Other cell lines tested did not develop STX140 resistance. RT-PCR and immunoblot analysis demonstrated that breast cancer resistance protein (BCRP) expression is dramatically increased in A2780.140 cells. The cells are cross-resistant to the most structurally similar bis-sulphamates, and to BCRP substrates, mitoxantrone and doxorubicin; but they remain sensitive to taxol, an MDR1 substrate, and to all other sulphamates tested. Sensitivity can be restored using a BCRP inhibitor, and this pattern of resistance is also seen in a BCRP-expressing MCF-7-derived cell line, MCF-7.MR. In mice bearing wild-type (wt) and BCRP-expressing tumours on either flank, both STX140 and mitoxantrone inhibited the growth of the MCF-7wt xenografts, but only STX140 inhibited growth of the MCF-7.MR tumours. In conclusion, STX140, a promising orally bioavailable anti-cancer agent in pre-clinical development, is highly efficacious in BCRP-expressing xenografts. This is despite an increase in BCRP expression in A2780 cells in vitro after chronic dosing with STX140.

The Prediction of Risk In Syncope using ECG characteristics (PRISE) pilot study: can heart rate variability be used to predict risk in patients presenting to the emergency department with syncope?

AIM: This study was conducted as a feasibility pilot for the Prediction of Risk In Syncope using ECG characteristics (PRISE) study. The secondary aim was to determine whether heart rate variability (HRV) characteristics may be useful to distinguish low and high-risk syncope patients. METHODS: Adult patients presenting to the emergency department (ED) with syncope over a one-month period underwent a 5-minute 12-lead ECG. Study patients were assigned high, medium or low-risk status according to the ED's existing syncope guidelines as well as one of four likely diagnostic categories. ECG signals from all patients were then analysed and time domain HRV characteristics were derived using WelchAllyn's Cardioperfect interpretation software. A control group of patients was also recruited. RESULTS: Over a 4-week period in July 2007, 32 patients were recruited into the study group and 23 into the control group. ECG tracings of five study group patients were not suitable for analysis. According to the ED's existing syncope guidelines there were nine low-risk, 12 medium-risk and six high-risk patients with diagnostic categories as follows: postural hypotension, five; vasovagal, 16; cardiac, five and other, one. Patients with cardiac syncope had greater mean values for all HRV characteristics except NN number and NN minimum; however, with overlapping confidence intervals. Low-risk patients were more likely to be younger than medium and high-risk patients. No HRV parameters showed any significant differences. CONCLUSIONS: Measuring HRV in the acute ED setting is feasible. If patients with cardiac and neurocardiogenic syncope have different HRV characteristics then it could be useful to determine a patient's underlying cause of syncope in the ED, which would allow earlier decision-making.

Inhibition of steroid sulfatase activity in endometriotic implants by STX64 (667Coumate): a potential new therapy.

Endometriosis is a common and debilitating condition of women in their reproductive age that is associated with pain and infertility. Current medical treatments are only partially effective and associated with wide-ranging side effects. New understanding of local estrogen production by endometriotic tissue and the availability of powerful suppressing drugs may herald a new era in the treatment of this condition.

2-Methoxyoestradiol-3,17-O,O-bis-sulphamate and 2-deoxy-D-glucose in combination: a potential treatment for breast and prostate cancer.

Drug combination therapy is a key strategy to improve treatment efficacy and survival of cancer patients. In this study the effects of combining 2-methoxyoestradiol-3,17-O,O-bis-sulphamate (STX140), a microtubule disruptor, with 2-deoxy-D-glucose (2DG) were assessed in MCF-7 (breast) and LNCaP (prostate) xenograft models in vivo. In mice bearing MCF-7 xenografts, daily p.o. administration of STX140 (5 mg kg(-1)) resulted in a 46% (P<0.05) reduction of tumour volume. However, the combination of STX140 (5 mg kg(-1) p.o.) and 2DG (2 g kg(-1) i.p.) reduced tumour volume by 76% (P<0.001). 2-Methoxyoestradiol-3,17-O,O-bis-sulphamate also reduced tumour vessel density. 2-Deoxy-D-glucose alone had no significant effect on tumour volume or vessel density. A similar benefit of the combination treatment was observed in the LNCaP prostate xenograft model. In vitro the degree of inhibition of cell proliferation by STX140 was unaffected by oxygen concentrations. In contrast, the inhibition of proliferation by 2DG was enhanced under hypoxia by 20 and 25% in MCF-7 and LNCaP cells, respectively. The combination of STX140 and 2DG in LNCaP cells under normoxia or hypoxia inhibited proliferation to a greater extent than either compound alone. These results suggest that the antiangiogenic and microtubule disruption activities of STX140 may make tumours more susceptible to inhibition of glycolysis by 2DG. This is the first study to show the benefit of combining a microtubule disruptor with 2DG in the two most common solid tumours.

The in vivo properties of STX243: a potent angiogenesis inhibitor in breast cancer.

The steroidal-based drug 2-ethyloestradiol-3,17-O,O-bis-sulphamate (STX243) has been developed as a potent antiangiogenic and antitumour compound. The objective of this study was to ascertain whether STX243 is more active in vivo than the clinically relevant drug 2-methoxyoestradiol (2-MeOE2) and the structurally similar compound 2-MeOE2-3,17-O,O-bis-sulphamate (STX140). The tumour growth inhibition efficacy, antiangiogenic potential and pharmacokinetics of STX243 were examined using four in vivo models. Both STX243 and STX140 were capable of retarding the growth of MDA-MB-231 xenograft tumours (72 and 63%, respectively), whereas no inhibition was observed for animals treated with 2-MeOE2. Further tumour inhibition studies showed that STX243 was also active against MCF-7 paclitaxel-resistant tumours. Using a Matrigel plug-based model, in vivo angiogenesis was restricted with STX243 and STX140 (50 and 72%, respectively, using a 10 mg kg(-1) oral dose), thereby showing the antiangiogenic activity of both compounds. The pharmacokinetics of STX243 were examined at two different doses using adult female rats. The compound was orally bioavailable (31% after a single 10 mg kg(-1) dose) and resistant to metabolism. These results show that STX243 is a potent in vivo drug and could be clinically effective at treating a number of oncological conditions.

Inhibition of steroid sulphatase activity via the percutaneous route: a new option for breast cancer therapy.

Steroid sulphatase (STS) inhibitors have been developed primarily for the treatment of hormone-dependent breast cancer, but may also have utility for the treatment of a number of androgen-dependent skin conditions. STS regulates the hydrolysis of steroid sulphates, such as oestrone sulphate (E1S) and dehydroepiandrosterone sulphate, (DHEAS). Liberated oestrone (E1) can be converted to biologically active oestradiol (E2) while dehydroepiandrosterone (DHEA) can undergo reduction to testosterone or aromatisation to E1. In this study the ability of the STS inhibitor STX64 (BN83495) and its N,N-dimethyl analogue (STX289) to inhibit liver and skin STS when applied orally or topically to nude mice was examined. Oral administration at 1 and 10 mg/kg resulted in almost complete inhibition of skin and liver STS. When applied topically to the dorsal neck region at 1.0 and 10 mg/kg not only skin but, unexpectedly, also liver STS was effectively inhibited. An investigation into the metabolism of these two compounds by HepG2 liver carcinoma cells, with high-performance liquid chromatography (HPLC) analysis, was also undertaken. In the presence of HepG2 cells a similar degree of desulphamoylation of STX64 (68%) or de-N, N-dimethylsulphamoylation of STX289 (66%) occurred over a 3h period. In the absence of cells, however, STX289 was resistant to de-N, N-dimethylsulphamoylation whereas STX64 was completely desulphamoylated, demonstrating the more favourable pharmaceutical profile of STX289 for development for topical application. It is concluded that both STX64 and STX289 are not only effective inhibitors of skin STS, but also liver STS when applied topically. These findings suggest that it may be possible to develop a formulation for the percutaneous administration of STS inhibitors, but also that this class of compound may have therapeutic potential for the treatment of a number of skin disorders.

Extracellular influences on tumour angiogenesis in the aged host.

Whether tumours are epithelial or non-epithelial in origin, it is generally accepted that once they reach a certain size all solid tumours are dependent upon a vascular supply to provide nutrients. Accordingly, there is great interest in how the extracellular environment enhances or inhibits vascular growth. In this minireview, we will examine key extracellular components, their changes with ageing, and discuss how these alterations may influence the subsequent development of tumour vasculature in the aged host. Because of the tight correlation between advanced age and development of prostate cancer, we will use prostate cancer as the model throughout this review.

Inhibition of steroid sulphatase activity in endometriotic implants by 667 COUMATE: a potential new therapy.

BACKGROUND: Local biosynthesis of estrogens is thought to be important for the maintenance and growth of endometriotic implants. In addition to the formation of estrogen via the aromatase pathway, steroid sulphatase (STS), which is responsible for the hydrolysis of estrogen sulphates, may be an important source of estrogens in endometriosis. METHODS: Eutopic and ectopic endometrial samples from 14 women with minimal or mild (MM) endometriosis and from 13 women with moderate to severe (MS) endometriosis were analysed for aromatase and STS activities. RESULTS: Aromatase and STS activity were detected in all samples. STS enzyme activity in both eutopic and ectopic endometrium was considerably higher and less variable than aromatase activity. Moreover, STS, but not aromatase, activity in endometriotic implants correlated with the severity of the disease (mean +/- SEM: 203 +/- 38 nmol/4 h/g wet weight tissue in MM disease versus 423 +/- 44 nmol/4 h/g wet weight tissue in MS endometriosis, P < 0.001). The STS inhibitor 667 COUMATE almost completely blocked STS activity (>99%) in both eutopic and ectopic tissues. CONCLUSIONS: The high levels of STS activity detected in ectopic endometrium and the correlation with severity of disease suggest that STS inhibitors could be useful for the treatment of endometriosis.

The therapeutic potential of a series of orally bioavailable anti-angiogenic microtubule disruptors as therapy for hormone-independent prostate and breast cancers.

Therapies for hormone-independent prostate and breast cancer are limited, with the effectiveness of the taxanes compromised by toxicity, lack of oral bioavailability and drug resistance. This study aims to identify and characterise new microtubule disruptors, which may have improved efficacy relative to the taxanes in hormone-independent cancer. 2-Methoxy-3-O-sulphamoyl-17beta-cyanomethyl-oestra-1,3,5(10)-triene (STX641), 2-methoxy-3-hydroxy-17beta-cyanomethyl-oestra-1,3,5(10)-triene (STX640) and 2-methoxyoestradiol-3,17-O,O-bis-sulphamate (STX140) were all potent inhibitors of cell proliferation in a panel of prostate and breast cancer cell lines. STX641 and STX640 significantly inhibited tumour growth in the MDA-MB-231 xenograft model. STX641 inhibited both in vitro and in vivo angiogenesis. Despite good in vivo activity, STX641 was not as potent in vivo as STX140. Therefore, STX140 was evaluated in the prostate hormone-independent PC-3 xenograft model. STX140 had superior efficacy to docetaxel, 2-MeOE2 and bevacizumab. In contrast to vinorelbine, no significant toxicity was observed. Furthermore, STX140 could be dosed daily over a 60-day period leading to tumour regression and complete responses, which were maintained after the cessation of dosing. This study demonstrates that STX641 and STX140 have considerable potential for the treatment of hormone-independent breast and prostate cancer. In contrast to the taxanes, STX140 can be dosed orally, with no toxicity being observed even after prolonged daily dosing.

Measurement of estrone sulfate in postmenopausal women: comparison of direct RIA and GC-MS/MS methods for monitoring response to endocrine therapy in women with breast cancer.

BACKGROUND: High concentrations of estrone sulfate (EIS) are present in serum of pre- and postmenopausal women. Most assays for this estrogen conjugate involve enzyme hydrolysis and chromatographic purification prior to RIA. We have compared concentrations of serum EIS in postmenopausal women measured by direct RIA or GC-MS/MS methods. PATIENTS AND METHODS: We analysed serum EIS concentrations using a direct 'ultrasensitive' RIA. Serum EIS concentrations were also measured by GC-MS/MS in which estrone conjugates are isolated using a solid-phase technique after which enzyme hydrolysis is employed to liberate estrone prior to GC-MS/MS analysis. RESULTS: We analysed 32 serum samples collected from 8 postmenopausal women participating in a Phase I trial of the steroid sulfatase inhibitor 667 COUMA TE. Concentrations of E1S were 998+/-86 pmol/l (mean +/- sem) and 912+/-114 pmol/l as measured by direct RIA and GC-MS/MS methods respectively. There was a highly significant correlation (r=0.96, p<0.001) between concentrations of EIS measured by the different methods. CONCLUSION: We conclude that the direct 'ultrasensitive' RIA for the measurement of serum EIS provides a reliable method for assaying serum concentrations of this estrogen conjugate and should be useful in monitoring the response to endocrine therapy in postmenopausal women with hormone-dependent breast cancer.

Tibolone and its delta-4, 7alpha-methyl norethisterone metabolite are reversible inhibitors of human aromatase.

Tibolone is used for the treatment of climacteric symptoms and osteoporosis in menopausal women. After ingestion, it is rapidly converted to a number of metabolites including 3alpha- and 3beta-hydroxy derivatives and the delta-4, 7alpha-methylnorethisterone (7alpha-MeNET) metabolite, which is rapidly cleared from circulation. Tibolone and some of its metabolites act in a tissue-selective manner to inhibit steroid sulphatase (STS) and 17beta-hydroxysteroid dehydrogenase Type 1 (17beta-HSD1) activities but also stimulate steroid sulphotransferase and 17beta-HSD2 activities. In the present study we have examined whether the ability of tibolone and its 7alpha-MeNET metabolites to regulate the activities of enzymes involved in oestrogen formation or inactivation extends to another key enzyme involved in oestrogen synthesis, the aromatase, which converts androstenedione to oestrone. Using JEG-3 choriocarcinoma cells, which have a high level of aromatase activity, tibolone and 7alpha-MeNET, but not the 3alpha- or 3beta-hydroxy metabolites, were found to inhibit aromatase activity in intact cells and also lysates prepared from these cells (up to 61% inhibition at 10muM). An investigation into the nature of aromatase inhibition by these compounds revealed that they inhibit aromatase activity by a reversible mechanism. Tibolone and 7alpha-MeNET also inhibited aromatase activity in MCF-7 breast cancer cells, which have a much lower level of aromatase activity than JEG-3 cells. It is concluded that, in addition to inhibiting STS and 17beta-HSD1, tibolone and 7alpha-MeNET may exert some of their tissue-selective effects in regulating oestrogen synthesis by also inhibiting aromatase activity.

In vivo inhibition of angiogenesis by sulphamoylated derivatives of 2-methoxyoestradiol.

Drugs that inhibit growth of tumours and their blood supply could have considerable therapeutic potential. 2-Methoxyoestradiol-3,17-O,O-bis-sulphamate (2-MeOE2bisMATE) has been shown to inhibit the proliferation of MCF-7 (ER+) breast cancer cells and angiogenesis in vitro. 2-MeOE2bisMATE and its analogue, 17-Cym-2-MeOE2MATE, were investigated for their ability to inhibit in vivo angiogenesis and tumour growth. The mouse Matrigel plug assay for angiogenesis was used to investigate the effect of compounds on neovascularisation and was quantified using a FITC-dextran injection technique. Nude mice bearing tumours derived from MCF-7 cells were used to assess efficacy on tumour growth. Tumour sections were stained for VEGFR-2 and Ki67 to assess tumour angiogenesis and cell proliferation respectively. Matrigel plugs supplemented with basic fibroblast growth factor resulted in increased neovascularisation over 7 days. Oral administration of 2-MeOE2bisMATE for 7 days at 10 or 50 mg kg(-1) significantly reduced neovascularisation to or below control levels respectively. 17-Cym-2-MeOE2MATE at 20 mg kg(-1) was equally effective. 2-MeOE2bisMATE, dosed daily for 21 days, caused a 52% reduction in tumour growth at 5 mg kg(-1) and 38% regression at 20 mg kg(-1). 17-Cym-2-MeOE2MATE (20 mg kg(-1)) reduced tumour growth by 92%. Immunohistochemistry revealed a reduction in angiogenesis and proliferation. Matrigel plug and tumour imaging after FITC-dextran injection indicated that 2-MeOE2bisMATE caused a marked disruption of vasculature. These sulphamoylated oestrogen derivatives have been shown to be potent inhibitors of angiogenesis in vivo. This, together with their ability to inhibit tumour growth, indicates the potential of this new class of drugs for further development for cancer therapy.