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Aim: The role of oestrogen metabolism and action in colorectal cancer (CRC) is controversial. An extensive review of the current literature, encompassing epidemiological evidence, systemic and peripheral oestrogen concentrations, 17β-hydroxysteroid dehydrogenase (17β-HSD) and aromatase in CRC, steroid sulphatase (STS)/oestrone sulphotransferase (EST) and in vitro and in vivo genomic effects was therefore undertaken., Methods: A literature search (key words: colorectal cancer, oestrogen, oestrogen receptor, 17β-HSD, STS, organic anion transporter) was performed using Embase, Medline, and Pubmed and papers were evaluated on scientific relevance on an individual basis., Results: Epidemiological data highlights that premenopausal women, or postmenopausal women taking hormone replacement therapy, are significantly less likely than males to develop CRC. This implies that oestrogen signalling is most likely involved in CRC physiology and aetiology. Little is known about oestrogen metabolism in the colon. However, the expression of 17β-HSD, STS, and EST, enzymes involved in oestrogen metabolism, have shown prognostic significance. Evidence also suggests that protective effects are modulated through oestrogen receptor beta, although which metabolite of oestrogen, oestradiol (E2) or oestrone (E1), is more active remains undefined. To complicate matters, the changes in the peripheral ratios of these enzymes, oestrogens and receptors most likely influences CRC progression., Conclusion: Epidemiological evidence, now supported by in vitro and in vivo studies, strongly associates oestrogen action and metabolism with CRC. Initially protective against CRC, once developed, results suggests that oestrogens increase proliferation. Consequently, hormone-ablation therapy, already successful against breast and prostate cancer, may be effective against CRC.

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyse the 17-position reduction/oxidation of steroids. 17β-HSD type 3 (17β-HSD3) catalyses the reduction of the weakly androgenic androstenedione (adione) to testosterone, suggesting that specific inhibitors of 17β-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia. STX2171 is a novel selective non-steroidal 17β-HSD3 inhibitor with an IC(50) of ∼200 nM in a whole-cell assay. It inhibits adione-stimulated proliferation of 17β-HSD3-expressing androgen receptor-positive LNCaP(HSD3) prostate cancer cells in vitro. An androgen-stimulated LNCaP(HSD3) xenograft proof-of-concept model was developed to study the efficacies of STX2171 and a more established 17β-HSD3 inhibitor, STX1383 (SCH-451659, Schering-Plough), in vivo. Castrated male MF-1 mice were inoculated s.c. with 1×10(7) cells 24 h after an initial daily dose of testosterone propionate (TP) or vehicle. After 4 weeks, tumours had not developed in vehicle-dosed mice, but were present in 50% of those mice given TP. One week after switching the stimulus to adione, mice were dosed additionally with the vehicle or inhibitor for a further 4 weeks. Both TP and adione efficiently stimulated tumour growth and increased plasma testosterone levels; however, in the presence of either 17β-HSD3 inhibitor, adione-dependent tumour growth was significantly inhibited and plasma testosterone levels reduced. Mouse body weights were unaffected. Both inhibitors also significantly lowered plasma testosterone levels in intact mice. In conclusion, STX2171 and STX1383 significantly lower plasma testosterone levels and inhibit androgen-dependent tumour growth in vivo, indicating that 17β-HSD3 inhibitors may have application in the treatment of hormone-dependent prostate cancer.

Estrogens and androgens are instrumental in the maturation of many hormone-dependent cancers. Consequently, the enzymes involved in their synthesis are cancer therapy targets. One such enzyme, steroid sulfatase (STS), hydrolyses estrone sulfate, and dehydroepiandrosterone sulfate to estrone and dehydroepiandrosterone respectively. These are the precursors to the formation of biologically active estradiol and androstenediol. This review focuses on three aspects of STS inhibitors: 1) chemical development, 2) biological activity, and 3) clinical trials. The aim is to discuss the importance of estrogens and androgens in many cancers, the developmental history of STS inhibitor synthesis, the potency of these compounds in vitro and in vivo and where we currently stand in regards to clinical trials for these drugs. STS inhibitors are likely to play an important future role in the treatment of hormone-dependent cancers. Novel in vivo models have been developed that allow pre-clinical testing of inhibitors and the identification of lead clinical candidates. Phase I/II clinical trials in postmenopausal women with breast cancer have been completed and other trials in patients with hormone-dependent prostate and endometrial cancer are currently active. Potent STS inhibitors should become therapeutically valuable in hormone-dependent cancers and other non-oncological conditions.

A chimeric approach is used to discover microtubule disruptors with excellent in vitro activity and oral bioavailability; a ligand-protein interaction with carbonic anhydrase that enhances bioavailability is characterised by protein X-ray crystallography. Dosing of a representative chimera in a tumour xenograft model confirms the excellent therapeutic potential of the class.

Background: Oxygen supplies capable of supporting transtracheal jet ventilators can be lifesaving. There is not much information about which oxygen sources (readily available inside and outside operating rooms) have sufficient driving pressure for transtracheal jet ventilation., Methods: We measured driving pressures (upstream or residual oxygen pressure) in a specially designed jet ventilation test system with a 2.25-mm (14-gauge) i.v. catheter. High-pressure oxygen sources evaluated included wall-mounted (Puritan, Allied Health, Precision, and Datex-Ohmeda) and anesthesia machine auxiliary oxygen flowmeters and oxygen flush valves from anesthesia machines (Draeger Narkomed 2B, Narkomed 4, Datex-Ohmeda Excel, and Datex-Ohmeda Modulus)., Results: All 4 types of wall-mounted oxygen flowmeters, opened past their highest scale settings (15 L/min), delivered sufficient working pressures (range, 103-282 kPa; 16-41 psi). Working pressures from auxiliary oxygen flowmeters mounted on Datex-Ohmeda machines were adequate to support jet ventilation (range, 189-248 kPa; 27-36 psi), whereas those on tested Draeger machines did not supply sufficient pressure for jet ventilation: Narkomed 2B, 14-28 kPa (2-4 psi); Narkomed 4, 24-28 kPa (3-4 psi). Working pressures delivered by oxygen flush valves on tested Draeger machines were adequate to support jet ventilation, ranging from 96 to 117 kPa (14-17 psi), whereas pressures generated by tested Datex-Ohmeda flush valves were not (ranging from 50 to 62 kPa, 7-9 psi)., Conclusion: Oxygen sources other than dedicated jet ventilator connectors to high-pressure pipeline oxygen may supply adequate working pressure, but each type of oxygen source needs testing to ensure that it supplies adequate working pressure.

Dissolved oxygen (DO) is one of the most important environmental variables of water quality, especially for marine life. Consequently, oxygen is one of the Chemical Quality Elements required for the implementation of European Union Water Framework Directive. This study uses the example of the Ria Formosa, a meso-tidal lagoon on the south coast of Portugal to demonstrate how monitoring of water quality for coastal waters must be well designed to identify symptoms of episodic hypoxia. New data from the western end of the Ria Formosa were compared to values in a database of historical data and in the published literature to identify long-term trends. The dissolved oxygen concentration values in the database and in the literature were generally higher than those found in this study, where episodic hypoxia was observed during the summer. Analysis of the database showed that the discrepancy was probably related with the time and the sites where the samples had been collected, rather than a long-term trend. The most problematic situations were within the inner lagoon near the city of Faro, where episodic hypoxia (<2 mg dm(-3) DO) occurred regularly in the early morning. These results emphasise the need for a balanced sampling strategy for oxygen monitoring which includes all periods of the day and night, as well as a representative range of sites throughout the lagoon. Such a strategy would provide adequate data to apply management measures to reduce the risk of more persistent hypoxia that would impact on the ecological, economic and leisure uses of this important natural resource.

Many anticancer drugs target microtubules and induce apoptosis. However, improved microtubule-targeting drugs, such as STX140 and STX641, are being developed. These compounds induce cell cycle arrest and apoptosis in a variety of tumour cells. The mechanisms that induce apoptosis and the key mediators involved are elucidated in this study. Results demonstrate that STX140 and STX641 depolarise mitochondrial bioenergetics and activate caspase 3/7 in A2780, LNCaP and MCF-7 cancer cells. Furthermore, both compounds cause a significant reduction in the expression of survivin and XIAP. This work details the temporal organisation of apoptosis induced by two microtubule disruptors and highlights the role that the down-regulation of survivin and XIAP may play in this process.

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are responsible for the pre-receptor reduction/oxidation of steroids at the 17-position into active/inactive hormones, and the 15 known enzymes vary in their substrate specificity, localisation, and directional activity. 17beta-HSD Type 3 (17beta-HSD3) has been seen to be over-expressed in prostate cancer, and catalyses the reduction of androstenedione (Adione) to testosterone (T), which stimulates prostate tumour growth. Specific inhibitors of 17beta-HSD3 may have a role in the treatment of hormone-dependent prostate cancer and benign prostate hyperplasia, and also have potential as male anti-fertility agents. A 293-EBNA-based cell line with stable expression of transfected human 17beta-HSD3 was created and used to develop a whole cell radiometric TLC-based assay to assess the 17beta-HSD3 inhibitory potency of a series of compounds. STX2171 and STX2624 (IC(50) values in the 200-450nM range) were two of several active inhibitors identified. In similar TLC-based assays these compounds were found to be inactive against 17beta-HSD1 and 17beta-HSD2, indicating selectivity. A novel proof of concept model was developed to study the efficacy of the compounds in vitro using the androgen receptor positive hormone-dependent prostate cancer cell line, LNCaPwt, and its derivative, LNCaP[17beta-HSD3], transfected and selected for stable expression of 17beta-HSD3. The proliferation of the parental cell line was most efficiently stimulated by 5alpha-dihydrotestosterone (DHT), but the LNCaP[17beta-HSD3] cells were equally stimulated by Adione, indicating that 17beta-HSD3 efficiently converts Adione to T in this model. Adione-stimulated proliferation of LNCaP[17beta-HSD3] cells was inhibited in the presence of either STX2171 or STX2624. The compounds alone neither stimulated proliferation of the cells nor caused significant cell death, indicating that they are non-androgenic with low cytotoxicity. STX2171 inhibited Adione-stimulated growth of xenografts established from LNCaPwt cells in castrated mice in vivo. In conclusion, a primary screening assay and proof of concept model have been developed to study the efficacy of 17beta-HSD3 inhibitory compounds, which may have a role in the treatment of hormone-dependent cancer. Active compounds are selective for 17beta-HSD3 over 17beta-HSD1 and 17beta-HSD2, non-androgenic with low toxicity, and efficacious in both an in vitro proof of concept model and in an in vivo tumour model.

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.

Steroid sulfatase (STS) regulates the hydrolysis of steroid sulfates to their unconjugated forms. Estrone sulfate and dehydroepiandrosterone sulfate can be hydrolyzed by STS to estrone and dehydroepiandrosterone, respectively, with these steroids being the precursors for the synthesis of more biologically active estrogens or androgens. A number of potent STS inhibitors have now been developed including STX64, which entered a phase I trial for the treatment of postmenopausal women with advanced metastatic hormone-dependent breast cancer. The results from this phase I trial were encouraging, suggesting that STS inhibitors may also have a role in the treatment of other hormone-dependent cancers including those of the endometrium, ovary, and prostate. In this paper the potential use of STS inhibitors to treat these hormone-dependent cancers is reviewed. In addition, results from in vitro studies show that Ishikawa endometrial cancer cells, OVCAR-3 ovarian cancer cells, and LNCaP prostate cancer cells all possess significant STS activity. Furthermore, STS activity in these cells can be almost completely inhibited by STX64 or the second-generation STS inhibitor, STX213. Results from these investigations therefore suggest that STS inhibitors could have therapeutic potential for the treatment of a range of hormone-dependent cancers.

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 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.

Purpose: The production of E2 is paramount for the growth of estrogen receptor-positive breast cancer. Various strategies have been used, including the use of enzyme inhibitors against either aromatase (AROM) or steroid sulfatase (STS), in an attempt to ablate E2 levels. Both these enzymes play a critical role in the formation of estrogenic steroids and their inhibitors are now showing success in the clinic., Experimental Design: We show here, in a xenograft nude mouse model, that the inhibition of both enzymes using STX681, a dual AROM and STS inhibitor (DASI), is a potential new therapeutic strategy against HDBC. MCF-7 cells stably expressing either AROM cDNA (MCF-7(AROM)) or STS cDNA (MCF-7(STS)) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of either androstenedione (A(4)) or E2 sulfate and bearing either MCF-7(AROM) or MCF-7(STS) tumors were orally treated with STX64, letrozole, or STX681. Treatment was administered for 28 days. Mice were weighed and tumor measurements were taken weekly., Results: STX64, a potent STS inhibitor, completely blocked MCF-7(STS) tumor growth but failed to attenuate MCF-7(AROM) tumor growth. In contrast, letrozole inhibited MCF-7(AROM) tumors but had no effect on MCF-7(STS) tumors. STX681 completely inhibited the growth of both tumors. AROM and STS activity was also completely inhibited by STX681, which was accompanied by a significant reduction in plasma E2 levels., Conclusions: This study indicates that targeting both the AROM and the STS enzyme with a DASI inhibits HDBC growth and is therefore a potentially novel treatment for this malignancy.

The steroid sulfatase (STS) enzyme plays a pivotal role in the formation of biologically active steroid hormones. Its involvement in the hydrolysis of estrone sulfate and dehydroepiandrosterone sulfate to estrone and dehydroepiandrosterone, respectively, is an important step in the formation of estradiol and androstenediol, both of which are estrogenic steroids that can stimulate tumor growth. Consequently, as STS is widely distributed throughout the entire body, it has a substantial influence on hormone-dependent cancer mitogenesis. It is a useful prognostic marker of disease as a significant majority of breast tumors over-express the enzyme and there are indications of STS having a role in prostate cancer. This knowledge has led to the development of potent STS inhibitors for use as anti-cancer agents. There are now several steroidal and non-steroidal STS inhibitors available. New in vivo models, using ovariectomized female nude mice, have been developed to pre-clinically test these inhibitors. These studies have demonstrated the excellent efficacy and effect of STS inhibitors on breast carcinoma development. Recently, 667 COUMATE, an irreversible type of inhibitor which utilizes a phenol sulfamate ester as its active pharmacophore, has completed a Phase I clinical trial in postmenopausal women with breast cancer. These studies have indicated the potential clinical benefit for the use of STS inhibitors. Most pre-clinical and clinical studies have focused on breast cancer as the target for STS inhibition. However, there are other hormone-dependent malignancies, such as endometrial and prostate cancer, that could in the future be treated with these new potent STS inhibitors.

Breast cancer is the leading cause of cancer deaths among women worldwide. The theory of targeting both cancer cells directly and their blood supply has significant therapeutic potential. However, to date, there are few clinically successful single agents that meet these criteria. 2-Methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE) and 2-ethylestradiol-3,17-O,O-bis-sulfamate (2-EtE2bisMATE) are potent inhibitors of proliferation in a range of cancer cells. The work presented here demonstrates the potent in vitro and in vivo effects of these compounds. They cause apoptosis via the intrinsic mitochondrial pathway in both MDA-MB-231 breast cancer cells and endothelial cells. Furthermore, they are potent anti-angiogenic inhibitors in vivo, as shown by their ability to reduce endothelial staining in MDA-MB-231 xenograft tumors. We have developed a novel, flow cytometry based, ex vivo method which shows in cells recovered from MDA-MB-231 tumors treated with 2-MeOE2bisMATE and 2-EtE2bisMATE an increase in intra-tumoral G(2)-M arrest and apoptosis. The degree of apoptosis inversely correlates to tumor volume. Further in vivo studies reveal that both 2-MeOE2bisMATE and 2-EtE2bisMATE are orally bioavailable and extremely efficacious when compared to clinically tested drugs. As these compounds are anti-proliferative against breast cancer and endothelial cells they have the potential to be potent, dual acting clinical drugs of the future.

Estrogenic steroids, such as estradiol, are known to play a crucial role in the development and growth of hormone-dependent breast cancer. Steroid sulfatase (STS) inhibitors that can prevent the biosynthesis of these steroids via the sulfatase pathway offer therapeutic potential. We show here the in vivo profile, including the efficacy in a xenograft breast cancer model and pharmacokinetics, of three potent STS inhibitors. MCF-7 cells stably over-expressing STS cDNA (MCF-7STS) were generated. Ovariectomised, MF-1, female nude mice receiving subcutaneous injections of estradiol sulfate (E2S) and bearing MCF-7STS xenografts, were orally treated with the STS inhibitors STX64, STX213, and STX1938. Treatment was administered once weekly at a dose of 1 mg/kg for 35 days during which animals received E2S thrice weekly. Mice were weighed and tumor measurements taken weekly. Furthermore, the pharmacokinetics for STX213 was determined in rats. STX213 and STX1938 exhibited potent STS inhibition in vivo. However, STX1938 demonstrated a greater duration of activity. In vehicle treated nude mice receiving E2S, tumor volumes increased by 260% after 35 days compared to day zero. STX64 (1 mg/kg) failed to reduce tumor growth when given once weekly. STX213 and STX1938 (once weekly, 1 mg/kg) significantly inhibited (P < 0.05) tumor growth over this same time period. These compounds completely inhibited liver and tumor STS activity and significantly reduced the levels of plasma E2. This study indicates that the STS inhibitor, STX213, exhibits excellent efficacy and pharmacokinetics and therefore offers a potentially novel treatment for hormone-dependent breast cancer.

The past few years have seen an increase in the reported incidence of endometrial carcinoma, one of the most frequently diagnosed malignancies of the female genital tract. Estrogen production is vital for the mitogenesis of endometrial tumors. Inhibition of steroid sulfatase (STS), an enzyme responsible for the synthesis of steroids with estrogenic properties, may represent a novel therapeutic target for this type of cancer. This study investigates the effects of STX64 (also known as 667Coumate and BN83495) and STX213, two potent STS inhibitors, on hormone-dependent endometrial cancer cell growth in vivo. When tested in intact mice with endometrial cancer xenografts, STX64 had limited effect on tumor growth. In contrast, the microtubule disruptor STX140 reduced tumor growth by 55%. In a hormone-dependent endometrial xenograft model in ovariectomized mice, both STX64 and STX213 given orally, daily at 1 mg/kg significantly inhibited tumor growth by 48 and 67%, respectively. However, when given orally at 1 mg/kg once weekly, only STX213 still inhibited tumor proliferation. At a higher dose of STX64 (10 mg/kg, orally, daily), a greater tumor growth inhibition of 59% was observed. Liver and tumor STS activity was completely inhibited in all daily treatment groups. Plasma estradiol (E2) levels were also significantly decreased. A significant correlation was observed between plasma E2 concentrations and STS activity, indicating the importance of circulating E2 on tumor growth. This novel study demonstrates for the first time that STS inhibitors are potent inhibitors of endometrial cancer growth in nude mice.

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer., ((c) 2008 Wiley-Liss, Inc.)

Unlabelled: This study characterises two recently developed anticancer agents in vitro and in vivo, 2-methoxyoestra-1,3,5(10), 16-tetraene-3-carboxamide (IRC-110160) and STX140., Materials and Methods: Hormone-dependent (MCF-7), hormone-independent (MDA-MB-231) and P-glycoprotein overexpressing (MCF-7Dox) cells were used for proliferation experiments. For the tumour efficacy studies, female nude mice were inoculated with MDA-MB-231 cells., Results: IRC-110160 is a potent inhibitor of both MCF-7 and MDA-MB-231 cell proliferation. Furthermore, the potency of IRC-110160 was unaffected by the over-expression of the P-glycoprotein drug efflux pump. IRC-110160 and 2-methoxyoestradiol-3,17-O,O-bis-sulfamate (STX140) induced apoptosis in a similar timeframe in the MDA-MB-231 cell line, but only STX140 caused G2/M arrest in these cells. In the MDA-MB-231 xenograft model 300 mg/kg p.o. (daily) of IRC-110160 and 20 mg/kg p.o. STX140 (daily) both completely inhibited tumour growth; however some toxicity was observed with IRC-110160. After 28 days of daily dosing STX140 (20 mg/kg p.o.) had minimal effect on the white blood population of mice with tumours. The masking of STX140 from white blood cells may be due to its interaction with carbonic anhydrase II (CAII) in the red blood cells. In contrast to STX140, IRC-110160 does not inhibit CAII. These studies highlight the activity of two orally bioavailable anti-cancer agents one of which, STX140, may offer a significant clinical advantage over existing drugs as a common dose limiting factor, haemotoxicity, may be minimised.

The synthesis, SAR, and preclinical evaluation of 17-cyanated 2-substituted estra-1,3,5(10)-trienes as anticancer agents are discussed. 2-Methoxy-17beta-cyanomethylestra-1,3,5(10)-trien-3-ol ( 14), but not the related 2-ethyl derivative 7, and the related 3- O-sulfamates 8 and 15 display potent antiproliferative effects (MCF-7 GI 50 300, 60 and 70 nM, respectively) against human cancer cells in vitro. Investigation of the SAR reveals that a sterically unhindered hydrogen bond acceptor attached to C-17 is most likely key to the enhanced activity. Compound 8 displayed significant in vitro antiangiogenic activity, and its ability to act as a microtubule disruptor was confirmed. Inhibitory activity of the sulfamate derivatives against steroid sulfatase and carbonic anhydrase II (hCAII) was also observed, and the interaction between 15 and hCAII was investigated by protein crystallography. The potential of these multimechanism anticancer agents was confirmed in vivo, with promising activity observed for both 14 and 15 in an athymic nude mouse MDA-MB-231 human breast cancer xenograft model.

Unlabelled: There is a continued need for orally bioavailable anticancer compounds that exhibit good efficacy against breast cancer. STX140, a derivative of 2-methoxyestradiol (2-MeOE2), has been shown to have excellent oral bioavailability and significantly reduces tumor growth. A new micronized formulation of STX140 has now been developed and its pharmacokinetics (PK) in rats and effect on MDA-MB-231 breast cancer growth in nude mice was investigated., Materials and Methods: For the PK studies, female Wistar rats were treated orally with STX140 in two separate vehicles (10% tetrahydrofuran (THF) in propylene glycol (PG) or 0.5% methyl cellulose (MC) in saline) and plasma samples taken for high performance liquid chromatography analysis over 48 h. For the tumor efficacy studies, female nude mice were inoculated with MDA-MB-231 breast cancer cells and then treated orally with a range of doses of STX140., Results: The PK studies demonstrated that the THF/PG vehicle resulted in a greater oral bioavailability of STX140 compared to the 0.5% MC vehicle. However, this was not translated to the tumor efficacy studies where STX140 at 20 mg/kg in either vehicle caused a significant reduction in tumor volume., Conclusion: The new micronized formulation of STX140 is orally bioavailable and efficacious at inhibiting MDA-MB-231 breast tumor growth.

The endocrine system and its steroids have long been thought to be instrumental in the etiology of breast cancer. A large proportion of cancerous breast tissues have been shown to express estrogen (ER), androgen (AR) and progesterone (PR) receptors. It is through these receptors that steroid hormones can exert their mitogenic effects. The local biosynthesis of estrogens is believed to play an integral part in the development of hormone-dependent breast cancer and recent studies on the use of inhibitors to block this steroid production has yielded an improvement of prognosis in breast cancer patients. Consequently, the understanding of the enzymes involved in the synthesis and metabolism of estrogens in breast cancer is paramount to treating this malignancy. This review examines the biological and clinical relevance of three key endocrine enzymes: steroid sulfatase (STS), aromatase (Arom), and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type-1. The importance of the over expression and increased activity of these enzymes in breast tissue and on breast cancer is discussed. Importantly, the intratumoral biosynthesis of estrogens is examined in detail. The effects of new inhibitors of these enzymes on the growth of hormone-dependent breast cancer will also be investigated. First and second generation STS inhibitors and third generation aromatase inhibitors are showing significant promise, whereas inhibitors for 17beta-HSD type-1 are still at an early stage. However, such endocrine therapy that is currently being explored has shown promising results for patients with hormone-dependent breast cancer.

Purpose: The aim of these studies was to characterize the action of STX140 in a P-glycoprotein-overexpressing tumor cell line both in vitro and in vivo. In addition, its efficacy was determined against xenografts derived from patients who failed docetaxel therapy., Experimental Design: The effects of STX140, Taxol, and 2-methoxyestradiol (2-MeOE2) on cell proliferation, cell cycle, and apoptosis were assessed in vitro in drug-resistant cells (MCF-7(DOX)) and the parental cell line (MCF-7(WT)). Mice bearing an MCF-7(DOX) tumor on one flank and an MCF-7(WT) tumor on the other flank were used to assess the in vivo efficacy. Furthermore, the responses to STX140 of three xenografts, derived from drug-resistant patients, were assessed., Results: In this study, STX140 caused cell cycle arrest, cyclin B1 induction, and subsequent apoptosis of both MCF-7(DOX) and MCF-7(WT) cells. Taxol and 2-MeOE2 were only active in the MCF-7(WT) parental cell line. Although both STX140 and Taxol inhibited the growth of xenografts derived from MCF-7(WT) cells, only STX140 inhibited the growth of tumors derived from MCF-7(DOX) cells. 2-MeOE2 was ineffective at the dose tested against both tumor types. Two out of the three newly derived docetaxel-resistant xenografts, including a metastatic triple-negative tumor, responded to STX140 but not to docetaxel treatment., Conclusions: STX140 shows excellent efficacy in both MCF-7(WT) and MCF-7(DOX) breast cancer xenograft models, in contrast to Taxol and 2-MeOE2. The clinical potential of STX140 was further highlighted by the efficacy seen in xenografts recently derived from patients who had failed on taxane therapy.

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.

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.

The effect of EGF and gefitinib on two EGFR-positive human bladder cancer cell lines has been investigated using array-based gene expression profiling. The most prominent transcript, increased up to 6.7-fold by EGF compared with controls in RT112 cells, was human early growth response protein 1 (hEGR1). This induction was prevented by gefitinib. The hEGR1 mRNA in EGF-treated samples was reduced in the presence of gefitinib, as was hEGR1 protein in cell lysates. In the RT4 cells, hEGR1 expression was halved in the presence of EGF and gefitinib in combination. In bladder tumour samples, there was a significant correlation between hEGR1 mRNA detected by RT-PCR and EGFR detected by ligand binding, (P=0.042). The induction by EGF of the hEGR1 gene, mRNA and protein in RT112 cells, and its inhibition by gefitinib, together with the detection of hEGR1 mRNA in bladder tumours, suggests that hEGR1 may be important in the EGFR growth-signalling pathway in bladder cancer and should be further investigated for its prognostic significance and as a potential therapeutic target.

Purpose: Steroid sulfatase (STS) inhibitors that can decrease or prevent the biosynthesis of estrogenic steroids via the sulfatase route may play an important role in the treatment of breast cancer. We compare the in vivo efficacy of two potent STS inhibitors, STX64 and STX213, in a xenograft breast cancer model., Experimental Design: MCF-7 cells stably expressing STS cDNA (MCF-7STS) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of estradiol sulfate (E2S) and bearing both MCF-7STS and wild-type MCF-7 (MCF-7WT) tumors were orally treated with STX64 and STX213. Treatment was given for 49 days followed by a recovery period of 35 days in which animals received only E2S. Mice were weighed, and tumor measurements were taken weekly., Results: STX64 and STX213 exhibited potent STS inhibition in vivo. However, STX213 showed a greater duration of activity. In vehicle-treated nude mice receiving E2S, tumor volumes increased 5.5-fold for MCF-7WT and 3.8-fold for MCF-7STS after 49 days compared with day 0. MCF-7WT tumor growth was reduced by 56% by STX213 over the dosing period, and subsequent growth was retarded during the recovery period. All treatments fully inhibited growth of MCF-7STS tumors, and recovery of these tumors was significantly retarded (P<0.01). All compounds completely inhibited liver and tumor STS activity. Additionally, STS mRNA expression in the MCF-7STS tumors directly correlated with the corresponding STS enzyme activity., Conclusions: This study indicates that STS inhibitors attenuate hormone-dependent human breast cancer growth and therefore offer a potentially novel treatment for this condition.

Nerve growth factor (NGF) is a potent mediator of inflammatory hyperalgesia, in addition to roles in the development and maintenance of neurons. We provide evidence for the novel concept that microvascular endothelial cells play a critical primary role in NGF-mediated events that lead to inflammatory hyperalgesia in rat skin. We show that, surprisingly, neutrophils are not directly activated by NGF, although local administration of NGF mediates thermal hyperalgesia via mechanisms involving concomitant neutrophil accumulation. The co-administration of actinomycin D with NGF negated both neutrophil accumulation and thermal hyperalgesia, indicating the dependence of NGF on local de novo protein synthesis. More significantly, an antibody against the endothelial-derived adhesion molecule ICAM-1 also blocked neutrophil accumulation and thermal hyperalgesia. Finally the ability of NGF to stimulate ICAM-1 in human cultured umbilical vein endothelial cells is shown. We propose that NGF acts primarily to activate endothelial cells and that this response is essential for the ensuing neutrophil accumulation and hyperalgesia. The findings reveal a central role of the endothelial cell in initiating NGF-dependent inflammatory hyperalgesia and emphasize the importance of further investigations aimed at examining the feasibility of new therapeutic strategies that target NGF.

Nerve growth factor (NGF) regulates the survival and development of specific populations of neurones and is involved in wound healing. A further area of study relating to the role of neurotrophins in the mature animal has concerned the possibility that NGF may be a pivotal mediator of inflammation and pain. It has previously been shown that injection of intradermal NGF can result in a neutrophil-dependent hyperalgesia in the rat. The purpose of the present study was to examine the pathological consequence of NGF injected intradermally into mature rat skin and to examine further the role of neutrophils. Standard histopathology techniques (H & E) were employed to determine inflammatory cell counts. Circulating neutrophils were depleted using an anti-rat neutrophil antiserum and results were compared to treatment with vehicle controls. Saline-pretreated rats exhibited normal circulating neutrophil numbers and the dorsal skin showed a significant increase of neutrophil and macrophages at 3 and 5 h and lymphocytes at 5 h after NGF treatment. By comparison, skin sites from neutrophil-depleted rats did not demonstrate a significant increase in neutrophil and macrophage accumulation after NGF administration. All NGF-treated sites, independent of pretreatment, demonstrated abnormal muscle fibre morphology and proliferation of the muscle sarcolemmal nuclei after NGF injection, indicative of tissue injury. In addition, oedema and some fibroplasia were also noted. Furthermore, fibrin production was increased at 3 and 5 h after NGF administration. It is suggested that NGF has a damaging effect on rat muscle which is independent of accumulating neutrophil and other inflammatory cells. In conclusion, the findings indicate a link between NGF-induced neutrophil and macrophage accumulation, as the increase in dermal macrophages was not observed in neutrophil-depleted rats. The results also suggest that NGF can have a profound effect on rat muscle and that this effect may be related to muscle regeneration., (Copyright 2002 John Wiley & Sons, Ltd.)

Patient safety depends on the skills, vigilance, and judgment of trained individuals working as members of a clinical team that includes anesthesiologists, surgeons, nurses, and technicians. Now, as never before, safe outcome depends both on better knowledge and better management. This requires organization of caregivers, who may be strangers from diverse disciplines, into teams. One can drill an individual to work safely alone. One can rehearse a series of scenarios with small groups (who regularly work together) to improve performance. But what does one do with an unrehearsed group, called together in an emergency from several different disciplines, usually including Anesthesia. These people may not know each other, their roles, their special skills, and may even be hazy about each other's goals. Rapid organization of such an ad hoc team becomes a critical priority where patient safety is at stake. The way by which such an ad hoc team from several disciplines can rapidly be helped to function effectively together is by teaching all the "strangers" the principles of Crisis Resource Management. These principles are not as well-presented in a written text or lecture format, as one cannot introduce the sense of urgency that emotionally charges and changes the impact. We believe the best teacher is experience gained in a realistic simulated environment using a model driven, full human simulator. This simulated environment is safe for both patient and trainee.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.

Background and Objectives: In two patients, one scheduled for epidural anesthesia and the other for placement of a spinal catheter for operative procedures, severe postdural puncture headache developed and was refractory to conservative therapy., Methods: The first patient had several unintentional dural punctures, and the second underwent a planned dural puncture with an 18-gauge needle for insertion of a 20-gauge catheter. When neither patient responded to conservative therapy following development of postdural puncture headache, an infusion of adrenocorticotropic hormone (ACTH) was given prior to consideration of epidural blood patching., Results: Both patients obtained complete and permanent relief from their headaches., Conclusion: A single treatment with ACTH may offer an alternative therapy in the treatment of postdural puncture headache.

although the waveform derived from a peripheral pulse monitor or pulse oximeter may resemble an arterial pressure waveform, it is in fact a visualization of blood volume change in transilluminated tissue caused by passage of blood: an indication of perfusion or blood flow. Most currently available pulse oximeters indicate this flow, but few display it in usable form. Since adequate tissue blood flow is a prerequisite for normal metabolic activity, it is a parameter that should merit a place in standard anesthesia or intensive care monitors. That the peripheral tissue blood flow is not routinely displayed may be in part due to the difficulty in quantifying data obtained: flow is not accurately measured as simply as pressure, even by invasive means. It is in the pattern of the waveform that beat-to-beat changes in stroke volume can be better seen than measured, or in the interaction of ventilation and circulation that tests general circulatory performance. The origin and interpretation of these changes are discussed and illustrated with examples. We indicate how new physiological tests of autonomic function and cardiac preload can be developed using pulse plethysmography. The importance and application of the Valsalva effect on the waveform is emphasized. This effect is particularly applicable for monitoring adequate fluid loading and the action of vasodilator drugs, which are both important in anesthesia. Differences between the arterial pulse pressure wave and tissue flow wave are discussed, as well as the cause of certain artifacts, including the wandering dicrotic notch.

A 43-year-old white female underwent orthotopic liver transplantation in 1992 for cirrhosis related to primary sclerosing cholangitis. Pre-transplantation protein S (PS) studies revealed a discrepancy between PS activity and free PS antigen consistent with type II PS deficiency. Since the presence of activated protein C (APC) resistance has been reported to interfere with PS activity assays resulting in an apparent type II PS deficiency, we retrospectively tested a pre-transplantation frozen plasma sample for APC resistance. The sample was found to have an abnormal APC resistance ratio (APCR-R) of 1.71. Follow up testing 2 1/2 years post-transplantation revealed correction of the APC resistance phenotype (normalization of the APCR-R to 2.79). Analysis of DNA extracted from lymphocytes revealed the patient to be heterozygous for the FV mutation associated with APC resistance (FV Leiden). Hereditary APC resistance was confirmed by family studies which revealed the presence of APC resistance and heterozygous FV Leiden in her son. Although the patient's post-transplantation plasma FV is normal, her platelet FV remains heterozygous for FV Leiden. To what extent, if any, platelet FV Leiden in the absence of plasma FV Leiden may contribute to a predisposition to thrombosis is unknown.

An international registry was established by the Subcommittee on von Willebrand Factor of the SSC/ISTH on the treatment of patients with types of von Willebrand disease (vWD) unresponsive to DDAVP infusion. Data was collected on 76 surgical events in 64 patients from 19 treatment centers. Thirty-three non-mucosal, 12 mucosal, 10 orthopedic and 21 dental procedures were reported. In the 76 surgical events, 14 cases prophylactically received cryoprecipitate while 62 received factor VIII (FVIII) concentrate. Surgical hemostasis was reported as satisfactory, good, or excellent in 75 of the 76 cases. Post-infusion bleeding times were measured in only three of 14 surgical events treated with cryoprecipitate. All three cases had a reduction but not correction of the bleeding time. The post-infusion bleeding time was measured in 27 of 62 cases in which FVIII concentrates were used. The bleeding time time was normalized in 15, reduced but not normalized in eight, and not changed from baseline in four. Data was also collected from 16 treatment centers on 50 serious bleeding events in 35 patients. These included 19 gastrointestinal, 15 other mucosal, four central nervous system, seven orthopedic, and five other mucosal, four central nervous system, seven orthopedic, and five other bleeds. Eleven cases received cryoprecipitate and 39 received FVIII concentrate as primary therapy. The efficacy of treatment was considered good or excellent in 49 of 50 cases. Post-infusion bleeding times were measured in only 15 of the 50 reported bleeding events. The post-infusion bleeding time was normalized in six, decreased but not normalized in eight, and not changed from baseline in one. In this retrospective survey, FVIII concentrates were subjectively as efficacious as cryoprecipitate in both the surgical setting and for the treatment of severe bleeds in patients with types of vWd unresponsive to DDAVP. Since the bleeding time was monitored during therapy in only a minority of these cases, a definitive relationship between the efficacy of therapy and normalization or reduction of the bleeding time in these two clinical settings cannot be established from this study. A prospective study on the use of FVIII and/or vWf concentrates in these clinical settings is necessary to address this issue.

A term infant had a life-threatening inferior venal caval thrombosis during the first 24 hours of life. The plasma from the infant and his mother was found to be resistant to activated protein C and to be heterozygous for the factor V mutation (FV Leiden) associated with this disorder. The presence of this hereditary disorder should be considered in infants with thrombosis and in infants with conditions predisposing them to thrombosis.

The prevalence of the Factor V (FV) mutation associated with activated protein C resistance (FV Leiden) and its significance as a genetic risk factor for venous thrombosis have necessitated the development of a simple, rapid, and accurate assay for its detection. The polymerase chain reaction with sequence specific primers (PCR-SSP) provides a powerful technique for the discrimination of alleles resulting from single base substitutions. PCR amplification was performed using a sense primer complementary to both FV alleles coupled with either of two antisense allele specific primers, one complementary to the normal FV allele and one complementary to the FV Leiden allele. PCR conditions were developed that favored amplification only in the case of perfect complementation between template DNA and allele specific primer. The FV genotype was assigned based on whether or not each allele specific primer set produced an amplified product. Assignment of genotypes correlated 100% with those determined by the method of PCR amplification followed by Mn1I digestion. PCR-SSP allows the rapid and accurate identification of carriers of the Factor V Leiden mutation by a simple PCR reaction without the need for the usual post-amplification specificity step.

An international registry was established on the reproductive health of women with types of von Willebrand disease (vWd) unresponsive to DDAVP. Data was collected on 44 women from 16 treatment centers in nine countries. Severe menorrhagia requiring blood product therapy occurred at least once in 80% of the women for whom data was reported. Most of the reported episodes occurred prior to the diagnosis of vWd and/or the use of oral contraceptive (OC) therapy. OC therapy was clinically effective in the treatment of chronic menorrhagia in 22 of 25 (88%) women treated. Two of the women, however, were unable to tolerate chronic OC use and a third became refractory to treatment. Hysterectomy was performed in 10 of the 44 women (23%). The reported indication for six of the procedures was menorrhagia. Seventy percent were performed at two treatment centers, suggesting different thresholds for the performance of the procedure. There were 69 pregnancies reported in 31 of the 44 women. Fifteen of the pregnancies resulted in spontaneous abortions. The incidence of miscarriage was 22% and appeared clustered, with 10 of 15 of the miscarriages occurring in just four of the women.

Obstacles to the establishment of school-based clinics are investigated to determine possible strategies for the future. The current social environment necessitates a health care model that provides increased access and improved outcomes for children in particular. The advanced registered nurse practitioner is an integral part of the school-based clinic. Although opposition to school-based clinics has been well organized in the past, proponents are learning new ways to deal with this challenge.

Lumbar epidural analgesia was administered to 60 ASA class 1 & 2 patients with 3 ml test dose of 1.5% lidocaine and bolus of 20 ml of 0.5% lidocaine containing 0.5 microgram/kg sufentanil. Bilateral decreased lumbar cold perception was accepted as evidence of analgesia despite persisting pinprick sensation in thoracic dermatomes. Oxygen saturation (SpO2), respiratory rate, cardiovascular parameters and leg muscle strength were monitored throughout and until 1 hour afterwards. Midazolam provided light sedation and atropine bradycardia control. Verbal communication was maintained. ESWL could start within 6-10 minutes of bolus, with analgesia adequate in 86% of patients, the rest being "rescued" with 5-10 ml 0.5% lidocaine or analgesic doses (20-30 mg IV) of ketamine. Leg weakness developed in 14%, with 1 patient fully paralyzed. All resolved within 1 hour. Topical urethral analgesia was used in males where cystoscopy preceded ESWL. Phenylephrine was required once for nild systolic hypotension, otherwise blood pressures were stable. Two of 4 patients experiencing pruritus needed naloxone relief. Itching appeared in skin recovering from sensory block while visceral analgesia persists. Excessive respiratory depression was not seen.

The major objective of the present study was to determine the effects of a partial structure of the lipid A moiety of gram-negative lipopolysaccharide, monophosphoryl lipid A (MLA), on endotoxin-induced mortality and disseminated intravascular coagulation (DIC) in rats. A second objective was to examine the role of polymorphonuclear neutrophil invasion to visceral organs, including lung, liver, heart, and kidney in the pathogenesis of the compromised multiorgan function which occurs in endotoxic shock. Finally, a third aim was to determine if the potential protective effects of MLA might be mediated via inhibiting neutrophil invasion to various visceral organs. Male Sprague-Dawley rats (220-260 g) were fasted over night and used the following day. In control rats, endotoxin (S. abortus equi LPS, 15 mg/kg, i.v.) produced a 89% mortality at 48 hr following its administration, and gross pathological and laboratory signs of DIC at 3 hr after injection. The latter included increased serum fibrin(ogen) degradation products (FDP, 24.00 +/- 7.81 vs. 0 micrograms/ml, P < .05), prothrombin time (PT, 16.20 +/- 1.12 vs. 13.03 +/- 0.20 sec, P < .05), and activated partial thromboplastin time (APTT, 32.70 +/- 3.83 vs. 20.11 +/- 0.60 sec, P < .05), and decreased plasma fibrinogen (233.2 +/- 41.6 vs. 406.3 +/- 23.2 mg/dl, P < .05) as well as evidence of gross visceral hemorrhage. Pretreatment with MLA (5 mg/kg) for 24 hr produced a marked reduction in endotoxin-induced mortality at 48 hr (0% versus 89% in controls) and inhibited all of the manifestations of DIC produced by endotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Eleven women with hyperprolactinemic amenorrhea were treated with a combined estrogen/progestogen preparation (Loestrin 30) for 3 months as hormone replacement therapy because of estrogen deficiency, with a view to protection against osteoporosis. Serum prolactin levels rose during the 1st month of treatment (p < 0.05) but did not rise significantly further during the 2nd and 3rd months. The levels rose in proportion to pretreatment levels by 28% (median), and fell significantly but not completely during the 1-week treatment-free intervals. After the study period, prolactin values appeared to remain stable in those women who continued longer on treatment, and returned to around pretreatment values in those who stopped. In one woman there was radiological evidence of pituitary tumor growth during treatment. This study shows that estrogen/progestogen treatment in standard contraceptive dosage usually leads to only moderate and non-progressive stimulation of pituitary activity in women with hyperprolactinemic amenorrhea, but occasional excessive growth of a prolactinoma can occur and treatment needs to be monitored. Women with relatively high prolactin levels seem to be at particular risk. Safer variations of estrogen therapy such as lower dosage or combination with a protective low dose of a dopamine agonist should also be considered.

A recombinant human von Willebrand factor (vWF) cDNA fragment library was constructed in lambda gt11 for the localization of anti-vWF monoclonal antibody epitopes. Twelve of 21 monoclonal antibodies screened identified epitopes expressed in lambda gt11 as beta-galactosidase fusion proteins. By sequence analysis, these antigenic determinants were localized to segments ranging from 17 to 105 amino acids in length. Four epitopes apparently shared by more than one antibody were identified, suggesting the presence of immuno-dominant epitopes within vWF. Monoclonal antibody C3, which blocks factor VIII (FVIII) binding to vWF, bound to the same epitope previously identified by a second monoclonal antibody which also blocks this function, suggesting that this region may be at or near the vWF/FVIII binding domain. Three antibodies recognize the same region within the vWF A2 repeat. Mutations near this region appear to be responsible for Type IIA von Willebrand's disease. The co-localization of these antibodies suggests that this domain might be exposed on the surface of vWF, consistent with its apparent increased sensitivity to plasma proteases.

The monoclonal anti-factor VIII (FVIII) antibody C4 has previously been reported to inhibit the binding of purified FVIII to immobilized von Willebrand factor (vWF). The binding area of C4 was identified to be within fifteen amino acid residues (1670-1684) based on the ability of a synthetic FVIII peptide consisting of amino acid residues 1670-1684 to completely inhibit the binding of C4 to FVIII. We now report the further localization of the binding region of C4 to within eight amino acid residues (1677-1684) of FVIII light chain. Nine new overlapping FVIII peptides were synthesized based on the amino acid sequence of the acidic region of FVIII light chain and tested, along with seven previously tested peptides, for the ability to inhibit C4 binding to FVIII in an ELISA assay. Three synthetic FVIII peptides 1670-1684, 1675-1690, and 1677-1684 demonstrated dose dependent inhibition of C4 binding to FVIII. The three reactive peptides contain residues 1677-1684 in common. Since C4 can completely inhibit the binding of FVIII to vWF, this report further localizes an eight amino acid residue region of FVIII which may be important in the mediation of vWF binding.

The factor VIII (FVIII) binding regions of the monoclonal anti-FVIII inhibitory antibody C5 and a human FVIII inhibitor antibody have previously been reported to be contained within amino acid residues 351-365 of FVIII. Localization of the binding regions of these two antibodies was based on their reactivity with four synthetic FVIII peptides. Nineteen synthetic FVIII peptides spanning the entire acidic region of the FVIII heavy chain have now been evaluated for the ability to inhibit the binding of C5 to FVIII in an ELISA assay. The smallest peptide tested that inhibited C5 binding to FVIII consisted of residues 351-361. Those peptides that were able to inhibit C5 binding in the ELISA assay were also able to neutralize the FVIII inhibitory activity of C5 in plasma. The FVIII inhibitory activity of two human FVIII inhibitor antibodies was also partially neutralized by peptides from this region. Evaluation of the pattern of peptides reactive with the three antibodies indicates that the binding regions of these antibodies are in very close proximity to each other, but are not identical. Their respective binding regions are contained within residues 351-361 (C5), 354-362 (inhibitor 1), and 342-354 (inhibitor 2). These results suggest that this 20 amino acid segment of the acidic region of the heavy chain of FVIII may be functionally important in the expression of FVIII procoagulant activity.

The relatively recent ability to obtain highly purified factor VIII (FVIII) preparations from plasma products, the cloning of the FVIII gene, and the expression of recombinant FVIII have provided the basis for significant advancements in the understanding of the structure-function relationships of FVIII. Evaluation of the molecular structure of FVIII has revealed the presence of domains of significant internal amino acid sequence homology as well as homology with similar structural domains of factor V. Specific proteolytic cleavage sites have been identified in the molecule and the use of site directed mutagenesis has identified those proteolytic cleavage sites required for the activation of FVIII. Deletion and substitution variants of FVIII as well as the precise epitope mapping of FVIII antibodies which inhibit the procoagulant function of the protein or its binding to von Willebrand factor have provided insight into the identification of regions of FVIII which are required for normal function.