The effect of treating mammary tumor-bearing rats with 2-methoxyestradiol (2-MeE2) on the urinary excretion of 12 phytoestrogens was investigated and compared with the changes in urinary excretion of estradiol metabolites. Alterations of excretion were registered for isoflavonoids, lignans and coumestans. However, due to large variations statistical significant differences were found only for two lignans, i.e. significant increases of enterodiol and matairesinol. Since the single components of phytoestrogens showed diverse alterations, excretions were expressed also by the ratio of total isoflavonoids to total lignans and compared with the estrogen ratios 2-hydroxyestrone to 16 alpha- hydroxyestrone and A-ring to D-ring metabolites. The ratio of isoflavonoids to lignans was consistently decreased, whereas both ratios of estradiol metabolites were highly increased. The latter effect is probably due to demethylation of 2-methoxyestrone resulting in high catechol estrogen levels in urine.These results suggest that the high levels of catechol estrogens, produced by 2-MeE2 treatment, may have influenced the urinary excretion pattern of phytoestrogens. (c) 2006 Elsevier Ltd. All rights reserved.

Lange Zeit galt Oxytocin als ein reines Schwangerschaftshormon, das von der Hypophyse zur Wehenstimulation und Milchejektion abgegeben wird. Zahlreiche wissenschaftliche Arbeiten liegen heute vor, die über Oxytocinfunktionen auch außerhalb der Schwangerschaft berichten. So ist bekannt, dass Oxytocinrezeptoren fast ubiquitär im Organismus vorkommen, Oxytocin auch außerhalb des Gehirns gebildet wird und Oxytocin in verschiedenen Organen spezifische Wirkungen auszuüben in der Lage ist. Obwohl der Großteil der Erkenntnisse auf Ergebnissen von Tierexperimenten basiert, gibt es bereits eine Reihe von Studien, die auf Oxytocinwirkungen auch beim Menschen hinweisen.

The present study investigated the influence of the endogenous estradiol metabolite 2-methoxyestradiol (2ME) on the growth of methyl-nitroso-urea (MNU)-induced mammary carcinoma in the rat. 2ME was administered by means of subcutaneously implanted osmotic pumps for a period of 4 weeks. The dosages of 2ME were 1 and 5mg/kg per day, the control animals received saline. At the low dosage of 2ME a stimulation of tumor growth was observed, whereas at the high dosage an inhibition was found. The urinary excretion of 15 estradiol metabolites revealed that 2ME triggered strong changes in estrogen metabolism in the organism. Our data show that 2ME may elicit both stimulation and inhibition of tumor growth depending on the dosage used, a fact which should be considered in case of therapeutic use.

Up to now only two placebo-controlled intervention studies exist which evaluate the effect of hormone replacement therapy (HRT)on clinical endpoints using sufficient sample sizes. The negative outcomes of these studies such as increased cardiovascular and breast cancer risk led to extrapolations onto the complete therapeutic HRT field. This is unscientific considering pharmacological issues. In both studies only one HRT preparation was tested, conjugated equine oestrogens (CEE) combined with medroxyprogesterone acetate (MPA), using one fixed dosage combination. CEE is a variable mixture containing over 200 chemical substances, at least 10 oestrogen components and steroids with progestogen or androgen properties. Since all menopausal symptoms are caused by oestradiol deficiency it seems reasonable to replace the human oestrogen which is pharmacologically by no means comparable with the biological extract CEE. Concerning the combination with a progestogen negative cardiovascular effects are of importance and might be more predominant with MPA than with other progestogens. In addition low-dose HRT regimens should be tested, whereby transdermal application forms of both oestrogens and progestogens seem of special advantage.

For a long time, oxytocin was regarded as a pregnancy hormone released by the hypophysis to stimulate labour and milk ejection. In the present survey, data have been collected from the literature to show the spectrum of the hitherto known functions of oxytocin outside pregnancy. It is now known that oxytocin receptors can occur almost ubiquitously in the organism, that oxytocin is also formed outside of the brain and that oxytocin has functions in a number of organs. In the first part of the survey, stimuli that contribute to an increase in oxytocin release are compiled. In the second part, details are given on the individual oxytocin targets. Although the majority of findings are based on the results of animal experiments, there are already a number of studies that indicate similar effects of oxytocin in humans. According to the current state of knowledge, oxytocin appears to be involved in functions in the following organs: male and non-pregnant female reproductive tract, pancreas, cardiovascular system, kidney, brain and breast. There are indications that oxytocin may also have actions in other organs. There continues to be a considerable need for research into oxytocin in order to better understand the physiological and pathophysiological actions and to be able to derive possible therapeutic uses. Further light on the spectrum of functions of oxytocin may be cast by the possibility of the use of oxytocin antagonists.

Estradiol can be metabolized to substances eliciting different, partly opposite effects even at low concentrations as shown in own investigations, e. g., regarding (anti)angiogenic actions. Specific anticancerogenic effects are ascribed to 2-hydroxyestrone and particularly 2-methoxyestradiol. In contrast, 16alpha-hydroxyestrone and the 4-hydroxyestrogens may be genotoxic under certain circumstances. Furthermore there are indications that endogenous production of proliferation-stimulating metabolites is raised in some cancers. Especially the urinary excretion of 2-hydroxyestrone to 16alpha-hydroxyestrone was investigated showing in own and other clinical studies a lower ratio in postmenopausal women with breast cancer. Research is ongoing inasfar the determination of estradiol metabolites also in blood or directly in the breast tissue by means of sensitive laboratory methods may allow predictive statements. However, it has be to consider that estradiol metabolism can be influenced by external factors such as nutrition, smoking, sports and drugs such as L-thyroxine and H2-antagonists. We were able to demonstrate that estradiol metabolism during estradiol treatment depends on the application mode and might be differently influenced by addition of the various progestins. Whether the investigation of gene polymorphism of enzymes, which are involved in estradiol metabolism, may be helpful for the assessment or treatment of risk patients, to our opinion needs further research.

Endogenous estradiol metabolism results in metabolic products that are still capable of exerting various biological, partially estrogen-antagonistic actions. This indicates that the effects of estradiol in carcinogenesis may depend on individual variations of metabolic breakdown of estradiol. The aim of this paper is to review and discuss the available data relating to stimulatory and inhibitory properties of estradiol metabolites on carcinogenesis. Results of main D-ring metabolites and main A-ring metabolites are presented. There are indications that the endogenous production of growth influencing estradiol metabolites may be elevated in neoplasias. Some results in this respect are available for stimulating tumor growth for the D-ring metabolite 16-hydroxyestrone and the A-ring metabolites 4-hydroxyestrone and 4-hydroxyestradiol. Inhibitory effects exist for the A-ring metabolite 2-methoxyestradiol (2-ME). So far, only a few metabolites have been studied closely for their influence on carcinogenesis. There is also a dearth of data on the intracellular metabolism of estradiol in neoplastic tissues. Knowledge of the metabolites may reveal new approaches to diagnosis and treatment of malignant diseases. 2-ME has already shown actions in pharmacological dosages which led already to a first trial to prove its suitability for treating human breast cancer.

Relaxin is a peptide hormone of the insulin family with pleiotropic effects [1]. While the physiological role of relaxin during human reproduction has been studied for many years, very recent findings indicate that due to its vasodilatory, cardioprotective, chronotropic, and central hemodynamic properties, relaxin may also have important implications on the cardiovascular system [2, 3].

The estradiol metabolism may be of clinical relevance in the pathophysiology of various diseases; the increase in D-ring metabolites over A-ring metabolites in breast cancer patients is of special interest. Since estrogen therapy has been blamed for increasing the risk of breast cancer, the effects of hormonal replacement therapy (HRT) and oral contraception were investigated on the ratio of the main D-ring metabolite, 16alpha-hydroxyestrone (16-OHE1), to the main A-ring metabolite, 2-hydroxyestrone (2-OHE1). In our study, hormone replacement therapy (HRT) in postmenopausal women consisted of administration of estradiol valerate either with or without addition of the progestin dienogest. In the second part of the study, women of reproductive age received ethinylestradiol plus dienogest or ethinylestradiol plus norethisterone acetate as oral contraceptives (OC). 2-OHE1 and 16-OHE1 were measured by enzyme immunoassay in 8 h night-urine collected before and after 3 months of hormone administration. With HRT, that is, estradiol valerate or estradiol valerate plus dienogest, the ratios before treatment were 0.47 and 0.60; after 3 months, they were 0.54 and 0.52, respectively. There were no significant differences. With oral contraception, that is, ethinylestradiol plus dienogest or norethisterone acetate, the ratios before administration were 0.62 and 0.68, vs. 0.31 and 0.54 after 3 months, respectively. The ratio after ethinylestradiol and dienogest was significantly lower after treatment. HRT and OC in the estrogen-progestin combinations tested did not impose any negative effects on estradiol metabolism--they did not elicit a higher D-ring metabolism, which is considered to increase breast cancer risk.

We present a sensitive homologous radioimmunoassay (RIA) for the quantitative determination of human relaxin (hRLX) in human serum, plasma, seminal plasma, and urine. This assay is based on a rabbit antiserum which was generated using recombinant hRLX-2 as immunogen. Using 125I-hRLX-2 as tracer and a total incubation time of 20 - 24 hours the radioimmunoassay showed linearity in a range of 60 - 4000 ng/l, a lower detection limit of 38 ng/l and a mean recovery rate of 98.5%. Intraassay variation was 4.0% (mean = 526 ng/l) and 11.9% (mean = 2368 ng/l), and interassay variation 10.7% (mean = 256 ng/l) and 13.1% (mean = 2368 ng/l). Using hRLX-2 hexapeptides on polystyrene pins, epitopes recognized by the hRLX-2 specific rabbit antiserum were determined experimentally, and compared to predicted epitopes. Both methods led to comparable results. The antiserum, recognizing different epitopes, showed no cross-reactivity with human insulin, hZn-insulin, hIGF-I, hIGF-II, human inhibin alpha-subunit, two different forms of seminal plasma inhibin like peptide, spermolaxin, ubiquitin, prolactin, LH, FSH and hCG.

PURPOSE:Estrogen replacement in postmenopause can elicit vasodilatory effects which may be important for cardiovascular protection. Progestin addition can antagonise this beneficial effect, but until now, only a few studies have been performed on this issue. In the present study the effect of estradiol as well as of added norethisterone acetate (NETA), a C19-progestin, on the renal excretion of various biochemical markers which can reflect vasoactive actions was investigated.METHODS:37 postmenopausal women were treated for one sequential estrogen/progestin treatment cycle, i.e. two weeks with estradiol alone followed by two weeks with an estradiol/progestin combination. Both oral (n = 20) as well as 'complete' transdermal (n = 17) hormone substitution was applied, and the excretion of the following vasoactive substances or the stable metabolites, respectively, were measured in nocturnal urine prior to treatment, after 14 and 28 days: cGMP, which can reflect the production of nitric oxide, prostacyclin, thromboxane and serotonin.RESULTS:The excretion of cGMP was increased with both forms of administration during the estrogen phase as well as during the consecutive estrogen/progestin treatment. The prostacyclin-thromboxane quotient increased during estrogen phases, but decreased significantly by the addition of oral, but not with transdermal NETA, reflecting possible vasoconstrictory effects. Serotonin excretion increased, but this only was significant after the oral estrogen-only phase (2 weeks treatment), and after one cycle of complete transdermal treatment (4 weeks treatment), respectively.CONCLUSION:The observed effects can be explained by vasodilatory actions during the estrogen phases which can be maintained or even increased during the consecutive estrogen/progestin treatment suggesting a time-dependent beneficial estrogen effect. However, oral progestin addition may antagonise this effect already within two weeks of treatment whereby the prostacyclin-thromboxane quotient seems to be the most sensitive marker surrogating on vasoconstrictory progestin action.

Background The 'sartans' are antagonists of the angiotensin type 1 (AT(1)) receptor that are mainly used for treatment of hypertension. Little is known about AT(1)-independent effects of these substances and interactions with other drugs used for prevention of cardiovascular diseases. Postmenopausal estradiol-replacement therapy has been shown to exert beneficial antiathero-sclerotic properties by inhibiting oxidation of low-density lipoprotein (LDL).Objective In the present study, the effects of valsartan alone and in combination with 17 beta-estradiol on the oxidation of isolated human LDL were investigated.Methods Oxidation of LDL, which was triggered by copper (II) chloride, was monitored spectrometrically at 234 nm. The test substances were added in vitro.Results Valsartan alone increased the duration of resistance of LDL to oxidation by 75.3 +/- 5.7 min at 5 mu mol/l and by 138.2 +/- 8.1 min at 10 mu mol/l. 17 beta-estradiol alone delayed the onset of oxidation of LDL by 75.7 +/- 5.1 min at 1 mu mol/l. With the combination of 5 and 10 mu mol/l valsartan with 1 mu mol/l estradiol the time to onset of oxidation of LDL was increased by 142.8 +/- 4.9 and 215.3 +/- 6.9 min, respectively.Conclusions There has been demonstrated an antioxidative effect of valsartan that was additive to that of 17 beta-estradiol. Thus this combination has the potential to be useful in the treatment of postmenopausal women with hypertension. Coron Artery Dis 11:347-349 (C) 2000 Lippincott Williams & Wilkins.

Fluvastatin, an agent of a class of lipid-lowering drugs, the "statins", significantly enhanced prostacyclin synthesis at the concentrations of 0.1 microM and 1 microM and significantly reduced endothelin production at the concentrations 0.01, 0.1 and 1 microM in cell cultures of human umbilical endothelial veins. Since prostacyclin is a vasodilator and endothelin a vasoconstrictor, fluvastatin may have a significant effect on hemodynamics by favoring the balance towards vasodilation. This mechanism may contribute to the prevention of cardiovascular diseases.

Objectives: To determine the progestational efficacy of continuous treatment with various doses of dienogest, combined with oestradiol valerate, on the basis of endometrial histology, effect on climacteric symptoms and bleeding profile in postmenopausal women. Methods: Patients were randomised to one of five fixed-combination treatments, oestradiol valerate 2.0 mg plus dienogest 0.5, 1.0, 2.0, 3.0 or 4.0 mg. Efficacy was assessed by endometrial biopsy, menstrual charts and change in climacteric symptoms. Results: The endometrium was classified as atrophic in 20.0, 31.3, 25.0, 55.6 and 57.1% of patients in the 0.5, 1.0, 2.0, 3.0 and 4.0 mg dienogest groups, respectively. The frequency of uterine bleeding was dose-dependent. The most favourable bleeding profile was seen in the 3.0 mg dienogest group, whereas the lower doses of dienogest had advantages with respect to the efficacy of the combined preparation. Conclusions: Dienogest 2.0 and 3.0 mg are the optimal doses for combination with 2.0 mg oestradiol valerate for continuous-combined hormone replacement therapy.

The available literature on estrogen metabolism and estrogen metabolites involved in carcinogenesis is reviewed. Endogenous estradiol metabolism leads to metabolic products that can have various, and, to some extent, contrary, biologic effects. Thus, there are numerous research findings on the stimulation and inhibition of cancer growth by estrogen metabolites. Furthermore, there are indications that, in certain types of cancer, the production of growth-stimulating estradiol metabolites is increased. There are also reports on substances that can influence estradiol metabolism. So far, only a few estradiol metabolites have been examined with respect to their influence on the development and growth of cancer. It is presumed that other metabolites can also intervene directly or indirectly in the cancer process, but there is a great lack of research in this area. An understanding of the actions of estradiol metabolites may open up new avenues for the therapy of malignant diseases. Although little is known about the biologic effects of most of the estradiol metabolites, the reported actions of certain estradiol metabolites already justify clinical investigations on their possible beneficial uses in tumor therapy.

Dominance of estradiol metabolism at the D-ring over the A-ring metabolism may play a role in the pathophysiology of human breast carcinogenesis. Currently, the influence of progestins on breast cancer risk is debated when added to postmenopausal estradiol replacement therapy. However, nothing is known about the action of progestins on estradiol metabolism. Therefore, the effect of oral and transdermal estradiol/norethisterone acetate (NETA) was investigated on the ratio of the main D-ring metabolite 16alpha-hydroxyestrone (16-OHE1) to the main Aring metabolite 2-hydroxyestrone (2-OHE1). The ratio of 16-OHE1 to 2-OHE1 after transdermal hormone replacement therapy (HRT) was 0.43 before treatment, 0.35 after estradiol and 0.52 after estradiol + NETA. The ratio after oral HRTwas 0.94 before treatment, 0.86 after estradiol and 2.30 after estradiol + NETA. Because of the high variations, no statistical significance could be calculated. Since there was a tendency to an increase after oral estradiol + NETA treatment, the individual patient profiles were examined. Here, three patients in the oral treatment group showed a significant increase of the ratio after the estradiol/NETA phase. In conclusion, transdermal NETA in HRT did not elicit any change in estrogen metabolism after 2 weeks' treatment. However, oral NETA may in some cases have an impact on estradiol metabolism which should be further evaluated.

The effects of 14 estradiol metabolites on the proliferation of cultured endothelial cells of human umbilical cord veins were examined and compared with that of their parent substance estradiol. The relationship between dosage and effect was tested over the pharmacological concentration range of 10−8 to 10−5 M. Estradiol showed a biphasic behaviour, in the form of stimulation at low concentrations and inhibition at the highest concentration. All 10 A-ring metabolites tested stimulated the growth of the endothelial cells at the lower concentrations. At the highest concentration, the 5 A-ring metabolites: 2-hydroxyestrone, 2-hydroxyestradiol, 2-hydroxyestriol, 4-hydroxyestrone and 4-hydroxyestradiol caused significant inhibitions. Except for the 2-hydroxyestradiol, methylation of these metabolites resulted in the loss of the proliferation inhibiting effect. The D-ring metabolites showed no marked effects compared to the A-ring metabolites except for 16α-hydroxyestrone which had an inhibiting effect from 10−7 to 10−5 M. Our results show that estradiol metabolites can influence the growth of vascular endothelial cells in the concentration range tested. While the antiproliferative action of 2-methoxyestradiol has been known for some time this study is the first to show the potential capacity of non-methylated metabolites of the A-ring metabolism in inhibiting endothelial proliferation. This may open up new clinical pharmacological aspects in the anti-angiogenetic treatment of tumors.

Objectives: Can transdermal (n = 20) and oral(n = 20) estradiol substitution influence the urinary excretion of vasoactive substances in postmenopausal women? Method: The vasoactive substances prostacyclin and thromboxane, cyclic guanosine monophosphate,which can reflect the systemic NO production, serotonin, relaxin, insulin and melatonin were measured in nocturnal 8-hour urine before and after 2 and 4 weeks' estradiol treatment. Results: The excretion of prostacyclin and thromboxane, calculated as a prostacyclin/thromboxane quotient, was shifted towards higher prostacyclin production. Only minor changes could be registered for the cyclic guanosine monophosphate excretion. The production of serotonin, relaxin and insulin was increased only after transdermal treatment with estradiol. For melatonin no changes could be observed. Conclusions: Hormone substitution therapy with estradiol in postmenopausal women is able to increase the urinary excretion of various vasoactive substances, both after transdermal and oral application, indicating a vasodilatory estrogenic action. Transdermal administration was more effective, although lower dosages were applied. An explanation may be that transdermal estradiol elicits continuous and constant effects on estrogenic target organs. Copyright (C) 2000 S. Karger AG. Basel.

Conjugated equine oestrogens (CEE) have been used for several decades for postmenopausal hormone replacement. The preparation is in the form of an extract from the urine of pregnant mares containing the effective substances, i.e. oestrogen metabolites. This was one of the earliest commercial preparations available for postmenopausal hormone replacement. Treatment with extracts of biological material has become less common nowadays because it is difficult for them to fulfil the high specifications for pharmacological products. The preferred aim now is the use of monosubstances with clearly defined pharmacokinetic and pharmacodynamic features. There are many uncertainties in the characterization of CEE. The British Pharmacopoeia, Martindale, which in general provides comprehensive information on drugs, gives little data on CEE [1]. It is known that the preparation contains oestradiol metabolites as well as equilin metabolites which are specific to horses. However, there is still a dearth of knowledge about the exact components of the urine extract. Hitherto unidentified components have been recognized recently in the extract [2]. These are delta-8-oestrone and its metabolites; furthermore 16α-hydroxy metabolites and catechol metabolites of equilin are also allegedly present. The pharmacological properties of these substances are still largely unknown and it is likely that other, previously unidentified steroids are also present. It is now known that oestrogen metabolites possess effects other than their oestrogenicity [3]. Thus, some have opposing effects: for instance, D-ring metabolites may exert proliferation-stimulating effects on cells, but A-ring metabolites proliferation-inhibiting effects. In general, D-ring metabolites still have an oestrogenic effect, while A-ring metabolites may even show antioestrogenic effects. There has been recent interest in oestrogen metabolites because there are indications that they can be involved in the development of hormone-dependent tumours, e.g. breast cancer [3]. Zhang et al. have shown that equilin metabolites have toxic properties with carcinogenic potential through the formation of quinones [4]. A special carcinogenic risk factor may be the formation of semiquinone-adducts with DNA [5]. Equilin metabolites appear to have stronger carcinogenic effects than comparable oestradiol metabolites [2]. In the 1980s, Diczfaluzy [6] expressed the view that it was not only the quantatively dominant metabolites that were important but probably also those less frequently present. There is still need for information in this field. It is long-term therapy with hormone replacement (HRT) that causes most concern in relation to the development of breast cancer. Because CEE has been available for longer than most other preparations, it predominates in the majority of studies which have shown this connection. This emphasizes the importance of establishing exactly what substances with carcinogenic potential are contained in the extract. It cannot be assumed that other HRT preparations share the same potential for an increase in associated breast cancer with long-term use. Because the extract from mares’ urine contains equilins that occur only in horses and are a foreign substance for humans, the description ‘natural oestrogens’ is really not correct. Also, the claim that the CEE extract exerts its positive effects by virtue of its mixture of components has no scientific basis [2]. The overall effect on postmenopausal women may be the resultant of different, partly competing, effects of metabolites and the effect of individual components may be masked. Oestradiol is now available in pure form, crystalline or as oestradiol valerate, and it seems more sensible to replace the oestradiol deficiency by giving the exact hormone rather than an extract of uncertain composition. Since CEE offers no advantage over physiological oestradiol, it is hard to understand why substitution with a pharmacologically poorly defined oestrogen mixture is still practised. From the point of view of clinical pharmacology, the use of an oestrogen extract should now be considered inappropriate. Received 7 September 1999, accepted 4 January 2000. Correspondence: Professor Dr T. H. Lippert, Section of Clinical Pharmacology, Department of Obstetrics and Gynaecology, University of Tuebingen, 72076 Tuebingen, Germany

We have studied the effects of the AT1-receptor antagonist valsartan and of the natural estrogen 17 beta-estradiol on the proliferation of human coronary artery smooth muscle cells. The substances were tested in the concentration range of 10(-8) to 10(-5) M, each alone and in equimolar combinations. Whereas 17 beta-estradiol significantly inhibited proliferation at 10(-6) and 10(-5) M, valsartan did not have a significant effect at any of the concentrations tested. In equimolar estradiol/valsartan combinations, valsartan did not antagonize the antiproliferative effect of 17 beta-estradiol. Thus combined treatment of hypertensive post-menopausal patients with valsartan and estradiol is likely to have no impact on the estradiol-induced inhibition of smooth muscle cell proliferation. Med Sci Res 27:169-170 (C) 1999 Lippincott Williams & Wilkins.