M. Hümpel

8-Prenyl naringenin is a potent ERα selective phytoestrogen present in hops and beer

1. IntroductionPhytoestrogens are compounds found in leafy plants andfungi that exhibit estrogenic activity both in vivo and invitro. Chemically they refer to the classes of isoflavones,flavanones, coumestrans and resorcylic acid lactones. Cir-cumstantial evidence suggests that phytoestrogens ingestedwith food have beneficial effects in the prevention of breastcancer, post-menopausal syndrome and atherosclerosis.However, the potency of most of these compounds is rel-atively low compared to 17 -estradiol. Recently, isolationof a potent phytoestrogen (8-prenyl naringenin (8-PN))from the heart wood of an indigenous tree in Thailand(Anaxagorea luzonensis A. Gray) was reported [1]. 8-PNwas also found in Compositae [2] and identified as the estro-genic component in hops (Humulus lupulus L.) and beer [3].We studied in vitro receptor binding of both natu-rally found 8-PN enantiomers 2S(−)8-PN and 2R(+)8-PNcompared to endogenous mammalian estrogens and otherphytoestrogens, using recombinant human ER /ER .Asastriking result we found that both 8-PN enantiomers showhigh affinity and strong selectivity for ER .2S(−)8-PNexhibits an overall higher affinity for both receptors than2R(+)8-PN.Using a mammalian cell-based transient transactivationassay, we were able to demonstrate that 8-PN is the strongestplant-derived ER agonist identified so far, being about 10times more potent than coumestrol and approximately 100times more potent than genistein. Surprisingly and in clearcontrast to genistein, 8-PN is a much weaker agonist ofER than of ER .

In vivo conversion of norethisterone and norethisterone acetate to ethinyl etradiol in postmenopausal women

Previous studies with postmenopausal women receiving oral doses of norethisterone-containing preparations have shown that a small fraction of the dose is converted metabolically to ethinyl estradiol and may be detected in the peripheral blood. To investigate the extent and the dose dependence of this conversion in more detail, we performed a study with 24 postmenopausal women who received single oral doses of 5 mg norethisterone as well as 5 and 10 mg norethisterone acetate with a washout phase of 2 weeks between each treatment. After each treatment, blood was collected at regular intervals and the concentrations of norethisterone and ethinyl estradiol were analyzed in the serum samples by a specific radioimmunoassay and by gas chromatography/mass spectrometry, respectively. Ethinyl estradiol was present in the serum samples of all women following treatment with norethisterone acetate and, except for four cases, also after treatment with norethisterone. The conversion ratio of norethisterone acetate to ethinyl estradiol was 0.7 +/- 0.2% and 1.0 +/- 0.4% at doses of 5 and 10 mg, respectively. This corresponded to an oral dose equivalent of about 6 micrograms ethinyl estradiol per milligram of norethisterone acetate. For norethisterone, a conversion ratio of 0.4 +/- 0.4% was found at a dose of 5 mg, which corresponded to an oral dose equivalent of about 4 micrograms ethinyl estradiol per milligram of norethisterone. Although it cannot be excluded that in individual cases, even higher doses of ethinyl estradiol may be produced by conversion, it is concluded that at therapeutic doses of the progestogens, the exposure to metabolically derived ethinyl estradiol is probably of little clinical significance not only in fertile women using oral contraceptive combination preparations containing norethisterone and ethinyl estradiol, but also in postmenopausal women who receive oral doses of estradiol for estrogen replacement. The estrogenic effects of metabolically derived ethinyl estradiol on the liver (eg. synthesis of transport proteins) are very likely more than compensated due to the androgenic activity of norethisterone.

Investigations of pharmacokinetics of ethinyloestradiol to specific consideration of a possible first-pass effect in women

Ethinyloestradiol-3H was given intravenously and orally to four and three women, respectively, in a dose of 60 micrograms and 3 mg. To another three female volunteers, 100 micrograms of ethinyloestradiol was administered by both routes in succession. Drug concentration in plasma and total radioactivity in plasma, urine and faeces were measured for different periods of time. Intraindividual comparison of the area under the drug level vs. time curve after intravenous and oral administration of 100 micrograms showed that ethinyloestradiol is subject to an about 60% first-pass effect in women. The time course of ethinyloestradiol concentration in plasma can be described by a 3-compartment model after intravenous injection and by a 2-compartment model after oral administration, because an early disposition phase with a half-life of about 15 minutes only becomes visible after i.v. injection. On an average, the terminal half-life of unchanged ethinyloestradiol level and total radioactivity was calculated to be about 1 day. However, a high variability was found with this parameter as well as with the rate and degree of elimination in urine.

Kinetics and biotransformation of lormetazepam

A specific and sensitive radioimmunoassay has been developed for a new benzodiazepine, lormetazepam. After intravenous injection, lormetazepam levels in plasma fell in three (α, β, γ) dispositional phases, two of them (α, β) mainly reflecting different distribution processes. The terminal (γ) phase correlated well with the rate of renal elimination of glucuronides. Oral doses were completely absorbed with widely varying absorption half‐lifes (t½s) amounting to an average of 0.67 ± 0.53 hr. Dose‐dependent maximum plasma levels were reached in about 2 hr. Lormetazepam undergoes first‐pass metabolism of about 20% of an oral dose. Total clearance was in the range of 200 ml/min. There was a trend toward slower terminal disposition phase in elderly subjects. In younger subjects, the terminal phase t½ was about 10 hr. Lormetazepam glucuronide peak plasma levels were reached by about 6 hr. Thereafter, the level fell in one (elimination) phase, with a t½ of 12 hr in young subjects and with a significantly (p < 0.05) different t½ of about 20 hr in the elderly. Renal clearance was calculated as about 30 to 40 ml and did not show an age‐dependent difference. Recovery of lormetazepam glucuronide with urine amounted to 70% to 80% of the dose during 72 hr after intravenous injection in both age groups.

8-Prenylnaringenin, a Novel Phytoestrogen, Inhibits Angiogenesis In Vitro and In Vivo

8‐Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three‐dimensional collagen gel within which they form capillary‐like tubes, we demonstrate that 8‐prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC50 of between 3 and 10 μM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8‐prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8‐prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co‐added. These findings suggest that 8‐prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component. J. Cell. Physiol. 199: 98–107, 2004© 2003 Wiley‐Liss, Inc.

Tissue specificity of 8-prenylnaringenin: protection from ovariectomy induced bone loss with minimal trophic effects on the uterus.

Plant secondary metabolites with estrogenic activity (phyto-estrogens) have been studied in the past as a potential alternative to classical hormone-replacement therapy (HRT) in menopausal women. No final verdict on the efficacy of soy or red clover based pharmaceutical preparations has been reached despite numerous clinical studies. We have studied the novel and most potent phyto-estrogen 8-prenylnaringenin (8-PN) in adult ovariectomized rats, an established animal model to mimic hormone dependent osteoporosis in menopausal women. Our results demonstrate that 8-PN can completely protect from ovariectomy induced bone-loss while exhibiting minimal, (dose independent) trophic effects on uterus and endometrium. It is estimated that at equivalent bone protective doses of 17beta-estradiol and 8-PN, the phyto-estrogen has a 10-fold lower stimulatory effect on uterus and endometrium. The bone tissue specific effect of 8-PN was confirmed in a transgenic reporter mouse model (ERE-Luc mice). Here we also found pronounced estrogenic activity in prostate. Present results add important aspects to the pharmacological profile of 8-PN and position this compound as an interesting alternative new candidate for treatment of peri- and postmenopausal symptoms.

The pharmacokinetics and biotransformation of the new benzodiazepine lormetazepam in humans. I. Absorption, distribution, elimination and metabolism of lormetazepam-5-14C.

SummaryThe pharmacokinetics and metabolism of the new benzodiazepine lormetazepam were investigated in five male volunteers using thel4C-abelled drug (position 5). Lormetazepam was administered intravenously and orally, at a dose of 0.2 and 2 mg respectively, to each of the test subjects.Measurements of total radioactivity showed that the drug was absorbed completely and eliminated almost exclusively by the renal route. Maximum plasma level of active ingredient and total radioactivity were observed about 2 hours and 5 hours following oral administration. As early as 30 min following oral administration, concentration of active ingredient amounted to 80% of the maximum values. After both treatments the terminal half-life of total radioactivity and lormetazepam glucuronide in plasma corresponded to the half-life of elimination in urine of about 13 hours.After enzymatic hydrolysis with β-glucuronidase/arylsulphatase, an average of 90% of total radioactivity from various urine and plasma samples was extractable with ether. Extracts from plasma contained only unchanged drug, indicating free and conjugated lormetazepam as ingredients of total radioactivity. Extracts from urine could be separated into lormetazepam and its N-demethylation derivative lorazepam. The relative amount of excreted lorazepam conjugate was demonstrated to be time-dependent, probably due to enterohepatic circulation. Since less than 6% of the total dose was demethylated by both routes of administration, it can be assumed that lormetazepam is the active product.

Terminal half-lives in plasma and bioavailability of norethisterone, levonorgestrel, cyproterone acetate and gestodene in rats, beagles and rhesus monkeys☆

Abstract Norethisterone, levonorgestrel, cyproterone acetate and gestodene have been used for a long time in oral contraception and other indications, or are in the process of development for such indications. However, very little is known concerning the bioavailability and plasma levels of un-metabolized gestagens in the animal species used for chronic toxicity testing and pharmacological investigation. In this study the gestagens were administered intravenously, subcutaneously and orally to rats, beagles and rhesus monkeys. The drug plasma levels were determined by specific radioimmunoassay. The half-life of the terminal disposition phase was calculated following intravenous administration, and the extent of bioavailability was determined from the area under the drug level curves following subcutaneous and oral administration. The terminal half-lives of a particular compound in different animal species differed considerably. Furthermore, comparison of the different gestagens showed large variations in this parameter in all the animal species. In addition, inter-animal species comparison of a particular substance, and comparison of different substances in a single species, also showed great differences in bioavailability. The results are compared with the corresponding parameters in man. This investigation illustrates the fundamental problems inherent in the extrapolation of the results of toxicity studies and pharmacological investigations in animals, to man. The best tolerance and the lowest degree of pharmacological effect seem to occur where the bioavailability of a gestagen is poor and its terminal half-life short.

Comparison of Serum Ethinyl Estradiol, Sex-Hormone-Binding Globulin, Corticoid-Binding Globulin and Cortisol Levels in Women Using Two Low-Dose Combined Oral Contraceptives

The study included 69 women taking a desogestrel (n = 30)- or gestodene (n = 39)-containing low-dose combined oral contraceptive for at least 3 months. Group size was calculated to detect a difference in mean values of 80% of 1 standard deviation (alpha = 0.05, beta = 0.1). Seven serum samples were obtained up to 4 h, and 1 sample 24 h, after drug intake on 1 day between the 10th and the 21st day of the cycle. The concentrations of sex-hormone-binding globulin (SHBG), corticoid-binding globulin (CBG) and cortisol were measured in a 0- to 4-hour serum pool by radioimmunoassay. Ethinyl estradiol (EE2) levels were analyzed in single and pooled samples using anti-EE2-6 beta-carboxymethyloxime-bovine serum albumin antiserum. The area under the curves (AUC) up to 4 and 24 h and Cmax and tmax were evaluated. Statistical analysis (analysis of covariance) did not reveal a dependence of values on duration of treatment or day of cycle. Both treatments resulted in almost identical values for all parameters evaluated. The mean levels of SHBG, CBG and cortisol were in the range of 186-226 nmol/l, 89-93 mg/l and 280-281 micrograms/l, respectively. Mean maximum EE2 levels of 106-129 pg/ml were found 1.6-1.8 h after pill intake and AUC0-4 h accounted for 329-374 pg.h.ml-1. The recently reported differences in serum EE2 and CBG levels between two groups of 11 women each treated with desogestrel- and gestodene-containing pills, respectively, could not be confirmed.

Development of antibody — mediated extraction followed by GC/MS (antibody/GC/MS) and its application to iloprost determination in plasma☆

A new analytical principle has been developed combining the features of both radioimmunoassay and GC/MS. Its application in eicosanoid analysis was tested with the prostacyclin analogue, iloprost. The iloprost antibody, generally employed in RIA measurements, was coupled to Sepharose 4B and used as stationary phase for extraction of the drug. Variations in recovery were corrected by using deuterated iloprost as an internal standard. The samples were derivatized and quantitated by negative-ion chemical ionization-mass spectrometry. Reproducibility was 2.3 % at the 50 pg/ml level and the limit of detection was 5 pg for 1 ml samples. With plasma volumes of up to 20 ml, 0.25 pg/ml could be determined. Antibody/GC/MS proved superior to radioimmunoassay due to its higher specificity and sensitivity and superior to GC/MS with conventional clean-up procedures because of a higher sample capacity.