The effects of oestrogens and progesterone on oestrogen receptors in female rat liver.

The administration of oestradiol-17 beta or ethynyloestradiol as well as the synthetic progestogen norethisterone acetate resulted in translocation of the oestrogen receptor. Progesterone and the synthetic progestogen (+)-norgestrel were ineffective. The increases in nuclear oestrogen receptor content 1 h after injection of each steroid were similar but different subsequently. The increase with oestradiol-17 beta extended for 3--6 h and for at least 9 h with ethynyloestradiol. With norethisterone acetate, nuclear content was still increased after 24 h. Oestrogen injection resulted in cytosol receptor depletion and a 'deficit' in receptor content extending for 6 h, whereas norethisterone acetate-induced translocation was quantitative. With injections of norethisterone acetate + ethynyloestradiol the increase at 1 h and retention of the nuclear receptors were similar to that with norethisterone acetate alone. In contrast, the depletion of cytosol receptor and its restoration were similar to that seen with ethynyloestradiol alone, suggesting that norethisterone acetate did not interfere with the oestrogen receptor replenishment. Specific binding in vitro of [3H]oestradiol-17 beta in liver cytosols was inhibited by (+)-norgestrel and norethisterone acetate, but not progesterone, at concentrations of 10--100 microM. Nuclear receptors present after norethisterone acetate injection bound oestrogen with high affinity (Kd = 1.52 nM), similar to receptors of oestrogen-injected animals. In the uterus, differential retention of nuclear receptors in response to oestrogens is associated with different cellular responses. The differences in the response of the receptor system in liver to the various steroids suggests that the corresponding tissue responses may also be dissimilar. These results are discussed in relation to the problems of liver dysfunction in oral-contraceptive users.     

Irreversible protein binding of norethisterone (norethindrone) epoxide.

14,15-3H-Norethisterone-4 beta, 5 beta-epoxide, a metabolite of norethisterone, was incubated with several proteins and nucleic acids. After 30 min incubation 0.19 nmol of the epoxide were irreversibly bound per mg albumin which contains free sulfhydryl groups; proteins without SH-groups, such as concanavalin A, gamma-globulin, DNA and RNA, did not irreversibly bind norethisterone epoxide. A superoxide (O2) generating enzyme system comprised of xanthine oxidase and hypoxanthine was capable of catalyzing the irreversible binding of the parent compound, norethisterone, to albumin, indicating that an oxidation product was formed which reacted with the protein. When norethisterone epoxide was incubated for 60 min with hepatic microsomes of rats in absence of NADPH, about 2.0 nmol of the epoxide were irreversibly incorporated per mg microsomal protein. This binding was increased to 5.2 nmol by addition of a NADPH regenerating system. Addition of glutathione and cytosol decreased only the NADPH-dependent protein binding; phenobarbital pretreatment of rats induced this NADPH-dependent binding of norethisterone epoxide to microsomal protein by a factor of 2. In presence of NADPH, binding of the epoxide to microsomal protein depended on substrate concentration used. The results indicate that norethisterone epoxide is able to chemically react with proteins. In addition, hepatic microsomal enzymes convert the epoxide to another metabolite which also can react with proteins.     

Synergistic effects of ethynylestradiol and norethisterone on the formation and lengthening of uterine lace in immature rabbits]

We have shown, through a test proceeding from the McPhail test, in the immature rabbit female, the existence of succession and simultaneity synergisms between ethynylestradiol and pure or impure norethisterone, given by oral route. The estrogen, administrated before the progestative, is able to potentialize the action of the latter on the building of the endometrial lace in 5 days, more than on the lengthening of it in 15 days. Ethynylestradiol, absorbed with norethisterone, strengthens the effects of the latter on the second phenomenon more than on the first one. Absolute and relative doses of the estrogen and of the progestative differ only lightly according as norethisterone is pure or not pure, because of the more potent estrogenicity of the impure norethisterone.     

Doping in sport—2. Quantification of the impurity 19-norandrostenedione in pharmaceutical preparations of norethisterone

The finding of measurable amounts of 19-norandrostenedione in norethisterone tablets prompted us to develop an assay to quantify this steroid. 19-Norandrostenedione is an anabolic steroid whose use in sport is prohibited by the World Anti-Doping Agency (WADA). The assay was developed using isotope dilution and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of 19-norandrostenedione in norethisterone formulations, with [3,4-¹³C₂]-19-norandrostenedione as the internal standard. The results showed amounts up to 1.01 ± 0.01 μg (mean ± S.E.M.) per tablet in those containing 5 mg of norethisterone or norethisterone acetate (0.02%, w/w) and up to 0.5 ± 0.01 μg (mean ± S.E.M.) per tablet (0.05%, w/w) in oral contraceptive tablets containing 0.35-1.5 mg of norethisterone or norethisterone acetate. No tablet tested exceeded the British Pharmacopoeia limit of 0.1% for this impurity.     

Testing synergism between ethinyl estradiol and norethisterone on the histamine-induced deciduoma and vaginal mucification of castrated rats]

Through the histaminic deciduoma test and a vaginal mucification test, we have endeavoured to show that, in spayed female rats there exists a possibility of synergism between ethynylestradiol and pure norethisterone per os. No synergism could be shown in the former test, owing to, or so it seems, a lack of deciduogenic power proper to norethisterone. The latter test showed, indeed, that ethynylestradiol can, in low quantities, bring about a synergism on the mucifying properties of norethisterone, whereas, in large quantities, it creates an antagonism on the aforesaid properties.     

Genotoxicity evaluation of norethisterone acetate

Genotoxic evaluation of a commonly used progestogen, norethisterone acetate, was undertaken using a combination of short-term in vitro and in vivo assays. The clastogenic potentiality of norethisterone acetate was evident from the chromosome aberrations and sister chromatid exchanges induced both with and without S9 mix in cultured human lymphocytes and also from the increased frequency of micronuclei formation and sister chromatid exchanges in mice. However, in the Ames Salmonella assay, both with and without S9 mix and in host-mediated assay, norethisterone acetate was unable to cause any significant increase/decrease in the His⁺ revertants/plate.     

Long-acting contraceptive agents: Structure activity relationships in a series of norethisterone and levonorgestrel esters

A large number of esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13β-ethyl-17α-ethynyl-17β-hydroxygon-4-en-3-one) were synthesized and tested for biological activity. The test employed in these studies was the duration of estrus suppression in cycling mature rats. In the norethisterone series several esters exhibited duration of activity comparable to that of norethisterone enarthate. In the levonorgestrel series the butanoic, cyclobutylcarboxylic and cyclopropylcarboxylic esters were longer acting than medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3, 20-dione) when prepared as aqueous microcrystalline suspensions.     

Binding of the gestagens norethisterone and levonorgestrel in blood of various species

This study investigates the binding of 2 widely used contraceptive steroids, levonorgestrel and norethisterone, by plasma from various animal species and compares the results to those obtained with human plasma. Equilibrium dialysis of plasma samples and polyacrylamide gel electrophoresis were performed as previously described. The plasma samples were diluted with phosphate-buffered saline on the percentage of levonorgestrel and norethisterone bound in comparison to human plasma. The concentration of total protein and albumin was measured colorimetrically in each sample. An ammonium sulphate precipitation technique measured the level of sex-hormone binding globulin (SHBG). Results of the equilibrium dialysis show that binding of levonorgestrel and norethisterone in plasma was similar in adult female rhesus monkeys and baboons to that of humans with both high-affinity and low-affinity classes of binding sites. The dissociation constants of the high-affinity class for levonorgestrel was 4-fold lower than that for norethisterone in all 3 primates, indicating levonorgestrel was more tightly bound. Total protein and albumin concentrations were also the same in all 3 primates. SHBG levels in female monkeys and baboons however were 3-4 times those found in normal human females. Although differences exist in the binding of the 2 gestagens between human, baboon, and rhesus monkey plasma, there are no significant differences in the metabolism of the gestagens in the 3 primates. Overall, the results indicate that in the human, baboon, and rhesus monkey, binding of norgestrel and norethisterone occur mainly to SHBG, which had a greater affinity for norgestrel than for norethisterone, and to a lesser extent, albumin. Differences in the binding of gestagens between human and nonprimate species (rat, dog, rabbit) studied suggest that only baboon and rhesus monkeys may be considered appropriate animal models for extrapolation of results of contraceptive studies to humans.     

Doping in sport: 3. Metabolic conversion of oral norethisterone to urinary 19-norandrosterone

The detection of 19 norandrosterone (19-NA) in a competitor's urine sample is taken as prima facie evidence of administration of nandrolone or other 19-norsteroid but a potential problem is that administration of norethisterone, a progestogen used for menstrual disorders and for hormonal contraception, also results in the excretion of 19-NA that can exceed the laboratory reporting threshold of 2 ng/mL. The contribution of norethisterone to urinary 19-NA with and without 19-norandrostenedione, a known norethisterone tablet impurity, requires evaluation. Preparations containing, either <2 ng or 1 μg 19-norandrostenedione impurity per 5 mg of norethisterone, administered to female volunteers (n = 10) in doses comparable to those used for menstrual disorders (5 mg three times daily for 10 days), resulted in maximal 19-NA concentrations of 51 and 63 ng/mL, respectively. The maximal concentration of 19-NA, 2 h post-administration of a single 1 μg dose of 19-norandrostenedione, was 2.4 ng/mL. These results prove unequivocally that norethisterone is metabolized to 19-NA and that there is only a minor contribution from the impurity 19-norandrostenedione. Administration to women (n = 30) of a single contraceptive tablet containing norethisterone (1 mg) with one of the highest proportions of the impurity 19-norandrostenedione (∼0.5 μg, 0.05%, w/w) resulted in a urinary 19-NA concentration of 9.1 ng/mL, with a maximum concentration ratio of 19-NA to the norethisterone metabolite 3α,5β-tetrahydronorethisterone of 0.36. We provide data that should remove the need for time-consuming follow-up investigations to consider whether doping with 19-norandrogens has occurred.     

A sensitive solid phase enzymeimmunoassay for norethisterone (norethindrone) in saliva and plasma

A sensitive, solid phase enzymeimmunoassay suitable for determining norethisterone in small aliquots of plasma (10 μl) and saliva (100 μ1) has been developed. A solid phase antiserum raised against a norethisterone-llα-hemi-succinyl/bovine serum albumin conjugate was prepared by coupling to cyanogen bromide activated cellulose. A norethisterone/horseradish peroxidase conjugate was used as enzyme label, o?-phenylenediamine/hydrogen peroxide being the substrate for colour development. The assay had a lower limit of sensitivity of 3 pg/assay tube and satisfied accepted validation criteria. Norethisterone concentrations determined by enzymeimmunoassay and by a well established radioimmunoassay were in excellent agreement in both plasma (r = 0.993, n = 20) and saliva (r = 0.989, n = 15). Plasma and salivary norethisterone concentrations determined in healthy volunteers reached peak values at about 1 hour after administering a norethisterone-containing oral contraceptive preparation. The maximum values achieved in saliva (775–1430 pmol/l) were only approximately 3% of those observed in plasma. Since salivary norethisterone concentrations reflected those in plasma, they may be useful in fertility control programmes and pharmacokinetic studies.     

Different effects of oestradiol benzoate and norethisterone on the blood flow and mineral content in rat bones.

In three experiments (2 on females, 1 on males), we determined the blood flow in the tibia and the distal part of the femur, together with cardiac output (by means of 85Sr-microspheres), tibial bone density and tibial ash weight related to bone volume. We found that 1) the bone blood flow always fell significantly after oestradiol benzoate, 2) no change occurred after norethisterone in doses corresponding to those of oestradiol benzoate, but the blood flow showed a tendency to fall after doses one order higher (it decreased significantly in one case only), 3) the density of the tibia and tibial ash weight related to bone volume rose nonsignificantly after oestradiol benzoate, but fell (mostly statistically significantly) after norethisterone. The lowering of the bone mineral indexes in rat bones after norethisterone is a surprising and potentially significant finding requiring further verification.     

Microbiological hydroxylation of norethisterone.

Hydroxylation of norethisterone by a large number of fungi has been investigated. 1alpha-Hydroxy-, 6beta-hydroxy-, 10beta-hydroxy-, 10beta,11beta-dihydroxy-15alpha-and 15beta-hydroxy-derivatives were formed from norethisterone. The microbiological dehydrogenation of 10beta-hydroxy-norethisterone resulting in 10beta,17beta-dihydroxy-17-ethynyl-1,4-estradien-3-one was also observed. The structure of transformation products was established by chemical and spectroscopical methods.     

Radioimmunoassay system for norethisterone using 3H- and 125I-labelled radioligands.

A radioimmunoassay procedure is outlined for norethisterone, a synthetic progestagen. This assay uses both tritiated and iodine-125 labelled radioligands and may serve as a model for assays of synthetic steroids for which no tritiated radioligand exists. Male volunteers took a single oral dose of 10 mg of norethisterone acetate (SH 420). Plasma hormone levels were then measured at various time intervals. The degree of binding of iodine-125 labelled radioligand to antiserum even at low serum dilution was always greater than 80%. Using antinorethisterone-11 alpha-BSA serum, triated norethisterone and norethisterone-3-OCMO-iodine -125-iodohistamine radioligands give comparable results of adequate specificity, precision, accuracy and sensitivity when used to analyze crude ether extracts of the plasma samples. The chromatographic step is unnecessary for specific analysis. Iodine-125 labelled ligands may be useful for the determination of other synthetic steroids.     

Progesterone and norethisterone have different effects on tachykinin-like immunoreactivity in rat cortex and striatum

OBJECTIVE: The purpose of this study was to investigate the effects of progesterone and the most commonly prescribed synthetic progestogen, norethisterone, on regional immune-like reactivity of neuropeptide Y (NPY), substance P (SP), neurokinin A (NKA) and neurotensin (NT) in brains of female ovariectomized estradiol-substituted rats. Results: Norethisterone+estradiol-treated rats had 44% lower SP levels compared with estradiol-only-treated in frontal cortex and 20% lower NKA levels in comparison with progesterone+estradiol-treated in frontal cortex. Progesterone+estradiol-treated rats had 66% lower SP levels in striatum in comparison with both estradiol-only-treated and norethisterone+estradiol-treated. No significant results were found for NPY and NT. CONCLUSION: Progesterone and the synthetic progestogen, norethisterone, have different effects on SP- and NKA-like immunoreactivity in rat cortex and striatum.The effects of NET on SP- and NKA-like immunoreactivity in frontal cortex may contribute to the mood effects ascribed to this progestogen in clinical usage.     

Effect of long-term treatment of norethisterone (a progestogen-only contraceptive) on salivary gland activity of the cycling albino rat

Effect of long-term treatment of norethisterone (a progestogen-only contraceptive) on the salivary glands of the cycling albino rats was evaluated from histomorphic and karyokinetic standpoints. Norethisterone increased concentration of zymogen granules in the serous acini and also of mucoid material in the mucous-containing acini in the submaxillary gland. In the parotid gland, the acini were hypertrophied, accompanied by cytoplasmic degranulation. In addition, the mitotic cells were seen in both the glands after the treatment. It is suggested that norethisterone perhaps stimulates the salivary gland activity of the rats.     

Effects of estradiol, progesterone, and norethisterone on regional concentrations of galanin in the rat brain.

Concentrations of immunoreactive galanin were compared in eight gross brain regions of ovariectomized female rats treated with either estradiol, estradiol + progesterone, estradiol + norethisterone, or placebo. Higher concentrations with estradiol treatment compared with placebo were found in the pituitary (357%), frontal cortex (162%), occipital cortex (174%), hippocampus (170%), and median eminence (202%). A more profound difference with addition of progesterone or norethisterone was seen in the pituitary (529% and 467%, respectively). Sex steroids, particularly estradiol, modulate galanin concentrations not only in reproductive, but also in nonreproductive, brain regions.     

Reproductive endocrine effects of intranasal administration of norethisterone to adult female rhesus monkeys (Macaca mulatta)

Intranasal administration of norethisterone at a daily dose of 9 micrograms between Days 5 and 14 of the menstrual cycles blocked ovulation in 10 out of 17 adult female monkeys. Serum concentrations of hormones indicated that ovulation was blocked due to a suppression of the mid-cycle, oestradiol-induced LH surge. Ovarian follicular activity in the treated menstrual cycles was not affected by norethisterone but there was a marked delay in the onset of the mid-cycle oestradiol surge in most of the treated animals. The duration of the menstrual cycle length after the oestradiol peak was significantly reduced in all the treated monkeys, indicative of a shortened luteal phase.     

Effects of Progestogen Oral Contraception with Norethisterone on Blood Clotting and Platelets

The effects on clotting tests and platelet function of six months'' continuous administration of the 19-norsteroid, progestogen-only contraceptive, norethisterone, have been studied in four groups of women. In a group of women who have not previously taken oral contraceptive no acceleration of clotting or platelet factors was found, but in contrast a tendency to reduced coagulability was observed. Women who had previously been taking combined oestrogen-progestogen preparations showed reduced clotting and platelet parameters when norethisterone was substituted. No changes in clotting or platelets were found in women who changed from 17-acetoxysteroid progestogen chloramadinone acetate or in a group of women started postpartum.     

Lack of aromatisation of the 3-keto-4-ene metabolite of tibolone to an estrogenic derivative

Tibolone is used for the treatment of climacteric symptoms in postmenopausal women. It is metabolised in a tissue-specific manner so that while some metabolites exert estrogenic effects on bone and the CNS, others are thought to protect the breast and endometrium from estrogenic stimulation. Tibolone is a 7alpha-methyl derivative of 19-norethynodrel. Since the introduction of synthetic progestagens for therapeutic use there has been considerable controversy as to whether they can undergo aromatisation to give rise to the potent estrogen, ethinylestradiol. In this study, we examined whether the delta-4-ene (7alpha-methyl norethisterone) metabolite of tibolone, which has a similar delta-4-ene A-ring structure to that of the estrone precursor, androstenedione, could undergo aromatisation to the potent estrogen, 7alpha-methyl ethinylestradiol. For these studies, JEG-3 choriocarcinoma cells were employed as they have a very high level of aromatase activity. TLC and HPLC procedures were developed to separate phenolic from non-phenolic compounds and were initially used to confirm that JEG-3 cells readily aromatised androstenedione to estrogens (up to 74%). The aromatisation of androstenedione to estrogens by these cells could be completely blocked with the potent aromatase inhibitor letrozole. When [(3)H] 7alpha-methyl norethisterone was incubated with JEG-3 cells no evidence for its conversion to [(3)H] 7alpha-ethinylestradiol was obtained. Radioactivity detected on the TLC plate or HPLC fractions where standard 7alpha-methyl ethinylestradiol was located, revealed that similar levels were present when 7alpha-methyl norethisterone was incubated with culture medium alone or with JEG-3 cells in the absence or presence of letrozole. From these investigations, it is concluded that 7alpha-methyl norethisterone does not undergo aromatisation to an estrogenic derivative.     

Norethisterone and levonorgestrel esters: a novel synthetic method

Aliphatic, alicyclic and arylcarboxylic esters of norethisterone and levonorgestrel were prepared in a one-step synthesis and in near-quantitative yield using trifluoroacetic anhydride.