Molecular insights into how SHBG dimerization exerts changes on ligand molecular recognition.

Sex hormone binding globulin (SHBG) is a homodimeric glycoprotein and is the major carrier protein for sex steroids in plasma, regulating sex hormone availability in most vertebrate groups. Although it was initially thought that human dimeric SHBG bound a single ligand at the homodimer interface, studies demonstrated that dimeric SHBG binds a ligand to each subunit with similar affinity. In fact, the findings from recent experimental studies suggest that ligand binding to the SHBG dimer involves a complex allosteric mechanism involving conformational changes that limit observations of the presence of allosteric regulation. Therefore, we combined structural data with molecular dynamics simulations using Molecular Mechanics Generalized-Born Surface Area (MMGBSA) to dissect the structural and energetic basis for molecular recognition between five ligands whose affinities and binding positions on SHBG are known, i.e., 3ß,17α-diol; 3ß,17ß-diol; DHT; norgestrel (NOG); and estradiol (E2), and monomeric and dimeric SHBG. Protein-ligand complexes, involving dimeric SHBG saturated with two ligands on each subunit, reproduce the experimental affinity tendency and allow the observation that dimerization exerts disparate effects on binding affinity, characteristic of negative cooperativity for E2, DHT, and NOG, whereas 3ß-17α-diol and 3ß-17ß-diol lack allostery.

Metabolite Identification, Reaction Phenotyping, and Retrospective Drug-Drug Interaction Predictions of 17-Deacetylnorgestimate, the Active Component of the Oral Contraceptive Norgestimate.

Ortho Tri-Cyclen, a two-drug cocktail comprised of ethinylestradiol and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450 (P450), recombinant uridine 5'-diphospho-glucuronosyltransferases, and human liver microsomes in the presence and absence of selective P450 inhibitors were conducted. It was found that CYP3A4 plays a key role in NGMN metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm value, the predicted plasma concentration versus time area under the curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of the reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm value of 1. The data presented in this paper will lead to better understanding and management of NGMN-based drug-drug interactions when norgestimate is coadministered with CYP3A4 modulators.

Norelgestromin/ethinyl estradiol intravenous infusion formulation optimization, stability and compatibility testing: A case study to overcome polysorbate 80 interference in chromatographic analysis.

Norelgestromin/ethinyl estradiol is a progestin/estrogen combination hormonal contraceptive indicated for the prevention of pregnancy in women. The very poor solubility and wettability of these drugs, along with their high potency (adsorption issues), give rise to difficulties in designing intravenous (IV) formulations to assess absolute bioavailability of products containing both drugs. The purpose of this study was to develop an IV formulation, evaluate its stability under different conditions and evaluate its compatibility with IV sets for potential use in absolute bioavailability studies in humans. Also, a selective high-performance liquid chromatography (HPLC) method for quantification of ethinyl estradiol and norelgestromin in polysorbate 80 matrix was developed and validated. Norelgestromin/ethinyl estradiol IV solution was prepared using sterile water for injection with 2.5% ethanol and 2.5% polysorbate 80 as a cosolvent/surfactant system to obtain a final drug solution of 25µg ethinyl estradiol and 252µg norelgestromin from a concentrated stock drug solution. The stabilities of the concentrated stock and IV solutions were assessed after storing them in the refrigerator (3.7±0.6°C) and at room temperature (19.5±0.5°C), respectively. Additional studies were conducted to examine the stability of the IV solution using an Alarias(®) low sorbing IV administration set with and without an inline filter. The solution was allowed to drip at 1mL/min over a 60min period. Samples were obtained at the beginning, middle and end of the 60min duration. The chemical stability was evaluated for up to 10 days. Norelgestromin and ethinyl estradiol concentration, purity, and degradant levels were determined using the HPLC method. The norelgestromin/ethinyl estradiol IV formulation met the chemical stability criteria when tested on day 1 through day 9 (216h). Norelgestromin concentrations assayed in stock and IV solutions were in the range of 90.0-98.5% and 90.9-98.8% after 9 days, respectively. As for ethinyl estradiol, the assayed concentrations were in the range of 91.8-100.9% and 92.7-100.8% for the stock and IV solutions, respectively. The administration set was found to be compatible with both drugs; the assayed concentrations were in the range of 99.2-100.3% for norelgestromin and 96.3-102.7% for ethinyl estradiol, but the inline filter showed some adsorption of ethinyl estradiol; where the assayed concentrations were in the range of 98.1-99.8% for norelgestromin and 95.9-97.4% for ethinyl estradiol. The present study provided evidence supporting the suitability of an intravenous formulation for norelgestromin/ethinyl estradiol using ethanol/polysorbate 80 as a cosolvent/surfactant system. Both IV and concentrated stock solutions when stored at room temperature and refrigeration, respectively, were found to be chemically stable up to 9 days. These results indicated that this formulation is chemically stable and can be used over the time period tested. This IV formulation can be used to evaluate the absolute bioavailability of products containing norelgestromin and ethinyl estradiol provided that microbial testing of the IV formulation is performed.

Simultaneous determination of estrogens (ethinylestradiol and norgestimate) concentrations in human and bovine serum albumin by use of fluorescence spectroscopy and multivariate regression analysis.

The endocrine disruption property of estrogens necessitates the immediate need for effective monitoring and development of analytical protocols for their analyses in biological and human specimens. This study explores the first combined utility of a steady-state fluorescence spectroscopy and multivariate partial-least-square (PLS) regression analysis for the simultaneous determination of two estrogens (17α-ethinylestradiol (EE) and norgestimate (NOR)) concentrations in bovine serum albumin (BSA) and human serum albumin (HSA) samples. The influence of EE and NOR concentrations and temperature on the emission spectra of EE-HSA EE-BSA, NOR-HSA, and NOR-BSA complexes was also investigated. The binding of EE with HSA and BSA resulted in increase in emission characteristics of HSA and BSA and a significant blue spectra shift. In contrast, the interaction of NOR with HSA and BSA quenched the emission characteristics of HSA and BSA. The observed emission spectral shifts preclude the effective use of traditional univariate regression analysis of fluorescent data for the determination of EE and NOR concentrations in HSA and BSA samples. Multivariate partial-least-squares (PLS) regression analysis was utilized to correlate the changes in emission spectra with EE and NOR concentrations in HSA and BSA samples. The figures-of-merit of the developed PLS regression models were excellent, with limits of detection as low as 1.6×10(-8) M for EE and 2.4×10(-7) M for NOR and good linearity (R(2)>0.994985). The PLS models correctly predicted EE and NOR concentrations in independent validation HSA and BSA samples with a root-mean-square-percent-relative-error (RMS%RE) of less than 6.0% at physiological condition. On the contrary, the use of univariate regression resulted in poor predictions of EE and NOR in HSA and BSA samples, with RMS%RE larger than 40% at physiological conditions. High accuracy, low sensitivity, simplicity, low-cost with no prior analyte extraction or separation required makes this method promising, compelling, and attractive alternative for the rapid determination of estrogen concentrations in biomedical and biological specimens, pharmaceuticals, or environmental samples.

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular-Docking Method.

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular-docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three-dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular-docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH3 in R1 had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8-anilino-1-naphthalenesulfonic acid, was changed with norgestrel addition.

Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay.

The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and progesterone bioassays to conduct a structure-activity study assessing the intrinsic androgenic potential of commercially available progestins and their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17alpha-Hydroxyprogesterone and 19-norprogesterone derivatives with their bulky 17beta-substituents were strong progestins but generally weak androgens. 17alpha-Ethynylated derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17alpha-ethynyl group of each of these progestins produces 17alpha-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17alpha-hydroxyprogesterone and 19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.

Tracking of enantioselective solubility of rac-norgestrel in the presence of cyclodextrin by a CD spectroscopic method.

Dissolution of hydrophobic rac-norgestrel in aqueous gamma-cyclodextrin (gamma-CyD) and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CyD) solutions was investigated, and enantioselective dissolution was observed. (-)-Norgestrel, the eutomer molecule, was dissolved to a greater extent using each of the CyDs, although the effect was more significant in the case of HP-gamma-CyD. A circular dichroism (CD) spectroscopic method based on measurement of the anisotropy factor was applied for the determination of the enantiomer ratio. The concentration and the enantiomer ratio of norgestrel were determined indirectly in octanol after extraction. Optical rotation dispersion (ORD) measurements could confirm that neither the free CyDs nor their inclusion complexes could get into the organic phase during transport to octanol. Only the norgestrel molecules were able to get into the organic phase, although the enantiomer ratio remained the same as was obtained in the aqueous CyD solution.

Monolithic silica columns with chemically bonded beta-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals.

An enantioselective silica rod type chiral stationary phase (CSP) is presented; a novel combination of the well known enantiomer separation properties of beta-cyclodextrin and the unique properties concerning the flow behavior of silica monoliths. Two different synthesis routes are described, and it was found that the in situ modification of a plain silica rod column turned out to be the best. The chromatographic behaviour of the beta-cyclodextrin silica rod was studied and compared with a very similar commercially available beta-cyclodextrin bonded particulate material (ChiraDex). Even if the amount of beta-cyclodextrin bound to the silica rod was only about half of the amount of beta-cyclodextrin bound to ChiraDex) particles, good resolutions were achieved for a set of chiral test components like Chromakalin, Prominal, Oxazepam, Methadone and some other drugs. By taking advantage of the unique features of the silica rods relating to their flat H/u (Van Deemter) curves, fast enantiomer separations could be demonstrated.

Chemical and analytical characterization of related organic impurities in drugs.

A system is proposed for the classification of related organic impurities in drugs and drug products including among others (separated and non-separated) intermediates, various kinds of by-products, among them products of different side reactions, epimeric/diastereomeric, enantiomeric impurities, impurities in natural products, and finally degradation products. Examples are taken mainly from the author's own experience and from among the named impurities in the European Pharmacopoeia with focus on impurities in hydrocortisone, prednisolone, enalapril maleate, lisinopril, ethynodiol diacetate, pipecuronium bromide, cimetidine, and ethynylsteroids. The methodological aspects of impurity profiling from the detection to the identification/structure elucidation and quantitative determination of impurities are briefly summarized.

[Capillary electrochromatography for chiral separation and purity testing of pharmaceutical drug substances]. / A kapilláris elektro-kromatográfia (CEC) tanulmányozása gyógyszerhatóanyag királis elválasztásara és tisztasági vizsgálatára.

Chiral separation of D,L-Norgestrel was successfully performed by cyclodextrin-modified capillary electrochromatographic (CEC) method. Optimized CEC conditions for purity testing were achieved: Hypersil C-18 packed capillary and 10 mM 2-[N-morpholino]-ethanesulfonic acid buffer (MES) and 40 v/v % methanol as background electrolyte containing 10 mM gamma-cyclodextrin at pH = 5.5. The resolution was found to be strongly dependent on the concentration of chiral selector gamma-cyclodextrin, applied voltage and temperature of the capillary. Our results represent a simple, fast and reproducible method for the separation of D,L-Norgestrel and purity testing of D-Norgestrel. It can be concluded that cyclodextrin modified HPLC, HPCE and CEC offer inexpensive, attractive and reliable chiral separation methods.

Determination of circular dichroism and ultraviolet spectral parameters of norgestimate- and other Delta(4)-3-ketosteroid oxime isomers via normal phase HPLC method.

The oxime formation reaction of therapeutical progestogen (levonorgestrel, levonorgestrel acetate, norethisterone), androgen (methyltestosterone, testosterone phenylpropionate) and anabolic (nortestosterone phenylpropionate) Delta(4)-3-ketosteroids has been investigated. The ketosteroid-hydroxylamine reaction was monitored by reversed phase HPLC system. It was established, that under the experimental conditions applied the oxime formation was complete within 2 h. The reaction leads to the formation of Z and E oxime isomers. The isomers of norgestimate (levonorgestrel 17-acetate oxime) and other Delta(4)-3-ketosteroid oximes have been separated by a new normal phase HPLC method. The identification (elution order assignation) and determination of the formation ratio of the isomers have been performed by 1H NMR spectroscopy on the basis of the chemical shift differences of 4-H signals. The on-line CD and UV spectra of the pure oxime isomers were recorded and then molar ellipticities and absorbances of the isomers were calculated in the wavelength range of 200-300 nm via parameter estimation method.

Estradiol and norgestimate: a review of their combined use as hormone replacement therapy in postmenopausal women.

The focus of this review is hormone replacement therapy (HRT) with continuous administration of micronised, oral 17beta-estradiol 1 mg/day (herein referred to as continuous estradiol) plus micronised, oral norgestimate 90 microg/day administered for 3 days then withdrawn for 3 days in a 6-day repeating sequence (herein referred to as intermittent norgestimate). According to data from randomised, comparative trials of 1 year's duration, continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day relieves climacteric symptoms (vasomotor symptoms and vulvovaginal atrophy) in postmenopausal women. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day appeared as effective as estradiol 1 mg/day alone or continuous estradiol 2 mg/day plus continuous norethisterone acetate 1 mg/day in the treatment of postmenopausal women with vasomotor symptoms. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day was as effective as continuous estradiol 1 mg/day in causing the maturation of vaginal epithelial cells. In a randomised, double-blind study, bone mineral density (BMD) increased to a significantly greater extent and the rate of bone turnover was slower in postmenopausal women treated with continuous oral estradiol 1 mg/day plus intermittent norgestimate 90 microg/day than in placebo-treated patients. Two randomised, double-blind studies indicated that the addition of norgestimate 90 microg/day to continuous estradiol 1 mg/day did not attenuate the beneficial effects of estradiol on lipid parameters. Continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day was associated with increases in mean serum high density lipoprotein (HDL)-cholesterol levels and decreases in total cholesterol, low density lipoprotein (LDL)-cholesterol and lipoprotein (a) levels, compared with baseline. There was no statistically significant increase in triglyceride levels. In comparative trials, continuous oral estradiol 1 mg/day plus intermittent oral norgestimate 90 microg/day was well tolerated. Headache, breast pain or discomfort, abdominal pain or discomfort, uterine bleeding, dysmenorrhoea, oedema, nausea and depression were the most commonly reported adverse events. Continuous estradiol 1 mg/day plus intermittent oral norgestimate 90 microg/day was associated with a favourable uterine bleeding profile that improved over time. In a randomised trial, 80% of women were free from bleeding (irrespective of spotting) during month 12 of treatment. Norgestimate 90 microg/day was effective in protecting postmenopausal women against induction of endometrial hyperplasia by continuous estradiol 1 mg/day. In conclusion, data from a limited number of randomised studies indicate that HRT with continuous estradiol 1 mg/day plus intermittent norgestimate 90 microg/day is effective in relieving climacteric symptoms, increasing BMD and slowing the rate of bone turnover in postmenopausal women. This HRT regimen is well tolerated and is associated with a similar incidence of adverse events to that reported in recipients of continuous estradiol 1 mg/day. The norgestimate component of the regimen provides good endometrial protection and is associated with a favourable bleeding profile. Long-term studies investigating the associated risk of breast cancer and thromboembolic events in recipients of continuous estradiol plus intermittent norgestimate are needed. In the meantime, continuous oral estradiol plus intermittent oral norgestimate can be regarded as an effective new option for HRT in postmenopausal women.

Norgestimate.

PIP: Norgestimate (NGM) is one of a class of newer, more selective progestins. The authors describe its structure and biologic activity, contraceptive effectiveness, cycle control, metabolic effects, carbohydrate metabolism, coagulation factors, and clinical side effects. NGM-containing oral contraceptives (OC), either as a monophasic or triphasic formulation, perform as well as pills using older progestins. Androgenic side effects, however, are somewhat lower with NGM, but not enough to be of in clinical importance. Clinicians who believe in the potential for clinical consequences associated with changes in lipid and carbohydrate metabolism should make NGM their first choice OC progestin. Although no study has ever demonstrated a long-term superiority of one OC over another with respect to atherogenesis, there is some evidence that changes in HDL and LDL cholesterol, especially over a 20-year period secondary to pill use, may play a role in accelerating or retarding atherosclerosis. There are also multiple influences upon cardiovascular risk, including direct effects of estrogens upon the endothelium. The prudent healthcare professional should therefore choose, with the patient, the preparation which has the most favorable impact upon lipid metabolism.^ieng

Desogestrel, norgestimate, and gestodene: the newer progestins.

OBJECTIVE: To review and compare the newer progestins desogestrel, norgestimate, and gestodene with regard to chemistry, pharmacokinetics, efficacy, and tolerability. DATA SOURCES: Primary literature on desogestrel, norgestimate, and gestodene was identified from a comprehensive MEDLINE English-literature search from 1984 through 1994, with additional studies selected by review of the references. Indexing terms included progestins, desogestrel, gestodene, norgestimate, levonorgestrel, and norgestrel. STUDY SELECTION: Only human clinical and pharmacokinetic trials performed in Europe, Canada, and the US were included. DATA EXTRACTION: All available data from human studies were reviewed; both comparative and noncomparative studies were included because of the paucity of direct comparative information available. DATA SYNTHESIS: The newer progestins were designed to minimize the adverse effects (e.g., acne, hirsuitism, nausea, carbohydrate and lipid metabolism changes) observed with older oral contraceptives (OCs) while maintaining efficacy and good menstrual cycle control. Desogestrel, norgestimate, and gestodene have minimal amounts of androgenicity and antiestrogenic potential. All of these agents are pharmacokinetically similar to older agents: they are highly bioavailable when administered orally, hepatically metabolized, and obtain steady-state concentrations after 8-10 days of continuous administration. The newer agents have similar Pearl Indexes and slightly better cycle control. Furthermore, the new progestins appear to cause fewer adverse effects, such as acne and hirsuitism, and similar rates of weight gain, blood pressure changes, and lipid and carbohydrate metabolism changes. CONCLUSIONS: Desogestrel, norgestimate, and gestodene appear to offer clinical advantages because of their decreased androgenicity. Women whose cycles are currently well controlled with other OCs should not be switched to a newer progestin. However, any of the combination OC products that contain these progestins may be prescribed for women intolerant of older agents or to first-time users of OCs. The newer progestins appear to be efficacious and offer similar cycle control, improved safety and tolerability profiles, and comparable price with the older agents.

PIP: The objective was to review and compare the chemistry, pharmacokinetics, efficacy, and tolerability of the newer progestins desogestrel, norgestimate, and gestodene. Data sources were primary literature on desogestrel, norgestimate, and gestodene identified from a comprehensive MEDLINE English-literature search from 1984 through 1994, with additional studies selected by review of the references. Only human clinical and pharmacokinetic trials performed in Europe, Canada, and the US were included. All available data from human studies were reviewed; both comparative and noncomparative studies were included. The newer progestins were designed to minimize the adverse effects (e.g., acne, hirsutism, nausea, blood pressure elevation, carbohydrate and lipid metabolism changes, hemostatic changes) observed with older oral contraceptives (OCs) while maintaining efficacy and good menstrual cycle control. Desogestrel, norgestimate, and gestodene have minimal amounts of androgenicity and antiestrogenic potential. All of these agents are highly bioavailable when administered orally, hepatically metabolized, and obtain steady-state concentrations after 8-10 days of continuous administration. These agents have similar Pearl Indexes and slightly better cycle control than older agents. They appear to cause fewer adverse effects such as acne and hirsutism, and similar rates of weight gain, blood pressure changes, and lipid and carbohydrate metabolism changes. Desogestrel, norgestimate, and gestodene appear to offer clinical advantages because of their decreased androgenicity; however, available data are based on relatively small studies of short duration. Women whose cycles are currently well controlled with other OCs should not be switched to a newer progestin. However, any of the combination OC products that contain these progestins may be prescribed for women intolerant of older agents or to first-time users of OCs because of their apparent efficacy, improved cycle control, superior safety, tolerability, and comparable prices. Patients who have significant acne or hirsutism with older products and diabetic women may experience clinically significant benefit with the newer agents.

Effect of temperature on separation of norgestrel enantiomers by high-performance liquid chromatography.

The influence of mobile phase composition, concentration of beta-cyclodextrin and temperature on the high-performance liquid chromatographic separation of norgestrel was studied. In studies of the effect of temperature on the enantioselectivity of (+/-)-norgestrel, acetonitrile-water (25:75, v/v) modified by the addition of beta-cyclodextrin (14 mM) was applied as the mobile phase. Enantiomers were detected using UV detection at 240 nm. The capacity factors were measured over a wide range of column temperatures from -5 to 70 degrees C.

Progestin-rhenium complexes: metal-labeled steroids with high receptor binding affinity, potential receptor-directed agents for diagnostic imaging or therapy.

In order to investigate the possibility of developing diagnostic imaging agents for steroid-positive tumors that are labeled with the readily available radionuclide technetium-99m, we prepared four conjugate systems in which a progestin is linked to a metal chelate system. Three of these are bis-amino bis-thiol (BAT or N2S2) systems and are linked through carbon-21 of progesterone or the 17 alpha- or 11 beta-position of a nortestosterone type progestin. The fourth, an amino-amido-thiol-alcohol chelate (N2OS) system, is linked at the 16 alpha,17 alpha-positions of a dihydroprogesterone. As a model for technetium-labeled complexes, all four chelate systems were converted to their oxo-rhenium complexes. Of the four possible diastereomers in the 16 alpha,17 alpha-system, only one was isolated, while of the four possible diastereomers in the other systems, a syn pair and an anti pair (linker methylene vs rhenium-oxo, relative to the N2S2 plane) were separated in the 17 alpha-substituted series, a syn pair was isolated in the 21-substituted series, and a syn pair and the two individual anti diastereomers were separated in the 11 beta-substituted series. In competitive radiometric receptor binding assays, the 21-, 17 alpha-, and 16 alpha,17 alpha-linked systems had low affinity for the progesterone receptor (less than 0.3% that of promegestone (R5020) or 2% that of progesterone). By contrast, the two anti diastereomers of the 11 beta-linked system had affinities that were 10% and 44% that of R5020 (or 64% and 283% that of progesterone) and the syn pair had an affinity 25% that of R5020 (or 161% that of progesterone). The latter finding indicates that it is possible to prepare metal-labeled steroids that retain high affinity for steroid receptors. These and related systems, when complexed with radioactive metals, may be useful in vivo as receptor-directed agents for diagnostic imaging or therapy of steroid receptor-positive tumors.

Pharmacologic and pharmacokinetic characteristics of norgestimate and its metabolites.

Biotransformation, pharmacologic, and pharmacokinetic studies of norgestimate and its metabolites indicate that 17-deacetyl norgestimate, along with the parent drug, contributes to the biologic response. The postulated metabolic pathway, which is based on the identification of urinary products had indicated that three metabolites of norgestimate, 17-deacetyl norgestimate, 3-keto norgestimate, and levonorgestrel, might participate in the response. The pharmacologic evaluation of these metabolites demonstrates that only 17-deacetyl norgestimate has a pharmacologic profile consistent with that of norgestimate, and significant concentrations of this metabolite have been measured in the serum of women after the administration of norgestimate. These studies indicate that 17-deacetyl norgestimate contributes to the pharmacologic response to norgestimate.

In vitro binding of progesterone to receptors in the human endometrium and the myometrium.

High affinity, low capacity progesterone binding receptors have been identified in the cytosol fractions of the human endometrium and the myometrium. The endometrial and the myometrial progesterone binding proteins had sedimentation coefficients of 4.5 S and 4.1 S, respectively. Analysis of the bound steroids revealed that, along with progesterone, small amounts of its metabolites (20alpha-hydroxy-4-pregnene-3-one, 5alpha-pregnane-3,20-dione, 5alpha-pregnane-20alpha-ol-3-one) were also bound to the receptor proteins. Among the steroids studied for ligand specificity, 5alpha-pregnane-3,20-dione showed the highest competition for progesterone binding sites. Progestational steroids, like chlormadinone acetate and norgestrel, did not compete for the progesterone receptors. The endometrial and the myometrial progesterone binding receptors were thermolabile and protein in nature. The molecular weight of the endometrial progestrone binding protein was about 60,000-67,000 with a molecular (Stokes) radius of 33 A and the frictional ratio of 1.26. The myometrial progesterone binding protein had a molecular weight of 56,000-58,000 with a molecular (Stokes) radius of 31 A and a frictional ratio of 1.23. The binding of corticosterone to the myometrial cytosol was only 22-34%, whereas with progesterone it was 70-95%. A study of the immunoabsorption of the plasma proteins from the endometrial and the myometrial cytosol suggested the presence of specific progesterone binding receptors in the cytosol that were different to plasma proteins. The association constant of progesterone for the endometrial progesterone receptor was 1.9 x 10(9) M(-1) and for the myometrial progesterone receptor it was 1.4 x 10(9) M(-1), values that are higher than the association constant of progesterone for corticosteroid binding globulin, which is 7 x 10(8) M(-1). The evidence suggested that the human endometrial and the myometrial progesterone binding proteins are different to the corticosteroid-binding globulin.