Optimization of the preparation of fluorine-18-labeled steroid receptor ligands 16alpha-[18F]fluoroestradiol (FES), [18F]fluoro furanyl norprogesterone (FFNP), and 16beta-[18F]fluoro-5alpha-dihydrotestosterone (FDHT) as radiopharmaceuticals.

Fluorine-18-labeled steroid receptor tracers, 16α-[(18)F]fluoroestradiol (FES), [(18)F]fluoro furanyl norprogesterone (FFNP), and 16ß-[(18)F]fluoro-5α-dihydrotestosterone (FDHT), are important imaging tools for studies of breast and prostate cancers using positron emission tomography (PET). The automated production of these ligands with high specific activity (SA) as radiopharmaceuticals requires modification and optimization of the currently reported methods. [(18)F]FES with high SA was synthesized in over 60% radiochemical yield (RCY) at the end of synthesis (EOS) using a small amount of precursor (1) (as low as 0.3 mg) and 1 M H2SO4 for deprotection of the intermediate (2). [(18)F]FFNP was synthesized in up to 77% RCY at EOS using the triflate precursor (4) at room temperature or in 25% RCY using the mesylate precursor (6) at 65°C. Both methods are highly reproducible and afford high SA. [(18)F]FDHT was synthesized by radiofluoride incorporation at room temperature, reduction with NaBH4 , and deprotection with HCl/acetone, giving [(18)F]FDHT in up to 75% yield (RCY). All of these methods can be easily translated to automated production. The information provided here will aid in the development of automated production of these steroid receptor tracers with high or improved yields, optimal SA, and ease of processing for research and clinical use.

Single-dose pharmacokinetics of Nestorone, a potential female-contraceptive.

A synthetic progestin Nestorone is being developed for female-contraception. This study was conducted to determine the distribution, metabolism, and excretion of tritium-labeled Nestorone ((3)H Nestorone) in adult female rats. Rats were injected subcutaneously (S.C.) with a single dose of 400 microCi (3)H Nestorone/kg BW. Its distribution and concentrations in blood, plasma and other tissues were determined at defined times. The excreta were examined for elimination of (3)H Nestorone. Radioactivity in all samples was analyzed by liquid scintillation counter. Metabolite profiling was performed by HPLC and LC/MS analysis of the plasma, urine, and feces samples. Following subcutaneous injection of (3)H Nestorone, the mean peak concentrations of radioactivity (C(max)) in the blood and plasma were 58.1 and 95.5 ng equiv. (3)H Nestorone/g, respectively, at 2-h postdose (T(max)). Thereafter, the concentration of drug steadily declined through 96-h postdose with a terminal elimination half-life (t(1/2)) of 15.6 h. (3)H Nestorone-derived radioactivity was widely distributed in most tissues by 0.5 h and attained a mean maximal concentration by 2-h postdose. Approximately, 81.4% and 7.62% of the administered dose was excreted via feces and urine, respectively. In vivo metabolism of (3)H Nestorone resulted into a total of 19 metabolites. Among them, two metabolites viz., 17alpha-deacetyl-Nestorone (M9) and 4,5-dihydro-17alpha-deacetyl-Nestorone (M19) were identified by HPLC and LC/MS analysis. Metabolite profiling of plasma samples showed that most of the circulating radioactivity was associated with unchanged parent drug, and M19. The M19 was a major metabolite in the profiled urine and feces samples. Presence of large proportion of drug/drug-related material in feces suggested that the biliary excretion is a main elimination route of (3)H Nestorone. The distribution, metabolism, and excretion profiles of (3)H Nestorone obtained in this study provide a fairly good insight about its fate in women.

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.

Accelerated stability studies on 16-methylene-17 alpha-acetoxy-19-nor-pregn- 4-ene-3,20-dione (Nestorone).

The stability of the contraceptive steroid, Nestorone (16-methylene-17 alpha-acetoxy-19-nor-pregn-4-ene-3,20-dione) in the solid state and in aqueous solutions, was investigated using reverse-phase high-performance liquid chromatography. In the solid state, whether as a powder or when it is incorporated into Silastic implants, the steroid does not undergo detectable degradation even under severe experimental conditions. In solution, the drug undergoes slow degradation that is dependent on temperature and pH of the medium. The decomposition is defined by first-order mechanism. As expected, the reaction rate increases with increasing storage temperature. The linearity of the Arrhenius plot indicates that there is no change in the reaction mechanism within the temperature range studied. In alkaline media, the drug degrades at a faster rate through hydrolytic rather than an oxidative mechanism. The major hydrolytic degradation product, 16-methylene-17 alpha-hydroxy-19-nor-pregn-4-ene-3,20-dione, was separated and identified by mass spectrometry.

Progestin radiopharmaceuticals labeled with technetium and rhenium: synthesis, binding affinity, and in vivo distribution of a new progestin N2S2-metal conjugate.

We have prepared and evaluated three metal conjugates of a progestin-monoamine-monoamide (MAMA') bisthiol chelate system. These conjugates of rhenium and technetium-99 and -99m, are structural analogs of the bisamino-bisthiol (BAT) conjugates we have described recently, but the MAMA' chelate, being more polar than the BAT system, gives a conjugate that is much less lipophilic, having an octanol-water partition coefficient that is nearly 80-fold lower. In competitive binding assays, the Re- and 99Tc-MAMA'-progestin conjugates bind to the progesterone receptor with affinities greater than that of progesterone itself, and in a direct binding assay, the equilibrium dissociation constant (Kd) of the 99mTc-MAMA' conjugate was 0.97 nM. As is typical for 11 beta-substituted progestins, these conjugates also have substantial binding affinity for glucocorticoid receptors. In tissue distribution studies in immature female rats, the progestin-99mTc-MAMA' conjugates show selective uptake for principal target tissue (such as uterus) over that of blood and nontarget tissue (such as muscle); these uptake ratios reach maximum levels of 5 and 4, respectively. Uptake by fat, liver, and kidney is quite high; however, only the uptake in uterus is displaceable upon coinjection of the selective progestin ORG2058. Metabolism studies show that the radioactivity in the uterus is essentially unmetabolized out to 4 h, while liver activity is completely due to metabolites. Other tissues show an intermediate fraction of unmetabolized conjugates that decreases with time. The in vivo behavior of the progestin-99mTc-MAMA' conjugate is similar to that of the labeled BAT conjugate: its uptake selectivity is somewhat greater than that of the BAT conjugate, but its target tissue uptake is lower. Factors that may be responsible for limiting the target tissue uptake properties of these conjugates are their moderate affinity for progesterone receptor, their substantial binding to glucorticoid receptors, and their large overall molecular size.

Structure-activity and structure-affinity relationships of 19-nor-progesterone derivatives in rat uterus.

19-nor-progesterone (19NP) is a potent progestagen which possesses a high affinity for the progesterone receptor (PgR). In contrast, 17 alpha-hydroxylated-progesterone (17OHP) shows no hormonal activity and does not compete with progesterone (P) for the PgR. The aim of the present work was to analyse in parallel the structure-affinity and the structure-activity relationships for new molecules obtained by modifications of 19NP and 17OHP. The attachment of a 17 alpha-hydroxyl group on 19NP led to a dramatic decrease in both affinity and activity for the end-product, 17 alpha-hydroxylated-19-nor-progesterone (17OH-19NP). The further addition of a methyl group combined with the formation of a double-bound at C6 on 17OH-19NP results in nomegestrol (NOM), the relative affinity of which remained low. Negligible activity was also associated with this affinity in comparison to the parent 19NP. Strikingly, the protection of the free 17 alpha-hydroxyl group of NOM by an acetate led to a potent progestin with high affinity for PgR. It is concluded that the sum of the modifications brought into the 17OHP-19NP molecule reestablishes both affinity and activity of the original 19NP molecule. The same conclusion holds if P is considered as the parent compound, as already stated in the literature.