Nestorone® as a Novel Progestin for Nonoral Contraception: Structure-Activity Relationships and Brain Metabolism Studies

Abstract

Nestorone® (NES) is a potent nonandrogenic progestin being developed for contraception. NES is a synthetic progestin that may possess neuroprotective and myelin regenerative potential as added health benefits. In receptor transactivation experiments, NES displayed greater potency than progesterone to transactivate the human progesterone receptor (PR). This was confirmed by docking experiments where NES adopts the same docking position within the PR ligand-binding domain (LBD) as progesterone and forms additional stabilizing contacts between 17α-acetoxy and 16-methylene groups and PR LBD, supporting its higher potency than progesterone. The analog 13-ethyl NES also establishes similar contacts as NES with Met909, leading to comparable potency as NES. In contrast, NES is not stabilized within the human androgen receptor LBD, leading to negligible androgen receptor transactivation. Because progesterone acts in the brain by both PR binding and indirectly via binding of the metabolite allopregnanolone to γ-aminobutyric acid type A receptor (GABAAR), we investigated if NES is metabolized to 3α, 5α-tetrahydronestorone (3α, 5α-THNES) in the brain and if this metabolite could interact with GABAAR. In female mice, low concentrations of reduced NES metabolites were identified by gas chromatography/mass spectrometry in both plasma and brain. Electrophysiological studies showed that 3α, 5α-THNES exhibited only limited activity to enhance GABAAR-evoked responses with WSS-1 cells and did not modulate synaptic GABAARs of mouse cortical neurons. Thus, the inability of reduced metabolite of NES (3α, 5α-THNES) to activate GABAAR suggests that the neuroprotective and myelin regenerative effects of NES are mediated via PR binding and not via its interaction with the GABAAR.

Figure 1.

Structures of NES and its tentative metabolites identified by in vitro metabolism studies. Reference metabolites were custom synthesized and used for metabolite profiling, PR transactivation, and GABA_A receptor binding studies.

Figure 2.

PR and AR transactivation activity in response to progestins. HEK-293T cells transiently expressing hPR (A) or AR (B) were incubated for 16 h with progesterone (Prog), levonorgestrel (LNG), NES, 13 ethyl NES (13NES), or norethindrone (Noret). The cell extracts were assayed for luciferase and β-galactosidase activities. The PR and AR transactivation activity was determined by the luciferase activity normalized by the β-galactosidase activity. Values are the means ± SEM of 3 independent experiments performed in triplicate. GraphPad Prism software was used for curve fitting and calculation of the EC₅₀ values.

Figure 3.

Docking of NES within the PR and AR ligand-binding cavity. (A, B) Accommodation mode of NES within the PR LBD (PDB ID: 1A28). The α-helices of the PR LBD are depicted as gray ribbons. (C, D) NES accommodation within the AR LBD (PDB ID: 2AMA). The α-helices of the AR LBD are depicted as blue ribbons. Some residues in the binding pockets are shown with their carbon, oxygen, nitrogen, and sulfur atoms colored in gray, red, blue, and green, respectively. The carbon and oxygen atoms of the ligand NES are colored in gold and red, respectively. The figure panels were generated using the Dino package (DINO: Visualizing Structural Biology 2002) (http://www.dino3d.org). The view in B and D was obtained by applying 90° rotations (around the x and y axis) on the view of A and C.

Figure 4.

NES metabolism in plasma and brain of female mice after NES administration. NES and its metabolites were measured by GC/MS/MS. Steroid concentrations were measured in plasma after administration of NES at 200 µg (A) and 10 µg (B) and in brain at 200 µg (C) and 10 µg (D) at different postadministration time points. Steroid concentrations are expressed as ng/mL ± SEM or ng/g ± SEM.

Figure 5.

NES metabolism in plasma and brain of female mice after NES administration at 10 and 200 µg/mouse. NES metabolites were measured by GC/MS/MS. Steroid concentrations are measured in plasma after administration of NES at 200 µg (A) and 10 µg (B) and in brain at 200 µg (C) and 10 µg (D) at different postadministration time points. Steroid concentrations are expressed as ng/mL ± SEM or ng/g ± SEM.

Figure 6.

The Nestorone metabolite 3α, 5α-THNES has little effect on GABA_ARs. (A) Illustrated are the GABA (3 µM)-induced inward currents recorded from a representative WSS-1 cell in the absence (black trace) and presence (gray trace) of the neurosteroid 3α, 5α-THPROG (100 nM). Note the large enhancement of the GABA-evoked response produced by this neurosteroid. (B) The nestorone metabolite 3α, 5α-THNES (100 nM) produced only a modest increase in the GABA (3 µM)-evoked current. (C) Bar chart summarizing the effect of 3α, 5α-THPROG (100 nM; n = 4) and 3α, 5α-THNES (100 nM, n = 10; 1 µM, n = 5) on the peak amplitude of the GABA-evoked response. **P < 0.01; ***P < 0.001 (Student t test). (D, E) The black traces illustrate averaged mIPSCs recorded from representative mouse cortical pyramidal neurons under control conditions. Superimposed upon these control recordings are representative mIPSCs recorded from cortical neurons obtained from brain slices incubated for ∼2 h in 3α, 5α-THPROG (100 nM) (D) and 3α, 5α-THNES (100 nM) (gray traces) (D). (F) Bar chart summarizing the effect of 3α, 5α-THPROG (100 nM; n = 5 neurons) and 3α, 5α-THNES (100 nM, n = 4 neurons; 1 µM, n = 4 neurons) on the control (n = 25 neurons) mIPSC decay time [quantified as the τw the weighted time constant of decay (ms)]. ^###P < 0.001 (1-way ANOVA). Note the large prolongation produced by 3α, 5α-THPROG (100 nM), whereas 3α, 5α-THNES (100 nM) was inert in this respect. The data for the control τw and the τw in the presence of 3α, 5α-THPROG (100 nM) is reproduced from Brown et al. (32).