Novel, orally active selective progesterone receptor modulator CP8947 inhibits leiomyoma cell proliferation without adversely affecting endometrium or myometrium

Abstract

Uterine leiomyomas are highly prevalent and often symptomatic, but current medical therapies are limited. A novel, potent, selective, orally active therapy is needed. The goal of these studies was to determine the progesterone receptor (PR) specificity and activation, endometrial response, and impact on leiomyoma cell proliferation and extracellular matrix (ECM) production of the novel non-steroidal selective progesterone receptor modulators (SPRMs) CP8863 and CP8947. In vitro progestational activity was assessed by alkaline phosphatase assay and ER-α expression. In vivo progestational activity was assayed by the McPhail assay. Proliferation and gene expression studies were performed in immortalized human leiomyoma and myometrial cells. Both CP8863 and CP8947 were highly selective for progesterone receptor (PR) but not for ER-α, AR, and GR. Both compounds induced alkaline phosphatase comparably to progesterone, while CP8947 induced ER-α in leiomyoma cells but not myometrial cells. CP8947 was progestational in rabbit endometrium. Nanomolar CP8947 treatment inhibited human leiomyoma but not myometrial cell proliferation. Extracellular matrix components were decreased in leiomyoma cells, including COL1A1 and COL7A1 at nanomolar concentrations. CP8947 was a potent novel non-steroidal SPRM that was selective for PR, demonstrated progestational activity in endometrium, inhibited leiomyoma cell proliferation and decreased ECM component production, without disrupting myometrial cell proliferation.

Figure 1

Chemical structure of CP8863 (left) and its metabolite CP8947 (right). These novel compounds are not structural derivatives of known steroids, but have a unique structure among the selective progesterone receptor modulators.

Figure 2

Effects of graded concentrations of CP8863 (A) and CP8947 (B) on estrogen receptor-alpha (ER-α) mRNA levels in immortalized human leiomyoma and patient-matched myometrial cells as assessed by real time RT-PCR. Comparative expression of the two compounds and cell types were assessed at 24 hours. (A) In the absence of treatment, ER-α expression was comparable between myometrial and leiomyoma cells. However, 10nM concentration (10⁻⁸M) of CP8863 induced increased ER-α transcripts in myometrial cells, and at 1nM concentrations in leiomyoma cells. Increasing concentration of CP8863 to 10μM did not result in further ER-α expression in myometrial cells, while there was a greater expression of ER-α in leiomyoma cells only 1μM concentration. At concentrations of 10uM CP8863, ER-α expression dramatically decreased in both cell types. *, P< 0.05 vs. untreated myometrial cells or untreated leiomyoma cells. (B) In the absence of treatment, ER-α expression was comparable between myometrial and leiomyoma cells. In myometrial cells, CP8947 did not induce ER-α expression at concentrations ranging from 1nM to 10μM. However, 1nM concentration (10⁻⁹M) of CP8947 dramatically induced ER-α expression in leiomyoma cells. Increasing concentration of CP8947 resulted in greater expression of ER-α in leiomyoma cells at concentrations up to 1μM. At 10μM CP8947, ER-α expression dramatically decreased. *, P< 0.05 vs. untreated leiomyoma cells.

Figure 3

Effects of CP8863 and CP8947 on expression of alkaline phosphatase activity in T47D breast cancer cells. Induction of alkaline phosphatase activity was assessed by measurement of the hydrolysis of p-nitrophenyl phosphate (PNPP). Alkaline phosphatase activity was measured following treatment for 20 hours without compound, with 10 nM progesterone alone, or with increasing concentrations of either CP8863 (A) or CP8947 (B), as indicated. Progesterone alone, CP8863, and CP8947 effectively induced alkaline phosphatase activity 10 nM treatment concentrations. Alkaline phosphatase induction by CP8863 approached progesterone induction levels, while CP8947 demonstrated 60% potency relative to 10nM progesterone, even at 1 μM concentration. In competition assays with progesterone, T47D cells were treated with 10 nM progesterone for 20 hours in the presence of increasing concentrations of either CP8863 (C) or CP8947 (D), as indicated. Concomitant treatment of progesterone and CP8863 demonstrated an additive induction at concentrations of 100nM or greater, while CP8947 partially inhibited progesterone-mediated alkaline phosphatase stimulation at 500nM concentrations. * = p<0.01 relative to untreated cells. ** = p<0.01 relative to progesterone-treated cells.

Figure 4

Progestational effects of CP8863 and CP8947 on rabbit endometrium assayed by McPhail Index after estradiol priming. In the absence of compound, no progestational influence was noted (X, red line). In the presence of progesterone, maximal progestational histologic changes were noted (●, green line). (A) When either CP8863 (yellow) or CP8947 (blue) were provided subcutaneously, there was a dose-dependent increase in progestational histologic findings that were comparable. (B) With oral dosing, the progestational effect of CP8947 was greater than CP8863 at maximal dose, approaching the progestational effect demonstrated with progesterone. (C) In competition studies using 10nM progesterone and increasing oral concentrations of either CP8863 or CP8947, neither compound demonstrated anti-progestational effect, even at concentrations as high as 3mg/rabbit/day. * = p<0.01 relative to untreated animals. **= p<0.01 relative to progesterone-treated animals.

Figure 5

Effects of graded concentrations of CP8863 and CP8947 on the number of viable cultured human myometrial and leiomyoma cells, as assessed by sulforhodamine-B method. At concentrations ranging from 1nM to 10μM, CP8863 had no discernable effect on either myometrial (A) or leiomyoma (B) cell viability. CP8947 also had no discernable impact on myometrial cell proliferation (C). However, compared with untreated control leiomyoma cultures, 48-h treatment with 1nM (10⁻⁹M) CP8947 significantly decreased the number of viable leiomyoma cells. Results represent the mean + SE of at least three independent experiments performed in triplicate. * = p<0.025 relative to untreated cells).

Figure 6

Effects of graded concentrations of CP8947 on the number of viable cultured human myometrial and leiomyoma cells, as assessed by sulforhodamine-B method after 72 hours treatment. At concentrations ranging from 10pM to 1μM, CP8947 inhibited myometrial cell proliferation only at 1μM treatment concentrations. However, compared with untreated control leiomyoma cultures, 72-h treatment with 0.1nM (10⁻¹⁰M) CP8947 significantly decreased the number of viable leiomyoma cells. Results represent the mean + SE of at least three independent experiments performed in triplicate. * = p<0.025 relative to untreated cells).

Figure 7

Effects of graded concentrations of CP8863 and CP8947 on caspase-3 expression (A), caspase-7 expression (B), and TRAIL induction in cultured human leiomyoma cells. Compared with untreated control cells, treatment with CP8863 induced caspase-3 activity at 100nM (10⁻⁷M) concentration, and stimulated further induction in a dose-dependent manner, while CP8947 had no discernable impact on caspase-3 expression at treatment concentrations as high as 10μM (10⁻⁵M). Caspase-7 was induced by CP8863 and CP8947 at 1μM concentration (B). TRAIL was induced with 100nM CP8947 treatment and 1μM CP8863 treatment (C). Results represent the mean + SE of at least three in triplicate. * = p<0.01 relative to untreated controls).

Figure 8

Effects of graded concentrations of CP8947 on COL1A1 and COL7A1 expression in human leiomyoma and patient-matched myometrial cells, as assessed by real time RT-PCR analysis. COL1A1 expression in human myometrial cells (A) was unaffected by CP8947 treatment to concentrations as high as 10μM (10⁻⁵M). Leiomyoma cells treated with CP8947 initially demonstrated a dramatic increase in COL1A1 expression by 24 hours at concentrations as low as 1nM, and a rapid diminution of COL1A1 expression to expression level seen in untreated myometrial cells by 48 h (B). COL7A1 expression in human myometrial cells was increased with 1nM treatment concentrations, but decreased by CP8947 treatment at concentrations of 1μM or greater (C), while expression was inhibited at concentrations of 1nM (10⁻⁹M) in human leiomyoma cells. Results represent the mean + SE of at least three in triplicate. * = p<0.01 relative to untreated myometrial cells. ** = p<0.01 relative to untreated leiomyoma cells.