113P Queen Elizabeth II Conference Centre London
BPS Winter Meeting 2010

 

 

In vitro and in vivo characterisation of two contrasting progesterone receptor antagonists

Amy Brown1, Kirsty Bess4, Peter Bungay3, Karl Gibson2, Nick Pullen1. 1Internal Medicine, Pfizer Global Research and Development, United Kingdom, 2World Wide Medicinal Chemistry, Pfizer Global Research and Development, United Kingdom, 3PDM, Pfizer Global Research and Development, United Kingdom, 4Primary Pharmacology Group, Pfizer Global Research and Development, United Kingdom.

 

Progesterone receptor (PR) signalling is involved in the secretory transformation of the uterine endometrium. Antagonising this process may prove to be a beneficial treatment for certain gynaecological disorders. Here, we describe and contrast the pharmacological properties of steroidal and non-steroidal PR antagonists, RU-486 and PF-02413873 (4-{[3-Cyclopropyl-1-(mesylmethyl)-5-methyl-1H-pyrazol-4-yl]oxy}-2,6-dimethylbenzonitrile) respectively, in in vitro and in vivo assays.

In cynomolgus macaques, compared with vehicle treated animals, RU-486 (20mg/kg/day) showed a 34% (p<0.003) reduction in endometrial thickness and an 87% (p<0.001) reduction in BrdU incorporation as a marker of proliferation. In contrast PF-02413873 (5 and 20 mg/kg/day) induced 43% (p<0.001) and 56% (p<0.001) reduction in endometrial thickness as well as 15% and 39% (p<0.015) reduction in BrdU incorporation, respectively. The pharmacological mechanism of action of RU-486 and PF-02413873 was evaluated in two in vitro assays, gene transcription and nuclear translocation.

Gene Transcription

Both RU-486 and PF-02413873 inhibited agonist (EC80 progesterone) induced alkaline phosphatase production in the breast cancer cell line, T47D. At high concentrations (>1µM), PF-02413873, in contrast to RU-486, also displayed an apparent agonism (Table 1). To explore this further, Schild experiments were conducted with Lew and Angus non linear regression analysis1 where dextral displacement of a progesterone concentration response curve was observed for both antagonists (Table 1).

Gene Transcription Nuclear Translocation
Agonist Antagonist Agonist
EC50 nM (±SEM) Emax (±SEM) IC50 nM (±SEM) pKb (±SEM) EC50 nM (±SEM) Emax (±SEM)
Progesterone 2.52 (±0.12) 97 (±0.97) 3.69 (±0.60) 107 (±3.56)
RU-486 NOA NOA 0.25 (±0.07) 11.59 (±0.06) 0.78 (±0.22) 85 (±5.13)
PF-02413873 >1000 ≥20 20.00 (±4.03) 6.34 (±0.06) 2541 (±517.2) 93.1 (±8.01)

Table 1 Mean or geometric mean of in vitro data. n≥3, NOA – no observable agonism.

Nuclear Translocation

Experiments were conducted using a recombinant cell line incorporating the DiscoveRx(TM) PR nuclear translocation PathHunter(TM) technology. When increasing concentrations of RU-486 and PF-02413873 were incubated alone with this cell line, they induced nuclear translocation (Table 1). RU-486 induced nuclear translocation at concentrations similar to those that inhibited gene transcription. PF-02413873 induced nuclear translocation at high concentrations (>1µM) similarly to those that displayed agonism in the gene transcription assay.

While the observed effects of RU-486 and PF-02413873 on endometrial thickness in the macaque appear to be similar, the mechanism of action of RU-486 and PF-02413873 appear to be different. RU-486 facilitates nuclear translocation, yet acts as a competitive antagonist on gene transcription whilst, in contrast, PF-02413873 appears to have complex pharmacology, behaving as a neutral antagonist at low concentrations while agonises PR function and facilitates nuclear translocation at high concentrations. Further work is needed to fully elucidate the mechanism of action of PF-02413873.

 

Lew, M.J. and Angus, J.A. Trends Pharmacol Sci, 1995. 16(10): 328-37.