**5. Pre-clinical effects of PR antagonists in pre-clinical models**

The study of the effects of PRAs in pre-clinical models has shed light on the site of and mechanism of action of PRAs in normal reproductive physiology and disease. The classical models for quantifying PRA activity are the modified assay in juvenile rabbits according to McPhail (McPhail, 1933), the induction of luteolysis in the guinea pig (Elger et al., 2000) and the inhibition of decidualisation in the rat. For the purposes of this review, I have focussed on the pre-clinical models which have been used to support a role for PR in the pathogenesis and treatment of endometriosis, principally focussing on studies in the rodent and macaque. Many data have been acceded from studies in the normal animal, but several experimental models of endometriosis have been also developed in normal as well as immune compromised rodents and non-human primates (D'Hooghe et al., 2009; Grummer, 2006; Laschke & Menger, 2007). In these models, cystic ectopic endometrial lesions develop following the transplantation of syngeneic or human uterine endometrial tissue under the control of ovarian estradiol. Measuring temporal changes in the size of these lesions and their proliferative capacity as well other aspects of the disease presentation is a powerful pre-clinical yardstick for testing the efficacy of experimental drugs.

### **5.1 Rodent**

164 Endometriosis - Basic Concepts and Current Research Trends

Dose dependent suppressed cell proliferation and tumour latency in a Nmethyl-N-nitrosoureainduced mammary carcinogenesis rat model

Intrauterine delivery of CDB-2914 suppressed endometrial growth and menstruation in artificially cycled Rhesus

macaques

Dose dependent interruption of

the macaque

ORG-31710 administered with desogestrel reduced the

incidence of unscheduled vaginal bleeding c.f. desogestrel alone in the macaque

Partial agonist properties in McPhail's assay. Marginal labourinducing activity during mid-pregnancy and ineffective in inducing preterm parturition in the guinea pig. Abolition of menstrual cyclicity and induction of endometrial atrophy in the macaque

pregnancy in the rat and reduction in tumour mass in a DMBAinduced rat mammary tumour model. Dose dependent inhibition of endometrial growth in

(Attardi et al., 2002; Brenner et al., 2010; Wiehle et al., 2011)

(Attardi et al., 2002; Brenner et al., 2010; Gainer & Ulmann, 2003)

(Afhüppe et al., 2010; Fuhrmann et al., 2000; Slayden et al., 2001)

(DeManno et al., 2003; Elger et al., 2000; Fensome et al., 2008)

(Kloosterbo er et al., 1994; 2000; Verbost et al., 2005)

nM; T47D functional IC50=11 nM1 GR binding IC50=17 nM IC50=130 nM AR binding IC50=288

nM; T47D functional

GR binding IC50=18 nM; functional IC50=73.8 nM AR binding IC50 = 65

GR binding IC50 = 16

AR binding IC50 = 54

AR functional IC50=6.1 nM

MR functional IC50=1.6 μM ER functional IC50=

GR functional IC50=85

affinity equivalent to

Table 1. (continuation) Pharmacological properties of key non-steroidal and steroidal PRAs 1 In these assays, the activity of RU-486 was PR binding IC50=9 nM; T47D IC50=7.6 nM; GR binding IC50=10 nM; IC50=5.9 nM; AR binding IC50=45 nM; 2 In these assays, the activity of RU-486 was PR binding IC50=0.028 nM; GR binding IC50=2.2 nM; AR binding IC50=10 nM

30 fold lower GR relative binding affinity c.f. RU-486

CDB-4124 / proellex <sup>N</sup>

CDB-2914 / ulipristal <sup>N</sup>

ZK-230211 / linaprostone

J-867 / asoprisnil

ORG-31710

O

O

O

O

O

H H H

H H H

H H H

H H H O O

O O

CF2CF3 OH

OMe <sup>N</sup> HO OMe PR functional IC50=0.2 nM

O

H H H

MeO <sup>O</sup> PR binding IC50=19

nM

nM1

nM

nM2

nM

1.9 μM

RU-486.

<sup>N</sup> PR relative binding

<sup>O</sup> PR binding IC50 =

<sup>O</sup> PR binding IC50=7

IC50=7 nM

0.0036 nM

Mouse knockout studies have elegantly described differences in the function of PR-A and PR-B. Both PR null mutation (PRKO) and selective disruption of the PR-A isoform (PRAKO)

Progesterone Resistance and Targeting

growth in the macaque.

**a b**

the Progesterone Receptors: A Therapeutic Approach to Endometriosis 167

facilitate endometrial growth. Studies in ovariectomised (OVX) and E2-supplemented macaques, have been perhaps even more revealing with respect to the mechanism driving this effect, especially the direct as well as indirect effects of PRAs on the hypothalamicpituitary-gonadal axis and endometrium. Firstly, RU-486 has been shown to suppress the estrogen-induced LH surge in the OVX macaque (Wolf et al., 1989), underwriting a role for PR in regulating the hypothalamic-pituitary axis in higher species as suggested by early studies in knockout mice (Conneely et al., 2001). Furthermore, as RU-486 does not appear to blunt GnRH-induced LH secretion in the macaque (Heikinheimo et al., 1995), this has suggested that PRAs directly block ovarian folliculogenesis. In intact macaques with PRA doses that are too low to block ovulation or in the OVX/E2 macaque, steroidal PRAs that include can also directly suppress the effects of estrogen on the endometrium by inhibiting cell proliferation and thickness suggesting that PR regulates reproductive function at multiple points (Brenner et al., 2010; DeManno et al., 2003; Hodgen et al., 1994; Ishwad et al.,

1993; Slayden et al., 1998; 2001; 2006; Wolf et al., 1989; Zelinski-Wooten et al., 1998).

While many studies have been commonly undertaken by oral or systemic administration of PRAs, principally RU-486, some studies have also been undertaken by local, intrauterine administration, such as those with CDB-2914 and ZK-230211(Brenner et al., 2010; Nayak et al., 2007). In each case, the intrauterine administration resulted in the characteristic inhibition of normal menstrual bleeding, atrophy of endometrial spiral arterioles and functionalis thickness, consistent with observations from systemic administration (Figure 2). Unfortunately neither of these studies were supported with a confirmation of drug exposure, comparing the local versus systemic exposure to demonstrate that the effects were mediated by a local site of action and not an indirect effect. Nonetheless the data support others which suggest that PRAs can work locally to block estrogen effects on endometrial

**Blank-IUS CDB-2914-IUS Blank-IUS CDB-2914-IUS**

The mechanism of attenuation of estradiol effects on the endometrium is not well understood, and although steroidal PRAs appear to block cell proliferation in various *in vitro* cell-based systems, the concentrations needed for this effect are considerably greater than those which elicit the effect *in vivo* (Freeburg et al., 2009a; Goyeneche et al., 2007; Murphy et al., 2000; Ohara et al., 2007; Wu & Guo, 2006). One clue to a potential mechanism

Fig. 2. (a) Induction of endometrial atrophy by CDB-2914-intrauterine system (IUS) versus blank-IUS (taken from (Brenner et al., 2010) with permission); E, endometrium; Myo, myometrium (original magnification ×25). (b) AR staining on endometrial tissue samples taken from macaques treated with CDB-2914-IUS or blank-IUS (original magnification ×340).

in the mouse leads to a failure of ovulation due to disabled follicular rupture in response to gonadotrophin stimulation (Lydon et al., 1995; Mulac-Jericevic & Conneely, 2005). The histological characterisation of uteri from PRKO mice confirmed extensive epithelial hyperplasia (Lydon et al., 1995). In contrast, the stromal compartment was distinctly oedematous and infiltrated with neutrophils and macrophages. While these data strongly support the notion of PR in suppressing ER function in the uterus, the cystic dilation, epithelial hyperplasia and associated inflammation are also histological hallmarks of endometriosis, especially those characterised in rodent disease models (Bruner et al., 1997; Grummer, 2006; Hull et al., 2003; Vernon & Wilson, 1985). The endometrial epithelial hyperplasia observed in the uteri of PRAKO mice was similar to that observed for PRKO mice suggesting that PR-B is unable to compensate for the loss of PR-A (Mulac-Jericevic et al., 2000). In contrast, ovarian and uterine response to E2/P4 appear to be normal in PR-B knockout mice, whereas mammary lobuloalveolar development was markedly reduced due to decreased ductal and alveolar epithelial cell proliferation (Mulac-Jericevic et al., 2003). Taken together these findings demonstrate the extremely important role PR plays in regulating ovarian function and spatiotemporal cell growth in different tissue compartments in response to E2/P4 in the mouse.

PRKO mice have been used to explore the role of PR in the development and growth of ectopic lesions in a syngenic mouse model of endometriosis (Fang et al., 2004). In this study, the volumes of PRKO lesions collected from animals treated with E2 were approximately 20% larger than those from corresponding wild-type animals. Additionally, the effects of P4 on PRKO lesions were ablated compared with those from wild-type animals, underscoring the important role that PR plays in regulating E2-dependent cell proliferation in the rodent.

Whilst the evaluation of gene ablation on eutopic and ectopic endometrial cell growth has been revealing, the studies of pharmacological modulation contrast these observations to a certain extent as both progestogens and PRAs reduce ectopic endometrial cell proliferation and disease burden in pre-clinical rodent models of endometriosis (Bruner-Tran et al., 2006; Chwalisz et al., 1998; Katayama et al., 2010; Katsuki et al., 1998; Stoeckemann et al., 1995). An explanation of this phenomenon compared with the phenotype of PRKO animals has been revealed by studies with PRAs in the non-human primate.

#### **5.2 Macaque**

Given the evolutionary and physiological proximity of the macaque menstrual cycle with the human, many groups have evaluated the role of PR and the effects of PRAs on the macaque endometrium. Most data revealing the effect of PRAs on the endometrium have come from studies evaluating the effects of steroidal PRAs. When administered acutely after the mid-cycle LH surge, or during the progesterone phase in artificially cycled animals, PRAs impair the effects of progesterone on endometrial arborisation and induce an early menstruation. In the intact macaque, animals undergo an anovulatory amenorrhoea under the influence of continuous steroidal PRA exposure (Brenner et al., 2010; Slayden et al., 2001). In these animals, the endometrium is characterised by decreased wet weight, thickness and mitotic activity. The endometrium undergoes a characteristic atrophy and compaction of the stroma, glandular apoptosis as well as degeneration of the endometrial spiral arterioles. These effects are characteristically anti-estrogenic in nature, and yet the effects occur in the presence of mid-follicular levels of E2, levels that should be sufficient to

in the mouse leads to a failure of ovulation due to disabled follicular rupture in response to gonadotrophin stimulation (Lydon et al., 1995; Mulac-Jericevic & Conneely, 2005). The histological characterisation of uteri from PRKO mice confirmed extensive epithelial hyperplasia (Lydon et al., 1995). In contrast, the stromal compartment was distinctly oedematous and infiltrated with neutrophils and macrophages. While these data strongly support the notion of PR in suppressing ER function in the uterus, the cystic dilation, epithelial hyperplasia and associated inflammation are also histological hallmarks of endometriosis, especially those characterised in rodent disease models (Bruner et al., 1997; Grummer, 2006; Hull et al., 2003; Vernon & Wilson, 1985). The endometrial epithelial hyperplasia observed in the uteri of PRAKO mice was similar to that observed for PRKO mice suggesting that PR-B is unable to compensate for the loss of PR-A (Mulac-Jericevic et al., 2000). In contrast, ovarian and uterine response to E2/P4 appear to be normal in PR-B knockout mice, whereas mammary lobuloalveolar development was markedly reduced due to decreased ductal and alveolar epithelial cell proliferation (Mulac-Jericevic et al., 2003). Taken together these findings demonstrate the extremely important role PR plays in regulating ovarian function and spatiotemporal cell growth in different tissue compartments

PRKO mice have been used to explore the role of PR in the development and growth of ectopic lesions in a syngenic mouse model of endometriosis (Fang et al., 2004). In this study, the volumes of PRKO lesions collected from animals treated with E2 were approximately 20% larger than those from corresponding wild-type animals. Additionally, the effects of P4 on PRKO lesions were ablated compared with those from wild-type animals, underscoring the important role that PR plays in regulating E2-dependent cell proliferation in the rodent. Whilst the evaluation of gene ablation on eutopic and ectopic endometrial cell growth has been revealing, the studies of pharmacological modulation contrast these observations to a certain extent as both progestogens and PRAs reduce ectopic endometrial cell proliferation and disease burden in pre-clinical rodent models of endometriosis (Bruner-Tran et al., 2006; Chwalisz et al., 1998; Katayama et al., 2010; Katsuki et al., 1998; Stoeckemann et al., 1995). An explanation of this phenomenon compared with the phenotype of PRKO animals has

Given the evolutionary and physiological proximity of the macaque menstrual cycle with the human, many groups have evaluated the role of PR and the effects of PRAs on the macaque endometrium. Most data revealing the effect of PRAs on the endometrium have come from studies evaluating the effects of steroidal PRAs. When administered acutely after the mid-cycle LH surge, or during the progesterone phase in artificially cycled animals, PRAs impair the effects of progesterone on endometrial arborisation and induce an early menstruation. In the intact macaque, animals undergo an anovulatory amenorrhoea under the influence of continuous steroidal PRA exposure (Brenner et al., 2010; Slayden et al., 2001). In these animals, the endometrium is characterised by decreased wet weight, thickness and mitotic activity. The endometrium undergoes a characteristic atrophy and compaction of the stroma, glandular apoptosis as well as degeneration of the endometrial spiral arterioles. These effects are characteristically anti-estrogenic in nature, and yet the effects occur in the presence of mid-follicular levels of E2, levels that should be sufficient to

been revealed by studies with PRAs in the non-human primate.

in response to E2/P4 in the mouse.

**5.2 Macaque** 

facilitate endometrial growth. Studies in ovariectomised (OVX) and E2-supplemented macaques, have been perhaps even more revealing with respect to the mechanism driving this effect, especially the direct as well as indirect effects of PRAs on the hypothalamicpituitary-gonadal axis and endometrium. Firstly, RU-486 has been shown to suppress the estrogen-induced LH surge in the OVX macaque (Wolf et al., 1989), underwriting a role for PR in regulating the hypothalamic-pituitary axis in higher species as suggested by early studies in knockout mice (Conneely et al., 2001). Furthermore, as RU-486 does not appear to blunt GnRH-induced LH secretion in the macaque (Heikinheimo et al., 1995), this has suggested that PRAs directly block ovarian folliculogenesis. In intact macaques with PRA doses that are too low to block ovulation or in the OVX/E2 macaque, steroidal PRAs that include can also directly suppress the effects of estrogen on the endometrium by inhibiting cell proliferation and thickness suggesting that PR regulates reproductive function at multiple points (Brenner et al., 2010; DeManno et al., 2003; Hodgen et al., 1994; Ishwad et al., 1993; Slayden et al., 1998; 2001; 2006; Wolf et al., 1989; Zelinski-Wooten et al., 1998).

While many studies have been commonly undertaken by oral or systemic administration of PRAs, principally RU-486, some studies have also been undertaken by local, intrauterine administration, such as those with CDB-2914 and ZK-230211(Brenner et al., 2010; Nayak et al., 2007). In each case, the intrauterine administration resulted in the characteristic inhibition of normal menstrual bleeding, atrophy of endometrial spiral arterioles and functionalis thickness, consistent with observations from systemic administration (Figure 2). Unfortunately neither of these studies were supported with a confirmation of drug exposure, comparing the local versus systemic exposure to demonstrate that the effects were mediated by a local site of action and not an indirect effect. Nonetheless the data support others which suggest that PRAs can work locally to block estrogen effects on endometrial growth in the macaque.

Fig. 2. (a) Induction of endometrial atrophy by CDB-2914-intrauterine system (IUS) versus blank-IUS (taken from (Brenner et al., 2010) with permission); E, endometrium; Myo, myometrium (original magnification ×25). (b) AR staining on endometrial tissue samples taken from macaques treated with CDB-2914-IUS or blank-IUS (original magnification ×340).

The mechanism of attenuation of estradiol effects on the endometrium is not well understood, and although steroidal PRAs appear to block cell proliferation in various *in vitro* cell-based systems, the concentrations needed for this effect are considerably greater than those which elicit the effect *in vivo* (Freeburg et al., 2009a; Goyeneche et al., 2007; Murphy et al., 2000; Ohara et al., 2007; Wu & Guo, 2006). One clue to a potential mechanism

Progesterone Resistance and Targeting

486 and other steroidal PRAs.

the Progesterone Receptors: A Therapeutic Approach to Endometriosis 169

assessment, PF-02413873 also appears to have a different pharmacological profile from RU-

While these interesting observations are important in the context of normal endometrial physiology, few studies have been undertaken in macaques with endometriosis to build translational understanding to disease. Menstruating primates, such as the baboon and the macaque, develop spontaneous endometriosis and ectopic lesions that are histologically identical to the human disease (D'Hooghe et al., 2009). For many researchers, the proximity of this model to the human condition has made this the model of choice for the assessment of interventional agents the endometriosis. Spontaneous disease is acquired with a similar time course as experienced by the human female, developing slowly over a period of years and is not easily diagnosed without laparoscopy. Consequently, researchers have used intraperitoneal inoculation of autologous menstrual or endometrial tissue to develop an

The only study evaluating the effect of a steroidal PRA, RU-486, in a non-human primate model of endometriosis was reported by Grow *et al* (1996). This study was undertaken in a surgical induction model of endometriosis and disease allowed to develop prior to dosing. A baseline measure of burden (peritoneal lesion area) was undertaken and then macaques were treated with either RU-486, leuprolide or vehicle for one year. Both RU-486 and leuprolide induced an anovulatory amenorrhoea and reduced peritoneal disease levels to a similar levels, >75%, compared with the vehicle control group. The authors additionally evaluated the effect of RU-486 and leuprolide on bone mineral density as revealed by dual x-ray absorptiometry. Consistent with the post-menopausal levels of E2 achieved, leuprolide induced a 0.035 g/cm2 reduction in bone mass compared with +0.1 g/cm2 for vehicle control and +0.25 g/cm2 for the RU-486 treated animals. These data support earlier observations that steroidal PRAs are able to suppress endometrial cell growth whilst maintaining bone-

experimental model of endometriosis that is similar to that observed in women.

**6. Clinical evaluation in healthy women and women with endometriosis** 

RU-486 was originally developed for emergency contraception, however early observations with lower doses than those used clinically, indicated that when given acutely during the luteal phase, RU-486 would facilitate the onset of menstruation by the upregulation of endometrial prostaglandins and given chronically, RU-486 would delay menses (Hapangama et al., 2002; Shoupe et al., 1987). The effects of RU-486 on the ovarian cycle and endometrium appear to be dose dependent, that is low doses interfere with estrogen function and disrupt endometrial growth (Croxatto et al., 1993; Narvekar et al., 2004), but higher doses additionally suppress follicular development by impairing gonadotrophin secretion (Gemzell-Danielsson et al., 1996; Liu et al., 1987; Spitz et al., 1993; 1994). These observations strike a resounding chord with those data acceded in the macaque described earlier. The potential value of PRAs as alternative contraceptives to current combined or progestin-only pills have been long recognised and evaluated in a number of different dosing and delivery strategies (Baird et al., 2003; Brown et al., 2002; Chabbert-Buffet et al., 2007; Heikinheimo et al., 2007; Lakha et al., 2007; Nayak et al., 2007). Whilst no pregnancies were reported after 200 months in women who received 2-5 mg RU-486 daily (Brown et al., 2002), lower doses appeared to be less effective (Croxatto et al., 1998). Similar observations on the suppression of ovulation and the normal menstrual cycle have also been made with

sparing mid-follicular levels of E2 (Heikinheimo et al., 1995).

has emerged from observations of elevated endometrial androgen receptor (AR) expression (Narvekar et al., 2004; Slayden & Brenner, 2003) following PRA administration and the known effects of AR modulators (e.g. danazol) on endometrium (Rose et al., 1988).

These observations have been functionally evaluated further (Slayden & Brenner, 2003) in which OVX/E2 macaques were continuously treated with ZK-137316 or together with the AR antagonist, flutamide, for 28 days. Flutamide reversed the inhibitory effects of ZK-137316 on the E2/OVX endometrium, restoring levels of endometrial proliferation and thickness to control levels (Table 2).


Table 2. Morphometric assessment of androgen receptor blockade of ZK-137316 effects on OVX/E2 macaquesa (Adapted from (Slayden & Brenner, 2003)) aAll values represent mean ± SE; b*p*<0.05 compared with values in the same row; cstromal cells/10,000 μm2; dmitotic cells/1000 epithelial cells

That flutamide did not appear to inhibit the PR activity of ZK-137316 (i.e. ZK-137316 induced menstruation in E2/P4 artificially cycled animals in the presence of flutamide), suggested that the endometrial anti-proliferative effects of steroidal PRAs like ZK-137316 are mediated by a mechanism involving AR. However, despite this extremely important observation, the seminal Slayden publication has not been followed up further. For instance, it is not clear what ligand is driving the AR effect, as testosterone levels do not appear to be altered in ZK-137316 treated animals, or how the signal is transduced through AR; if it is genomic or non-genomic. If AR is inducing a genomic effect, what are the transcripts that are altered and confer the inhibitory effect on PRA? Other important and, as yet, unaddressed questions also include whether these effects are only manifested only by the steroidal class of PRAs, but the observation that RU-486 can elevate endometrial AR expression in women goes some way to understanding the translational significance of the macaque findings (Narvekar et al., 2004).

Non-steroidal classes of PRAs have also been studied in a similar way in the macaque. Of the novel class of cyanophenoxypyrazoles, PF-02367982 dose-dependently inhibited the progesterone-mediated aborisation of the endometrium and delayed menses induction when dosed for 20 days from the start of the menstrual cycle. PF-02367982 also increased AR protein expression in a similar manner to that observed by RU-486 and the non-steroidal PRA, WAY-255348 (de Giorgio-Miller et al., 2008; Fensome et al., 2008). These data are consistent with other non-steroidal PRAs that have been assessed, such as WAY-255348 (Fensome et al., 2008). More recently, PF-02413873 a more potent PRA than PF-02367982 (Table 1) has been shown to reduce endometrial cell proliferation and thickness in intact macaques dosed for 10 days from the start of menstruation (Howe et al., 2011). In this study, however, AR expression was not appreciably altered with PF-02413873 treatment compared with RU-486. While this may be, in part, due to the timepoint for the comparison and

has emerged from observations of elevated endometrial androgen receptor (AR) expression (Narvekar et al., 2004; Slayden & Brenner, 2003) following PRA administration and the

These observations have been functionally evaluated further (Slayden & Brenner, 2003) in which OVX/E2 macaques were continuously treated with ZK-137316 or together with the AR antagonist, flutamide, for 28 days. Flutamide reversed the inhibitory effects of ZK-137316 on the E2/OVX endometrium, restoring levels of endometrial proliferation and

(0.1 mg/kg i.m.)

45.5 ± 3.4 142.3 ± 63.7b 54.0 ± 4.6

Table 2. Morphometric assessment of androgen receptor blockade of ZK-137316 effects on OVX/E2 macaquesa (Adapted from (Slayden & Brenner, 2003)) aAll values represent mean ± SE; b*p*<0.05 compared with values in the same row; cstromal cells/10,000 μm2; dmitotic

That flutamide did not appear to inhibit the PR activity of ZK-137316 (i.e. ZK-137316 induced menstruation in E2/P4 artificially cycled animals in the presence of flutamide), suggested that the endometrial anti-proliferative effects of steroidal PRAs like ZK-137316 are mediated by a mechanism involving AR. However, despite this extremely important observation, the seminal Slayden publication has not been followed up further. For instance, it is not clear what ligand is driving the AR effect, as testosterone levels do not appear to be altered in ZK-137316 treated animals, or how the signal is transduced through AR; if it is genomic or non-genomic. If AR is inducing a genomic effect, what are the transcripts that are altered and confer the inhibitory effect on PRA? Other important and, as yet, unaddressed questions also include whether these effects are only manifested only by the steroidal class of PRAs, but the observation that RU-486 can elevate endometrial AR expression in women goes some way to understanding the translational significance of the

Non-steroidal classes of PRAs have also been studied in a similar way in the macaque. Of the novel class of cyanophenoxypyrazoles, PF-02367982 dose-dependently inhibited the progesterone-mediated aborisation of the endometrium and delayed menses induction when dosed for 20 days from the start of the menstrual cycle. PF-02367982 also increased AR protein expression in a similar manner to that observed by RU-486 and the non-steroidal PRA, WAY-255348 (de Giorgio-Miller et al., 2008; Fensome et al., 2008). These data are consistent with other non-steroidal PRAs that have been assessed, such as WAY-255348 (Fensome et al., 2008). More recently, PF-02413873 a more potent PRA than PF-02367982 (Table 1) has been shown to reduce endometrial cell proliferation and thickness in intact macaques dosed for 10 days from the start of menstruation (Howe et al., 2011). In this study, however, AR expression was not appreciably altered with PF-02413873 treatment compared with RU-486. While this may be, in part, due to the timepoint for the comparison and

E2/ZK-137316 (0.1 mg/kg i.m.) +

Flutamide (2 mg/kg s.c.)

known effects of AR modulators (e.g. danazol) on endometrium (Rose et al., 1988).

thickness to control levels (Table 2).

Stromal compactionc

cells/1000 epithelial cells

macaque findings (Narvekar et al., 2004).

E2 alone E2/ZK-137316

Weight (mg) 360 ± 32 64 ± 10b 265 ± 92 Thickness (mm) 3.3 ± 0.4 1.1 ± 0.3b 2.2 ± 0.6

Mitotic indexd 6.3 ± 0.6 0.3 ±0.3b 5.2 ± 3.8

assessment, PF-02413873 also appears to have a different pharmacological profile from RU-486 and other steroidal PRAs.

While these interesting observations are important in the context of normal endometrial physiology, few studies have been undertaken in macaques with endometriosis to build translational understanding to disease. Menstruating primates, such as the baboon and the macaque, develop spontaneous endometriosis and ectopic lesions that are histologically identical to the human disease (D'Hooghe et al., 2009). For many researchers, the proximity of this model to the human condition has made this the model of choice for the assessment of interventional agents the endometriosis. Spontaneous disease is acquired with a similar time course as experienced by the human female, developing slowly over a period of years and is not easily diagnosed without laparoscopy. Consequently, researchers have used intraperitoneal inoculation of autologous menstrual or endometrial tissue to develop an experimental model of endometriosis that is similar to that observed in women.

The only study evaluating the effect of a steroidal PRA, RU-486, in a non-human primate model of endometriosis was reported by Grow *et al* (1996). This study was undertaken in a surgical induction model of endometriosis and disease allowed to develop prior to dosing. A baseline measure of burden (peritoneal lesion area) was undertaken and then macaques were treated with either RU-486, leuprolide or vehicle for one year. Both RU-486 and leuprolide induced an anovulatory amenorrhoea and reduced peritoneal disease levels to a similar levels, >75%, compared with the vehicle control group. The authors additionally evaluated the effect of RU-486 and leuprolide on bone mineral density as revealed by dual x-ray absorptiometry. Consistent with the post-menopausal levels of E2 achieved, leuprolide induced a 0.035 g/cm2 reduction in bone mass compared with +0.1 g/cm2 for vehicle control and +0.25 g/cm2 for the RU-486 treated animals. These data support earlier observations that steroidal PRAs are able to suppress endometrial cell growth whilst maintaining bonesparing mid-follicular levels of E2 (Heikinheimo et al., 1995).
