Importance of the alkylaminoethoxy side-chain for the estrogenic and antiestrogenic actions of tamoxifen and trioxifene in the immature rat uterus

Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,

291

21 ( 1982) 291-306 Ltd.

IMPORTANCE OF THE ALKYLAMINOETHOXY SIDE-CHAIN FOR THE ESTROGENIC AND ANTIESTROGENIC ACTIONS OF TAMOXIFEN AND TRIOXIFENE IN THE IMMATURE RAT UTERUS V.C. JORDAN

and B. GOSDEN

Department of Human Oncology, Wisconsin Clinical Cancer Center, University of Wisconsin, Madison, WI 53792 (U.S.A.) Received

10 February

1982; revision

received

16 April

1982; accepted

6 May 1982

The estrogenic and antiestrogenic properties of tamoxifen and trioxifene were compared with their phenolic derivatives (ICI 77949 and LY 126412, respectively) without the alkylaminoethoxy side-chain. Trioxifene was a more potent antiestrogen than tamoxifen in immature rat uterine weight tests and both compounds were partial estrogen agonists. Removal of the side-chain from tamoxifen to produce ICI 77 949 converted the compound from a partial estrogen agonist to a full estrogen agonist. Tamoxifen, trioxifene and ICI 77949, produced an estradiol-like increase in uterine progesterone receptor concentrations (as determined by sucrose density gradient analysis) and a dose-related, estradiol-like, increase in the size and shape of uterine luminal epithelial cells. In contrast, removal of the side-chain from trioxifene to produce LY 126412 converted the compound from a partial estrogen agonist, with antiestrogenic properties, to one with a very low affinity for the estrogen receptor and no biological activity in viva at the daily doses tested (l-64 pg). This was established by uterine wet weight tests, histological examination of luminal epithelial cells and determination of progesterone receptor concentrations. The alkylaminoethoxy side-chain is not only necessary for the antiestrogenic properties of both tamoxifen and trioxifene but also essential for the effective estrogen receptor binding and pharmacological actions of trioxifene. Keywordv:

estrogen

receptor;

progesterone

receptor;

uterine

histology.

The introduction of the non-steroidal antiestrogen tamoxifen for the treatment of advanced breast cancer has focused attention on the pharmacology of this group of drugs (Sutherland and Jordan, 1981). The non-steroidal antiestrogens inhibit the binding of [3H]estradiol by estrogen target tissues (e.g., uterus, vagina and pituitary gland) in vivo (Roy et al., 1964; Perry et al., 1973; Jordan and Dowse, 1976) and inhibit the binding of [3H]estradiol to the estrogen receptor in vitro (Korenman, 1970; Skidmore et al., 1972). However, at present, the precise molecular

0303-7207/82/0000-0000/$02.75

0 Elsevier Scientific

Publishers

Ireland,

Ltd.

292

V.C. /ordun, B. Gosden

mechanism of action of antiestrogens is unknown. There is renewed interest in developing novel antiestrogens because tamoxifen has been so successful as a unique therapy for breast cancer. While the new compounds might have clinical applications they also provide a valuable opportunity to study the structure-activity relationships of the antiestrogenic ligand. The potent non-steroidal antiestrogens are related to a structural derivative of the weak estrogen triphenylethylene. In contrast, one new compound trioxifene, with reported antiestrogenie (Jones et al., 1979; Black and Goode, 1980) and antitumor (Rose et al., 1981) properties, diverges from the usual triphenylethylene-type of structure (Fig. 1). In this study the estrogenic, antiestrogenic and estrogen receptor binding properties of tamoxifen have been compared with trioxifene. Since all of the potent antiestrogens have an alkylaminoethoxy side-chain we have determined the impact of removal of the side-chain from tamoxifen and trioxifene. The synthesis of progesterone receptor and uterine histology are also indicators of the stimulant activity of estrogenic and antiestrogenic compounds so these assays were included in the comparative studies to examine the actions of the test compounds in the rat uterus. The results demonstrate the fundamental importance of the alkylaminoethoxy side-chain for antiestrogenic activity.

MATERIALS

AND

METHODS

Reagents LY 126412 and LY 133314 mesylate (trioxifene mesylate) were gifts from Lilly Research Laboratories, Indianapolis, IN; ICI 77949 and ICI

R’

R ICI

77.949

H

ICI

46,474

CH,CH&,

(TAMOXIFEN)

LY ,w %

126412

H

L Y 126770

CH,

LY

CH,CH,

133314

N 3 IMESYLATE)

(TRIOXIFENE)

Fig. 1. The formulae of compounds

used in the study.

Tamoxifen and trioxifene actions

293

46474 (tamoxifen) were gifts from ICI Ltd. (Pharmaceutical Division), Alderley Park, Macclesfield, Cheshire (England). The formulae of compounds under study are shown in Fig. 1. Estradiol-17P, diethylstilbestrol and Sa-dihydrotestosterone (DHT) were obtained from Sigma Chemical CO., St. Louis, MO: [6,73H]Estradiol-17~ (60 Ci/mmole in toluene:ethanol 9: I, 98% pure) was obtained from the Radiochemical Centre, Amersham (England) and [ 3H]R5020 (promogestone-17e[Me-3H) 87 Ci/mmole in ethanol 98% pure), non-radiolabelled R5020 and [ i4C]ovalbumin (9.94 pCi/mg) from the New England Nuclear Co. (NEN), Boston, MA. The buffer used for t3H]estradiol-17~ binding experiments was TEM (10 mM Tris, 1.5 mM EDTA, 5 mM monothioglycerol pH 7.4 at 4“C) and for progesterone receptor determinations was phosphate buffer (50 mM sodium phosphate, 1 mM monothioglycerol, 10% glycerol and 20 mM sodium molybdate pH 7.4). All constituents of the buffers were purchased from Sigma Chemical Co., St. Louis, MO. Dextran-coated charcoal (DCC) (2.5%) was purchased from Wien Laboratories, Succasunna, NJ.

Immature rat uterine weight tests All injections were made in 0.1% ml peanut oil. Oily solutions of LY 126412, trioxifene, ICI 77949, tamoxifen and estradiol- 17@or combinations of LY 126412, trioxifene and tamoxifen with estradiol-17/I were prepared by taking aliquots from freshly prepared ethanolic solutions, adding the required volume of peanut oil and evaporating the ethanol under a stream of purified nitrogen (oxygen free) on a warm heating mantle. Immature female rats (40-50 g, 22 days old, of the Sprague-Dawley strain) from Ring Animal Laboratories, Inc., Oregon, WI, were randomly divided into groups of at least 8 animals. To determine estrogenic activity, compounds were injected subcutaneously in the loose skin behind the head, on 3 consecutive days and on the 4th day animals were killed by stunning and cervical dislocation. To determine antiestrogenic activity, injections contained a standard dose (0.32 pg daily) of estradiol17/3 and various doses of LY 126412, trioxifene and tamoxifen. Uteri were dissected free of fat, expelled of intraluminal fluid and weighed immediately on a Roller Smith torsion balance. Selected tissue was collected on ice and used for the determination of [‘H]R5020 binding, whilst some was fixed in WARF solution (Glacial acetic acid: 40% formaldehyde: 95% ethanol: distilled water 1: 9 : 10: 20 v/v) and then prepared for histological examination by hematoxylin-eosin staining. Luminal epithelial cell heights, from mid-horn sections, were determined

294

V. C. Jordan, B. Gosden

with an ocular micrometer The results were expressed

at 400 X , calibrated in pm.

with a stage micrometer.

Inhibition of [‘HI estradiol binding in vitro Immature rat uteri were frozen and stored at - 70°C until use. Pooled uteri were homogenized with a Polytron tissue homogenizer (Brinkmann Instruments, Wesbury, NY) (2 X 10 set, setting 2, with ice-water cooling) in a ratio of 1-2 uteri per ml of TEM buffer. The homogenate was centrifuged at 1OOOOOg for 1 h (4°C) in a Sorvall OTD65 ultracentrifuge using a T-865 fixed angle rotor. The pooled cytosol was stored on ice/water prior to use, then 200-~1 aliquots were incubated at 30°C for 30 min with different concentrations of estradiol-17P, trioxifene, tamoxifen, ICI 77946 or LY 126412 administered in 10 ~1 ethanol and 100 ~1 [ 3H]estradiol-17P solution in TEM buffer (final concentration 5X IOh M). Parallel incubations of cytosol (200 pl), [3H]estradiol solution (100 ~1) and either 10 ~1 ethanol or 10 ~1 of ethanolic diethylstilbestrol (to give an incubate concentration of 5 X 1O-6 M) were used to determine the total specific binding of [3H]estradiol by subtraction. All tubes were cooled in ice/water for at least 15 min, then 500 ~1 of a suspension of Dextran coated charcoal (0.25% in TEM) was added to each. They were allowed to a stand for 20 min at 4°C with occasional shaking, then centrifuged at 2000g (4’C) for 10 min. The supernatants were decanted into scintillation vials and radioactivity determined using lo-ml ACS scintillant (Amersham, Arlington Heights, IL 60005). The inhibition curves for each compound were plotted and the relative binding affinities (RBA’s E, = 100) calculated from the following relationship: RBA=

100X

molar concentration molar concentration

of estradiol for 50% inhibition of specific binding of competitor for 50% inhibition of specific binding

Determination of [‘HI R.5020 binding in vitro Uteri from treated immature rats were homogenized in phosphate buffer as described previously (2-4 uteri/ml) and centrifuged at 100000 g to prepare cytosol. Sucrose density gradient analysis was used to establish [ 3H]R5020 binding. Cytosol (370 ~1) was incubated for 2 h (4°C) with 10 ~1 ethanol containing Scr-dihydrotestosterone (final concentration of lo-’ M), 10 ~1 ethanol containing [ 3H]R5020 (to a final concentration of lop8 M) and either 10 ~1 ethanol (for controls) or 10 ~1 ethanol containing nonradioactive R5020 (for a final concentration of 10e6 M) to determine specific and nonspecific binding. Tubes were incubated for 2 h at 4°C unbound ligands were adsorbed onto charcoal pellets (prepared by centrifugation of 1 ml of 0.5% DCC at 4000g and decanting the

295

Tamoxifen and trioxifene actions

LYl26412

‘4/

’ 0.0 I

Daily

I

I

I

0. I

I .o

IO

I

100

Dose of Compounds (lug)

Fig. 2. The estrogenic effects of the compounds in the 4-day immature rat uterine weight test. Increasing daily doses of estradiol-17/3 (A); tamoxifen (0); ICI 77949 (0); trioxifene (0) and LY 126412 (W) were administered SC in 0.1 ml peanut oil for 3 days. On the 4th day animals were killed by stunning and cervical dislocation, uteri were dissected free of fat, expelled of intraluminal fluid and weighed on a torsion balance. 8 rats per group.

supernatant), agitated vigorously with a Vortex mixer and incubated for 20 min at 4’C. After centrifugation at 4000g for 15 min, 200 ~1 of supernatant were layered onto 5-25s sucrose gradients prepared in phosphate buffer. 10 ~1 [‘4C]ovalbumin sedimentation marker (3.6s) was added and tubes were centrifuged for 16 h (4’C) at 250000 g in a Sorvall OTD65 ultracentrifuge using an AH650 swinging bucket rotor. Successive loo-p1 fractions were collected into 3-ml ACS scintillant by needle puncture at the bottom of the tube and radioactivity determined. Cytosol protein concentrations were determined by the method of Lowry et al. (195 1) using BSA for the standard curve. RESULTS Uterine weight tests Dose-response curves of all compounds were compared with estradiol in the 4-day immature rat uterine weight test (Fig. 2). Compared with estradiol, tamoxifen and trioxifene were both partial estrogen agonists of similar potency. Removal of the aminoethoxy side-chain from tamoxifen to form ICI 77949 converted the compound from a partial estrogen

KC.

296

Jordan,

B. Gosden

agonist to a full estrogen agonist. The potency of ICI 77949 was, however, about 1% of that observed with estradiol. In contrast removal of the aminoethoxy side-chain from trioxifene to form LY 126412 converted the compound from a partial estrogen agonist to one with apparently no activity at the doses tested (l-64 pg daily). Compound LY 126770 was similarly not active at increasing uterine weight at the doses tested (l-64 pegdaily) (data not shown). The ability of trioxifene, tamoxifen and LY 126412 to inhibit the full estrogenic effect of 0.32 pg of estradiol daily, were compared in the 4-day immature rat uterine weight test (Fig. 3). Trioxifene was approximately 5 times more potent than tamoxifen as an antiestrogen. However, LY 126412 was inactive as an antiestrogen at the doses tested (l-64 pg daily). Histology 2 uteri from each of the treatment groups in Fig. 2 were taken for

120-

; IIOv) +I gloo2 .$

go-

B b B 800) .5 & 70S 60

I IO

Daily

Dose of Compounds

I

100

(lug)

Fig. 3. The antiestrogenic effects of trioxifene (M); tamoxifen (A) and LY 126412 (0) in the 4-day immature rat uterine weight test. Increasing daily doses of the compounds were administered with a standard dose of estradiol-17P (0.32 rg) and compared with estradiol alone. Injections were made subcutaneously in 0.1 ml of peanut oil for 3 days, on the 4th day animals were killed by stunning and cervical dislocation, uteri were dissected free of fat, expelled of intraluminal fluid and weighed on a torsion balance. 8 rats per group.

Tamoxifen

and trioxifene

291

actions

histological examinations. 5 serial sections from the midline of each uterine horn were compared. In general the estrogenic compounds estradiol and ICI 77949 seemed to increase all cell types in the uterus, i.e., luminal epithelial, stromal and myometrial. In contrast the histology of the uteri from tamoxifen and trioxifene treated animals showed that the luminal epithelial cells were stimulated and increased in size but there was not a general increase in the other cell types. The sizes of the luminal epithelial cells for each of the compounds were compared over their dose-response curves. The daily administration of increasing doses of estradiol for 3 days, produced a corresponding increase in the mean height of luminal epithelial cells: 20.2 _t 0.6 pm (0.02 pg estradiol), 43.5 * 1.2 pm (0.08 pg estradiol), 62.5 -C 1.7 pm (0.32 pg estradiol) and 60.9 k 1.4 pm (1.28 pg estradiol). These data were compared with the dose response curves of test compounds in Table 1. Irrespective of whether the compound was a full estrogen in the uterine weight test (estradiol or ICI 77949) or a partial estrogen (tamoxifen or trioxifene) there was a comparable increase in the size of luminal epithelial cells. Once the uterus had attained its maximal growth potential for a given compound, supramaximal doses caused a differential increase in the size of the luminal epithelial cells. For a given compound, the increase in the size of the luminal epithelial cells was in very good agreement with results obtained with uterine weight data (Fig. 2). No increases in the luminal epithelial cells were observed in uteri from animals treated with LY 146412 (Table 1) which was consistent with uterine weight findings (Fig. 2). Inhibition of [ “Hjestradiol binding in vitro The relative ability of increasing concentrations

of the compounds

to

Table 1 The effects of compounds after 3 days of treatment Compound administered

on uterine luminal

epithelium

Daily dose of compound 1.0

cell heights (am)

in immature

rats

(pg)

4.0

16.0

64.0

Tamoxifen

17.5kO.4

27.4-cO.7

66.0*2.5

64.8 * 2.5

ICI 71949

15.3*0.4

20.3-t

1.3

32.8-cO.8

56.9f

1.6

Trioxifene

27.5 * 1.O

53.32

1.5

61.2*

59.6*

1.6

LY 126412

13.1kO.5

14.4kO.2

Values represent

mean*

SEM for 20 determinations.

1.5

13.7kO.3 Control

12.8kO.2

value was 12.9 pmk0.28.

298

V. C. Jordan, B. Gosden

inhibit the binding of [3H]estradiol to estrogen receptors derived from immature rat uteri were compared with estradiol. All compounds tested produced a concentration related inhibition of [ 3H]estradiol binding (Fig.4). Both tamoxifen (RBA, 2.4) and ICI 77949 (RBA 1.6) had a similar ability to inhibit the binding of [ ‘Hlestradiol. However, trioxifene (RBA, 20.8) was a much more potent inhibitor of [3H]estradiol binding than tamoxifen. Removal of the alkylaminoethoxy side-chain from trioxifene to form LY 126412 (RBA 0.2) caused a dramatic decrease in the ability of the compound to inhibit [ 3Hlestradiol binding. Determination of uterine progesterone receptors Sucrose density gradient analysis was used to compare and contrast the ability of estradiol and the test compounds to increase the concentration of progesterone receptor in the rat uterus after 3 daily treatments. All the results were converted to an equivalence based on 1 mg of cytosol protein. The amounts of protein actually layered on the gradients ranged

Fig. 4. Inhibition of the binding of [3H]estradiol to rat uterine estrogen receptors by estradiol-17b (A), trioxifene (O), tamoxifen (0), ICI 77949 (0) and LY 126412 (m). Uteri were homogenized (2X 10 set bursts of a Polytron tissue homogenizer) in TEM buffer (10 mM Tris, 1.5 mM EDTA, 5 mM monothioglycerol pH 7.4 at 4’C) with l-2 uteri/ml. Cytosol was prepared by centrifugation for 1 h at 100000 g (4’C) in a Sorvall OTD-65 ultracentrifuge using a T-865 fixed angle rotor. Cytosol(200 cl) was incubated with various concentrations of the compounds administered in 10 ~1 of ethanol and [ ‘Hlestradiol-178 in 100 ~1 of TEM buffer (to give an incubation concentration of 5 X lOK9 M) for 30 min at 30°C. After cooling to 4°C free ligand was removed with a dextran-coated charcoal suspension (500 ~1, 0.25%), by incubation at 4°C for 20 min followed by centrifugation at 2000 g (4’C) for 10 min. The supematants were counted and results were represented as a percentage of specific [ ‘Hlestradiol binding in controls.

299

Tamoxifen and trioxifene actions

between 0.5 mg (control) to 1.8 mg (estradiol-treated). The specific binding of [ 3H]R5020 was increased (controls) or decreased (treatments) to standardize the results as if 1 mg cytosol protein had been layered on each gradient. This conversion was undertaken because it was unreasonable to compare progesterone receptor contents of cytosol prepared from uteri of different weights. However, it should be emphasized that there

A

3.6s 1

FRACTION

NUMBER

Fig. 5. Comparison of sucrose density gradients (5-25%) of uterine cytosols from immature rats treated for 3 days with: (A) vehicle (A), estradiol-17j3, 0.2 pg daily (0), 0.08 pg daily (W), 0.32 cg daily(O), and 1.28 pg daily (A); (B) vehicle (A), tarnoxifen 25 pg daily (Cl), tamoxifen 50 pg daily (W), tamoxifen 100 gg daily (0), estradiol-17/3 1.28 pg daily (A) and ICI 77494 50 pg daily (0); (C) vehicle (A), tamoxifen 25 pg daily (O), tamoxifen 100 pg daily (0), trioxifene 25 pg daily (W), trioxifene 100 pg daily (Cl) and estradiol-178 0.08 pg daily (A); (D) vehicle (A), LY 126412 64 pg daily (m) and ICI 77949 64 pg daily (0). Progesterone receptor was identified as [‘H]R5020 binding peaks. [ “C]Ovalbumin sedimentation standard (3.6s) was added before centrifugation. Equivalent DPM are specific DPM per’mg cytosol protein. For experimental detail see the methods section.

V.C. Jordan,

B. Gosden

3.6s 1

FRACTION

NUMBER

Fig. 5B.

were no major changes in the relative sizes of the [3H]R5020 binding peak in the 8-10s region of the gradients with the standardization calculations. A dose-response experiment for estradiol is shown in Fig. 5A. It is clear that maximal increases in progesterone receptor concentration occur at doses between 0.08 and 1.28 pg of estradiol daily. The abilities of the test compounds to increase the concentration of progesterone receptor in the 8s region were compared with estradiol. Tamoxifen (25, 50 and 100 pg daily) and ICI 77949 (50 ,ug daily) produced similar increases in progesterone receptor concentration compared with estradiol (1.28 pg daily) (Fig. 5B). It is clear that increasing daily doses of tamoxifen, at the top of the partial agonist dose response curve (Fig. 2), did not cause an additional increase in the concentration of progesterone receptor. In another experiment, no differences were observed between the ability of either tamoxifen (25 or 100 pg daily) or

Tamoxifen

and irioxifene actions

301

3.6 S

BOTTOM

FRACTION

NUMBER

Fig. 5C.

trioxifene (25 or 100 pg daily) to increase uterine concentration of progesterone receptor (Fig. 5C). A comparison of ICI 77949 (64 pg daily) with LY 126412 (64 pg daily) demonstrated that whereas ICI 77949 increased the concentration of uterine progesterone receptor, LY 126412 essentially did not increase the progesterone receptor concentration above that observed in untreated control uteri (Fig. 5D). Although these experiments to determine [3H]R5020 binding in uteri after three daily injections of test compounds to groups of rats were conducted over several weeks, the reproducibility was excellent (Figs. 5A-D).

DISCUSSION The aim of this study was to compare and contrast the uterine actions of tamoxifen and trioxifene with their related derivatives (ICI 77949 and

V.C. Jordan, B. Gosden

D

3.6s 1

ITTOM

IO

TOP

FRACTION NUMBER Fig. 5D.

LY 126412, respectively) without the aminoethoxy side-chain. In the uterine weight test, tamoxifen and trioxifene have similar antiestrogenic and partial estrogenic properties. Although binding affinity of a compound for the estrogen receptor is not considered to be either a good predictor of potency or biological activity in vivo, it should be pointed out that trioxifene has a higher relative binding affinity for the estrogen receptor than tamoxifen (Black et al., 198 1; Rose et al., 198 1; Fig. 4) and possibly as a result, trioxifene is a more potent antiestrogen than tamoxifen. It is clear that the alkylaminoethoxy side-chain is essential for the antiestrogenic activity of both tamoxifen and trioxifene. However the effect of removal of the side-chain from tamoxifen and trioxifene produces a dramatically different effect on the properties of the product. Removal of the side-chain from tamoxifen to form ICI 77949 makes the molecule a full estrogen which is consistent with the extensive literature

Tamoxifen and trioxifene actions

303

published on substituted triphenylethylenes (Robson and Schonberg, 1937, 1942; Emmens, 1942, 1947; Thompson and Werner, 1951). A similar structure-activity relationship is found with the bicyclic antiestrogens based on 3,4_dihydronaphthalene (Lednicer et al., 1966). Nafoxidine (U-l 1100A) is an antiestrogen in the immature rat (Duncan et al., 1963) but complete removal of the aminoethoxy side chain to form U-l 1854 (l-phenyl-6-methoxy-2-phenyl-3,4-dihydronaphthalene) results in full estrogenic activity in this species (Lednicer et al., 1966; Jordan et al., 198 1). The structure of trioxifene, though essentially similar to that of nafoxidine, differs in that a ketone links the phenyl ring substituted with the alkylaminoethoxy side-chain to the 3,4_dihydronaphthalene ring. Unlike the situation with nafoxidine, removal of the aminoethoxy sidechain from trioxifene to produce LY 126412 results in a molecule with no estrogenic or antiestrogenic activity at the doses tested in the rat uterus. Nevertheless, increasing concentrations of LY 126412 inhibit the binding of [3H]estradiol to rat uterine estrogen receptors in vitro. This result suggests that LY 126412 might possibly produce estrogen-like changes in the uterus if much higher doses were used in future studies. Another interesting point when considering structure-activity relationships is that the presence of a phenolic hydroxyl in many antiestrogens (including tamoxifen) increases the binding affinity for the rat uterine estrogen receptor (Jordan et al., 1977; Hayes et al., 1981; Black et al., 1981) and pharmacological activity (Jordan et al., 1977; Coezy et al., 1982). However, it is apparent from the present findings that the phenolic hydroxyl has to be in a specific position in the molecule so that there is high affinity binding at the site on the receptor normally occupied by the 3 phenolic hydroxyl of estradiol and the side-chain is in a certain position in space (Jordan et al., 1981). The 2 phenols in the present study (ICI 77 949 and LY 126412) did not have improved receptor binding and the loss of the side-chain was accompanied by a loss of antiestrogenic activity at the doses tested. Progesterone receptor synthesis is an indicator of estrogen action in estrogen target tissues (Feil et al., 1972; Milgrom et al., 1973; Rao et al., 1973; Philibert and Raynaud, 1974). Similarly the antiestrogens related to triphenylethylene increase the level of progesterone receptor in the rat (Koseki et al., 1977; Dix and Jordan, 1980a, b) and hamster uterus (Leavitt et al., 1977) in vivo and in MCF7 breast cancer cells in vitro (Horwitz et al., 1978; Eckert and Katzenellenbogen, 1982). The synthetic progestational agent R5020 was used to determine progesterone receptors in uterine cytosols because this ligand shows high binding affinity for the progesterone receptor but does not show significant binding to plasma

304

V. C. Jordan,

B. Gosden

steroid binding components (Philibert and Raynaud, 1973). However, R5020 does show some affinity for androgen and glucocorticoid receptors (Raynaud, 1977). Dihydrotestosterone (lo-’ M) was included in the assay to block any androgen receptors in the rat uterine cytosols. Walters and Clark (1977) have verified that R5020 does not bind significantly to cortisol inhibitable proteins in rat uterine cytosols at concentrations below 2 X lo-* M (1 X IO-* M R5020 was used in the present study). Be that as it may, the level of specific glucocorticoid receptor found in the rat uterus is extremely low (< 5 fmoles/mg wet weight) and, unlike the progesterone receptor, is not inducible with estrogen (Panko et al., 1981). In the present studies, sucrose density gradient analysis was used to compare and contrast the relative ability of test compounds to increase the progesterone receptor content of rat uteri. Sodium molybdate (20 mM) was included in all buffers to stabilize the steroid hormone receptor in the high molecular weight form (Anderson et al., 1980; Gaubert et al., 1980; Hawkins et al., 1981). Without the molybdate the peaks on the sucrose gradients were diffuse and reproducibility was poor (data not shown). Tamoxifen and trioxifene increased the concentration of progesterone receptor in the uterus but the compound of special interest LY 126412 was, by comparison with ICI 77949, unable to increase progesterone receptor concentration. While estrogen increases uterine wet weight and progesterone receptor levels it is important to appreciate that the uterus is composed of different cell types: myometrial, stromal, glandular and luminal epithelial cells. The luminal epithelial cells appear to be the most responsive to the actions of estrogen (Kaye et al., 1972; Mukku et al., 1981) and changes in the size of the epithelial cells can be sensitive indicators of biological action (Clark et al., 1978a, b; Clark and Guthrie, 1981). We were very interested to find that the size of the luminal epithelial cells is related to the dose of tamoxifen, trioxifene, ICI 77949 or estradiol used and the degree of stimulation of the uterus. At maximal stimulation for a given compound (irrespective of whether the compound is a full or a partial estrogen) there is an equivalent increase in the size of luminal epithelial cells. This conclusion is supported by the recent finding that the estrogenic cis isomer of clomiphene, zuclomiphene and the tram isomer of clomiphene, enclomiphene produce similar dose related increases in epithelial cell height (Clark and Guthrie, 1981). However, this conclusion contrasts with earlier reports which demonstrate that unlike estrogens, antiestrogens cause an enormous increase in the size of lurninal epithelial cells (Kang et al., 1975; Clark et al., 1978a, b). The reason for the difference in the results is probably related to the different experimental designs. Previous studies compared histology at different times,

Tamoxifen and trioxifene actions

305

after a single administration of estrogen or antiestrogen so the rate of stimulation of the epithelium is being compared. In the present study multiple administrations were used and a comparison of different doses was made at a single time point when the equilibrium growth potential for that dose had been achieved. Another factor that is generally not considered in histology studies is the large amount of intrahuninal fluid that is present only in estrogen-stimulated uteri. The fluid causes distortion and stretching of the uterus and as a result produces a distortion of the cells of the lurninal epithelium. In these experiments all the fluid was expressed before fixing the tissue so that a true comparison of uteri stimulated with estrogen or antiestrogen could be made. In conclusion the present study was undertaken to compare and contrast the biological activity of tamoxifen and trioxifene with their respective derivatives ICI 77949 and LY 126412 without the aminoethoxy side-chain. It is clear that the aminoethoxy side-chain is not only important for the antiestrogenic properties of both tamoxifen and trioxifene but also essential for the biological activity of the trioxifene molecule over the same dose range.

ACKNOWLEDGEMENTS We would like to thank Dr. A.H. Todd (ICI Ltd.) and Dr. CD. (Eli Lilly) for providing the compounds used in this study.

Jones

REFERENCES Anderson, K.M., Phelan, J., Marogil, M., Hendrickson, C., and Economou, Steroids

S. (1980)

35, 273-280.

Black, L.J., and Goode, R.L. (1980) Life Sci. 26, 1453-1458. Black, L.J., Jones, CD., and Goode, R.L. (1981) Mol. Cell. Endocrinol. 22, 95- 103. Clark, J.H., and Guthrie, S.C. (1981) Biol. Reprod. 25, 667-672. Clark, E.R., Dix, C.J., Jordan, V.C., Prestwich, G., and Sexton, S. (1978a) Br. J. Pharmacol. 62, 442P-443P. Clark, J.H., Hardin, J.W., Padykula, H.H., and Cardasis, CA. (1978b) Proc. Natl. Acad. Sci. (U.S.A.) 75, 2781-2784. Coezy, E., Borgna, J.L., and Rochefort, H. (1982) Cancer Res. 42, 317-323. Dix, C.J., and Jordan, V.C. (1980a) J. Endocrinol. 85, 393-404. Dix, C.J., and Jordan, V.C. (1980b) Endocrinology 107, 2011-2020. Duncan, G.W., Lyster, SC., Clark, J.J., and Lednicer, D. (t963) Proc. Sot. Exp. Biol. Med. 112,439-442. Eckert, R.L., and Katzeneflenbogen, B.S. (1982) Cancer Res. 42, 139-144. Emmens, C.W. (1942) J. Endocrinol. 3, 1688173. Emmens, C.W. (1947) J. Endocrinol. 5, 170-173.

306

%C. Jordan,

B. IGosden

Feil, P.D., Glasser, S.R., Toft, D.O., and O’Malley, B.W. (1972) Endocrinology 91, 738-746. Gaubert, CM., Tremblay, R.R., and Dub&, J.Y. (1980) J. Steroid B&hem. 13, 931-937. Hawkins, E.F., Lieskovsky, G., and Markland, F.S. (1981) J. Clin. Endocrinol. Metab. 53, 456-458. Hayes, J.R., Rorke, E.A., Robertson, D.W., Katzenellenbogen, B.S., and Katzenellenbogen, J.A. (1981) Endocrinology 108, 164-172. Horwitz, K.B., Koseki, Y., and McGuire, W.L. (1978) Endocrinology, 103, 1742- 175 1. Jones, C.D., Suarez, T., Massey, E.H., Black, L.J., and Tinsley, F.C. (1979) J. Med. Chem. 22, 962-966. Jordan, V.C., and Dowse, L.J. (1976) J. Endocrinol. 68, 297-303. Jordan, V.C., Collins, M.M., Rowsby, L., and Prestwich, G. (1977) J. Endocrinol. 75, 305-316. Jordan, V.C., Clark, E.R., and Allen, K.E. (1981) in: Non-Steroidal Antioestrogens. Eds. R.L. Sutherland and V.C. Jordan (Academic Press, Sydney) pp. 31-57. Kang, Y.H., Anderson, W.A., and DeSombre, E.R. (1975) J. Cell Biol. 64, 682-691. Kaye, A.M., Sheratzky, D., and Lindner, H.R. (1972) Biochim. Biophys. Acta 261,475-486. Korenman, S.G. (1970) Endocrinology 87, 1119- 1123. Koseki, Y., Zava, D.T., Chamness, G.C., and McGuire, W.L. (1977) Steroids 30, 169- 178. Leavitt, W.W., Chen, J.J., and Allen, T.C. (1977) Ann. N.Y. Acad. Sci. 286, 210-219. Lednicer, D., Lyster, S.C., Aspergren, B.D., and Duncan, G.W. (1966) J. Med. Chem. 9, 172- 176. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (195 1) J. Biol. Chem. 193, 265-275. Milgrom, E., Thi, L., Atger, M., and Baulieu, E.E. (1973) J. Biol. Chem. 248, 6366-6374. Mukku, V.R., Kirkland, J.L., Hardy, M., and Stancel, G.M. (1981) Endocrinology 109, 1005-1010. Panko, W.B., Clark, J.H., and Walters, M.R. (1981) J. Receptor Res. 2, 29-45. Perry, K.W., Dipasquale, G., Ferguson, E., Pozzi, C., and Rassaert, CL. (1973) Contraception 7, 231-242. Philibert, D., and Raynaud, J.P. (1973) Steroids 22, 89-98. Philibert, D., and Raynaud, J.P. (1974) Endocrinology 94, 627-632. Rao, B.R., Weist, W.G., and Allen, W.M. (1973) Endocrinology 92, 1229-1235. Raynaud, J.P. (1977) in: Progesterone Receptors in Normal and Neoplastic Tissues. Eds: W.L. McGuire, J.P. Raynaud and E.E. Baulieu (Raven, New York), pp. 9-21. Robson, J.M., and Schonberg, A. (1937) Nature (London) 140, 196. Robson, J.M., and Schonberg, A. (1942) Nature (London) 150, 22-23. Rose, D.P., Fisher, A.H., and Jordan, V.C. (1981) Eur. J. Cancer 17, 893-898. Roy, S., Mahesh, B. and Greenblatt, R.B. (1964) Acta Endocrinol. Copenh. 47, 669-675. Skidmore, J., Walpole, A.L., and Woodbum, J. (1972) J. Endocrinol. 52, 289-298. Sutherland, R.L., and Jordan, V.C. (1981) Non-steroidal Antiestrogens. Academic Press, Sydney. Thompson, CR., and Werner, H.W. (195 1) Proc. Sot. Exp. Biol. Med. 77,494-497. Walters, M.R., and Clark, J.H. (1977) in: Progesterone Receptors in Normal and Neoplastic Tissues. Eds: W.L. McGuire, J.P. Raynaud and E.E. Baulieu (Raven, New York) pp. 271-285.