Pyrrolidino-4-Iodotamoxifen and 4-Iodotamoxifen, New Analogues of the Antiestrogen Tamoxifen for the Treatment of Breast Cancer1

[CANCER RESEARCH 51. 5851-5858. November I. 1991] Pyrrolidino-4-iodotamoxifen and 4-Iodotamoxifen, New Analogues of the Antiestrogen Tamoxifen for the Treatment of Breast Cancer1

S. K. Chander,2 R. McCague, Y. Luqmani, C. Newton, M. Dowsett, M. Jarman, and R. C. Coombes Medical Oncology L'nit, Depl. of Molecular and Cellular Science [S. K. C., Y. Â¿.,R. C. C.j, St George's Hospital Medical School, Ijmdon SW17ORE; Dept. of Academic Biochemistry [C.N., M. D.J, Royal Manden Hospital and Institute of Cancer Research, Fulham Road, London SH'3 6JJ; and CRC Drug Development Dept. [R. M., M.JJ, Institute of Cancer Research, Sutton, Surrey SM2 5PT, united Kingdom

ABSTRACT rapidly conjugated, metabolized, and excreted, which may ex plain their low potency as antitumor agents in animals (12-14). New tamoxifen analogues were tested for their antiproliferative activity The antiestrogen MER-25 displays no agonist activity, but the both in vitro and in vivo. Binding studies showed that both 4-iodotamoxifen and pyrrolidino-4-iodotamoxifen and 2.5-fold higher affinities for the use of this compound has been limited by its low potency and estrogen receptor compared with tamoxifen. Pyrrolidino-4-iodotamoxifen relatively high toxicity (15). was also 1.5-fold more effective in causing inhibition of estrogen-induced The precise mode of action of antiestrogens in breast cancer growth of MCF-7 cells compared with tamoxifen at IO"6 M. The 4- is not clearly resolved. The relative importance of estrogenic iodotamoxifen analogue was similar to tamoxifen in its inhibitory action and antiestrogenic mechanisms in inducing tumor regression at 1(1" M. Antiproliferative activities of these drugs were tested using remains uncertain (16). Nonetheless, the development of pure the nitrosomethylurea-induced rat mammary tumor model. Pyrrolidino- antiestrogens which display greater ER1 binding affinity than 4-iodotamoxifen caused regression in 92% of rats, whereas tamoxifen present compounds may be valuable in eliminating many of the caused regression in 75% of rats. The agonist activity of the analogues problems associated with drugs exhibiting partial agonist prop was determined using the immature rat and mouse uterotrophic assays. Both tamoxifen and 4-iodotamoxifen had similar partial agonist activity, erties. Comparative studies of a pure antiestrogen and tamoxi and this was greater than that seen with pyrrolidino-4-iodotamoxifen. fen may also provide a clearer understanding of the mode of Furthermore, pyrrolidino-4-iodotamoxifen caused a dose-dependent in action of the latter compound in breast cancer patients. hibition of estrogen-induced vaginal cornification, whereas tamoxifen and In this paper, we describe the properties of a novel tamoxifen 4-iodotamoxifen did not. These studies demonstrate that pyrrolidino-4- analogue which has improved ER binding compared with that iodotamoxifen is more effective than tamoxifen in inhibiting tumor regres of tamoxifen and has a greater antiproliferative activity both in sion and that its reduced uterotrophic activity and increased estrogen vitro and in vivo. Furthermore, it appears to show reduced receptor binding may give it significant clinical advantages over the parent agonist activity in the immature rat and mouse uterotrophic compound. assays.

INTRODUCTION MATERIALS AND METHODS The use of the antiestrogen, tamoxifen, is well established Animals for the treatment of breast cancer in both advanced disease (1) and adjuvant treatment of primary breast cancer (2-4). The Inbred virgin female (Ludwig/Wistar/Olac) rats bearing tumors in major advantage of tamoxifen over other antiestrogens has been duced with NMU were supplied by Olac, Oxon, United Kingdom. its low incidence of side effects (5). However, there are a number of serious drawbacks with the use of tamoxifen and other Chemicals antiestrogens, such as clomiphene and nafoxidine; (a) not all The antiestrogens used in this study were tamoxifen \\\-trans-\-\4- estrogen-responsive tumors respond; and (b) duration of action [2-(dimethylamino)ethoxy]phenyl|-l,2-diphenyl-butene||, 4-iodota is limited to an average of about 18 mo, and this may be related moxifen 11Â£-1-|4-[2-(dimethylamino)ethoxy]pheny] |-1-(4-iodophenyl)- to the partial agonist activity of the drugs. Clinical evidence of 2-phenyl-l-butene|| (17), and pyrrolidino-4-iodotamoxifen |E-l-(4-io- an estrogen-like stimulation of tumors by tamoxifen has been dophenyl)-1 -[4-(2-pyrrolidinoethoxy)phenyl]-2-phenyl-1 -butenel ( 18). observed in some breast cancer patients starting therapy (6-8). Structures are illustrated in Fig. 1. This tumor "flare" seen with tamoxifen appears to be transient Estrogen Receptor Binding Assay and is indicative of the estrogen sensitivity of the tumor; how ever, tumor regression subsequently occurs in most cases. Fur Competitive binding assays to measure the relative affinity of anti- thermore, patients who fail to respond to tamoxifen therapy or estrogens for rat uterine ER followed the method described by Wakeling gain resistance often subsequently respond to other agents (19). All stock antiestrogens were prepared in absolute ethanol, and 10- directed at estrogen deprivation, such as aromatase inhibitors fold dilutions were made in 10 HIMphosphate buffer, pH 7.4, containing (9-11). Tamoxifen-stimulated growth could occur in patients 1.5 HIMEDTA, 2.0 rnM 2/i-mercaptoethanol, and 0.1% bovine serum albumin. Cytosol was prepared from mature rat uterus by homogeni- as one form of drug resistance that results in therapeutic failure. zation in the above-described 10 mm phosphate buffer, pH 7.4, and The search for nonsteroidal antiestrogens which lack signif centrifuged at 100,000 x g for l h at 4"C. Aliquots of cytosol (100 u\) icant agonist activity has been only partially successful. Those were incubated with 50 ^1 of 10 n\i 17/S-[2,4,6,7-3H]estradiol (85 to antiestrogens which display weak or no agonist activity, for 110 Ci/mmol; Amersham, United Kingdom) in the absence or presence example, keoxifene and the related compound LY117018, are of an increasing concentration of drug (in 50 u\) for 16 h at 4Â°C.After incubation, a suspension (200 ÃŸ\)of dextran-coated charcoal [0.5% Received 7/30/90; accepted 8/27/91. dextran T-70:5% Norit A charcoal (wt/vol) in 10 HIMphosphate buffer, The costs of publication of this article were defrayed in part by the payment pH 7.4, without 0.1 % bovine serum albumin] was added, and incubation of page charges. This article must therefore be hereby marked advertisement in was continued for 15 min at 4Â°C.After removal of charcoal by centrif- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported by the Cancer Research Campaign. 2To whom requests for reprints should be addressed, at Dept. of Medical ' The abbreviations used are: ER, estrogen receptor: NMU. nitrosomethylurea; Oncology. Charing Cross Hospital Medical School. Fulham Palace Road. London PIT, pyrrolidino-4-iodotamoxifen; 4-IT, 4-iodotamoxifen; i.m.. intramuscular; W6 8RF. United Kingdom. E2B, cstradiol benzoate. 5851

TAMOXIFEN At the end of the 4-wk period, all palpable lesions were removed from -CH3 the mammary area and stored in liquid nitrogen. Oâ€”CH2â€”CH2â€”N Uterotrophic Studies C = C The method used has been previously described in detail (21). In brief, immature female Wistar rats (19 to 21 days, 40 to 50 g) were molecular wt, 371.3 obtained from the breeding unit at St George's Hospital Medical School and randomized into 5 rats/treatment group. All drugs used in the study were dissolved in peanut oil (Sigma, United Kingdom) prior to injection. Rats were dosed s.c. daily for 4 days. On Day 5 rats were sacrificed, the uteri were removed and dissected free of extraneous 4-IODOTAMOXIFEN tissue, fluid content was expelled, tissues were blotted, and the wet -CH, weight was recorded. The effect of tamoxifen, 4-IT, and PIT on uterine weight in immature Oâ€”CH2â€”CH2â€”N mice was also investigated as previously described (22). Immature C=C -CH3 female MF-1 mice (21 days, 10 to 11 g) were obtained from the breeding unit at St George's Hospital Medical School. Animals were randomized molecular wt. 497 into 5 mice/treatment group. All drugs used in the study were dissolved in peanut oil and dosed s.c. daily for 3 days. On Day 4, mice were sacrificed, and the uteri were removed and dissected free of extraneous tissue, fluid content was expelled, tissues were blotted, and the wet weight was recorded. PYRROLIDINO-4-IODOTAMOXIFEN Vaginal Cornification Studies

Oâ€”GHzâ€”CH2â€”N ^ The vaginal cornification assay has been described by Harper and Walpole (23). In brief, adult female Wistar rats were ovariectomized 14 days prior to the start of the experiment. On Day 15, rats were randomized into 2 experimental groups for the assessment of (a) molecular wt. 523.2 estrogenic activity and (b) antiestrogenic activity. Fig. 1. Structure of antiestrogens. Estrogenic Activity. Rats were randomized into 5 animals/group and dosed s.c. for 3 days. Vaginal cornification was assessed by taking smears each day from each rat for 5 days. Smears were classified as ugation, protein-bound tritiated estradici in the supernatant (200 ÃŸ\) cornified when composed of cornified and/or nucleated epithelial cells was measured by liquid scintillation counting. with no more than the occasional leukocyte. Antiestrogenic Activity. Rats were randomized into 5 animals/group. Cell Growth Studies Each group received 1.25 ng/kg of estradici plus appropriate drug s.c. Dosing was carried out for 7 days, and vaginal smears were examined MCF-7 cells were obtained from P. Dabre (Imperial Cancer Research daily from the first to eighth day of the experiment. Control animals Fund, London, England), and MDA-MB-231 cells were obtained from M. O'Hare (Institute of Cancer Research, Sutton, Surrey, United received 1.25 /Â¿g/kgofestradici only. The appearance, during this time, Kingdom). All stock cells were grown in Dulbecco's modified Eagle's of leukocytes in the smears from animals given the analogues was taken as an index of their antiestrogenic effect. medium with phenol red (Gibco, Ltd., Paisley, United Kingdom) sup plemented with 100 lU/ml of penicillin and 0.1 mg/ml of streptomycin, 2 mM L-glutamine (Sigma, Poole, United Kingdom), 10 nM estradici RESULTS (Sigma), 10 Mg/ml of insulin (Sigma), plus 5% myoclone fetal calf serum (Gibco) and cultured in an incubator with 5% CO2, 95% air and Estrogen Receptor Binding Assays. Estrogen receptor binding 95% humidity. Before cells were used in experiments, they were grown affinities of the new antiestrogens were compared to that of in RPMI-1640 medium without phenol red (Gibco), estradici, and insulin, plus 5% charcoal-stripped fetal calf serum for 7 days. In all experiments, 3 x IO4 cells/well (unless otherwise stated) were seeded 12000-, into 24-well plates and left to settle for 24 h. The medium was removed and replaced with fresh medium containing the appropriate drug at the 10000- stated concentration. Cells were treated with drug for 7 days, with E medium (containing appropriate drug) changed every 2 days. Cells were -o 8000 harvested by trypsinization (1% trypsin/versene solution) and counted using trypan blue (Sigma) on Days 3 and 7. Data were tested for S 6000 statistical significance using Student's ? test. .o

In Vivo Studies 2000- The NMU-induced rat mammary tumor model was used as described previously (20). In all studies, adult rats bearing tumors between 10 and 20 mm in diameter were randomized. All rats were treated with -11 -10 -9 -8 -7 -6 -5 daily i.m. injections of drugs or vehicle 5 days/wk for 4 wk. Tumor log drug concentration (M) measurements were made weekly for 4 wk by measuring two diameters Fig. 2. Competition of antiestrogens with estradiol for rat uterus ER. Rat at right angles with Vernier callipers. Tumor volume was estimated uterine cytosol was incubated with 10 nM [3H]estradioI and increasing concentra using the following formula tions of estradiol (â€¢),tamoxifen (A), 4-IT (V), and PIT (Â»).Each point represents the mean of 3 determinations. Bars have not been included because they were (%) D [(

25-r 50-45-f~ 20-o^J 40-ox 35-Â« 15-Ci 30-cI 25~c 10"cCO5-0 15-E Mra1 10- 5-1.M. -â€¢ 'V''â€¢/'/<'/'.â€¢X1-8T7/v'111vvrii-7 y/(Â° rlrl%.Y/. -5drug -6 -8-5drug -7 -6 (M)h25-r cone (M)45-~ cone

40-oH.w 20-o^S

15-ci 30-cg 25-O1 20-i 1Â°-â€¢E 15' 5 - E 10-5ririri-8

n-Ã¯-f/'x'â€¢/';:TiygÃ¬ri/Â¡ -8 -7 -6 -5drug-7 -6 drug cone (M) (M)5045~ cone c. 25

40oE r 20 o 35Â« 30CÃ¤ 25O1 meancell 20E

counts(x10"")II ^>OI3mean 10fe D00gj

cellc25-20-15-5-n i- <-< 0Ã•E

10 -

O --3E-I-i-: -r*,ri{-,\p^<-VA l ix/i estradici control estradici control Fig. 4. Effect of antiestrogens on the estrogen-stimulated growth of MCF-7 Fig. 3. Effect of antiestrogens on the growth of MCF-7 cells. A total of 3 x cells. A total of 3 x 10* MCF-7 cells/well were treated with medium containing IO4 MCF-7 cells/well were treated with an increasing concentration of drugs Id* M estradici and an increasing concentration of drug ranging from Id ' M to ranging from 10~*M to 10"* M. a, tamoxifen; b, 4-IT; c, PIT. d shows the effect 10"B M. a, tamoxifen; b. 4-IT; c, PIT; d, estradiol alone (10"* M) and control of estradiol itii " M) and control (0.1% ethanol) in the absence of added drug. (0.1% ethanol) in the absence of added drug. Medium was changed every 2 days. Medium was changed ever)' 2 days. Cell counts were made on 3 days (D) and 7 Cell counts were made on 3 days (D) and 7 days (^). Columns, mean of triplicate days (i i|. Columns, mean of triplicate determinations; bars, SEM. determinations; bars, SEM. 5853

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. NEW ANTIESTROGENS FOR THE TREATMENT OF BREAST CANCER tamoxifen. Receptor binding curves for the various agents are Table 2 Dose-response of PIT on the growth of NMl]-induced rat tumors All rats were given daily i.m. doses of drug dissolved in peanut oil or vehicle shown in Fig. 2. These were used to calculate the relative (control) for 5 days/wk for 4 wk. Tumor growth was recorded every wk for the 4- binding affinities. Tamoxifen, 4IT and PIT had ER binding wk period, by measuring two diameters at right angles with Vernier calipers. affinities of 5, 12.5, and 12.5, respectively, compared with that Tumor volume was estimated using the following formula of estradiol. The analogues 4-IT and PIT both showed 2.5-fold 0/6MW, x d2)3Â«] greater affinity for ER compared with tamoxifen. Effect of Analogues on the Growth of Breast Cancer Cells in Each of the animals was categorized into one of three groups: (a) those with 50% or greater regression; (b) those with 0 to 50% regression; (c) those with an Vitro. Fig. 3, A to C, shows the effect of varying concentrations increase. of the analogues alone on the growth of MCF-7 cells after 3 and 7 days. At 10~5M, all analogues were completely cytotoxic Treatment groups PIT PIT PIT after 3 days of treatment. The highest level of inhibition, Control (0.25 mg/kg) (1.0 mg/kg) (2.0 mg/kg) compared with that of the control, for all the analogues was observed at 10~6M after 3 days of treatment (tamoxifen, 38.7%; No. of rats 12 12 12 12 No. of initial tumors 18 18 17 18 4-IT, 35.2%; PIT, 58.7%). After 7 days of treatment at KT6 M (>:10 mm) No. of new tumors 22 30 there was a reduced but significant inhibition produced by all No. of rats showing to- 1387 the analogues, compared with the control (P = >0.001). At tal tumor regression doses of 10~8M and 10~7M after both 3 and 7 days of treatment >50% No. of rats showing to- 14 34 there was reduced by significant inhibition of growth. Although tal tumor regression PIT showed a reduction in the level of inhibition at 10~7 and <50% 10~" M after 3 and 7 days, these levels were significantly No. of rats showing to- 10 5 1 1 tal tumor progression different from those of tamoxifen and 4-IT (P= >0.001). With % of rats showing 8.3 25 66.7 58.3 estradiol alone (Fig. 3rf), a 2.4-fold increase in cell number was >50% tumor regres sion observed compared with the control. % of rats showing any 16.7 58.3 91.7 91.7 The effect of the analogues on the inhibition of estradiol- tumor regression stimulated growth of MCF-7 cells is shown in Fig. 4, a to c. Again, a dose of 10~5M was cytotoxic after 3 days of treatment. Maximal growth inhibition was seen at 10~6 M after 7 days of Table 3 Comparison of tumor response rates with tamoxifen, 4-IT, and PIT using the NMU-induced rat mammary tumor model treatment. At this dose, tamoxifen and 4IT produced 44% and All rats were given daily i.m. doses of 1.0 mg/kg of drug dissolved in peanut 48% inhibition, respectively, of the estradiol-induced prolifer- oil or vehicle (control) for 5 days/wk for 4 wk. Tumor growth was recorded every wk for the 4-wk period, by measuring two diameters at right angles with Vernier calipers. Tumor volume was estimated using the following formula (WMirf,xd,)ini

Each of the animals was categorized into three groups: (a) those with 50% or greater regression; (ft) those with 0 to 50% regression; or (c) those with an increase. Treatment groups Tamoxifen 4-IT PIT Control (1.0 mg/kg) (1.0 mg/kg) (1.0 mg/kg) No. of rats 12 12 12 12 No. of initial tumors 12 16 16 18 (>10 mm) No. of new tumors 2 3 3 4 No. of rats showing to- 0 5 1 7 tal tumor regression >50% No. of rats showing to- 0 4 days of incubation tal tumor regression <50% Fig. 5. Effect of adding estradiol to cells initially treated with analogues and No. of rats showing to- 12 3 5 0 estradiol to show reversal of inhibition. A total of 3 x 10* MCF-7 cells/well were tal tumor progression treated initially with medium containing vehicle (A), either estradiol alone (10~8 % of rats showing 0 42 8 58 M) (â€¢)or with 10"' M analogues for 3 days: tamoxifen (D); 4IT (O); PIT (â€¢). >50% tumor regres After 3 days (J) medium was replaced with medium containing only 10"' M sion estradiol for a further 4 days. Medium was changed every 2 days. Cell counts % of rats showing any 0 75 58 100 were made on Days 3 and 7. Each point is the mean of triplicate determinations. tumor regression Bars are not included because they were smaller than the data symbols.

Table 1 Effect ofantiestrogens on the growth ofMDA-MB-231 cells ation; PIT produced 65% inhibition. There was no significant Three x IO4 MDA-MB-231 cells/well were treated with medium containing difference between tamoxifen and 4-IT in their levels of inhi vehicle alone (control, 0.1% ethanol), estradiol (10~8 M), or estradiol plus ana bition at a dose of 1(T6 M (P = 0.155). However, a comparison logues at 10"' M. Medium was changed every 2 days. Cell counts x 10"' between tamoxifen and PIT showed that the latter was a signif icantly more effective inhibitor (by 1.5-fold) than tamoxifen (P = 0.006). At doses of IO"7 M and IO'8 M, tamoxifen and 4-IT DrugTamoxifen days20.5 days39.7 + estradiol Â±2.9" Â±0.8 did not have much effect on the estradiol-stimulated growth of 4-IT + estradiol 22.7 Â±1.2 39.0 Â±1.5 MCF-7 cells. However, PIT was still effective and produced PIT + estradiol 22.5 Â±0.9 41.2 Â±1.4 51% inhibition at 10~7M but was without effect at 10~8M. Estradiol alone 25.5 Â±0.9 41.2 Â±1.3 Control3 23.9 Â±1.17 41.5 Â±1.3 Reversal of Antiestrogen Inhibition by Estradiol. The inhibi " Mean Â±SEM. tory effects of the antiestrogens on the estrogen-stimulated 5854

300 a. 91.7% of rats at both 1-mg and 2.0-mg/kg doses. This was significantly different from the control (1.0 mg, P = 0.006; 2.0 250- mg, P = 0.0007). In a third comparative study among tamoxi TJ jaO fen, 4-IT, and PIT (Table 3) at doses of 1 mg/kg all three drugs oÃ200 - inhibited tumor progression compared with the control at 28 o o days (Kruskal-Walllis = >// = 8.61, P = < 0.02). The greatest 150- percentage of reduction in tumor volume was achieved in the order: PIT > tamoxifen > 4-IT. Although there was no signif 100- icant difference between tamoxifen and PIT in decreasing total tumor volume, in this particular study PIT was effective in 50 causing some degree of tumor regression in all rats, whereas O tamoxifen caused tumor regression in only 75% of animals; the 0.01 0.1 1 10 remaining 25% of rats showed tumor progression. Overall in drug concentration (mg/Kg) the three studies, PIT caused regression in 92% of rats, which 300-1 b. was significantly greater than that caused by tamoxifen. Uterotrophic and Antiuterotrophic Study. The effect of the 250- various agents, either alone or together with E2B, on uterine o weight in immature rats is illustrated in Fig. 6. E2B alone J3 produced a 3.7-fold increase in uterine weight. Tamoxifen given 01 200- o o alone s.c. at doses up to 10 mg/kg stimulated an average uterine 150- growth of 123 mg/100 g of body weight compared with the control of 65.2 mg/100 g of body weight (1.9-fold increase). 100 When administered together with E2B it failed to inhibit com pletely the Uterotrophic action of E2B. The 4-IT analogue also 50 resulted in partial agonist effects, as an increase in uterine 0J weight of 108 mg/100 g of body weight was recorded at the highest dose compared with the control 1.7-fold increase). 0.01 0.1 1 10 However, the increase in weight with 4-IT was less than that dose (mg/Kg) 300 n c observed with tamoxifen. Doses of 1 and 10 mg/kg of PIT alone both resulted in a 1.3-fold increase in uterine weight 250 compared with the control (l mg/kg, P = 0.018; 10 mg/kg, P = 0.006). When given with E2B it produced a dose-dependent o, 200-] o Table 4 Effect of 4-IT and PIT on tamoxifen Uterotrophic activity in immature rats Immature Wistar rats (21 days; 40 to 50 g) received 4 daily s.c. doses of vehicle 100 J alone (control), estradiol, or two doses of drugs alone at 1 and 10 mg/kg. Another set of rats received tamoxifen at 1 mg/kg plus increasing doses of either 4-IT or o PIT as indicated. On Day 5. rats were killed, and their uteri were weighed. .E 50 - Uterine wt Treatments (mg/100gofbody wt) Control 66.7 Â±3.5" 0.01 0.1 1 10 Estradiol (0.5 fig/kg) 204.8 Â±5.8 drug concentration (mg/Kg) Drugs alone (mg/kg) Fig. 6. Uterotrophic/antiulerotrophic responses in immature rats. Rats re ceived 4 daily s.c. doses of vehicle alone (lower open bar), 0.5 pg/kg of E2B (upper Tamoxifen hatched bar), or increasing doses of drug alone (broken line) or together with E2B 1 126.8Â± 1.6 (solid line). Each point is the mean of 5 determinations, a. tamoxifen; b. 4-IT; c, 10 130.7 Â±2.2 PIT. 4-IT 1 103.9 Â±3.1 growth could be reversed by removing the drug after 3 days 10 115.5 Â±3.5 (Fig. 5) and treating with 10~8 M estradici alone for 4 days. PIT This indicated that the inhibitory1 effect of the antiestrogens 1 97.0 Â±0.6 was reversible. 10 96.1 Â±1.1 Effect of Antiestrogens on MDA-MB-231 Cells. The effect of Tamoxifen (1 mg/kg) + 4-IT (mg/kg) at the various drugs on the growth of the ER-negative cell line doses given below MDA-MB-231 is illustrated in Table 1. None of the compounds 0.01 120.1 Â±5.8 0.1 105.4 Â±1.9 had any effect on cell numbers after 7 days compared with 1.0 103.9 Â±4.3 untreated controls. No growth stimulation of MDA-MB-231 10.0 107.8 Â±5.6 was observed with estradiol. Tamoxifen (1 mg/kg) + PIT (mg/kg) at In Vivo Studies. The NMU-induced rat mammary tumor doses given below model was used to investigate the antiproliferative activities of 0.01 120.5 Â±4.0 0.1 107.5 Â±4.1 the various antiestrogens. In a preliminary single-dose study, 1.0 103.6 Â±5.9 PIT produced tumor regression in 83% of rats. In a subsequent 10.0 100.2+ 1.8 dose-response experiment (Table 2), PIT caused regression in 1Mean Â±SEM of 5 determinations. 5855

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. NEW ANTIESTROGENS FOR THE TREATMENT OF BREAST CANCER _ 500 -i 4-IT were unable to block the E2B effect. PIT, however, showed a dose-dependent inhibition of the E2B stimulation but was

-o 400 unable to block the effect completely at the highest dose. .o Vaginal Cornification in Adult Ovariectomized Rats. Tables 5 and 6 display the results of the vaginal cornification studies. g 300 Tamoxifen displayed estrogenic activity as indicated by vaginal cornification at 0.5 mg/kg and was completely cornifying at 20 Â¿ 200- mg/kg. However, in contrast, 4-IT and PIT did not show any estrogenic activity at 0.5 mg/kg but did so at 5 mg/kg. Full 100 cornification was observed at 50 mg/kg with 4-IT and PIT. Control animals, given estradiol only, showed cornified smears from the fourth to the eighth day inclusive. At doses 0.01 0.1 1 10 much below those required to induce vaginal cornification, tamoxifen and 4-IT both displayed antiestrogenic activity. They drug concentration (mg/Kg) produced a dose-dependent inhibition of estradiol stimulation but were unable to completely block the effect at 10 mg/kg. However, PIT was able to completely block the estradiol- induced cornification in a dose-dependent manner. Inhibition â€¢¿ caused by PIT at 10 mg/kg was significantly different from Â§ 360 -I tamoxifen and 4-IT (P = 0.0009).

B a. 240- DISCUSSION The agents described here all belong to the triphenylethylene * Â» 120- group of antiestrogens. The data presented in this study indicate

0 J i Table 5 Estrogenic activity of antiestrogens using the rat vaginal cornification 0.01 0.1 1 10 assayDrug drug concentration (mg/Kg) of smears showing cornified dose epithelial cells/rat group (mg/kg/day)Tamoxifen from Daysinclusive5/15(33.3%r* 3 to 5

0.5 5.0 8/15(53.3)c 20.0 15/15 (100)'' 50.04-IT0.5 15/15(100)0/15(0)*

5.0 5/15(33.3)c 20.0 10/15 (66.6)" 50.0PIT0.5 15/15(100)0/15(0)*

5/15(33.3)r 0.01 0.1 1 5.0 20.0 10/15(66.6)'' drug concentration (mg/Kg) 50.0Control 15/15(100)0/15

Fig. 7. Uterotrophic/antiuterotrophic responses in immature mice. Mice re (0) ceived 3 daily s.c. doses of vehicle alone (lower hatched bar), 0.1 Â»ig/kgof EZB Estradiol (1.25 Mg/kg/day)No. 15/15(100) (upper hatched bar), or increasing doses of drug alone (solid line) or together with Â°Numbers in parentheses, percentage. E2B (broken line). Each point is the mean of 5 determinations; bars, SEM. a, * 4-IT and PIT are different from tamoxifen at 0.5 mg/kg (P = 0.014). tamoxifen; b, 4-IT; c, PIT. r No difference between any analogue at 5 mg/kg (P = 0.27). d 4-IT and PIT are different from tamoxifen at 20 mg/kg (P = 0.014). inhibition of the uterotrophic action of E2B. At 10 mg/kg, PIT completely blocked the effect of E2B. This study showed that Table 6 Antiestrogenic activity of antiestrogens using the rat vaginal cornification assay PIT had the lowest uterotrophic activity, which was signifi Inhibition by antiestrogens of vaginal cornification induced by estradiol in cantly lower by 1.4- and 1.27-fold compared with tamoxifen ovariectomized rats. The rats were dosed daily (Days 1 to 7 inclusive) with and 4-IT, respectively (P = 0.030). estradiol (1.25 Â»/g/kgs.c.)plus antiestrogens at the doses indicated. The ability of 4-IT and PIT to inhibit tamoxifen uterotrophic Total no. of smears showing activity was investigated, and the results are shown in Table 4. leukocytes/rat group from Although both PIT and 4-IT showed some effect on inhibiting tamoxifen-stimulated uterine weight, this was not significant (mg/kg/day)0.0 (0)" (P = 0.3 to 0.9 between 1 and 10 mg/kg, respectively). (0) (0) 0.005 1/25(4) 0/25 (0) 1/25 (4) Uterotrophic Activity in Immature Mice. The uterotrophic 0.05 4/25(16) 4/25(16) 7/25 (28) activities of the various antiestrogens are illustrated in Fig. 7. 0.110Tamoxifen0/2510/25(40) 5/25 (20) 15/25(60) 16/25(64)4-IT0/25 13/25(52)PIT0/25 25/25(100)* Tamoxifen alone was highly uterotrophic at 0.1 mg/kg, whereas " Numbers in parentheses, percentage. PIT and 4-IT showed minimal uterotrophic activity at this dose * PIT different from tamoxifen at 20 mg/kg (P = 0.0009); PIT different from but were highly uterotrophic at 10 mg/kg. Both tamoxifen and 4-IT at 20 mg/kg (P = 0.0001). 5856

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. NEW ANTIESTROGENS FOR THE TREATMENT OF BREAST CANCER that binding to the ER can be increased by modifying the parent showed that para-halogenated derivatives of tamoxifen show tamoxifen molecule. The two analogues tested both included reduced uterotrophic activity compared with tamoxifen. The an iodine atom at position 4 which has been shown to increase only antiestrogen so far described which lacks agonist activity the affinity for the ER (17). This modification was designed to is the steroidal antiestrogen ICI 164 384 (28). Previous com produce a tamoxifen analogue which would have similar prop parative studies of the uterotrophic and antiuterotrophic action erties to those of 4-hydroxytamoxifen. This is a metabolite of of antiestrogens with different degrees of intrinsic estrogenic tamoxifen which has about 100 times greater potency in vitro activity have clearly shown that their gross effect on the uterus than tamoxifen but is rapidly inactivated in vivo by glucuroni- represents a balance between estrogenic and antiestrogenic ac dation of the hydroxyl group (24, 25). It was expected that the tivity (29, 30). A compound devoid of estrogenic activity should iodine would improve the potency of tamoxifen in vitro because thus be capable of complete antagonism of estrogen action. of increased affinity for the receptor and in vivo because of an This appears to be the case with ICI 164 384. The advantage increased resistance to metabolism, since the iodine-containing of the tamoxifen analogue, PIT, over LY 117018 and nafoxi- compound cannot undergo glucuronidation at position 4. An dine, which also contain the pyrrolidino group, is that the latter additional theoretical advantage of this modification is that the are rapidly metabolized (31). It has recently been shown that isomerization of fra/is-4-hydroxytamoxifen to the m-form, both 4-IT and PIT are metabolized 4 times more slowly than which is associated with the estrogen agonist property of ta tamoxifen by rat hepatocytes.4 PIT may also have some advan moxifen, cannot occur in the iodine-substituted molecule. tages over the ICI 164 384 compound in terms of slow metabolic Our receptor binding studies confirm earlier results of in removal. Although PIT did show some agonist activity, its creased ER binding of 4-iodo-substituted derivatives of tamox marked inhibitory effect on tumor growth shows favorable ifen (17). The difference between 4-IT and PIT is that the latter comparison with the ICI 164 384 compound (32). agent has a pyrrolidino group instead of the dimethylamino The precise mode of action of pure antiestrogens remains to group found in the parent compound. The original aim of this be determined but probably involves the blocking of the TAF2 replacement was to prevent the conversion by the liver of the transcriptional activation function of the ER (33; for review, dimethylamino group to give desmethyl metabolite, which re see Ref. 34). Since PIT binds to the ER with greater affinity sults in the release of formaldehyde, which cannot occur with than tamoxifen, there should be a greater reduction in ER the pyrrolidino group. It is interesting to note that the transcriptional activation than is seen with tamoxifen. Studies LY117018 which has low agonist activity also contains the are currently in progress with PIT to assess its effects on the pyrrolidino substituent (26). levels of estrogen-induced transcripts. Effects on breast cancer cells in vitro indicated that PIT was at least 1.5-fold more effective in causing inhibition of estrogen- ACKNOWLEDGMENTS induced cell growth compared with tamoxifen at a dose of 1 fiM. The 4-IT analogue at 1 ^M had no additional advantage We thank Judith Bliss (Dept. of Epidemiology, Institute of Cancer over tamoxifen in this respect. Having demonstrated that the Research, Sutton SM2 5PT) for her help in the statistical analysis of new analogues bound to the ER and inhibited growth of breast the in vivo studies and for her help in proofreading this manuscript. cancer cells, it was important to establish their effects in vivo. These studies showed that 4-IT produced the lowest level of REFERENCES regression of endocrine-dependent rat mammary tumors. There was no statistical difference between tamoxifen and PIT in their 1. Furr, B. J., and Jordan, V. C. The pharmacology and clinical uses of tamoxifen. Pharmacol. Ther., 25: 127-205, 1984. ability to cause regression of tumors. However, in one particular 2. "Nolvadex" Adjuvant Trial Organization. Controlled trial of tamoxifen as study, it was observed that tamoxifen caused only 75% of rats single adjuvant agent in management of early breast cancer. Lancet, /: 836- 839. 1985. to show tumor regression, whereas PIT caused some tumor 3. "Nolvadex" Adjuvant Trial Organization. Controlled trial of tamoxifen as a regression in 100% of the rats. It is possible that the ability of single adjuvant agent in the management of early breast cancer. Br. J. Cancer, this drug to cause regression of all the animal tumors could 57:608-611, 1988. 4. Scottish Breast Cancer Trials Committee. Adjuvant tamoxifen in the man give it an important advantage in the future use of the com agement of operable breast cancer: the Scottish trial. Lancet, 2: 171-175, pound in the clinical management of breast cancer. 1987. 5. Mouridsen, H., Palshof. T., Patterson, J., and Battersby, L. Tamoxifen in The uterotrophic studies indicated that both tamoxifen and advanced breast cancer. Cancer Treat. Rep., 5: 131-141, 1978. 4-IT had partial agonist activity. The interesting finding from 6. Tormey, D. C., Simon, R. M., Lippman, M. E., Bull, J. M., and Meyer, C. this study was that PIT showed reduced agonist activity com E. Evaluation of tamoxifen dose in advanced breast cancer: a progress report. pared with that of tamoxifen and 4-IT. This may be at least Cancer Treat. Rep., 60: 1451-1459. 1976. 7. Mclntosh. I. H., and Thynne, G. S. Tumor stimulation by antiestrogens. Br. partly responsible for its apparent improved efficacy in the J. Surg.. Â«4:900-901. 1979. animal studies. Uterotrophic studies in immature mice dem 8. Plotkin, D., Lecher. J.. Jung. W. E., and Risen. P. J. Tamoxifen flare in advanced breast cancer. JAMA, 240: 2644-2646. 1978. onstrated that both 4-IT and PIT were only uterotrophic at a 9. Smith. 1. E., Harris, A. L., Morgan, M., Ford. H. T., Gazet, J. C., Harmes. dose which was 10 times higher than for tamoxifen. However, C. L., White, H., Parsons, C. A., Villardo, A., Walsh, G.. and McKinna, J. A. Tamoxifen versus aminoglutethimide in advanced breast carcinoma: a 4-IT was unable to block the E2B effect, but PIT in this respect randomized cross-over trial. BMJ. 283: 1432-1434, 1981. showed some antiuterotrophic activity. The reduced agonist 10. Murray, R. M. L., and Pitt, P. Aminoglutethimide in tamoxifen-resistant activity of 4-IT and PIT was further demonstrated in the vaginal patients: the Melbourne experience. Cancer Res., 42 (Suppl.): 3437s-3441s, 1982. cornification studies. In this case 4-IT and PIT had agonist 11. Buzdar. A. O., Powell, K. C., and Blumenschein, G. R. Aminoglutethimide effects at a dose 10 times higher than with tamoxifen. In the after tamoxifen therapy in advanced breast cancer: M. D. Anderson Hospital antiuterotrophic study, PIT produced a dose-dependent inhi experience. Cancer Res., 42 (Suppl.): 3448s-3450s, 1982. 12. Jordan. V. C., and Gosden, B. Inhibition of the uterotrophic activity of bition of estradici stimulation, whereas tamoxifen and 4-IT estrogens by the short acting antiestrogen LY 117018. Endocrinology, 113: were unable to block the estradiol-stimulated vaginal 463-468, 1983. cornification. 4 R. McCague, I. B. Parr, and B. P. Hayes, manuscript submitted for Our results are consistent with those of Allen et al. (27), who publication. 5857

13. Clemens. J. A., Bennett, D. R.. Black. L. J.. and Jones. C. D. Effect of a new 24. Jordan. V. C., and Allen, K. E. Evaluations of the antitumor activity of the antiestrogen, keoxifene(LY 156758). on growth of carcinogen-induced mam nonsteroidal antiestrogen, monohydrox\tamoxifen, in the DMBA induced mary tumors and on LH and prolactin levels. Life Sci.. 32:2869-2875. 1983. rat mammary carcinoma model. Eur. J. Cancer, 16: 239;-251, 1980. 14. Gottardis. M. M.. and Jordon. V. C. The antitumor actions of keoxifene and 25. McCague, R.. Parr, I. B., Leclerq, G., Leung, O-T., and Jarman, M. Metab tamoxifen in the /V-nitrosomethylurea-induced rat mammary carcinoma olism of tamoxifen by isolated rat hepatocytes. Identification of the glucu- model. Cancer Res.. 47: 4020-4024. 1987. ronideof 4-hydroxytamoxifen. Biochem. Pharmacol., 39: 1459-1465, 1990. 15. Lerner, L. J. The first non-steroidal antiestrogenâ€”MER 25. In: R. C. 26. Robertson. D. W., Katzenellenbogen. J. A., Hayes, J. R., and Katzenellen Sutherland and V. C. Jordan (eds.). Non-steroidal Antiestrogen. pp. 1-16. bogen. B. S. Antiestrogen basicity-activity relationships: a comparison of the Sydney: Academic Press. 1981. estrogen receptor binding and antiuterotrophic potencies of several analogues 16. Nicholson, R. L. Antiestrogens and breast cancer therapy. In: B. J. A. Furr of (Z)-l,2-diphenyl-l-|4-[2-(dimethylamino)ethoxy]phenyl|-l -butÃ¨ne (ta and A. E. Wakeling (eds.). Pharmacology and Clinical Uses of Inhibitor of moxifen, nolvadex) having altered basicity. J. Med. Chem., 25:167-171, Hormone Secretion and Action, pp. 60-86. London: Bailliere-Tindall. 1986. 1982. 17. McCague, R., Leclerq, G., Legros, N., Goodman, J., Blackburn. G. M., 27. Allen, K. E., Clark, E. R., and Jordan, V. C. Evidence for the metabolic Jarman. M., and Foster, A. B. Derivatives of tamoxifen. Dependence of activation of non-steroidal antiestrogens: a study of structure-activity rela antiestrogenity on the 4-substituent. J. Med. Chem., 32: 2527-2533. 1989. tionship. Br. J. Pharmacol.. 71: 83-91. 1980. 18. McCague, R. Halogenated tamoxifens. British Patent Application No. 28. Wakeling, A. E., and Bowler, J. Novel antiestrogens without partial agonist 8621908/1986. activity. J. Steroid Biochem., il: 645-653, 1988. 19. Wakeling, A. E. Anti-hormones and other steroid analogues. In: B. Green 29. McNab, M. W., Tallarida, R. J., and Joseph. R. An evaluation of tamoxifen and R. E. Leake (eds.). Steroid Hormones: A Practical Approach, pp. 219- as a partial agonist by classical receptor theoryâ€”an explanation of the dual 236. Oxford: IRL Press, 1987. action of tamoxifen. Eur. J. Pharmacol., 103: 321-326, 1984. 20. Wilkinson, J. R., Williams. J. C., Singh. D., Goss, P. E., Easton, D., and 30. Wakeling, A. E. Pharmacology of antiestrogens. In: B. J. A. Furr and A. E. Coombes, R. C. Response of nitrosomethylurea-induced rat mammary tumor Wakeling (eds.). Pharmacology and Clinical Uses of Inhibitors of Hormones to endocrine therapy and comparison with clinical response. Cancer Res., Secretion and Action, pp. 1-9. London: Bailliere-Tindall, 1986. 46:4862-4865, 1986. 31. Black. L. J., Jones, C. D., and Falcone, J. F. Antagonism of estrogen action 21. Wakeling, A. E., Valcaccia, B., Newboult, E. E., and Green, L. R. Steroidal with new benzothiophene-derived antiestrogen. Life Sci., 32: 1031-1036, antiestrogensâ€”receptor binding and biological response in rat uterus, rat 1983. mammary carcinoma, and human breast cancer cells. J. Steroid Biochem., 32. Wakeling, A. E., and Bowler, J. Biology and mode of action of pure anties 20: 111-120, 1984. trogens. J. Steroid Biochem., 30: 141-147, 1988. 22. Black. L. J., and Goode, L. Uterine bioassay of tamoxifen, trioxifene, and a 33. Wilson, A. P. M., Weatherill, P. J., Nicholson, R. L, Davies, P., and new estrogen antagonist (LY117018) in rats and mice. Life Sci.. 26: 1453- Wakeling, A. E. A comparative study of the interaction of estradici and the 1458. 1980. steroidal pure antiestrogen, ICI 164,384, with the molybdate-stabilized estro 23. Harper, M. J. K., and Walpole, A. L. A new derivative of triphenylethylene: gen receptor. J. Steroid Biochem., 35: 421-428. 1990. effect on implantation and mode of action in rats. J. Reprod. FÃ©rtil.,13: 34. Green, S. Modulation of estradiol receptor activity by estrogens and anti- 101-119. 1967. estrogens. J. Steroid Biochem. Molec. Biol., 37: 747-751, 1990.

5858

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1991 American Association for Cancer Research. Pyrrolidino-4-iodotamoxifen and 4-Iodotamoxifen, New Analogues of the Antiestrogen Tamoxifen for the Treatment of Breast Cancer

S. K. Chander, R. McCague, Y. Luqmani, et al.

Cancer Res 1991;51:5851-5858.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/51/21/5851

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/51/21/5851. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.