A Permanently Charged Tamoxifen Derivative Displays Anticancer Activity and Improved Tissue Selectivity in Rodents1

[CANCER RESEARCH 56. 4328-4331. October I. 1996] Advances in Brief

Anat Biegon,2 Marcus Brewster, Hadassa Degani, Emil Pop, Dalia Somjen, and Alvin M. Kaye

Phannos. Lui., Kiryat Weizmann. Rehovot 76326. Israel Â¡A.B.]: Pharmos Corporation, Alaclnta. Florida 32615 Â¡M.B., E. P.Â¡:Departments of Biological Regulation [H. D.Â¡and Molecular Genetics Â¡A.M. K.Â¡,Weidmann Institute of Science, Reiwvot 76100, Israel: and Endocrine Unit, Tel Aviv Medical Center, Tel Aviv 64381, Israel [D. SJ

Abstract Materials and Methods

A quaternized form of tamoxifen (TAM), tamoxifen methiodide (TMI), Chemistry and Materials. TAM-free base and 17/3-estradiol were ob was shown to demonstrate very low brain uptake compared to TAM and, tained from Sigma Chemical Co., St. Louis, MO. TMI was prepared as unexpectedly, was considerably less estrogenic than TAM in the uterus. described previously (12). Briefly, TAM was treated with neat methyl iodide, The agonist activity of TMI in the bone was similar to that of TAM. TMI generating the object compound. Chemical and isomerie purity were assessed manifested significant dose-dependent tumoricidal activity with a rapid using HPLC and were found to be >99%. Other spectrophotometric (UV and onset of action against MCF-7 human breast cancer implants in nude mice infrared), spectroscopic ('H and I3C nuclear magnetic resonance), and spec- and a mean reduction in tumor size of 60% over six weeks. trometric (mass spectromety) data were consistent with the assigned structure. Two formulations were used for animal experiments. Biodegradable pellets Introduction (estradiol, placebo, TMI, and TAM) were custom-made by Innovation Re search of America (Sarasota. FL). The treatment pellets contained either 5 mg TAM1 (1) is a triphenylbutylene derivative that binds to the estro TAM, 6.8 mg TMI, or no drug (placebo). The pellets contained, in addition to gen receptor and exerts both antiestrogenic and estrogenic effects, the active ingredient, cholesterol, microcrystalline cellulose, a-lactose, di- and depending upon the ambient estrogen concentration, the tissue, and tricalcium phosphate, calcium and magnesium sterate, and steric acid and were designed to release the incorporated drugs at a constant rate over a 21-day the species (2). In humans, TAM seems to be mainly an antagonist in the brain and breast cancer and estrogen-like in the bone, uterus, and period through bioerosion. Drug loading was confirmed by HPLC. The target plasma level after establishment of the zero-order release profile was 5 ng/ml. cardiovascular system (3-5). The activity of TAM against breast The second formulation was a parenteral system designed for i.p. administra cancer is thought to be mediated through competition for the estrogen tion of TMI. In this prototype dosage form, TMI was solubilized in a 50% receptor. In addition, there is growing evidence for other mechanisms aqueous 2-hydroxypropyl-ÃŸ-cyclodextrin (Roquette, Paris, France) solution to involving the cell membrane (see Ref. 6 for a review). The side effects a concentration of 1.0 mg/ml. of TAM, when compared to those of other chemotherapeutic agents, Brain and Blood Distribution. TAM (0.36 mg/kg) or equimolar TMI are relatively minor. However, TAM is currently being assessed as a (0.5 mg/kg) was solubilized in DMSO and injected into the tail vein of male prophylactic agent in healthy women at increased risk for the disease Sprague-Dawley rats (175-200 g) at a vehicle dose of 0.5 ml/kg. At 0.25, (7). The side effects most often cited by patients are hot flashes, 1, 2, and 4 h after drug administration, groups of animals (Â«= 6) were sometimes accompanied by other CNS-related symptoms (depression, sacrificed. Brain and blood samples were collected, weighed, and homog enized in one volume of deionized water. To this system were then added irritability, and memory problems) thought to reflect the antiestro 4.0 ml of ice-cold acetonitrile and 1.0 ml of saturated saline. The organic genic activity of the drug in the brain (see Ref. 4 for a review). phase, which separated under these conditions, was collected for HPLC Estrogen is important for the maintenance of short-term memory and analysis. The system configuration included a Waters Model 510 pump, a hippocampal dendritic spines in rodents (8), and estrogen deprivation Thermo Separation Products FL2000 spectrofluorimetric detector, a Spec (or blockade) has been recently shown to be associated with short- tra-Physics Model SP 8880 Autosampler, and a SpectraPhysics Model SP term memory deficits and increased risk of Alzheimer's disease in 4270 integrator. The assay used an Alltech. Inc. Altima C8 5-/j,m particle women (9, 10). Because quaternization of amines significantly re size column protected with a guard column and a mobile phase containing duces their brain penetration, conversion of TAM to a suitable qua 70% acetonitrile, 15% isopropyl alcohol, and 15% of an aqueous triethyl ternary ammonium salt may reduce or eliminate these adverse effects. ammonium acetate buffer (pH 4.5). The flow rate of 1.5 ml/min and all Another problem of TAM therapy is an increased susceptibility to determinations were performed at ambient temperature. TAM and TMI were converted to their phenanthrene photoconversion products on-line endometrial cancer as a result of uterine hyperplasia, due to the (post-column) by passage through a 5-m knitted teflon reaction coil ex agonist activity of the drug in the uterus (11). These observations led posed to a 15 W low-pressure Hg lamp (254 nm). The products were us to consider the potential use of permanently charged TAM deriv quantitated fluorimetrically (excitation at 256 nm, emission at 380 nm). atives in breast cancer therapy and prevention (12). Herein, we report CK Determinations. PrepubertÃ !female Wistar-derived rats (~70 g) were the brain uptake, tissue selectivity, and anti-breast cancer action of obtained from the breeding colony at the Weizmann Institute of Science and one such derivative, TMI, in mice and rats. were injected i.p with various doses of TAM or TMI. either alone or concom- itantly with 10 nmol 17ÃŸ-estradiol. Rats were killed 24 h later, and the

Received 6/19/96: accepted 8/14/96. diaphyseal bone (tibia) and uterus were excised for determination of CK The costs of publication of this article were defrayed in part by the payment of page activity as described previously (13). Ten to 15 animals/dose were used in the charges. This article must therefore be hereby marked advertisement in accordance with TAM and TMI groups (2-3 separate experiments on groups of 5), and n = 60 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by a grant (to A. M. K.) from the Israel Cancer Association. in the estradiol alone group because the same dose of estrogen was given in all A. M. K. is the Joseph Moss Professor of Molecular Endocrinology. Weizmann Institute experiments. of Science. In Vivo Antitumor Action. CD1-/W athymic female mice, eight weeks old, 2 To whom requests for reprints should be addressed. Phone: 00972-8-9409679; Fax: were obtained from the Weizmann Institute Science Department of Animal 00972-8-9409686. 1The abbreviations used are: TAM. tamoxifen: TMI, tamoxifen methiodide; CK, Services. MCF-7 human breast cancer cells (originally obtained from the creatine kinase: CNS, central nervous system: HPLC. high-performance liquid chroma- laboratory of Prof. M. Lippman at the NIH, Bethesda. MD) were cultured in DMEM with 6% PCS in 5% CO^ at 37Â°C(14). Cells were detached from their tography. 4328

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. IMI DISPLAYS ANTICANCER ACTIVITY IN RODENTS flasks with 0.03% EDTA in PBS and were implanted in the flank of mice at a concentration of 10 X IO6cells/0.5 ml. All animals were implanted with slow release pellets of 17ÃŸ-estradiol (0.72 mg/pellet, 60-day release; Innovation Research of America). When tumors reached an average size of 1 cm' (1-2 months after implantation), the estrogen pellets were replaced by either a placebo pellet (n = 9), a 5-mg 21-day release TAM pellet (H = 9). or a 6.8-mg (equimolar to 5 mg TAM) 21-day release TMI pellet (n = 10). Animals were observed for six weeks, and tumor volumes were measured one to two times per week, starting from the day of pellet replacement. The length, width, and height of the tumors were measured using calipers and multiplied to achieve a volume estimate. The percentage change in tumor volume relative to day 0 was computed for each animal at each time point. In a second study, animals were prepared as indicated above, i.e., estrogen-stimulated tumors were allowed to reach a size of I cm1, after which the estrogen pellets were removed. Mice were then treated with daily i.p. injections of vehicle (50% aqueous hydroxypropyl-ÃŸ-cyclodextrin) or TMI at doses of 2, 5. and 10 mg/kg/day for 21 days. Tumors were measured weekly as described above. Significant differ ences in means were assessed using ANOVA with post hoc ScheffÃ©test in which a was preset to 0.05.

Results and Discussion Because quaternization greatly curtails the ability of small lipophilic molecules to cross the blood-brain barrier, we proposed that CNS side effects of TAM could be prevented by limiting its access to the brain, hopefully without affecting the anticancer activity. This hypothesis was tested using TMI (Fig. 1). The i.v. dosing of TAM to rats provided brain and blood concen tration profiles over time in accordance with the lipophilic character of the drug. Initial brain levels were very high, whereas blood levels were relatively low (Fig. 2A). B The brain:blood ratio, as measured by areas under the concentration curves from 0-4 h. was 18. Conversely, administration of TMI gen erated high blood concentrations and relatively low brain levels, which resulted in a brain:blood ratio of 0.5. Thus, as reflected by the n respective brain:blood ratios (Fig. 2ÃŸ),the exposure of the brain to cc TAM was significantly reduced through quaternization of the parent â€¢¿o o compound. o TMI was tested for estrogenic as well as antiestrogenic activity in tu vivo in two peripheral tissues, uterus and bone. Estrogen produces a â€¢¿Â»-'o reliable dose-dependent increase in CK-specific activity in both tis C sues (13) that can be used as a marker. In the diaphyseal bone. TMI fi was comparable to TAM as an agonist when tested alone or as an m antagonist in the presence of injected estrogen (Fig. 3/4). Significant effects were attained early in the dose-response study ( 100 nmol). The antiestrogenic potency of the two drugs in the uterus 0.25 was also comparable; however, the agonist activity of TMI in this tissue was several-fold lower than that of TAM (Fig. 3/1) over an order Time (hr) of magnitude of drug concentrations (Fig. 3ÃŸ).In addition, the data Fig. 2. Blood and brain concentrations and hrain:hlood ratios as a function of time after indicate that both the agonist effect of estrogen is countered by the i.v. administration of TAM or TMI. A, time course in blood and brain. B. time course of applied antiestrogens and the estrogenic action of TAM and its me- the brain:blood ratio for the two compounds.

thiodide salt is negated by coadministration of an estrogen. These CH results demonstrate a complementary extinction of activity that cannot be explained by a simple analysis of the estrogen receptor. The mechanism of this mutual diminution of activity may be related to ancillary proteins including steroid receptor cofactors. Anti-breast cancer activity of TMI and TAM was examined in a nude mouse model relative to a placebo control (14). Sham-treated animals (blank pellets) showed continued growth of the tumor during the first month of observation. The animals treated with TMI showed evidence of antitumor activity as early as one week after implantation. Tamoxifen Tamoxifen Methiodido The tumors regressed in 9 of 10 mice. The remaining animal showed Fig. 1. Synthesis and structure of TMI. arrest but no regression. By the end of the six-week observation 4329

was identical to that of JAM when isolated estrogen receptors were examined, but that there was minimal binding when whole cells were assayed. In addition, incubation of human breast cancer-derived cells 160 in culture with the methiodide did not affect growth at concentrations in which TAM was effective. These authors concluded that the charged molecule was hindered, relative to TAM, in its passage through the cell membrane (a 5% penetration was measured), con sistent with its lack of in vitro activity (12). However, in vivo, we found that TMI demonstrated estrogenic as well as antiestrogenic activity in peripheral tissues and was efficacious as a tumoricidal

UTERUS BONE B ~ 80

nu u _c

CD IO a

0.1 0.2 0.5 20 40 50 Dose (limole/animai) Treatment (days) Fig. 3. Agonist and antagonist effects of TAM and TMI in uterus and bone. A. comparison of the effects of TAM and TMI in uterus and bone at a single (0. l-/Â¿mol)dose. B. dose response in the uterus. â€¢¿.TAM;Ã¼ TMI. period, a 60%- reduction of mean tumor volume was seen in the TMI-treated group (Fig. 44). In four of the animals, the tumor was no longer measurable at the last time point. Under the same conditions, TAM treatment resulted in significant inhibition of the growth rate at two to three weeks. Pub lished literature suggest that TAM gives some degree of variability in this assay with slowed tumor growth, tumoristasis. and tumor regres sion reported (15). At time points later than 21 days (the lifetime of the slow release pellet), the growth-inhibitory effect of TAM was no longer significant (Fig. 4A). Thus, TMI had a faster onset, more persistent action, and a more potent effect than TAM in this experi ment (Fig. 44). Daily injections of TMI (2, 5, or 10 mg/kg/day, given i.p. over 21 days) demonstrated that the effect of the drug is dose-dependent. The 2 mg/kg dose had no effect; an intermediate (tumoristatic) effect was achieved with the intermediate dose. The 10 mg/kg dose, chosen to replicate the pellet dose, produced a significant decrease in the tumor -50 volume, similar to that observed in the pellet study at the same time 10 15 20 25 period of 21 days (Fig. 4B). Treatment (days) The quaternization of TAM was initially undertaken to prevent the passage of the drug through the blood-brain barrier, thus preventing Fig. 4. Inhibition by TAM and TMI of growth of human breast cancer implanted in nude mice. A, antitumor effects of s.c. pellets of TAM and TMI. â€¢¿.blankpellets (n = 9); CNS-related side effects. As expected, the charged molecule was, X. TAM pellets (n = 9); A. TMI pellets (Â»i= 10). *, significantly different from control; indeed, largely excluded from the brain. In addition, this manipulation Â«*,significantly different from control and TAM. B. dose dependence of the effect of TMI seems to have changed the tissue selectivity of the molecule without administered i.p. daily on estrogen-dependent human breast cancer growth in nude mice. A significant overall drug effect was due to the two higher doses. 5 and 11)mg/kg, which eliminating its antitumor activity. Earlier studies with TMI in vitro were significantly better than 2 mg/kg and vehicle but not significantly diffÃ©rentfrom each (12) showed that its affinity (K^) for the cytoplasmic estrogen receptor other (posi hoc ScheffÃ©analysis, or = 0.05). 4330

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. TMI DISPLAYS ANTICANCER ACTIVITY IN RODhNTS agent in an animal model of human breast cancer. The discrepancy Acknowledgments between in vitro and in vivo findings may be related to the dynamics We thank R. Margalit for his expert technical assistance. of TMI distribution. It is possible that TMI was excluded from cells in the whole cell receptor binding assay described by Jarman el al. References

(12), but this procedure required less than l h to complete. In whole 1. Harper. M. !.. and Walpolc. A. L. A new derivative of triphcnylelhylene: effect on animal studies, TMI was present for longer times, providing ample implantation and mode of action in rats. J. Reprod. FÃ©rtil..13: 101-119. 1967. opportunity for equilibration into accessible tissues. 2. Lerner. L. J.. and Jordan. V. C. Development of antiestrogens and their use in breast cancer. Cancer Res.. 50: 4177-4189. 1990. In addition, it is possible that TMI acts by additional mechanisms 3. Jordan. V. C. A current view of tamoxifen for the treatment and prevention of breast that contribute to its antitumor activity in vivo but not in vitro. TAM cancer. Br. J. Pharmacol.. 110: 507-517, 1993. and other steroids, for example, have been shown to affect angiogen- 4. Love. R. R., Wiebe. D. A.. Newcomb. P. A.. Cameron. L.. Leventhal. H.. Jordan. V. C, Feyzi, J., and DeMets. D. L. 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Hormonal chemoprevention of cancer in women. Science (Washington DC). 259; 633-638. 1993. tein kinase C and phospholipases, both of which are inhibited by TAM 8. Woolley. C. S.. and McEwen. B. S. Roles of estradiol and progesterone in regulation (6). TMI is likely to inhibit such enzymes even more efficiently than of hippocampal dendritic spine density during Ihe estrous cycle in the rat. J. Cotnp. TAM because the presence of a positive charge has been shown to Neural., 336: 293-306. 1993. 9. Phillips. S. M., and Sherwin. B. B. Effects of estrogen on memory function in increase the potency of various lipophilic amines as protein kinase C surgically menopausa! women. Psychoneuroendocrinology. 17: 485-495. 1992. inhibitors (18). Indeed, TMI seems to be at least five times more 10. Paganini-Hill. A., and Henderson. V. W. Estrogen deficiency and risk of Alzheimer's potent than TAM as an inhibitor of membranal phospholipase C disease in women. Am. J. Epidemic].. 140: 256-261. 1994. 11. 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Effects of structural modifications of tamoxifen on phospholipase C and phosphoinositide kinase activities. Isr. J. Med. treated women, the lower activity of the methiodide may translate Sci., 31: 158, 1995. into a reduced risk of endometrial cancer. Thus, if the pattern of 20. Black. L. J.. Sato. M.. Rowley. E. R.. Magee. D. E.. Bekele. A.. Williams. D C.. activities for TMI reported here for rodents translates to the human, Cullinan. G. J.. Bendeele. R.. Kauffmann. R. F.. Bensch. W. R.. Frolik. C. A., Termine. J. D.. and Bryant. H. U. Raloxifene (LY139481 HO) prevenÃsbone loss and TMI may offer a breast cancer treatment and prevention alternative reduces serum cholesterol without causing uterine hypertrophy in ovariectomi/ed with the benefits, but without some of the drawbacks, of TAM. rats. J. Clin. Invest.. 93: 63-69. 1994.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. A Permanently Charged Tamoxifen Derivative Displays Anticancer Activity and Improved Tissue Selectivity in Rodents

Anat Biegon, Marcus Brewster, Hadassa Degani, et al.

Cancer Res 1996;56:4328-4331.

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