Permanently ionic derivatives of steroid hormones and their antagonists

The present disclosure relates to compounds of the general formulae: ##STR1## wherein DRUG is a steroid agonist or antagonist, a mixed agonist-antagonist, or a partial agonist, and to the use of such compounds as anti-inflammatory and anti-tumor agents.

FIELD OF THE INVENTION 
This invention relates to pharmaceutical compositions that are suitable for 
use in methods of treatment requiring steroid hormones or their 
antagonists, and to novel permanently ionic chemical compounds that may be 
used in such methods. 
BACKGROUND OF THE INVENTION 
In principle, certain types of steroid hormones used in therapeutic 
procedures might beneficially be excluded from entering the central 
nervous system (CNS). It would be advantageous to produce derivatives of 
such hormones that retain their action in peripheral tissues and organs 
but are devoid of CNS activity. Penetration into the CNS requires that a 
compound be sufficiently lipophilic to cross the blood brain barrier 
(BBB). Therefore, to prevent penetration across the BBB it will often 
suffice to create an ionic drug derivative, especially a derivative that 
has a large charged moiety attached. However, the receptors through which 
steroid hormones exert their action are intracellular. Thus, it is not at 
all obvious that a derivative which is incapable of penetrating the BBB 
can in fact cross the cell membrane to reach the appropriate receptors. In 
other words, the cell membrane also constitutes a lipophilic barrier which 
hinders the passage of charged molecules. 
Examples of categories of steroid receptor binding drugs that would 
beneficially be excluded from the CNS include: corticosteroids, which have 
been shown to produce neuronal loss; progestins, which are used as an 
adjunct to estrogen replacement therapy in order to prevent endometrial 
hyperplasia; and antiestrogens that are used predominantly in preventing 
or retarding the growth of tumors. 
The use of progestins as an adjunct to estrogen in hormone replacement 
therapy in peri- or postmenopausal women is predicated on their opposition 
to the effects of estrogen. While estrogen has highly desirable actions in 
the brain, bone, and cardiovascular system, unopposed estrogen may be 
undesirable, particularly for the endometrial lining of the uterus. 
Progestins effectively prevent the undesirable hyperplasia of the 
endometrium. However, in the CNS they induce depression and hot flushes by 
virtue of their antiestrogenic activity. The use of progestins limited to 
their peripheral activity would be advantageous. 
Corticosteroids are extremely useful in suppressing inflammatory reactions. 
Their clinical use is severely curtailed by undesirable side effects, 
especially during chronic administration (Sapolsky et al. (1985) J. 
Neurosci. 5, 1222-1227; Landfield (1987) Prog. Brain Res. 72. 279-300). 
Many of these adverse side effects could be avoided if these compounds 
were incapable of exerting their harmful action in the CNS. 
Pharmaceutical therapy for breast cancer consists currently of cytotoxic 
and hormonal agents. Hormonal therapy was developed because, in many 
women, the breast cancer cells have receptors for the steroid hormone 
estrogen. The growth of these estrogen receptor-positive cancer cells can 
be stimulated by estrogen. Antiestrogen therapy attempts to reduce or stop 
the synthesis of estrogen or to block the action of estrogen on the cancer 
cell. 
Among the hormonals, tamoxifen (U.S. Pat. No. 4,536,516) holds a preeminent 
position. Originally designed as an antiestrogen to treat breast cancer in 
patients with estrogen receptor-positive tumors, the drug was also found 
to slow the growth of breast cancer in women with estrogen 
receptor-negative tumors. Tamoxifen is, therefore, useful in most 
patients. The antiestrogen tamoxifen is particularly effective in delaying 
recurrence in breast cancer patients and in the palliative treatment of 
advanced metastatic breast cancer. It is also useful in the treatment of 
additional types of cancer including prostatic neoplasms (Litherland, S. 
et al. Cancer Treatment Reviews, 1988, 15: 183; Jordan, C., Br. J. 
Pharmacol., 1993, 110: 507). 
Antiestrogens, including tamoxifen, compete with estrogen for receptor 
sites in cancerous tissues. Occupancy of the receptor site by an 
antiestrogen fails to elicit the further transcriptional actions generated 
by estrogens and blocks their activity. It is generally believed that 
estrogens function by binding to the target cell cytosolic receptors then 
moving into the cell nucleus and in turn affecting DNA transcription. 
Tamoxifen and other antiestrogens also affect cellular, tumor, and organ 
responses by less direct mechanisms. Antiestrogens penetrate into the CNS 
and disrupt the normal feedback loops for hormonal balance 
(hypothalamus-pituitary axis) by blockading estrogen receptors in the 
anterior pituitary and hypothalamus. Often the physiological activity 
arising from altered circulating hormone levels is undesirable and leads 
to a variety of known side effects of antiestrogen administration. Hot 
flushes, which are CNS-mediated, are the most common side effect of 
tamoxifen (Jordan, C., ibid.). 
The actions of tamoxifen and other nonsteroidal antiestrogens are 
complicated further by their mixed agonist-antagonist nature. Tamoxifen 
has partial agonist (estrogenic) activity, and the degree of agonist 
versus antagonist (antiestrogenic) activity is a function of the target 
cell (Furr, B. et al., Pharmacology & Therapeutics, 1984, 24: 127). 
Tamoxifen has been shown to act mainly as an antagonist in breast and 
brain, while its agonistic activity is more apparent in bone and the 
cardiovascular system. 
Whereas it has been postulated that pure antiestrogenic compounds might be 
more effective antitumor agents, another school of thought asserts that it 
is advantageous to retain the partial estrogenic activity of these 
antitumor agents since agonistic estrogenic activity is of proven value in 
preventing osteoporosis, cardiovascular disorders, and postmenopausal 
symptoms such as hot flushes (Jordan, C., Br. J. Pharmacol., 1993, 110: 
507) and possibly age-related cognitive decline and depression (Sherwin, 
B., Psychoneuroendocrinology, 1988, 13: 345). In particular, it has been 
envisaged that antiestrogen therapy could be administered prophylactically 
to healthy women at high risk for developing breast cancer, and large 
prospective clinical trials are underway to test this concept. It would be 
very desirable to minimize the deleterious effects of estrogen deprivation 
(or antagonism) in this population. 
Considerable effort has been invested in the development of novel tamoxifen 
analogs presumed to have improved therapeutic potential, by virtue of 
increased selectivity as antiestrogenic compounds (e.g. U.S. Pat. No. 
4,973,755; EP 0 168,175) or higher affinity for the estrogen receptor (WO 
92/06068). 
In various cases there have been discrepancies between the activity of 
tamoxifen derivatives in vitro and in vivo. For example, Foster et al. 
(Anticancer Drug Design, 1986, 1: 245) describes the effect of various 
tamoxifen hydroxy-derivatives on the growth of MCF-7 breast cancer cell 
line in culture. Hydroxy tamoxifen derivatives that are highly active in 
vitro were found to be less active than tamoxifen in vivo against a 
DMBA-induced estrogen receptor-positive tumor in rats, and only slightly 
more active against a hormone dependent mammary tumor in mice. However, 
when 4-hydroxy-tamoxifen itself is administered in vivo, its polarity 
reduces its ability to cross the cell membrane, thereby reducing its 
access to estrogen receptors located in the cytoplasm. Indeed, in vivo 
tests indicate a 4-hydroxytamoxifen to be less active than the native 
tamoxifen (Foster et al., J. Med. Chem., 1985, 28: 1491). 
Jarman et al., Anticancer Drug Design, 1986, 1: 259-268 described the 
preparation and testing of tamoxifen as well as tamoxifen methiodide, 
ethyl bromide, and N-oxide. When tested in vitro, these derivatives were 
reported not to halt the proliferation of breast tumor cell lines grown in 
culture. The interpretation offered was that the quaternized analogs fail 
to enter the cells (Jarman, M. et al. ibid.; Canabrana, B., Hidalgo, A. 
Pharmacology, 1992, 329). It was predicted, therefore, that these 
compounds would be of no therapeutic value in vivo. 
SUMMARY OF THE INVENTION 
The present invention provides compounds that act as steroid agonists 
and/or antagonists in peripheral organs and tissues, as required, while 
virtually devoid of activity in the central nervous system. Contrary to 
the teachings of the prior art, it is shown herein that compounds that are 
rendered ionic can achieve their desired therapeutic action in their 
target cells, even when they may exert their effects intracellularly. 
According to the present invention it is now disclosed that, unexpectedly, 
ionic derivatives of the antiestrogen tamoxifen which were predicted to be 
of no value in vivo on the basis of their lack of activity in vitro are in 
fact more active in vivo than the parent compound. 
In view of the robust activity of tamoxifen and other antiestrogens in the 
brain, apart from the disruption of normal feedback loops for gonadotropic 
function in the hypothalamic and pituitary regions, it is often desirable 
to use antiestrogenic agents that do not cross into the CNS and brain. 
Such peripheral antiestrogens would, in general, exhibit reduced side 
effects during clinical use and particularly in premenopausal women. 
Hydrophilic compounds and particularly compounds with ionic charges 
(cationic or anionic) are often very poorly distributed into the CNS and 
brain since a lipophilic barrier (the blood-brain barrier or "BBB") 
exists. One method for creating a permanent charge on a drug is the 
incorporation of a quaternary ammonium salt (nitrogen with four carbon 
atoms attached). Tamoxifen and other antiestrogens that contain an amino 
group can be quaternized (converted to a quaternary ammonium group) 
resulting in a permanent positive charge on the parent molecule which 
should effectively reduce its penetration across physiological membranes 
that are inherently lipophilic and resistant to penetration of ions, 
particularly large ions. 
In those cases where the drug contains no appropriate amine group, a 
bridging group can be utilized in order to provide an ionizable amine or 
other ionic species. In the case of progestins bearing a hydroxyl group, 
such as 17-hydroxy progesterone or medroxypregesterone, a bridging group 
can be attached in the form of an ester or phosphate or any other suitable 
species that can provide a permanent ionic moiety. In the case of 
corticosteroids, the 21-hydroxyl similarly can serve to attach a bridging 
group for providing an ionic moiety. Thus, by utilizing a strategy 
involving bridging groups, steroid hormone agonists can be converted to 
permanently ionic derivatives according to the general precepts of the 
instant invention. 
It is an object of this invention to provide peripheral antiestrogens for 
clinical treatment of cancer and other diseases and pathological 
conditions. These peripheral antiestrogens will possess estrogen 
antagonist activity, and may possess partial estrogen agonist or mixed 
activity, but are limited in biodistribution by being excluded from the 
CNS and brain, thereby exhibiting reduced side effects and being 
beneficial for clinical use. Thus, a primary objective of the present 
invention is to provide novel compounds that retain antiestrogenic 
activity in tumor tissue, while rendering them incapable of penetrating 
into the brain. 
Another object of this invention and beneficial to clinical use is the 
comparatively elevated circulatory levels of these agents due to the fact 
that they are not sequestered in fat tissue, thereby allowing for more 
precise control of dosing. 
Yet another aspect of this invention is to provide for the formulation and 
drug delivery of the aforementioned peripheral antiestrogens. 
These and other objects of the present invention are achieved by providing 
compounds of the general formula: 
##STR2## 
wherein Y.sup.- is any non-toxic pharmaceutically acceptable anion, DRUG 
is a steroid antagonist, mixed agonist-antagonist, or partial agonist; X 
is a direct bond or --O--, NR.sub.6, --S--, --SO--, --SO.sub.2, or 
--PO.sub.3 --; R.sub.1 and R.sub.2 are independently H, alkyl of 1-10 
carbon, aralkyl of 7-16 carbons or aryl; R.sub.3, R.sub.4, R.sub.5 are 
independently branched or unbranched, cyclic or noncyclic alkyl of 1-10 
carbons, alkyl of up to 10 carbons atoms substituted by carboxy, hydroxy, 
alkoxy, halo, or nitro, branched or unbranched, cyclic or noncyclic 
arylalkyl of 7-16 carbons, aryl; n is 0-12, as well as by providing 
compounds according to the formula 
##STR3## 
wherein X is a direct bond or is --O--, --NR.sub.6 --, --S--, --SO--, 
--SO.sub.2 --, or --PO.sub.3 --; R.sub.1, R.sub.2, and R.sub.6 are 
independently H, alkyl of 1-10 carbons, aralkyl of 7-16 carbons, or aryl; 
n is 0-12; G is a moiety selected from the group consisting of --N(R') 
(R") (R'"), --(O)N(R') (R"), --S (R') (R"), and --P(R') (R") (R'"), 
wherein R' is alkyl of 1-10 carbon atoms, alkyl of up to 10 carbons atoms 
substituted by carboxy, hydroxy, alkoxy, halo, or nitro, cycloalkyl of 4-8 
carbon atoms, cycloalkyl-alkyl of 5-18 carbon atoms, or aralkyl of 7-16 
carbons atoms and R" and R'" are independently C.sub.1 -C.sub.7 alkyl and 
R" and R'" together with N may form a 4- to 8- membered ring; B is C.sub.p 
H.sub.2p+1, halo, nitro, or a moiety which is linked to the 2-position of 
the phenyl that is neither the phenyl linked to the same ethylene carbon 
as B nor the phenyl substituted by the radical containing the permanently 
ionic group G, said moiety being selected from the group consisting of 
--CH.sub.2 C(R.sub.1)(R.sub.2)-- and --CH.sub.2 --O--; L and M are 
independently 0-3; l, m, and p are independently 1-7; and Y is a 
pharmaceutically acceptable anion, provided that when G is --N(R') (R") 
(R'") or --(O)N(R') (R"), R' and R" cannot both be unsubstituted alkyl.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides novel pharmaceutical compositions that act 
as steroid agonists or antagonists or have mixed agonist-antagonist 
activity with the limitation that these molecules have a permanent ionic 
moiety that prevents their penetration into the CNS. Contrary to the 
teachings of the prior art, these permanently charged derivatives of drugs 
can achieve their desired therapeutic activity in target cells even when 
the receptors may be located intracellularly. 
Accordingly, the present invention provides novel pharmaceutical 
compositions that retain antiestrogenic activity in tumor tissue while 
being largely incapable of penetrating into the brain. More specifically 
compounds according to the present invention provide anti-tumor activity 
in the breast or other reproductive organs and additionally provide 
partial estrogenic activity in organs such as bone or the cardiovascular 
system where estrogen activity is beneficial. A most preferred embodiment 
of the present invention is devoid of CNS activity, due to its inability 
to cross the BBB, while simultaneously being efficacious as an anti-tumor 
agent (irrespective of the mechanisms involved) and as an estrogenic agent 
in non-tumor tissues. 
One aspect of the present invention is pharmaceutical compositions that 
have anti-tumor activity and that contain as an active ingredient a 
therapeutically effective quantity of a compound of the formula 
##STR4## 
wherein Y.sup.- is any non-toxic pharmaceutically acceptable anion, DRUG 
is a steroid agonist or antagonist, a mixed agonist-antagonist, or a 
partial agonist; X is a direct bond or --O--, NR.sub.6, --S--, --SO--, 
--SO.sub.2 --, or --PO.sub.3 --; R.sub.1 and R.sub.2 are independently H, 
alkyl of 1-10 carbon, aralkyl of 7-16 carbons or aryl; R.sub.3, R.sub.4, 
R.sub.5 are independently branched or unbranched, cyclic or noncyclic 
alkyl of 1-10 carbons, alkyl of up to 10 carbons atoms substituted by 
carboxy, hydroxy, alkoxy, halo, or nitro, branched or unbranched, cyclic 
or noncyclic arylalkyl of 7-16 carbons, aryl; n is 1-12 or is 0, in which 
case DRUG or the X moiety is directly attached to the quaternary nitrogen 
atom. 
A preferred subgeneric grouping of said pharmaceutical compositions 
contains as an active ingredient a therapeutically effective quantity of a 
compound of the formula 
##STR5## 
wherein antiestrogen is an estrogen antagonist, mixed agonist-antagonist, 
or partial agonist and the remaining variables are as defined above. 
As discussed in more detail hereinbelow, said pharmaceutical compositions 
will ordinarily contain a pharmaceutically acceptable diluent or carrier, 
for instance: a diluent comprising an aqueous cosolvent solution 
comprising a pharmaceutically acceptable cosolvent, a micellar solution 
prepared with natural or synthetic ionic or nonionic surfactants, or a 
combination of such cosolvent and micellar solutions; a diluent consisting 
essentially of a solution of ethanol, a surfactant, and water; a diluent 
consisting essentially of an emulsion comprising triglycerides, lecithin, 
glycerol, an emulsifier, an antioxidant, and water; or a carrier selected 
from the group consisting of corn starch, lactose, sucrose, sorbitol, 
talc, stearic acid, magnesium stearate, dicalcium phosphate, and gums. 
Said pharmaceutical compositions can be formulated as a tablet for oral 
dosage, or be otherwise prepared in unit dosage form. A typical daily 
dosage of said compound will be from about 0.01 to about 10 mg/kg body 
weight, more preferably, from about 0.05 to about 5 mg/kg body weight. 
Also contemplated according to the present invention are methods such as a 
method of treatment of tumors which comprises administering to a patient a 
therapeutically effective amount of a pharmaceutical composition as 
defined above. Thus the present compositions may be used for treating 
cancer of the breast, ovaries, uterus, or prostate, and for preventing or 
retarding the growth of cancer, malignant cells, or neoplasms, and for 
reducing or preventing the metastasis of cancer-cells. 
The antiestrogens 
Various classes of antiestrogens can be modified in accord with the 
precepts of the present invention. These include: (a) antiestrogens 
derived from triphenylethylene, such as tamoxifen, toremifene and 
clomiphene; (b) antiestrogens derived from diphenyl naphthalene, such as 
nafoxidine; and (c) antiestrogens derived from triphenyl ethanol, such as 
ethamoxytriphetol. 
Tamoxifen, which has the following formula, may be regarded as a 
triphenylethylenic antiestrogen. 
##STR6## 
Other triphenylethylenic antiestrogens include enclomiphene, 
##STR7## 
zuclomiphene, 
##STR8## 
nitromifene, 
##STR9## 
nafoxidene, 
##STR10## 
desmethyltamoxifen, toremifene, and desmethyltoremifene. 
Structurally similar antiestrogens include ethamoxytriphetol, 
##STR11## 
centchroman, 
##STR12## 
and trioxifene 
##STR13## 
The permanent ionic charge 
The modification of drugs to prevent their access to the CNS may be most 
conveniently accomplished by preparation of quaternary salts. Such 
compounds may be prepared by a variety of chemical reactions. In the case 
of antiestrogens containing an amino nitrogen side chain, such as 
tamoxifen and its analogues, one such method is to react the antiestrogen 
with an alkylating agent, in order to quaternize the nitrogen atom on the 
side chain. The alkylating agent can be an alkyl halide, tosylate, alkyl 
or dialkyl sulfate or any other appropriate moiety. The alkylation may be 
performed with or without addition of organic solvents, as appropriate, 
and may be carried out under cooling or at room temperature or with 
heating, as appropriate, to ensure that the reaction proceeds 
satisfactorily to completion. However, cooling is preferable whenever 
cis-trans isomerization is possible. The reaction may be monitored by 
standard analytical methods known to one skilled in the art including thin 
layer chromatography, high pressure liquid chromatography, nuclear 
magnetic resonance spectroscopy or any other suitable method. The 
resulting quaternary salt is purified by standard methods, known to the 
artisan, usually including at least one step involving recrystallization. 
The associated anion may be changed if desired by standard procedures such 
as ion-exchange columns. Pharmaceutically acceptable anions in accordance 
with the present invention include citrates, chlorides, bromides, iodides, 
tosylates, mesylates, sulfates, and in general any anions derivable from 
alkylating agents or analogues thereof and which are nontoxic. 
Pharmacology 
The compound provided can be formulated by any required method to provide 
pharmaceutical compositions suitable for administration to a patient. 
The novel compositions contain, in addition to the active ingredient, 
conventional pharmaceutically acceptable carriers, diluents and the like. 
Solid compositions for oral administration, such as tablets, pills, 
capsules or the like, may be prepared by mixing the active ingredient with 
conventional, pharmaceutically acceptable ingredients such as corn starch, 
lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, 
dicalcium phosphate and gums, with pharmaceutically acceptable diluents. 
The tablets or pills can be coated or otherwise compounded with 
pharmaceutically acceptable materials known in the art to provide a dosage 
form affording prolonged action or sustained release. Other solid 
compositions can be prepared as microscapsules for parenteral 
administration. Liquid forms may be prepared for oral administration or 
for injection, the term including subcutaneous, intramuscular, 
intravenous, and other parenteral routes of administration. The liquid 
compositions include aqueous solutions, with or without organic 
cosolvents, aqueous or oil suspensions, emulsions with edible oils, as 
well as similar pharmaceutical vehicles. In addition, the compositions of 
the present invention may be formed as encapsulated pellets or other 
depots, for sustained delivery. 
The active dose for humans is generally in the range of from 0.01 mg to 
about 10 mg per kg body weight, in a regimen of 1-4 times a day. However, 
administration at longer intervals may also be possible, for compounds or 
formulations having prolonged action. The preferred range of dosage is 
from 0.05 to 5 mg per kg body weight. It is evident to one skilled in the 
art that dosage form and regimen would be determined by the attending 
physician, according to the disease to be treated, method of 
administration, and the patient's general condition. It will be 
appreciated that the most appropriate administration of the pharmaceutical 
compositions of the present invention will depend first and foremost on 
the clinical indication being treated. The prophylactic treatment of 
healthy women at high risk for malignant breast tumors will necessitate a 
sustained maintenance dosage regimen. In contradistinction, the treatment 
of existing breast cancer will require higher doses at more frequent 
intervals. 
Biological activity 
The present invention provides novel medical uses for both known compounds 
(Jarman et al., Anticancer Drug Design, 1986, 1, 259) and the novel 
antiestrogen derivatives as described above. These compounds have 
unexpectedly been shown to possess improved anti-tumor activity when 
compared to tamoxifen. In addition, these compounds exhibit peripheral 
estrogenic and antiestrogenic activity in vivo, without appearing to 
influence the brain. Estrogenic, antiestrogenic, and anti-tumor activity 
are demonstrated in the Examples hereinbelow. 
It should be noted that in those instances where tumor metastases in the 
CNS may be suspected, the use of non-BBB penetrating compounds of the 
present invention should not be contraindicated, since tumors in the CNS 
cause local disruption of the BBB. Indeed, in cases of metastatic brain 
tumors the use of quaternized anti-tumor agents may achieve preferential 
drug delivery to the tumor due to the known disruption of the BBB at the 
tumor site. 
EXAMPLES 
In order to further illustrate the present invention, specific examples are 
given below. It is to be understood that the examples given are for 
illustration only and are in no way limiting. 
EXAMPLE 1 
Tamoxifen (2.0 g) and methyl iodide (13 ml) were mixed together and the 
mixture held at 0.degree. C. for 24 hrs. Ethyl acetate (15 ml) was added 
and the white solid collected by filtration, rinsed with ethyl acetate and 
dried to afford 2.7 grams of tamoxifen N-methyl iodide (tamoxifen 
methiodide). All operations were performed at 0.degree.-5.degree. C. to 
avoid cis-trans isomerization. 
EXAMPLE 2 
A mixture of tamoxifen (0.5 g) and ethyl iodide (1.5 ml) was stored for 24 
hrs at 0.degree.-5.degree. C. Ethyl acetate was added and the solid 
collected by filtration and rinsed. After drying under vacuum, 0.7 g of 
tamoxifen N-ethyl iodide were obtained. All operations were conducted at 
0.degree.-5.degree. C. 
EXAMPLE 3 
Analogous to Example 2, tamoxifen (0.5 g), propyl iodide (1.5 ml) and ether 
(3 ml) were reacted for one week at 0.degree.-5.degree. C. and the 
resulting white solid was collected by filtration to yield 0.41 g of 
tamoxifen N-propyl iodide. 
EXAMPLE 4 
Tamoxifen (0.5 g) and bromomethane (0.25 g) in ether (5 ml) were mixed and 
held at 0.degree.-5.degree. C. for 24 hours until the initial sticky 
precipitate converted to a white solid. The white solid was collected by 
filtration and rinsed with ether. After drying, 0.60 g of tamoxifen 
N-methyl bromide were obtained. 
EXAMPLE 5 
Effect of tamoxifen or tamoxifen methiodide on uterine weight in immature 
female rats: induction and blockade of uterine hypertrophy. Prepubertal 
female rats respond to treatment by estrogen and estrogen agonists by 
increase in uterine volume and weight. Antiestrogens are known to block 
this effect when administered in conjunction with the agonist. Induction 
of uterine hypertrophy in this model is considered a standard test for in 
vivo estrogenic activity. Blockade of the estrogen induced response is 
considered a standard test for antiestrogenic activity in vivo. 
Groups of female rats, 22-23 days old, were injected i.p. with 25%/75% 
ethanol/water (vehicle), tamoxifen (0.5 mg/animal) in 25%/75% 
ethanol/water suspension, or tamoxifen methiodide (0.4 mg/animal) in 
25%/75% ethanol/water suspension. The volume injected was 150 microL. The 
animals were sacrificed 24 h later and the wet weight of excised uteri 
determined. 
The results of the experiment are presented in FIG. 1. The results were 
subjected to one-way analysis of variance, and the results are indicated 
with standard error bars. Tamoxifen ("TAM") and tamoxifen methiodide 
("TAM-Q") significantly increased uterine weight. (Overall treatment 
effect: P&lt;0.0006, post-hoc analysis by Scheffe's test: tamoxifen vs. 
control p&lt;0.015, tamoxifen methiodide vs. control p&lt;0.0008). 
The difference between the native tamoxifen and the methiodide was not 
significant, but it should be noted that a 20% lower dose of the 
methiodide was injected, which in tamoxifen equivalents is even lower. 
This suggests that no biological activity was lost in the tamoxifen 
derivatization and some may even have been gained. 
EXAMPLE 6 
In vivo estrogen effects of tamoxifen methiodide on bone: induction of 
creatine kinase activity in the bone. Estrogens are known to increase the 
activity of the enzyme creatine kinase (CK) in a number of target organs, 
including the bone. Compounds increasing bone CK activity are predicted to 
preserve bone mass, i.e. have estrogen like anti-osteoporosis effects. 
Groups of female rats, 22-23 days old, were injected i.p. with 25% 
ethanol/75% water (vehicle), tamoxifen 0.5 mg/animal in 25% ethanol/75% 
water suspension, or tamoxifen methiodide 0.4 mg/animal in 25% ethanol/75% 
water suspension. The volume injected was 150 microL. 
Animals were sacrificed 24 h later. Tibia and femur were excised and the 
epiphyses and diaphyses of the long bones collected and washed thoroughly 
in cold saline. Tissues were homogenized and centrifuged at 12,000.times. 
g for 5 min. The supernatant following this centrifugation was used to 
assay CK activity (Somjen et al., Biochem., 1991, 277: 863). Enzyme 
activity was measured on a spectrophotometer at a wavelength of 350 nm. 
Protein was determined by the Coomasie blue method so that results could 
be expressed in units as CK specific activity in .mu.mol/min/mg protein. 
Both treatments nearly doubled CK activity in both tissues (FIG. 2). The 
increase was highly significant. The effect of tamoxifen methiodide ("TQ") 
on the diaphysis was actually larger than the effect of tamoxifen ("TM") 
itself, but this difference did not reach statistical significance. 
EXAMPLE 7 
Effects of tamoxifen methiodide on body weight changes: induction and 
blockade of changes in body weight in ovariectomized female rats. Body 
weight in female rats is tightly regulated by estrogen, which inhibits 
food and water intake through direct influence on the CNS thereby limiting 
growth. Ovariectomy results in a cumulative increase in food and water 
intake and body weight. Estrogen and its agonists prevent this effect of 
ovariectomy and antiestrogens reverse the outcome of estrogen. Thus, 
changes in body weight in ovariectomized females are a standard test for 
estrogenic and antiestrogenic activity. Compounds that do not cross the 
BBB are not expected to be effective in this model. 
Pellets containing either 5 mg tamoxifen or 7 mg tamoxifen methiodide were 
manufactured by Innovative Research of America (Toledo, Ohio). Pellets 
containing 100 .mu.g of estradiol-17 .beta. were also supplied by the same 
manufacturer. This study was designed to continue the evaluation of the 
antiestrogen effects of TAM and TAM-Q both in the central and peripheral 
compartments of the rat. Female Sprague-Dawley rats weighing 150-170 g 
were bilaterally ovariectomized and allowed a 9 day recovery period for 
the rat to become maximally sensitized to changes in the estrogen 
environment. Rats were administered either TAM, TAM-Q, estradiol or 
combinations of the antiestrogen with estrogen (6 animals per 
composition). All pellets were designed for release of the active compound 
over 21 days. The experimental groups are presented in Table 1. 
TABLE 1 
______________________________________ 
Animal Treatment Dose 
Group No. Surgery Therapy mg Drug/Animal 
______________________________________ 
1 Ovx TAM 15 
2 Ovx TAM-Q 21 
3 Ovx TAM/Estradiol 
15/0.2 
4 Ovx TAM-Q/Estradiol 
15/0.2 
5 Ovx Estradiol 0.2 
6 Ovx Placebo -- 
7 Intact Placebo -- 
______________________________________ 
The in vivo phase of the study is presented in FIG. 3. As can be seen from 
the results, TAM-Q, unlike TAM, does not block the central effects of 
estrogen on body weight in these animals, thus indicating that it is not 
capable of penetrating into the CNS. In contradistinction, as shown in 
FIG. 1, the quaternized compound (TAM-Q) is efficient in blocking the 
peripheral effect of estrogen on uterine weight confirming its peripheral 
antiestrogen effect. 
EXAMPLE 8 
Brain and plasma levels of tamoxifen methiodide. Adult rats, 4 per group, 
were injected with tamoxifen methiodide, 0.5 mg/kg or an equimolar dose of 
tamoxifen (0.36 mg/kg) i.v. in DMSO. Fifteen minutes later, animals were 
sacrificed and their sera and brains were collected, homogenized and 
analyzed by HPLC. 
The results (Table 2) show fast accumulation of tamoxifen in the brain and 
very low serum levels at this time point, probably due to sequestration 
into lipid compartments and elimination. Tamoxifen methiodide, on the 
other hand, was present in serum at concentrations of 1-2 .mu.g/ml while 
brain levels were below detection. These results indicate that the 
quaternization of tamoxifen did, indeed, render it incapable of 
penetration into the brain. 
TABLE 2 
______________________________________ 
Serum Brain 
______________________________________ 
Tamoxifen not detectable 680 .+-. 49 nanog/g 
below 100 nanog/ml 
Tamoxifen 1625 .+-. 110 nanog/ml 
not detectable 
methiodide below 100 nanog/ml 
______________________________________ 
EXAMPLE 9 
Anti-tumor activity of tamoxifen methiodide in mice: anti-tumor activity in 
nude mice implanted with human breast cancer cells. Human breast cancer 
cells can be grown in culture. Injection of such cells into the flank of 
genetically athymic nude mice, coupled with estrogen treatment, results in 
the emergence of a tumor within a few weeks. If left untreated, the tumors 
will grow over time even after withdrawal of the exogenous estrogen until 
death of the animals occurs. Tumor size is relatively easy to measure and 
the ability of compounds to halt, or most preferably to induce regression 
of tumor growth, is considered a test of their anti-tumor activity. 
MCF7 cells (human breast cancer derived cell line) were grown in culture. A 
total of 30 nude mice were each injected with 2.times.10.sup.6 tumor cells 
in the flank area. Concomitantly, a subcutaneous estrogen pellet was 
implanted in the back of the neck. Animals were observed for tumor 
emergence. When visible tumors were detected (2-4 weeks after 
implantation), their length, width and height were measured with calipers. 
The values recorded were multiplied to produce a volume measurement and 
noted as baseline (time 0) tumor size. At this point, the estrogen pellet 
was replaced by a blank pellet (9 animals/control group), a tamoxifen 
methiodide pellet containing a molar equivalent of 5 mg tamoxifen (10 
animals), or a tamoxifen 5 mg pellet (9 animals). The pellets were 
purchased from Innovation Research and were designed to provide 21 days of 
constant release. Animals were then monitored for tumor growth and general 
appearance at least once a week over the next six weeks. The changes in 
tumor size compared to time 0 were calculated as percent change as 
follows: 
##EQU1## 
Thus, total regression of the tumor will translate into a -100% change. 
Statistical analysis of variation with repeated measure revealed highly 
significant effects of treatment (p&lt;0.0001) and time (repeated measure, 
p&lt;0.0001) and a significant interaction (p&lt;0.0001). Post-hoc analysis was 
performed using the Scheffe F test with alpha preset to 0.05. 
The results of the experiment are summarized in FIG. 4, in which the symbol 
.circle-solid. indicates the results obtained with blank pellets, the 
symbol X indicates the results obtained with tamoxifen pellets, and the 
symbol .DELTA. indicates the results obtained with tamoxifen methiodide 
pellets. As can be seen therefrom, tamoxifen methiodide unexpectedly 
induced significant tumor regression as early as 10 days after 
implantation of the pellet. 
NOVEL COMPOUNDS 
As indicated hereinabove, certain of the compounds that can be used in the 
pharmaceutical compositions and therapeutic methods according to the 
present invention are known. The following novel compounds, however, 
constitute in themselves another aspect of the invention herein described. 
Permanently ionic compounds having the formulae 
##STR14## 
wherein Y- is any non-toxic pharmaceutically acceptable anion, DRUG is a 
steroid agonist or antagonist, mixed agonist-antagonist, or partial 
agonist; antiestrogen is an estrogen antagonist, mixed agonist-antagonist, 
or partial agonist; X is a direct bond or --O--, NR.sub.6, --S--, --SO--, 
--SO.sub.2 --, or --PO.sub.3 --; R.sub.1 and R.sub.2 are independently H, 
alkyl of 1-10 carbon, aralkyl of 7-16 carbons or aryl; R.sub.3, R.sub.4, 
R.sub.5 are independently branched or unbranched, cyclic or noncyclic 
alkyl of 1-10 carbons, alkyl of up to 10 carbons atoms substituted by 
carboxy, hydroxy, alkoxy, halo, or nitro, branched or unbranched, cyclic 
or noncyclic arylalkyl of 7-16 carbons, aryl; n is 1-12 or 0, provided 
that when DRUG or antiestrogen is tamoxifen or 
4-hydroxy-2-methyltamoxifen, R.sub.3 and R.sub.4 are methyl, and R.sub.5 
is methyl or ethyl, Y is not a halide. 
Preferred compounds according to the above formulae are those wherein 
antiestrogen is selected from the group consisting of tamoxifen, 
desmethyltamoxifen, toremifene, desmethyltoremifene, clomiphene, 
nafoxidine, and ethamoxytriphetol. 
Another grouping of novel compounds according to the present invention is 
those compounds having the formula 
##STR15## 
wherein X is a direct bond or is --O--, --NR.sub.6 --, --S--, --SO--, 
--SO.sub.2 --, or --PO.sub.3 --; R.sub.1, R.sub.2, and are independently 
H, alkyl of 1-10 carbons, aralkyl of 7-16 carbons, or aryl; n is 0-12; G 
is a moiety selected from the group consisting of --N(R') (R") (R'"), 
--(O)N(R') (R"), --S(R') (R"), and --P(R') (R")(R'"), wherein R' is alkyl 
of 1-10 carbon atoms, alkyl of up to 10 carbons atoms substituted by 
carboxy, hydroxy, alkoxy, halo, or nitro, cycloalkyl of 4-8 carbon atoms, 
cycloalkyl-alkyl of 5-18 carbon atoms, or aralkyl of 7-16 carbons atoms 
and R" and R'" are independently C.sub.1 -C.sub.7 alkyl and R" and R'" 
together with N may form a 4- to 8-membered ring; B is C.sub.p H.sub.2p+1, 
halo, nitro, or a moiety which is linked to the 2-position of the phenyl 
that is neither the phenyl linked to the same ethylene carbon as B nor the 
phenyl substituted by the radical containing the permanently ionic group 
G, said moiety being selected from the group consisting of --CH.sub.2 
C(R.sub.1) (R.sub.2)-- and --CH.sub.2 --O--; L and M are independently 
0-3; l, m, and p are independently 1-7; and Y is a pharmaceutically 
acceptable anion, provided that when G is --N(R') (R") (R'") or --(O)N(R') 
(R"), R' and R" cannot both be unsubstituted alkyl. 
Preferred compounds of this grouping are those wherein X is --O--; R.sub.1 
and R.sub.2 are H; n is 2; G is --N(R') (R") (R'"); B is CH.sub.3, C.sub.2 
H.sub.5, halo, nitro, or a moiety which is linked to the 2-position of the 
phenyl that is neither the phenyl linked to the same ethylene carbon as B 
nor the phenyl substituted by the radical containing the permanently ionic 
group G, said moiety being selected from the group consisting of 
--CH.sub.2 CH.sub.2 -- and --CH.sub.2 --O--; L and M are 0 or 1, l and m 
are 1, and p is 2; and Y is a pharmaceutically acceptable anion. 
These compounds have utility as peripheral antiestrogens effective in the 
clinical treatment of cancer and other diseases and pathological 
conditions. These peripheral antiestrogens will possess estrogen 
antagonist activity, and may possess partial estrogen agonist or mixed 
activity. The compounds, however, are limited in biodistribution by being 
excluded from the CNS and brain, thereby exhibiting reduced side effects. 
It is this limited biodistribution that significantly enhances the 
clinical usefulness of the present compounds. Another useful aspect of the 
compounds of this invention that enhances their attractiveness for 
clinical use is the comparatively elevated circulatory levels of these 
agents due to the fact that they are not sequestered in fat tissue, 
thereby allowing for more precise control of dosing. The use of the 
present compounds in the treatment of cancerous tumors has been 
demonstrated above. 
While the invention has been described in terms of various preferred 
embodiments, the skilled artisan will appreciate that various 
modifications, substitutions, omissions and changes may be made without 
departing from the spirit thereof. Accordingly, it is intended that the 
scope of the present invention be limited solely by the scope of the 
following claims.