JP2003528137A - Antiprogestin with partial agonist activity - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method of inhibiting progestin activity in a cell or a subject in need of inhibiting progestin activity, and a method of treating or preventing a progestin-dependent condition in a subject in need of treatment or prevention of a progestin-dependent condition. Features. The method includes administering to a cell or subject an effective amount of a C-17-derived dexamethasone antiprogestin. The present invention also provides a method for screening for a substance against progestin or antiprogestin activity, and a method for detecting a progestin, a progestin agonist, or an antiprogestin in a sample using the C-17-derived dexamethasone antiprogestin of the present invention.

Description

Detailed Description of the Invention

Approval This invention was made with in-house support by the National Institutes of Health. the government,
The present invention has certain rights.

FIELD OF THE INVENTION The present invention relates generally to the identification of classes of compounds that act as anti-progestins with partial agonist activity, and methods for their use.

BACKGROUND OF THE INVENTION The main effect of steroid hormones is to regulate the rate of transcription of genes that respond during development, differentiation, and homeostasis. The basic steps appear to be the same for all members of the steroid / nuclear receptor superfamily that bind ligand. After the ligand enters the cell and binds to its cognate intracellular receptor protein, the resulting receptor-ligand complex forms DN
It undergoes activation to gain increased affinity for A. This receptor-ligand complex then attaches to a specific biologically active DNA sequence called the hormone response element (HRE) and associates with various cofactors,
It is thought to affect the transcription mechanism for modifying the transcription rate of the target gene.

The human progesterone receptor (PR) exists as three distinct isoforms: PR-A, PR-B, and PR-C (Kastner et al., EMBO J.
9: 1603-1614, 1990; Wei et al., Mol Endo 10: 1.
379-1387, 1996). Among them, PR-A and PR-B are the most abundant. However, the ratio of PR-A to PR-B isoforms is not constant between target tissues, and this may alter the cellular response as the activity of each isoform may change.

Anti-steroidal agents are defined as compounds that block the action of agonist steroids. However, virtually all antisteroidal agents retain some agonist activity under the selected conditions. Partial agonist activity was once considered a drawback of anti-steroidal drugs when the goal was to synthesize compounds that blocked all of the activity of agonist steroids. More recently, it has become clear that partial agonist activity may be a highly desirable property.
Therefore, in endocrine therapy of disease states, it is advantageous to retain agonist activity in as many unaffected tissues as possible. For example, tamoxifen is currently widely used as an anti-estrogen in the treatment of breast cancer. This is because tamoxifen acts as an estrogen in bone tissue and therefore does not decalcify the patient's bone.

Most stroid receptors each bind several ligands that exhibit partial agonist activity, but there are very few compounds that exhibit partial progestin activity under a wide variety of conditions. The most commonly used anti-progestin, RU-486, shows partial agonistic activity only under selected conditions (Meyer et al., EMBO J 9: 3923-3932, 1990;
Jackson et al., Mol Endo 11: 693-705, 1997). R
A closely related derivative of RU-486 known as TI 3021-020 (RTI-020) has been described as a partial agonist in T47D human breast cancer cells. However, this compound does not produce CV-1 monkey kidney cells (which
It is not active in (used for studies on the mechanism of PR action) (
Wagner et al., Proc Natl Acad Sci USA 93:87.
39-8744, 1996).

Anti-progestin compounds with partial agonist activity are useful for treating a variety of progestin-related diseases and conditions. Such anti-progestin compounds also have numerous research applications. However, some known anti-progestins have only limited partial agonist activity, and there remains a need in the art for anti-progestins with broad partial agonist activity. . The present invention provides a method for inhibiting progestin activity based on the identification of a class of compounds having a broader range of partial agonist activity of progestin than previously known anti-progertin compounds. Overcoming the previous drawbacks in.

SUMMARY OF THE INVENTION In a first aspect, the invention features a method of inhibiting progestin activity in a subject in need of inhibiting progestin activity. This method is
Administering to the subject an effective amount of a -17-derived dexamethasone anti-progestin.

In a second aspect, the invention features a method of treating or preventing a progestin-dependent condition in a subject in need of treatment or prevention of the progestin-dependent condition. The method comprises administering to the subject an effective amount of a C-17-derived dexamethasone antiprogestin.

In preferred embodiments of the first and second aspects of the invention, the subject can be a mammal (eg, a human, domestic animal, livestock animal, or laboratory animal), and the subject The body can be female.

In various other embodiments of the first and second aspects of the invention, the method can be used for: to regulate menstruation; benign progestin-dependent conditions (eg, To treat or prevent endometriosis, leiomyoma, ovarian cyst, premenstrual syndrome, anemia, dysmenorrhea, or pelvic inflammatory disease; or, a progestin-responsive tumor (eg, breast cancer, ovarian cancer, Prostate cancer, endometrial cancer,
Cervical cancer, leiomyosarcoma, or meningioma).

In other embodiments of the first and second aspects of the invention, the method may be used to prevent pregnancy. For example, the C-17-induced dexamethasone antiprogestin can be administered before ovulation or before sexual intercourse, or after ovulation or after intercourse. In still other embodiments of the first and second aspects of the invention, the administration inhibits embryonic egg implantation in the subject, or the administration inhibits ovulation in the subject.

In yet another preferred embodiment of the first and second aspects of the invention, the method is a method for inducing cervical maturation in a female. Cervical maturation can be in preparation for labor and childbirth, or for dilatation and curettage. In yet another preferred embodiment of the first and second aspects of the invention, the administration is to induce elimination of embryos or fetuses from the subject.

In a third aspect, the invention features a method of detecting a progestin or progestin agonist in a sample. The method comprises the steps of: a) contacting a sample with a reaction mixture containing a progesterone receptor and a C-17-derivatized dexamethasone antiprogestin; and b).
Measuring progestin activity in the reaction mixture. Here, the progestin or progestin agonist in the sample is detected by an increase in progestin activity compared to the amount of progestin activity measured in the reaction mixture not contacted with the sample.

In a fourth aspect, the invention features a method of screening a substrate for progestin activity. The method comprises the steps of: a) contacting a substrate with a reaction mixture containing a progesterone receptor and a C-17-derivatized dexamethasone antiprogestin; and b) measuring progestin activity in the reaction mixture. Process. Here, a substrate having progestin activity is identified by an increase in progestin activity compared to the amount of progestin activity measured in a reaction mixture not contacted with the substrate.

[0016] In a fifth aspect, the invention features a method of screening a substrate for antiprogestin activity. The method comprises the steps of: a) contacting a substrate with a first reaction mixture containing a progesterone receptor and a progestin or progestin agonist; b) determining the progestin activity in the first reaction mixture. Measuring the progestin activity in the first reaction mixture, and c) the progestin activity in a second reaction mixture containing a progesterone receptor, a progestin, or a progestin agonist, and a C-17-induced dexamethasone antiprogestin. The process of comparing the quantity. A first that is at least about 20% of a decrease in progestin activity in the second reaction mixture compared to the amount of progestin activity in the second reaction mixture in the absence of anti-progestin.
Substrates having antiprogestin activity are identified by a decrease in progestin activity in the reaction mixture of.

In a sixth aspect, the invention features a method of detecting anti-progestin in a sample. The method comprises the steps of: a) contacting the sample with a first reaction mixture containing a progesterone receptor and a progestin or progestin agonist; b) determining the progestin activity in the first reaction mixture. Measuring the progestin activity in the first reaction mixture, and c) the progestin activity in a second reaction mixture containing a progesterone receptor, a progestin, or a progestin agonist, and a C-17-induced dexamethasone antiprogestin. The process of comparing the quantity. Progestin in the first reaction mixture that is at least about 20% of a decrease in progestin activity in the second reaction mixture compared to the amount of progestin activity in the second reaction mixture in the absence of anti-progestin Anti-progestin in the sample is detected by the decrease in activity.

In a seventh aspect, the invention features a method of inhibiting progestin activity in a cell in need of inhibiting progestin activity. This method is C-17
Administering an effective amount of an inducible dexamethasone antiprogestin to the cells.

In a preferred embodiment of the seventh aspect of the invention, the cells are mammalian cells (
For example, human, monkey, mouse, or rat cells). The cell can be a primate cell (ie, taken directly from a mammal), or the cell can be from an established cell line (eg, CV-1 or 1470.2).

In an eighth aspect, the invention features a method of detecting a gene whose expression is regulated by an antiprogestin. The method comprises the following steps:
a) contacting the progesterone receptor with a C-17-induced dexamethasone anti-progestin, wherein the progesterone receptor is present in a sample containing a gene positively or negatively regulated by the progesterone receptor; b) sample Measuring the expression level of the gene therein; and c).
Comparing the expression level of the gene in the sample with the expression level of the gene in the sample not contacted with the C-17-induced dexamethasone antiprogestin. C-
Its expression by increasing or decreasing in the expression level of a gene in a sample contacted with C-17-induced dexamethasone anti-progestin as compared to the expression level of the gene in a sample not contacted with 17-induced dexamethasone anti-progestin Detects a gene regulated by anti-progestin.

In a preferred embodiment of the eighth aspect of the present invention, the C-17-induced dexamethasone antiprogestin may be a partial antiprogestin (ie a partial progestin agonist). In another embodiment of the eighth aspect of the invention,
Samples not contacted with C-17-induced dexamethasone anti-progestin were R
It can be contacted with a pure progestin agonist such as 5020.

In still other embodiments of the eighth aspect of the invention, the progestin or progestin agonist is, for example, before, after, or at the same time C-17-induced dexamethasone antiprogestin is added to the sample, It can be added to the sample. In still other embodiments of the eighth aspect of the invention, the sample can be a mammal, cell, or cell extract.

In all preferred embodiments of the above aspects of the invention, the C-17-induced dexamethasone anti-progestin can be dexamethasone-21-mesylate or dexamethasone-oxetanone.

DETAILED DESCRIPTION OF THE INVENTION Dexamethasone (Dex) has a very low affinity for the progesterone receptor (PR) (Ojasoo et al., J Med Chem 31: 11160-1).
169, 1988) and activity (Vegetto et al., Mol Endocrino.
17: 1244-1255, 1993) is a strong glucocorticoid. The present invention provides the surprising discovery that the C-17 derivative of Dex acts as an antiprogestin with partial agonist activity. In detail, D
ex-21-myristate (Dex-Mes), which is an affinity label and antagonist for the glucocorticoid receptor (GR) (Simo
ns and Thompson, Proc. Natl. Acad. Sci. USA
78: 3541-35545, 1981)) and Dex-oxetanone (De).
x-Ox), which is a reversible anti-glucocorticoid (Pons and Si
mons, J Org Chem 46: 3262-3264, 1981; La.
montagne et al., Endcrinology 114: 2252-2263.
, 1984)) and PR in two different cell lines, including CV-1 cells.
It showed a large amount of partial progestin activity with both -A and PR-B.
Both Dex-Ox and Dex-Mes have an affinity for cell-free PR (which is consistent with their whole-cell effect caused by binding to intracellular PR proteins), and other It showed partial progestin activity under conditions where the reported partial progestin was inactive. In detail, both De
The x derivative is RU 486 or RTI 3021-02 under comparable conditions.
It was more active than either 0. These results are particularly significant since only a few partial agonists are present for PR. Furthermore, Dex-Me
s and Dex-Ox retained PR isoforms (PR-A or PR-B), enhancers, promoters, reporters, or partial agonist activity depending on the cells tested. Thus, Dex-Mes and Dex-Ox offer the promise as mixed progestin agonists in a wide variety of different laboratories and clinical settings than previously reported compounds.

The activity of Dex-Mes and Dex-Ox with PR was not completely expected. Dex itself, if present, has only a very small affinity for PR or activity with PR. Therefore, derivatives of Dex are expected to be similarly inactive. Obviously, this is Dex-Mes and Dex-Ox.
Is not depending on the conditions and shows an agonist activity of between 5% and 95% with PR. These results suggest that other D-ring derivatives of Dex should be similarly active as partial PR agonists.

Both the significant affinity of Dex-Ox and Dex-Mes for PR, and the appreciable partial progestin activity of these compounds in a wide variety of settings, differed between Dex-Ox and DEx-Mes. Suggest that it should be a particularly useful steroid in clinical and research applications. Dex-
In contrast to Ox, Dex-Mes is an electrophilic affinity label that does not covalently label PR but forms a covalent bond with the glucocorticoid receptor (S.
imons et al. Biol. Chem. 262: 9676-9680, 198.
7). Although both Dex-Ox and Dex-Mas are anti-glucocorticoids, the use of these compounds as anti-progestins shows that Dex-Ox and Dex-Mes usually exert significant amounts of partial glucocorticoid activity. As shown, it probably does not have the major side effects of blocking the action of glucocorticoids (
Mercier et al., J Steroid Biochem 25: 11-20,
1986; Szapary et al. Biol. Chem. 271: 30576 ~
30582, 1996). This is in contrast to RU 486, the most commonly used antiprogestin. RU 486 is a pure anti-glucocorticoid in a wide variety of cells. Therefore, Dex-Mes and Dex-
The use of Ox as an anti-progestin should be achieved with reduced side effects, as the lesser effects of the glucocorticoid receptor are also blocked. Therefore, C-17 derivatives of Dex, such as Dex-Mes and Dex-Ox, hold promise for partial progestin agonists useful in a wide variety of clinical and research settings where partial progestin activity is desired. provide.

Definitions In this specification and in the claims that follow, reference will be made to a number of terms which shall be defined to have the following meanings.

By “progestin” and “progestin agonist” is meant any compound (natural or synthetic) capable of binding to the progesterone receptor and stimulating progestin activity under physiological conditions. Examples of progestins and progestin agonists include progesterone and promegestone (R5020; Figure 2), respectively.

By “progestin activity” is meant the ability of progestins and progestin agonists to regulate specific physiological processes. Sample (eg,
The amount of progestin activity in an animal; tissue, blood, or urine derived from the animal; cells; or cell extract) can be determined, for example, by measuring:
Progesterone receptor (PR) (eg, PR-A, PR-B, or P
The amount of progestin (or progestin agonist) binding to R-C);
Amount of progestin-stimulated expression of a reporter gene regulated by progesterone receptors in whole cells or in a cell-free transcription system; amount of cell proliferation or gene expression stimulated by progestin; secretory phase of the menstrual cycle Amount of thickening of the endometrium wall during; ovulation amount; fertility (eg, as indicated by blastocyst implantation or childbirth); and / or amount of inhibition of cervical maturation prior to parturition. Relative progestin biological activity in a sample is determined by comparing the amount of progestin activity in a sample to that of a suitable control sample that presents a known amount of progestin activity. obtain.

By “increased progestin activity” is meant an increase in the level of progestin activity, as described above, for example as measured by: an increased amount of progestin bound to the progesterone receptor; total. Increased progestin-stimulated expression of reporter genes regulated by progesterone receptors in cells or cell-free transcription systems; or acceleration of progestin-dependent physiological processes such as cell proliferation. Preferably, the increase is at least 1.5 fold to 2 fold, more preferably at least 3 fold, and most preferably at least 5 fold.

By “reduced progestin activity” is meant a reduced level of progestin activity, as described above, for example as measured by: reduced amount of progestin bound to progesterone receptor; whole cells. Or reduced progestin-stimulated expression of a reporter gene regulated by the progesterone receptor in a cell-free transcription system; or delaying progestin-dependent physiological processes such as cell proliferation. Preferably, the reduction can be at least about 20% and greater (eg, at least about 2).
1% to 40%, 41% to 60%, 61% to 80%, 81% to 90%, or 91
% -100%). Pure anti-progestin results in a 100% reduction, although less reduction is often desired in compounds that are considered partial anti-progestins (ie, anti-progestins with partial agonist activity).

[0032]   By "modulate" is meant altered by either an increase or a decrease.

By “progestin-dependent disease” or “progestin-dependent condition” is meant a distress, disorder, or physiological condition that is progestin-dependent for its presence or is exacerbated by progestin activity. Progestin-dependent diseases include, for example, progestin-responsive tumors (defined below)
Endometriosis; dysmenorrhea; and premenstrual syndrome. Progestin-dependent conditions include, for example, ovulation; the presence of a secretory endometrium capable of supporting blastocyst implantation; inhibition of uterine contractions and / or cervical maturation; and pregnancy.

By “progestin-responsive tumor” is meant a tumor that contains a progesterone receptor and whose growth and / or metastatic potential is stimulated by the binding of the progestin to the progesterone receptor within the tumor.

By “anti-progestin” or “progestin antagonist” is meant any compound (natural or synthetic) that inhibits the activity of a progestin or progestin agonist. Previously known examples of antiprogestins are R
U 38,486 (RU 486; Figure 2). The antiprogestin of the present invention is
For example, a C-17 derivative of dexamethasone, as shown in Figure 2 and described herein. The anti-progestin or progestin antagonist may (but need not) have partial agonist activity of progestin.

By “dexamethasone derived from C-17”, the C- of dexamethasone
A chemical derivative of dexamethasone is meant, which contains a chemical group at its C-17 position that is different from the chemical group shown at the 17 position (Figure 2). As used herein, any C-17-derived dexamethasone also has antiprogestin activity. Preferred examples of C-17-derived dexamethasone include dexamethasone 21-mesylate (Dex-Mes; FIG. 2) and dexamethasone oxetanone (Dex-Ox; FIG. 2).

By a “partial agonist of progestin” or a “mixed agonist of progestin” in certain circumstances (eg, in certain types of cells)
An antiprogestin that exhibits some progestin activity is meant. For example, partial or mixed agonists of progestins can bind to and inhibit the ability of progesterone receptors to activate progestin-dependent gene expression in one cell type, but In a cell type of the above, in the binding to the progesterone receptor, the progesterone receptor is a progestin-dependent gene (for example, a gene encoding the progesterone receptor, a gene encoding α-lactalbumin, or a mouse mammary tumor virus (MMTV) promoter). Expression of a gene under the transcriptional regulation of). This is similar to tamoxifen's partial estrogen agonist or mixed estrogen agonist activity. They act as antiestrogens in breast cancer cells and as estrogen in bone cells.

By “sample” is meant an animal (ie, a warm-blooded animal (eg, a human, domestic animal, livestock animal, or research animal, as described herein below)).
Any body fluid (eg, without limitation, blood, urine, cerebrospinal fluid, serum, sputum, saliva, tears, joint synovial fluid, coelomic fluid, or lavage fluid), tissue, or organ obtained from an animal; A cell (either in an animal, taken directly from an animal, or maintained in culture or derived from a cultured cell line); a lysate (or a fraction of a lysate) or Cell-derived extracts; molecules derived from cells or cellular material (eg, DNA molecules used as transcription templates in cell-free transcription assays); or compounds tested for progestin or antiprogestin activity (Eg, test compound). These are assayed or analyzed for progestin or anti-progestin activity according to the methods of the invention.

“Regulation of gene expression level” or “modification in level of gene expression”
By is meant a change in the amount of transcription, translation, mRNA stability, or protein stability. As a result, the total amount of gene product (ie, mRNA or polypeptide) is increased or decreased.

By “subject” is meant any animal that has a progesterone receptor. In contrast, the C-17-derivatized dexamethasone antiprogestin of the present invention is administered for therapeutic or experimental purposes or to control pregnancy. For example, the subject may be a cold-blooded animal (eg, fish, reptile,
Or an amphibian) or the subject can be a warm-blooded animal (eg, a human, domestic animal, livestock animal, or research animal, as described herein). The analyte can also be a cell, or a DNA molecule in a cell-free environment, such as a DNA molecule used as a transcription template in a cell-free transcription assay.

By “reaction mixture” is meant any environment containing a progesterone receptor, and which can be used to measure the progestin or antiprogestin activity of a test substance, or the progestin or antiprogestin activity in a sample. . The reaction mixture contains, for example, microtiter plates containing progesterone receptors either in test tubes, or in the wells of tissue culture dishes, or bound to soluble forms or to a solid phase; bearing progesterone receptors. On a filter or polymer beads; or in an animal, animal tissue, cell, cell lysate, or cell extract containing the progesterone receptor.

An “effective amount” of an anti-progestin derived from C-17 indicates an effective amount therefor, such as a certain function of anti-progestin (eg inhibition of progestin activity; treatment of a progestin-dependent condition or An amount that is useful for effecting prevention is meant. The actual amount required, as described below, was derived from the recognized variables (eg, subject treated, reason for treatment, and specific C-17 used). Dexamethasone antiprogestin compound). Therefore, it is not possible to specify the actual "effective amount". However, as described below, a suitable "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

By “in need thereof” it is desired to treat, prevent, inhibit or regulate a progestin-dependent disease or condition (or regulate expression of a progestin-dependent gene). Alternatively, the C-17-derived anti-progestins of the invention for studying their pharmacological properties (including, but not limited to, safety, efficacy, or physiological effects). (Eg, humans, domestic animals, livestock animals, or laboratory animals, as described below), or cells, cell-free transcription systems, or DNA subjected to experimental administration of A molecule is meant.

By “treatment” is meant administering an anti-progestin compound of the invention to a subject, cell, or cell-free transcription system for: progestin dependence in a subject. Frequency or severity of symptoms of a progestin-dependent disease or condition in a subject; in order to stabilize or delay the progression of a progestin-dependent disease or condition in a subject; To reduce the severity and / or recurrence; or to regulate the expression of progestin-dependent genes in a subject, cell, or cell-free transcription system.

By “preventing” is meant minimizing the chance of a subject developing a progestin-dependent disease or condition, or delaying the onset of a progestin-dependent disease or condition in a subject. To be done. For example, the anti-progestin compounds of the invention can be administered to a female subject to minimize the chances of becoming pregnant. Have an inherited progestin-dependent disease or condition (eg, progestin-dependent breast cancer, progestin-dependent prostate cancer, progestin-dependent meningioma, endometriosis, or uterine fibrosis) For subjects belonging to the family, anti-progestin therapy may be initiated prior to the onset of the disease, thereby reducing the chances of subject being victim of the disease and / or initiating anti-progestin therapy. Delays the onset of the disease compared to when it has not already occurred.

[0046]   By "about" is meant ± 10% of the indicated value.

Therapeutic Uses of Antiprogestins Progesterone and other progestins have been shown to affect reproductive health and reproductive function,
And play a major role in other non-reproductive physiological processes. Based on our findings of these various biochemical pathways, where possible, it is now possible to treat, prevent, and prevent a wide variety of progestin-dependent diseases and conditions,
Alternatively, it may be desirable to use antiprogestin therapy for modulation. Compounds having antiprogestin activity are characterized by antagonizing the effects of progesterone or another progestin. As such, they can be used in any case where it may be desired to modulate progestin activity.

For example, the C-17-derived dexamethasone antiprogestin of the present invention is:
It can be used to regulate pregnancy during the female reproductive cycle of the animals of the invention. They are therefore used to control irregularities in the human menstrual cycle, or in commercial animals (eg fish, cows, sheep, pigs, goats, or chickens), laboratory animals (eg monkeys, chimpanzees). , Rats, mice, or guinea pigs) or domestic animals (dogs, cats, weasels, birds, rabbits, or horses) can be used to synchronize or suppress gestational age. They can also be used to reduce hypermenorrhea, and are therefore useful in treating or preventing anemia and dysmenorrhea. Regulation of the female reproductive cycle by the antiprogestins of the invention also includes
Provide useful treatments for the prevention or treatment of premenstrual syndrome, endometriosis, leiomyomas (uterine fibrosis), and / or ovarian cysts. Further, in subjects who have or have previously had an inflammatory disease of the pelvis (which may be scarred in the fallopian tubes), the progestins of the present invention It can be used to reduce the chance that pregnancy can occur.

The C-17-derived dexamethasone antiprogestins of the present invention can be used as a contraceptive agent for the prevention of pregnancy. Depending on the desired dose level and frequency, these may be, for example, daily, weekly, biweekly, or monthly to inhibit fertilization and / or fertilize into the lining of the endometrium. It can be administered to inhibit the implantation of eggs (or blastocysts). Thus, the antiprogestins of the invention can be administered before ovulation or after ovulation. Further, the anti-progestins of the invention can be administered prior to or after sexual intercourse to prevent pregnancy. The dose of anti-progestin that is effective for contraception is that amount that inhibits fertilization by inhibiting ovulation or implantation of a fertilized egg or blastocyst. Based on the knowledge in the art of progestin flow during the menstrual cycle and the role of progestins in the development of ovulating and secretory endometrium (ie, the endometrium that allows the implantation of blastocysts) The skilled artisan can readily ascertain the effective antiprogestin dose level and frequency used in the methods of the invention. For example, Bygdeman
Et al, Eur J Contracept Reprod Health Car
e 4: 103-107, 1999; Spitz and Robbins, Hum.
Reprod Update 4: 584-593, 1998; Grow et al.
Fertil Steril 69: 936-943, 1998; and Ze.
Linski-Woten et al., Hum Reprod 13: 2132-21.
38, 1998.

Anti-progestin must also be induced to mature the cervix prior to induction of labor (eg, during gestation, or post-gestation), or to remove a fetus whose birth has died. Can also be used. Antiprogestins are also used to terminate pregnancy by inducing the shedding of live embryos or fetuses from the uterus, or to promote cervical dilation prior to uterine curettage. Can be done. These applications include, for example, Mackenzie, A
nn Acad Med Singapore 22: 151-157, 199.
3; Sitruk-Ware et al., Contraception 41: 221-.
243, 1990; and Hingorani et al., Acta Obstet G.
ynecol Scan Suppl 149: 25-29, 1989.

Many types of tumors, such as breast, ovarian, prostate, endometrial, and cervical cancers, and leiomyosarcoma and meningioma have been shown to contain progestin receptors. .. These tumors are progestin-responsive,
That is, it proliferates when exposed to progestins. Conversely, the growth of such progestin-responsive tumors is inhibited by the use of anti-progestin compounds. Thus, the anti-progestins of the invention, whether benign or metastatic, are used to treat progestin-responsive tumors or to prevent the first onset or recurrence of cancer (especially in general Subjects known to have a greater than normal risk for such cancers compared to other populations (
For example, a subject who has an inherited risk for such cancer, or who has previously suffered from cancer)) can be used. The growth-inhibitory effect of antiprogestins on progestin-responsive tumors has been demonstrated, for example, by Etreby and L.
iang, Breast Cancer Res Treat 49: 109-
117, 1998; Klijn et al., Hum Reprod 9 supplement 1:18.
1-189, 1994; Baker, et al., J Steroid Biochem.
Mol Biol 37: 789-794, 1990; and Rose and Barnea, Oncogene 12: 999-1003, 1996.

Contraception, treatment of uterine maturation, embryo or fetus shedding, uterine leiomyoma (fibroids), endometriosis, and other conditions associated with the female reproductive cycle, as well as of various cancers. Further information regarding the use of antiprogestins for treatment (eg, dosage, frequency, and physical effects) can be found in, eg, US Pat. No. 6,015,805; and Jang and Benet, J. et al. Pharmacokinet Bi
opharm 25: 647-672, 1997.

Modes of Administration The C-17-derived dexamethasone antiprogestins of the present invention, and compounds identified using any of the methods disclosed herein, are pharmaceutically acceptable. It can be administered to a subject in a unit dosage form with a diluent, carrier, or excipient. By "pharmaceutically acceptable" is meant a material that is not biological or otherwise undesired. That is, these materials are derived from C-17 without causing any undesired biological effects or interactions in a deleterious manner with any of the components of the pharmaceutical composition contained therein. The dexamethasone antiprogestin may be administered to an individual. Conventional pharmaceutical practice can be used to provide suitable formulations or compositions for administering such compositions to a subject. For example, any suitable route of administration may be used, including but not limited to: intravenous, parenteral, transdermal,
Subcutaneous, intramuscular, intracranial, intraorbital, ophthalmological, intraventricular, intracapsular, intrathecal, cisternal, intraperitoneal, intranasal, rectal, vaginal, aerosol, or oral administration. Therapeutic formulations can be in the form of liquid solutions or suspensions; for oral administration, the formulations can be in the form of tablets or capsules; for intranasal administration, the formulations can be powders,
Nasal drops or in the form of an aerosol; for vaginal formulations, the formulation may be vaginal cream, suppository, or pessary; for transdermal formulations, the formulation may be It can be in the form of a cream or in the form of being distributed over a patch applied to the skin.

Methods well known in the art for making formulations are described in, for example, Remingto.
n: The Science and Practice of Pharma
cy (19th edition), A. R. Gennaro, Mack Publish
g Company, Easton, PA 1995. Formulations for parenteral administration may include, for example, excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems,
And liposomes. Formulations for inhalation may include excipients such as lactose, or alternatively aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate. Solution, or it may be an oily solution for administration in the form of drops or gel for the nasal cavity.

Dose The C-17-derived dexamethasone anti-progestin of the present invention is for inhibiting, or for treating, preventing, inhibiting, or modulating progestin activity in a subject in need thereof. Can be administered to a subject in an amount sufficient to modulate a progestin-dependent condition in a subject in need thereof. Those skilled in the art will appreciate that the optimum dose used will vary according to the individual treated, the particular compound used, and the route of administration chosen. Optimum dosages will also vary between individuals based on age, size, weight, sex, and physical condition. Methods for determining the optimal dose are described, for example, in Remington: The Sc
Ience and Practice of Pharmacy (19th Edition)
Ed., A. R. Gennaro, Mack Publishing Compan
y, Easton, PA 1995.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the disease treated, the species of animal, and the particular route of administration. The therapeutically effective amount can be determined by routine experimentation and by analogy with the amounts used to treat the same disease states with similar anti-progestin compounds. For example,
A unit dose of anti-progestin may preferably contain from 0.001 milligram (mg) to 1 gram of the active ingredient. Typically, for treatment of humans, the antiprogestins of the invention are administered in amounts ranging from about 0.001 to 10 mg / kg body weight, and preferably 0.1 to 3 mg / kg. Up to. The compounds may be administered from 1 to 4 times daily, preferably once or twice daily. Anti-progestins can also be administered once a week, once a month, or sporadically (eg, postcoitus), as is well known in the art. Examples of regimens for administration of anti-progestins include, for example, Bygdeman et al., Eur J Contracept.
t Reprod Health Care 4: 103-107, 1999,
And Spitz and Robbins, Hum Reprod Update
e 4: 584-493, 1998.

Efficacy The efficiency of administration of dexamethasone antiprogestin derived from a specific dose of C-17 depends on the medical history, signs, symptoms, and subjects in need of inhibition of progestin activity, or progestin. Treatment, prevention of addictive diseases or conditions,
It can be determined by assessing a particular aspect of the laboratory test of interest which is known to be useful in assessing the condition of a subject in need of inhibition, or modulation. These signs, symptoms, and laboratory tests of interest will be specific to those treated or prevented, as is known to any physician treating such subjects, or investigators conducting experiments in the field. Depends on the disease or condition.
For example, based on comparisons with appropriate control groups and findings of normal disease progression in the general population or in particular individuals, 1) show that the frequency or severity of recurrence in the subject has improved. 2) is shown to stabilize or delay the progression of the disease, or 3) diminishes the need for the use of other medications to treat the condition or disease Alternatively, if the need is eliminated, the particular treatment is considered effective.

In a specific example, in using an anti-progestin of the invention to treat a progestin-responsive tumor, inhibition of tumor growth or metastasis, or tumor shrinkage is efficacious by anti-progestin treatment. It is an index that is. Similarly,
A reduction in the signs or symptoms of a diagnosed progestin-dependent disease or condition (eg, endometriosis or uterine fibrosis) following anti-progestin treatment is indicative of treatment efficacy.

Diagnostic and Experimental Uses of Antiprogestins Progestins are involved in a variety of physiological processes. Although best studied for their role in the female reproductive cycle, and for diseases and conditions of the female reproductive organs (eg, breast, ovary, uterus, and cervix), progestins also demonstrate both normal physiology and disease. , Also plays a role in male animals. In particular, progestins may be involved in the development and / or progression of prostate cancer and meningioma. While levels of progestin in both male and female subjects influence and reflect the health and reproductive status of the subject, measurement of progestin and / or antiprogestin levels in the subject is
Allows diagnosis and / or monitoring of a progestin-dependent disease or condition in a subject. The C-17-derived dexamethasone antiprogestins of the present invention can be used in diagnostic assays for the determination of progestin and / or antiprogestin levels in such subjects. These diagnostic assays also provide progestin in a subject receiving treatment for the purpose of inhibiting or modulating progestin activity (eg, for contraception, controlling menstruation, or inducing cervical maturation), It can also be used to monitor levels of progestin agonists, and / or antiprogestins.

For example, to detect a progestin or progestin agonist in a sample (eg, tissue biopsy, blood, urine, cells, cell lysate, or cell extract) derived from a subject, the sample is progesterone. It is added to the reaction mixture containing the receptor (eg, in the wells of a microtiter plate). The C-17-derived dexamethasone antiprogestin of the present invention is added either before, after, or concurrently with the addition of the sample to the wells. One of skill in the art would use one of many receptor binding assays known to those of skill in the art (eg, the cell-free receptor binding assay described in Example V below) to determine the amount of progestin or progestin agonist activity. Know how to measure. For example, a relative increase in progestin activity was labeled (eg, radioactivity, colorimetric, compared to control wells containing no sample).
Or fluorescence) C-17-derived dexamethasone anti-progestin is indicated by a decrease in binding to the progesterone receptor in the sample wells. This indicates the relative level of progestin in the sample. Those skilled in the art will appreciate that similar assays can also be used in high throughput screens to identify compounds having progestin activity by using the test compound as a sample rather than a sample derived from the subject. .

Those of skill in the art will also appreciate that assays for progestin activity in a sample, or rapid screening to identify compounds with progestin activity, also include many known reporter gene assays (eg, Examples II-IV below. It can be carried out using one of the luciferase reporter gene assays described in. In these assays, the reaction mixture (eg, in a microtiter well) is labeled with a progesterone receptor, a reporter gene transcriptionally activated by the progesterone receptor (eg, the reporter gene described in Examples II-IV below). Gene), and any other factors required for transactivation of the reporter gene by the progesterone receptor.
The reporter gene and progesterone receptor can be present, for example, in an intracellular or cell-free extract. Microtiter wells also contain a C-17-derivatized dexamethasone antiprogestin of the invention. The C-17 derivatized dexamethasone anti-progestin can be introduced into the well either before, after, or at the same time as the sample or test compound is added to the well. To measure progestin activity in a sample or to identify a test substance that has progestin activity, the sample or test substance is added to microtiter wells and then the amount of activity of the reporter gene is measured. An increase in the activity of the reporter gene compared to the amount of the activity of the reporter gene in the control or microtiter wells lacking the sample or test compound is indicative of the progestin activity of the sample or test compound. Those skilled in the art are well aware that numerous variations to this basic reporter gene assay can be used in a similar fashion.

Anti-progestin activity can be similarly detected in a sample derived from a subject using, for example, a progesterone receptor binding assay as described above. In one example, the sample is added to a reaction mixture containing a progesterone receptor and a progestin or progestin agonist. The progestin activity in the mixture is measured (eg, by the amount of binding to the progesterone receptor) and a second, containing C-17-derivatized dexamethasone antiprogestin of the invention, in addition to the progesterone receptor and the progestin or progestin agonist. Compared to progestin activity in microtiter wells. The progestin activity in the second microtiter well is compared to the progestin activity in the third well, which contains the progesterone receptor and a progestin or progestin agonist but lacks the antiprogestin. A decrease in progestin activity in the first well that is at least about a 20% decrease in progestin activity in the second well, where the decrease in the second well is proportional to the third control well, The relative amount of anti-progestin activity in is shown. Similar assays can also be used in rapid screening to identify compounds with anti-progestin activity by using the test compound as a sample rather than a sample from the subject.

In addition, a reporter gene assay (eg, the reporter gene assay described in Example V below, or one of many other reporter gene assays known in the art) is assayed for anti-progestin activity. Can be used to measure antiprogestin activity in a sample or test compound. In certain examples, the sample or test compound is added to a reaction mixture (eg, in a microtiter well) containing a progesterone receptor-modulated progesterone receptor and a reporter gene, as described above. Progestins or progestin agonists are also added to the wells.
The amount of reporter gene activity in the wells is measured and reporter gene activity in a second well containing a progesterone receptor, a progesterone receptor responsive reporter gene, a progestin, or a progestin agonist, and a C-17 derivatized dexamethasone antiprogestin. Compared to the amount of. Second
Of the reporter gene activity in the first well that is at least about 20% decrease in the reporter gene activity of the wells (wherein the decrease in the second well is in the third well lacking antiprogestin). Of the reporter gene activity of
Indicates that the sample or test compound contains no antiprogestin activity.

The anti-progestin compounds of the invention can also be used to detect genes that are positively or negatively regulated by anti-progestin. For example,
Mammalian cells, or containing mammalian cell-free transcription or transcription / translation extracts (ie, to assist progesterone receptors, genes transcriptionally regulated by progesterone receptors, and transcription or transcription / translation) A sample (containing any other necessary factors) is contacted with a C-17 derivatized anti-progestin of the invention such that the anti-progestin binds to the progesterone receptor and one or more downstream genes. Regulate the expression of (the person skilled in the art may need to add exogenous progestin or progestin agonist to the sample, either before, after, or simultaneously with the addition of anti-progestin to the sample, or Understand that it may be desired). The pattern of mRNA and / or protein in the treated sample is compared to the pattern in the untreated control sample, and the increase or decrease of specific mRNA or protein in the treated sample is used in the experiment. MRNA or protein encoded by a gene that is positively or negatively regulated by a particular anti-progestin. The gene encoding the mRNA or protein is then isolated by well known methods (eg Velcules).
cu et al., Science 270: 484-487, 1995, as described in Serial Analysis of Gene Expressi.
on or using SAGE). A comparison of the expression patterns of genes that are regulated by anti-progestins in different types of cells or cell extracts (eg, liver-derived vs. ovarian-derived) allows for the analysis of one or more of the specific cell types. It provides valuable information on the physiological effects of a given antiprogestin on specific genes. This information can help in designing various therapeutic strategies that help inhibit the activity of progestins only in selected cell types.

Test Compound Generally, a novel drug having progestin or antiprogestin activity is
It can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
Those skilled in the field of drug discovery and development understand that the exact source of the test extract or compound is not critical to the screening procedure of the invention. Therefore,
Virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include plant-based extracts, fungal-based extracts, prokaryotic-based extracts, or animal-based extracts, fermentation broths, and synthetic compounds, as well as existing compounds. However, the present invention is not limited to these. Numerous methods are also random or directed to any number of compounds, including, but not limited to, sugar-based extracts, lipid-based extracts, peptide-based extracts, and nucleic acid-based extracts. Available to generate syntheses (eg, semi-synthetic or full-synthetic). A library of synthetic compounds is, for example, Brandon.
Associates (Merrimack, NH) and Aldrich C
commercially available from chemical (Milwaukee, WI). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available from Biotics (Sussex, UK), Xenova (Slough, UK).
), Harbor Branch Oceangraphics Instit
ute (Ft. Pierce, FL), and PharmaMar, U. S. A
． (Cambridge, MA) from a number of sources. further,
Natural and synthetically produced libraries are produced, if desired, according to methods known in the art, eg, by standard extraction and fractionation methods. Further, if desired, any library or compound is
It is easily modified using standard chemical, physical, or biochemical methods.

Further, those skilled in the art of drug discovery and development will be familiar with dereplicati
on) (eg, taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof), or replication of materials already known for their progestin or antiprogestin activity. It is easy to understand that the method for elimination of repeats should be used whenever possible.

If the crude extract is found to have progestin or anti-progestin activity, a further fraction of the positive derived extract will yield a chemical component that is responsive to the observed effect. It is necessary to separate. Thus, the purpose of the extraction, fractionation, and purification process is the careful characterization and identification of the presence of compounds in the crude extract that have progestin-mimicking or progestin-antagonizing activity. The same assay described herein for the detection of activity in a mixture of compounds can be used to purify the active ingredient and test its derivatives. Methods for fractionating and purifying such heterologous extracts are known in the art. If desired, compounds shown to be useful reagents for treatment can be chemically modified according to methods known in the art. Compounds identified as therapeutically valuable are subsequently analyzed using animal models for diseases or conditions in which it is desired to modulate progestin activity, as described herein. Can be done.

C-17 Derivatized Dexamethasone Anti-Progestin The C-17 derivatized dexamethasone anti-progestin used in the methods of the present invention is readily synthesized using techniques commonly known to synthetic organic chemists. Can be done. Suitable methods for synthesizing Dex-Mes and Dex-Ox are provided, for example, in the Examples below and are known in the art. Dex-Ox
And Dex-Mes can be further modified, for example, by replacing the oxygen of the 4-membered ring of Dex-Ox (FIG. 2) with: Sulfur (S), Nitrogen (N), or Carbon (C). Groups (including cyclic ester, amide, and thioloester groups); 3-membered rings having only C; lacking or containing one or two oxygen (O), S, or N groups, 5-7 Member rings (including cyclic ester, amide, or thioloester groups).

Guidance for the production of various C-17 derivatized dexamethasone is also provided, for example:
It can be found in: Rousseau et al., J Steroid Bioche.
m 18: 237-244, 1983 (see, eg, page 238, Table 1).
Giesen and Beck, Horm Metabol Res 14: 2;
52-256, 1982; Rousseau et al., Nature 279: 158.
-160, 1979 (see, eg, page 159, Table 1); Lifebvr
e et al., J Steroid Biochem 33: 557-563, 1989.
(See page 558, Tables 1 and 2, for example); and Lee and So
Liman, Science 215: 989-991, 1982. Each of the above articles is incorporated herein by reference in its entirety.

In addition, C-21 derivatized dexamethasone anti-progestin can be used in the methods of the invention. Such C-21 derivatized anti-progestin is, for example,
Simons et al., J Steroid Biochem 13: 311-322.
, 1980.

C-17 derivatized dexamethasone anti-progestins (eg, those that have both anti-progestin activity and partial agonist activity) that are particularly useful in the methods of the invention use methods well known to those of skill in the art. And can be easily identified. For example, numerous references are cited herein above and below to describe in vitro and in vivo methods for assessing the relative antiprogestin activity and partial agonist activity of compounds. It

The invention is described in more detail in the following examples, which are intended as an illustration only. Because many modifications and variations therein will be apparent to those of ordinary skill in the art.

[0073]   Example I: Materials and Methods   Unless otherwise noted, all operations were performed at 0 ° C.

[0074] Chemicals, buffers, and plasmids: [17-methyl ^- 3 H] Promeg
estone (R5020, 85 Ci / mmol) and non-radioactive R5020
Was obtained from NEN (Boston MA). Dex, Sigma (St
． Louis, MO). Dex-Ox (Pons and Simon
S., J Org Chem 46: 3262-3264, 1981) and De.
x-Mes (Simons et al., J Org Chem 45: 3084-308.
8, 1980) was prepared as previously described. RTI-3021-
020 is C.I. Edgar Cook (Research Triangle)
Institution, NC), kindly donated. MMTVLuc (p
LTR Luc) by Gordon Hager (NIH, Bethesda, M.
Obtained from D). Renilla Null Luciferase Reporter
Obtained from ega (Madison, WI).

Cell culture and transfection: Monolayer culture of COS-7 cells at 5%
Dulbecco's Modified Eagle Medium (DMEM; GIB) supplemented with bovine fetal serum
CO / BRL Life Technologies, Inc. ) At 37 ° C. with 5% CO _{2 in} each. 1470.2 mouse mammary adenocarcinoma cells (Pennie et al., Mol Cell Biol 15: 2125-2134).
, 1995) with 4.5 g of glucose / L (Quality Biologi).
cals, Inc) in DMEM. On the other hand, T47D cells were grown in Dulbecco's modified Eagle medium with high glucose (4.5 g / L, 2 mM glutamine, and 60 ng insulin) and with both 10% fetal bovine serum. 1470.2 cells with human PR-B construct, 1 μg M
MVL Luc, and 50 ng of Renilla null luciferase, pBS
Transfected total D resulting in 3 μg / 60 mm dishes with K ⁺ DNA
Using NA, the previously described calcium phosphate method (Szapary et al., J.
Biol Chem 271: 30576-30582, 1996) was used to transiently transfect. 24 hours after steroid treatment, cells were
× Passive Lysis Buffer (0.5 ml / dish; Promeg
a, Madison, WI). 20 μl of cell lysate was added to Prom
Dual-Lusiferase As from ega (Madison, WI)
It was used to assay for luciferase activity using the sey System and the method recommended by the supplier. CV-1 and T47
D cells were treated as described (Szaparry et al., J. Biol. Chem. 271.
: 30576-30582, 1996), human PR plasmids and 1 μg of reporter construct (GREtkCAT, GREtkLuc, or MMTVLuc) were transiently transfected.

Assay for steroid competition: 1 with human PR-B cDNA
Transient transfections of COS-7 cells in 0 μg / 60 mm dishes were performed as previously described (Szaparry et al. Mol Endocri.
Nol 7: 941-952, 1993). Cytosol of transfected cells containing steroid-free receptor was obtained by lysis of cells at -80 ° C and centrifuged at 15,000 xg (Simons et al., B.
iochemistry 23: 6876-6882, 1984). 30% cytosolic with 20 mM sodium molybdate was added to 2.5 nM [ ³ H] R5020 ± 2, 20, 200, or 2000 fold excess of non-radioactive competitor and 0. Incubated for 18 hours at ° C. Unbound [ ³ H] R5020 was removed by dextran-coated charcoal. Specific binding was calculated by subtracting the background dpm seen with 2000-fold excess of R5020 from total binding of [ ³ H] R5020 in the presence or absence of non-radioactive competitor. did. [ ³ H] R
The specific dpm obtained with 5020 and competitor was divided by the specific dpm in the absence of competitor and presented as a percentage of non-competing binding.

Steroids: FIG. 2 shows the structures of agonist and antagonist steroids used in our study. GR (Dex) and PR (R502
The structure of the agonist for 0) is shown above the bold line. Structures (Dex-Mes and Dex-Ox) for two novel partial anti-progestins of the present invention (
These were previously described as anti-glucocorticoids (Simons and Thompson, Proc. Natl. Acad. Sci. USA 78: 3.
541-3545, 1981); Pons and Simons, J Org C.
hem 46: 3262-3264, 1981; Lamontagne et al., En.
dcrinology 114: 2252-2263, 1984)) is shown below the bold line. The previously described anti-progestins (RTI 3021-020 and RU486) are also underlined.

Analysis of Data: Biological activity with subsaturating concentrations of agonist, or saturating concentrations of antagonist, was determined by saturating concentrations of agonist (30 nM R5020 unless otherwise indicated). Shown as a percentage of maximum activity when used. Fold induction with 30 nM R5020 was calculated as either chloramphenicol acetyl transferase (CAT) or luciferase activity calibrated with 30 nM R5020 divided by the basal activity obtained with ethanol. The individual values plotted were generally on average ± 20%.

Example II: Partial agonistic activity of Dex-Mes PR Three different cell lines (CV-1, T47D, and 1470.2) were tested for biological activity of various steroids. Used to 2 1470.2 cells
Most of the assays were selected for one reason. First, they have no endogenous PR, allowing induction by transient transfection of either the PR-A or PR-B isoforms. Second, 1470.2 cells provide a much larger dynamic range of transactivation versus concentration of transiently transfected PR than CV-1 cells (about 5 times the concentration of PR. 3 to 20 times more induction). Therefore, experiments in 1470.2 cells could be performed under some conditions where PR limits transactivation.

FIG. 1 is a control experiment showing the effect of PR concentration on all-trans activation and ploidy induction in transiently transfected 1470.2 cells.
Triplicate 1470.2 cells were treated with MMTVLuc as described in Example I above.
Transfections were performed with the reporter and the indicated amount of human PR-B plasmid and 50 ng Renilla plasmid as an internal control for transfection, then treated with EtOH ± 30 nM R5020. After determining the relative level of luciferase expression, the data was
It was calibrated for nilla expression and plotted both as total activity with 30 nM R5020 (filled circles) and as basal (EtOH) activity (open squares) versus fold induction over PR concentration. Error bars are SD of 3 plates
Indicates.

FIG. 3 Dex- in 1470.2 cells transiently transfected with PR.
The trans-activating activity of Mes is shown. Triplicate 1470.2 cells were transfected with the indicated amounts of PR-B plasmid as described in Figure 1 and EtOH.
± 30 nM R5020, 1 μM Dex-Mes, or 10 μM Dex-
Treated with Mes. All data were calibrated for Renilla expression.
The relative level of expression of total luciferase was then calculated using EtOH, 30 nM R
Plotted for 5020, and 10 μM Dex-Mes. Numbers in brackets above the bars for 30 nM R5020 indicate fold induction. The activity of Dex-Mes at 1 and 10 μM shown as percent of maximal induction by 30 nM R5020 at the two PR concentrations is shown in the inset graph. Error bars indicate SD of triplicate plates.

Transient transfection of human PR-B into 1470.2 cells allowed successful induction of the co-transfected reporter MMTVLuc by both R5020 and Dex-Mes. (Figure 3). R
The activity with Dex-Mes did not increase from 1 μM to 10 μM when expressed as a percentage of the maximum activity seen with the saturating concentration of 5020. This indicates that these were saturating concentrations of Dex-Mes. Dose response curves, as compared to 0.4nM for R5020, yielded _{EC 5 0} to about 350 nM. Interestingly, the percent agonist activity of Dex-Mes is
Increased with added PR (Szapary et al., J. Biol. Che), just as was observed for glucocorticoids with increasing amounts of GR.
m. 271: 30576-30582, 1996; Szapary et al., Mol.
Endocrinol 13: 2108-2121, 1999). Nevertheless, using each concentration of PR, maximal activity was 1 μM Dex-Mes.
Reached using. Therefore, Dex-Mes exhibits a partial agonist profile for PR-B. 1470.2 cells contain GR as determined by Scatchard analysis. However, as shown in Figure 3, this GR was inactive with the added Dex-Mes. Dex-Mes transactivating activity was only seen after addition of PR-B. Transiently transfected CV-
In other experiments in 1 cell, Dex-Mes was expressed in human PR-A and human PR-B, either GREtkCAT or MMTVLuc reporter.
It showed agonist activity between 5% and 50% with both inductions. Therefore, the partial agonistic activity of Dex-Mes using PR is dependent on the receptor form (PR-A or PR-B), enhancer, promoter, gene (GRE).
tkCAT and MMTVLuc), and cell type (1470.2 and CV-).
1 cell).

Example III: Partial agonistic activity of Dex-Ox PR Dex-Ox, another C17 derivative of Dex (FIG. 2), was also transiently transfected in 1470.2 cells. Had a partial agonistic activity for PR induction of MMTVLuc in. Figure 4 shows transiently transfected P
5 shows the transcriptional activity of Dex-Ox in 1470.2 cells using R. 3 in 14
70.2 cells were treated with the indicated amounts of PR-B as described for the experiment shown in FIG.
Plasmids were transfected, but without Renilla, and treated with EtOH ± 30 nM R5020 or 300 nM Dex-Ox.
All data were calibrated for protein levels in cell lysates. The relative levels of total luciferase expression were plotted for EtOH, 30 nM R5020, and 300 nM Dex-Ox. Numbers in brackets above the bars for 30 nM R5020 indicate fold induction. 30 nM D at two PR concentrations
The percent maximal induction by 30 nM R5020 achieved with ex-Ox is shown in the inset graph. Error bars indicate SD of triplicate plates.

FIG. 4 shows that Dex-Ox is a partial agonist, as increasing concentrations of Dex-Ox up to 3 μM did not increase all-trans activation at all. The dose response curve produced an EC ₅₀ of approximately 30 nM compared to 0.4 nM for R5020. Moreover, with Dex-Mes, the amount of agonist activity as a percentage of maximal transactivation by saturating concentrations of R5020 was higher in the presence of more PR. Again, the activity of Dex-Ox was due to the inactivity in the absence of transfected PR (Fig. 5A), and the added PR-.
It was not due to endogenous GR, as shown by the increased total activity with B (Fig. 5B). Dex-Ox was also used in CV-1 cells with GREtkCAT and in T47D cells with the GREtkLuc reporter, which contained endogenous PR-A and B, from 40% with PR-B. It showed an agonist activity of between 60%. Thus, with Dex-Mes, the partial agonist activity of Dez-Ox with PR is dependent on enhancers, promoters, genes, and cell types.

Example IV: Dex-Mes and Dex- in Comparison with Other Antiprogestins
PR agonist activity of Ox) The partial agonist RTI 3021-020 has been described to be inactive in CV-1 cells (Wagner et al., Proc. Natl. Ac.
ad. Sci. USA 93: 8739-8744, 1996), and the inventors have confirmed this. Furthermore, we have found that Dex-Mes and Dex
-Ox was found to exhibit much greater agonist activity than either RIT 3021-020 or RU 486 in CV-1 cells. A more detailed study was then carried out in 1470.2 cells in which a large inductive response could be observed with PR.

5A and 5B show Dex, Dex-Mes, D in 1470.2 cells without (FIG. 5A) or with (FIG. 5B) transfected PR.
Figure 4 shows a comparison of the relative partial progestin activity of ex-Ox and RTI 3021-020. Triplicate 1470.2 cells were transfected with 3 or 30 ng of PR-B plasmid as described for the experiment shown in FIG. 1 and EtOH ± R5020 (30 pM, 90 pM, or 30 nM), or 1 μM Dex. , Dex-Mes (DM), Dex-Ox, or RTI 3
021-020 (RTI). All data were calibrated for Renilla expression. The data in cells without the addition of PR (Fig. 5A) are shown as fold induction above basal levels. In the cells to which PR was added (FIG. 5B),
The activity of each steroid was determined to be 30 nM using each concentration of PR-B.
R5020 was plotted as a percentage of maximum activity. Error bars indicate SD of triplicate plates.

As shown in FIG. 5A, the only steroid that had some activity in the absence of transfected PR was Dex. This observation is consistent with the results in Figures 3 and 4. Because 1470.2 cells contain no PR, but GR
Because it includes. In addition, Dex-Mes (Szapary et al., J. Biol.
． Chem. 271: 30576-30582, 1996; Simons and Thompson, Proc. Natl. Acad. Sci. USA 78: 3
541-3545, 1981) and Dex-Ox (Lamontagne et al.,
Endcrinology 114: 2252-2263, 1984) is a GR.
Can be used with little or no agonist activity. Thus, the data in Figure 5A confirmed that none of the tested anti-progestins had any activity with endogenous GR.
When PR was added, Dex-Mes and Dex-Ox showed a significant amount of partial progestin activity, while anti-progestin RTI-3021-
020 was inactive. Therefore, Dex-Mes and Dex-Ox appear to have a broader mixed agonistic activity for PR than any other anti-progestin described further.

Example V Cell-Free Binding of Dex-Mes and Dex-Ox to PR To establish that Dex-Mes and Dex-Ox are true mixed agonists for PR, The inventors questioned whether each steroid could bind PR with high affinity. FIG. 6 shows cell-free competition of [ ³ H] R5020 binding by non-radioactive steroids. Duplicate samples of overexpressed human PR-B containing [ ³ H] R5020 and containing increasing concentrations of the indicated non-radioactive steroid were incubated, and Example 1 above. Were analyzed as described in. The remaining binding data observed was then presented as the percentage of uncompeted [ ³ H] R5020 binding and plotted as a function of concentration of non-radioactive steroid. Error bars indicate the range of duplicate samples.

The cell-free competitive binding experiments shown in FIG. 6 show 1.4% of R5020, respectively.
And binding affinities for Dex-Ox and Dex-Mes that are <0.3%. These affinities are comparable to Dex-Ox (1.3
%) And the Dex-Mes (0.11%) EC ₅₀ closely correlated. This correlation indicates that the partial agonist activity of Dex-Ox and Dex-Mes
It strongly supports the notion of driving them to R by binding.

Example VI: Anti-Progestin Activity of Dex-Mes and Dex-Ox To determine whether Dex-Mes and Dex-Ox have anti-progestin activity, we used a PR-based reporter. The ability of these steroids to antagonize the progestin-activated induction of the gene was evaluated. Triplicate cultures were transfected as described for the experiments shown in Figure 1 and incubated with the indicated concentrations of R5020 ± Dex-Mes or Dex-Ox. The absolute level of luciferase activity (± SD in triplicate values) was plotted. Considering the low affinity of Dex-Mes vs. Dex-Ox for PR, we find that 1 μM and 10 μM Dex-Mes, but only 1 μM to inhibit the action of 300 pM R5020. Dex-Ox of was used. Using high enough concentrations, each Dex derivative reduced the activity of 300 pM R5020 over that of the competitive steroid alone (FIG. 7). Therefore, Dex-Mes and Dex-Ox are anti-progestins with abilities similar to their affinity for PR.

Other Embodiments All publications and patent applications mentioned herein are incorporated by reference in their respective individual publications or patent applications in detail and individually. As such, they are hereby incorporated by reference to the same extent.

Although the present invention has been described in combination with its specific embodiments, further modifications are possible, and the present application covers any variations, uses, or adaptations of the invention that follow. It is intended that, in general, the invention and its principles, including such deviations from the disclosure of the invention, are known in the art to which the invention pertains or become common practice therein, And what may be applied to the essential features shown here above, and within the scope of the appended claims,
Is understood.

[Brief description of drawings]

FIG. 1 shows transiently co-transfected 147 treated with R5020.
FIG. 6 is a graph showing the effect of PR on all-trans activation and ploidy induction of a progestin-responsive reporter gene in 0.2 cells.

FIG. 2 is a diagram showing the structures of glucocorticoid agonists and progestin agonists, partial agonists, and antagonists.

FIG. 3 shows the progestin-responsive reporter gene Dex-M in 1470.2 cells co-transfected with the reporter gene and PR cDNA.
It is a graph which shows the transcriptional activity induced by es.

FIG. 4 shows the progestin-responsive reporter gene Dex-O in 1470.2 cells co-transfected with the reporter gene and PR cDNA.
3 is a graph showing transcriptional activity induced by x.

FIG. 5A: Dex, Dex-Mes, D with glucocorticoid receptor in 1470.2 cells not transfected with PR cDNA.
FIG. 6 is a graph showing the relative activity of ex-Ox and RTI 3021-020.

FIG. 5B shows 1470.2 transfected with various concentrations of PR cDNA.
Dex, Dex-Mes, Dex-Ox, and RTI 3021 in cells
Figure 9 is a graph showing the relative partial progestin activity of -020.

FIG. 6 shows [ ³ H] R5020 binding to PR by non-radioactive steroids.
5 is a graph showing the cell-free competition of FIG.

FIG. 7 is a graph showing the anti-progestin properties of Dex-Mes and Dex-Ox.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 15/04 A61P 15/04 171 171 15/08 15/08 15/18 15/18 35/00 35 / 00 C07J 5/00 C07J 5/00 21/00 21/00 C12Q 1/68 C12Q 1/68 Z G01N 33/53 G01N 33/53 AB (81) Designated country EP (AT, BE, CH, CY, DE) , DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, M , RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE. , DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL , TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Simon's, S. Stoney Junia United States Maryland 29817, Bethesda, Johnson Ave 5609 F Term (reference) 4B063 QA08 QQ08 QQ48 QQ94 QR48 4C086 AA01 AA02 DA10 DA13 MA01 MA04 NA14 ZA55 ZA81 ZA86 BB8603010101A01 KK12 LL01 MM03 NN02 PA03 PA05 PA09 PA13 PB02 QQ02 QQ07 QQ18

Claims (27)

[Claims] 1. A method of inhibiting progestin activity or treating or preventing a progestin-dependent condition in a subject in need of inhibition of progestin activity, treatment or prevention of a progestin-dependent condition, said method comprising: Comprising administering C-17-derivatized dexamethasone to the subject in an amount effective to inhibit progestin activity or to treat or prevent a progestin-dependent condition in the subject. 2. The method of claim 1, wherein the C-17-induced dexamethasone antiprogestin is dexamethasone-21-mesylate. 3. The method of claim 1, wherein the C-17-derived dexamethasone antiprogestin is dexamethasone-oxetanone. 4. The method of claim 1, wherein the subject is a mammal. 5. The method of claim 1, wherein the method is a method for controlling menstruation. 6. The method of claim 1, wherein the method is for treating or preventing a benign progestin-dependent condition. 7. The method of claim 1, wherein the method is for treating a progestin-responsive tumor. 8. The method of claim 1, wherein the method is a method for preventing pregnancy. 9. The method of claim 1, wherein said administration inhibits ovulation in said subject. 10. The method of claim 1, wherein the method is for inducing cervical maturation in females. 11. The method of claim 1, wherein said administration is performed to induce elimination of embryos or fetuses from said subject. 12. A method of detecting a progestin or progestin agonist in a sample, the method comprising: contacting the sample with a reaction mixture comprising: a) a progesterone receptor and a C-17-derivatized dexamethasone antiprogestin; And b) measuring progestin activity in the reaction mixture, wherein the progestin activity in the sample is increased by an increase in progestin activity as compared to the amount of progestin activity measured in the reaction mixture not in contact with the sample. Detecting a progestin or progestin agonist. 13. The method of claim 12, wherein the C-17-induced dexamethasone antiprogestin is dexamethasone-21-mesylate. 14. The method of claim 12, wherein the C-17-derived dexamethasone antiprogestin is dexamethasone-oxetanone. 15. A method of detecting anti-progestin in a sample, the method comprising: a) contacting the sample with a first reaction mixture comprising a progesterone receptor and a progestin or progestin agonist; b) Determining the progestin activity in the first reaction mixture; and c) the amount of progestin activity in the second reaction mixture comprising progesterone receptor, progestin or progestin agonist, and C-17-induced dexamethasone antiprogestin and the first. Comparing the progestin activity in a reaction mixture, wherein the amount of progestin activity in the second reaction mixture in the absence of the anti-progestin is compared to compare the amount of progestin activity in the second reaction mixture. At least about 20% reduction in progestin activity, Detecting the anti-progestin in the sample from a decrease in progestin activity in the first reaction mixture. 16. The method of claim 15, wherein the C-17-induced dexamethasone antiprogestin is dexamethasone-21-mesylate. 17. The method of claim 15, wherein the C-17-derived dexamethasone antiprogestin is dexamethasone-oxetanone. 18. A method of inhibiting progestin activity in a cell in need of inhibiting progestin activity, the method comprising administering to said cell an effective amount of a C-17-induced dexamethasone antiprogestin. ,Method. 19. The method of claim 18, wherein the C-17-induced dexamethasone antiprogestin is dexamethasone-21-mesylate. 20. The method of claim 18, wherein the C-17-derived dexamethasone antiprogestin is dexamethasone-oxetanone. 21. A method of detecting a gene whose gene expression level is regulated by an anti-progestin, which method comprises: a) contacting a progesterone receptor with a C-17-induced dexamethasone anti-progestin, wherein: Wherein the progesterone receptor is present in a sample containing a gene positively regulated or negatively regulated by the progesterone receptor; b) measuring the expression level of the gene in the sample; And c) comparing the expression level of the gene in a sample not contacted with the C-17-induced dexamethasone anti-progestin with the expression level of the gene in the sample, wherein the C-17 induction The expression level of the gene in the sample not in contact with dexamethasone antiprogestin and In comparison, detecting a gene whose expression is regulated by the anti-progestin is detected by an increase or decrease in the expression level of the gene in the sample that is contacted with the C-17-induced dexamethasone anti-progestin.
A method comprising the steps of :. 22. The method of claim 21, wherein the C-17-induced dexamethasone antiprogestin is dexamethasone-21-mesylate. 23. The method of claim 21, wherein the C-17-derived dexamethasone antiprogestin is dexamethasone-oxetanone. 24. The method of claim 21, wherein a progestin or progestin agonist is added to the sample prior to the measuring. 25. The method of claim 21, wherein the sample is cells. 26. The method of claim 21, wherein the sample is a cell extract. 27. The method of claim 21, wherein the sample is a mammal.