BACKGROUND OF THE INVENTION The steroid hormone progesterone is involved in the regulation of growth and development of mammary gland and uterus. Synthetic progestins and antiprogestins have been used or are in human clinical trials in treatment of endometrial and breast cancer, as combination oral contraceptive agents, and as adjuncts to estrogen in hormonal replacement therapy.

Produced in the ovaries, and passed into the blood stream, progesterone manifests biological activity primarily in those cells which express intracellular progesterone receptors (PRs). Upon binding to their hormonal ligands, the activated progesterone receptors bind with high affinity to specific DNA binding sites and activate transcription of the cis-linked genes. PR is a member of the steroid hormone receptor superfamily.

A typical steroid hormone receptor can be divided into six domains, A, B, C, D, E and F as indicated in Figure 7. The function of each domain is indicated by solid lines.

The N-terminal A/B domain contains a transactivation function.

The C region is responsible for DNA binding and receptor dimerization. The D region is a hinge region which allows the

protein to bend or alter conformation. The E region is important for dimerization, transactivation, intramolecular repression and ligand binding. DNA sequences responsive to steroid hormones have been termed hormone response elements (HRES).

Gronemeyer et al. (1987) EMBO J. 6: 3985-3994, cloned and sequenced chicken PR and described that both the N- terminal A/B region and the C-terminal hormone binding domain are required for transactivation function on MMTV promoter in HeLa cells. Carson et al. (1987) Mol. Endo. 1: 791-801, cloned and sequenced chicken PR, and conducted analysis to define the functional domains of the receptor. The authors state that a transcription activation function domain resides outside the hormone binding domain and that the C-terminal region may function as a"repressor".

The human progesterone receptor (hPR) occurs as two distinct forms within target cells, hPR-A and hPR-B (1). The two isoforms differ only in their N-terminal sequences. Form A lacks the amino terminal 164 amino acids present in form B. hPR-A and hPR-B differentially activate transcription of progesterone-responsive genes (Vegeto et al., Mol. Endo.

7: 1244-1255,1993; Wen et al., Mol. Cell. Biol. 14: 8356-8364, 1994, all incorporated by reference herein).

In the absence of hormone, hPR resides within the nuclei of target cells associated with a large macromolecular complex comprising heat shock protein 90 (hsp90), hsp70, hsp59 and possibly other proteins. Hormone binding disrupts this complex and initiates a cascade of events which lead ultimately to the association of a receptor dimer with

specific sequences located within the regulatory regions of target genes (2). It has been shown that hormone activated hPR can interact directly with the transcription apparatus by contacting transcription factor TFIIB (3) or BP binding protein TAF110 (4). In addition, proteins which facilitate indirect interactions of PR with the general transcription apparatus have been identified, i. e. co-activators (5).

Evans et al., U. S. Patent 5,071,773, incorporated by reference herein, describes an assay by which hormone receptors, ligands for such receptors, and proteins having transcription activating properties of a hormone receptor, can be detected. Generally, the assay involves using a cell containing both a DNA encoding a receptor protein, and a DNA encoding a hormone responsive element (e. g., a promoter) linked to an operative reporter gene. When a suitable hormone or ligand is provided to the cell, a receptor-hormone is formed and delivered to an appropriate DNA-binding region to thereby activate the hormone responsive element and cause expression of the reporter gene. The expression product of the reporter gene is detected by standard procedures known to one skilled in the art.

Webster et al., Cell 54: 199 (1988), used chimeric receptors to localize regions responsible for transcription activation function. The authors propose that a hormone is responsible for allowing a receptor to recognize a DNA response element, and that the hormone induces a transcription activation function in the hormone-binding domain.

SUMMARY OF THE INVENTION Within the scope of this invention, Applicant has identified a new class of PR ligands, PR-mixed agonists, which interact with PR in a manner distinct from either agonists or antagonists. Applicant has designed assays to screen for and/or identify agonists, antagonists and mixed agonists of PR. Applicant has discovered that the agonist, antagonist and mixed agonist activity of a PR-ligand can be predicted by the region within the PR ligand binding domain (PR-LBD) that the PR-ligand interacts with. PR ligands made, described or identified according to this invention may be used as contraceptive or used in hormone replacement therapy to treat uterine proliferation caused by estrogen treatment, endometriosis, fibroids, endometrial cancer, brain meningiomas, or breast cancer with little or no side effects.

Thus, in a first aspect, the present invention features novel mixed agonist of progesterone receptor which is capable of interacting with and binding both the agonist pocket of progesterone receptor and the antagonist pocket of progesterone receptor.

By"agonist pocket of progesterone receptor"is meant the portion of progesterone receptor at the C-terminus which binds with PR agonist, including, but not limited to, progesterone, and is required for stimulating the transcription activation activity of PR-WT and PR-722 by PR agonist. In the alternative, agonist pocket is meant the portion of progesterone receptor whose deletion or mutation destroys or diminishes the ability of a PR agonist to bind

progesterone receptor and activate the transcription activation activity thereof.

By"antagonist pocket of progesterone receptor"is meant the portion of progesterone receptor in and around the "lip-pocket"which binds with PR antagonist (7), including, but not limited to, RU486, and is required for both decreasing the transcription activation activity of PR-WT and stimulating the transcription activation activity of PR-UP-1 by PR antagonist. In the alternative, antagonist pocket is meant the portion of progesterone receptor whose deletion or mutation destroys or diminishes the ability of a PR antagonist to bind progesterone receptor and modulate the transcription activation activity thereof.

By"mixed agonist of progesterone receptor"is meant a compound or composition which when combined with progesterone receptor selectively activates PR in a certain cellular context but not others or selectively activates certain progesterone responsive promoters but not other progesterone responsive promoters. A mixed agonist of progesterone receptor also has one or more of the following characteristics: (1) binds to both agonist pocket and antagonist pocket of PR; (2) binds and activates a PR having intact and wild-type agonist pocket and antagonist pocket (e. g. PR-WT), a mutant PR missing or having a mutated agonist pocket (e. g. PR-UP-1), and a mutant PR missing or having a mutated antagonist pocket (e. g. PR-722); and (3) has agonist activity on PR-WT in the absence of progesterone but antagonist activity on PR-WT activated by progesterone. By "novel"is meant that the mixed agonist compound or composition is not known or used before this invention.

In a preferred embodiment, the mixed agonist is of the formula:

where the r groups are independently: rl is H, CF3, CH3 r2 is H, CF3, CH3 r3 is r7C=O, or' r4 is H, F, Cl, Br, I, NO2, CO2H, CO2r9, CHO, CN, CF3, CHZOH, optionally substituted C1-C6 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, alkynyl or alkenyl r-5 is H, F, Cl, Br, I, NO2, CO2H, CO2r9, CHO, CN, CF3, CH2OH, optionally substituted C1-C6 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, heteroarylmethyl, alkynyl or alkenyl r6 is optionally substituted C1-C12 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, heteroarylmethyl, alkynyl or alkenyl, including but not limited to 4-acetylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-cyanophenyl

r7 is H, CO2H, C02r9, CHO, CN, CF3, CH20H, optionally substituted C1-C6 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, heteroarylmethyl, alkynyl or alkenyl r8 is H, CF3, optionally substituted C1-C6 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, heteroarylmethyl, alkynyl or alkenyl r9 is H, CF3, optionally substituted C1-C6 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, heteroarylmethyl, alkynyl or alkenyl.

For example: r r2 r3 r4 rS r6 r7 r8 r9 *CH3 H CH3CO H Et 4-(CH3) 2NPh CH3---- *-- CH3 CH3CO H Et 4-(CH3) 2NPh CH3-- -- *H H CH3CO H Et 4-(CH3) 2NPh CH3---- H H OH CCCH3 Et 4-(CH3) 2NPh--H __ H H OH CCCH3 Et 4- (CH3CO) Ph--H CH3 H CH3CO H Et 4- (CH3 CO) Ph CH3---- H CH3 CH3CO H Et 4- (CH3CO) Ph CH3-- -- H H EtCO H Et 4-(CH3) 2NPh Et--__ H H HOCH2CO H Et 4- (CH3) zNPh HOCHZ---- *Cook compound In another preferred embodiment, the mixed agonist is of the formula :

wherein R1 =-H or-CH3 ; R2 =-OH or-COCH3 ; R3 =-CH2N3,- CH2OCH3,-OAc or-H; and R4-CnH2,, I ; n is an integer greater than 2.

The above described compounds may be prepared with methods known to those skilled in the art, e. g. U. S. Patent 4,954,490 entitled"11 (3-substituted progesterone analog", incorporated by reference herein.

In a second aspect, this invention features a progesterone receptor bound by a mixed agonist including, but not limited to, those described above in both the agonist pocket and antagonist pocket. The mixed agonist bound progesterone receptor adapts a conformation different from that adapted by an agonist bound PR or an antagonist bound PR.

In a third aspect, this invention features a method for screening for a mixed agonist of progesterone receptor by detecting a compound or composition which is capable of interacting with and binding both the agonist pocket of progesterone receptor and the antagonist pocket of progesterone receptor.

In a preferred embodiment, this method screens for a compound or composition which is capable of binding and activating the transcription activation activity of both PR- UP-1 and PR-722, or both PR-UP-1 and PR-WT.

In another preferred embodiment, the screening assay is conducted in cells (e. g. yeast cells or mammalian cells).

One cell contains a nucleic acid encoding progesterone receptor and another nucleic acid encoding a reporter gene operatively linked to a promoter. The progesterone receptor has an agonist pocket capable of activating transcription from the promoter when bound by progesterone (e. g. PR-WT or PR- 722). Another cell contains a nucleic acid encoding a second progesterone receptor and another nucleic acid encoding a second reporter gene operatively linked to a second promoter.

The second progesterone receptor either does not have an agonist pocket or has a mutated agonist pocket which is incapable of activating transcription from the second promoter. However, the second progesterone receptor has an antagonist pocket capable of activating transcription from the second promoter when bound by RU486 (e. g. PR-UP-1). A candidate compound is brought into contact with the first cell and the second cell and the level of transcription of the first reporter gene and second reporter gene is measured. An increased transcription of both the first reporter gene and the second reporter gene is indicative of the compound being a mixed agonist of progesterone receptor.

In other preferred embodiments, a mixed agonist is detected by its ability to compete with both an agonist (e. g. progesterone) and an antagonist (e. g. RU486) to bind with a

progesterone receptor having an agonist pocket (e. g. PR-WT or PR-722) and/or a progesterone receptor having an antagonist pocket but not having an agonist pocket or having a mutated agonist pocket (e. g. PR-UP-1 or PR-891). A mixed agonist is also detected by its ability to induce a conformation in PR which is different from that induced by an agonist (e. g. progesterone) or an antagonist (e. g. RU486). Such a conformation is probed by limited protease digestion assay.

In this assay, a ligand bound PR is digested by a protease (e. g. trypsin) before electrophoresis analysis on a gel. The protein gel patterns generated from different PR-ligand bound progesterone receptors are compared and a distinct pattern generated by a candidate compound as compared to those generated by either an agonist (e. g. progesterone) or an antagonist (e. g. RU486) is indicative of the candidate compound being a mixed agonist.

The methods described herein allow rapid screening for mixed agonists of progesterone receptor. The assays may be conducted in human derived cells or other eukaryotic cell lines, such as chicken and yeast cells.

An antagonist of progesterone receptor having a steroid backbone may be converted into a mixed agonist of progesterone receptor by adding or substituting a group to the 16a carbon position of the steroid backbone. Such a group is selected from F, Cl, Br, I, NO2, CO2H, C02r9, CHO, CN, CF3, CH20H, optionally substituted C1-C6 alkyl or perfluoroalkyl, optionally substituted allyl, arylmethyl, aryl, heteroaryl, heteroarylmethyl, alkynyl or alkenyl. An antagonist so

modified may be put through the assays described above to verify its mixed agonist activity.

Compounds that can be screened include compounds with a similar chemical structure to RTI 3021-020,3021-021, or 3021-022. Compounds described in this application may be screened for mixed agonist activity. Other compounds described in Ojasoo and Raynaud,"Steroid Hormone Receptors", In Comprehensive Medicinal Chemistry Volume 3; Emmett, J. C., Ed.; Pergamon Press; New York, 1990: pp: 1200-1207; Teutsch and Philibert,"History and Perspectives of AntiProgestins from the Chemist's Point of View", In Human Reproduction Vol. 9 (1) ; Edwards Ed.; Oxford University Press; Oxford, England, 1994; pp 12-31; Neef, et al. (1984) Steroids 44: 349-372; PCT US 93/03909, PCT US 93/10086, and PCT US95/16096 may also be screened by the assays of this invention. Those skilled in the art will readily recognize modifications and substitutions that can be made to compounds that can be screened for a mixed agonist profile.

While steroids and steroid analogues may exemplify agents identified by the present invention, Applicant is particularly interested in the identification of agents of low molecular weight (less than 10,000 daltons, preferably less than 5,000, and most preferably less than 1,000) which can be readily formulated as useful therapeutic agents.

Such agents can then be screened to ensure that they are specific to tissues with pathological conditions related to progesterone receptor with little or no effect on healthy tissues such that the agents can be used in a therapeutic or prophylactic manner. If such agents have some effect on

healthy tissues they may still be useful in therapeutic treatment, particularly in those diseases which are life threatening.

Once isolated, a candidate agent can be put in pharmaceutically acceptable formulations, such as those described in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990), incorporated by reference herein, and used for specific treatment of diseases and pathological conditions with little or no effect on healthy tissues.

Thus, in a fourth aspect, this invention features methods and compositions for treating a host having a progesterone related disease or pathological condition by administering to the host a composition containing a pharmaceutically effective amount of a mixed agonist of progesterone receptor and a pharmaceutically acceptable carrier. The host may be a human patient or an animal model of human progesterone related disease or pathological condition. The compositions of this invention are adapted to cure, improve or prevent one or more symptoms of the progesterone related disease or pathological condition in the host. A preferred composition is highly potent and selective with low toxicity. In this regard, those skilled in the art will recognize endometriosis as an example of a disease that can be treated with the methods and compositions of the present invention. Other disorders relating to progesterone receptor may also be treated with the methods and compositions of the present invention, including, but not limited to, uterine proliferation caused by administration of estrogen to

said host, endometrial cancer, breast cancer, fibroids, and brain meningiomas. The composition of this invention may also be used as contraceptive to prevent unwanted pregnancy.

By"pharmaceutically effective amount"is meant an amount of a pharmaceutical compound or composition having a therapeutically relevant effect on a progesterone related disease or pathological condition. A therapeutically relevant effect relieves to some extent one or more symptoms of endometriosis, uterine proliferation caused by administration of estrogen to said host, endometrial cancer, breast cancer, fibroids, and brain meningiomas, or other disorders in a patient or returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the diseases or pathological conditions.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and 1B are graphs showing normalized Gal activity in Saccharomyces cerevisiae transformed with an expression vector encoding either PR-WT (YEphPR) (Fig. 1A) or PR-UP-l (YEphPR-UP-1) (Fig. 1B) and a vector containing PRE-CYC1-ß-galactosidase reporter gene.

Transformed yeast cells were grown to exponential growth phase, whereupon ligands were added as indicated.

Following a 4 hr incubation, the cells were harvested and ligand induced transcriptional activity was measured. The data presented are from a representative experiment which has

been repeated independently several times. Each data point is an average of triplicate determinations.

Figures 2A and 2B are graphs showing relative alkaline phosphatase activity in T47D cells as a measurement of the agonist and antagonist activity of a series of PR-ligands. In Figure 2A, agonist activity of the PR-ligands were measured by the enzymatic activity of the PR responsive alkaline phosphatase gene product following exposure of T47D cells to the indicated ligands (10-9-10-6M) for 48 hrs. In Figure 2B, antagonist activity of the PR-ligands were measured by the alkaline phosphatase activity which resulted from treatment of cells with progesterone (10-7M) and increasing concentrations of competing ligands (10-8-10-6M) as indicated.

Following incubation with ligands, the cells were fixed and alkaline phosphatase activity was measured. The data from a representative experiment are shown. Each data point represents the average of triplicate determinations.

Figure 3 is a radiograph showing the result of a protease digestion assay. Radiolabelled (35S) PR-WT was produced in vitro using a coupled transcription/translation system. The resultant receptor was subsequently incubated for 10 minutes at room temperature with EtOH (lanes 1-4), 10 AM progesterone (lanes 5-7), 1 AM RTI 3021-020 (lanes 8-10) and 1 AM RU486 (lanes 11-13). Following PR-ligand addition, the complexes were incubated at room temperature in the absence (lane 1) or presence of trypsin at a concentration of 10 Hg/ml (lanes 2,5,8,11), 25 yg/ml (lanes 3,6,9,12) and 50 Ug/ml (lanes 4,7,10,13). The products of these digests were analyzed on a 12% denaturing polyacrylamide gel.

Figures 4A, 4B and 4C are graphs showing relative luciferase activity in monkey kidney fibroblasts (CV-1 cells) transfected with either an expression vector encoding PR-WT or a vector encoding PR-722 together with the MMTV-LUC reporter plasmid and a CMV--galactosidase expression vector to control for transfection efficiency. In Fig. 4A, transfected cells were incubated with no ligand (solid bar) or progesterone (10-7M) (hatched bar) for 48 hrs. In Fig. 4B, transfected cells were incubated with (B) progesterone alone (10-7M) (hatched bar) or progesterone (10-7M) and RU486 (10-6M) (open bar) for 48 hrs. In Fig. 4C, transfected cells were incubated with no ligand (solid bar) or RTI 3021-020 (10-7M) (stippled bar) for 48 hrs.

The cells were subsequently harvested and ferase and P-galactosidase activities were measured. The data are presented as normalized response, representing the absolute luciferase activity corrected for transfection efficiency by normalizing against the ß-galactosidase activity.

Figures 5A and 5B are graphs showing W binding of [3H] RU486 to PR-891 (Fig. 5A) or PR-wt (Fig. 5B) in the presence of other PR-ligands. Whole cell binding assays were performed as described in Materials and Methods. COS-1 cells were transfected using a single concentration (100 ng) of expression plasmids (SVhPR-A891 (Fig. 5A) or SVhPR-A (Fig.

5B). After 48 hrs the cells were incubated for an additional 4 hrs at 37°C with 1 nM [3H] RU486 alone or with the addition of increasing concentrations of unlabeled ligands. competition with unlabeled R5020 (g) was done at a single concentration (200 nM). [3H] RU486 was extracted from cells with ethanol and

counted. Values for competitive binding of unlabeled ligands were calculated as a W of total [3H] RU486 binding; set as 1000 in the absence of competition. At the 1000i value, [3H] RU486 binding to PR-891 was 23,340 dpm and 35,016 to PR-WT.

Figures 6A and 6B are graphs showing relative luciferase activity in T47-D breast cancer cells transfected with an MMTV-LUC reporter plasmid and a CMV--galactosidase expression vector to control for transfection efficiency. In Fig. 6A, the transfected cells were incubated with no ligand or various concentrations of either progesterone or RTI 3021-020 for 48 hrs. In Fig. 6B, the transfected cells were incubated with no ligand or 10-7M progesterone along with various concentrations of either RU486 or RTI 3021-020 for 48 hrs.

The cells were subsequently harvested and luciferase and ß-galactosidase activities were measured. The data are presented as normalized response, representing the absolute luciferase activity corrected for transfection efficiency by normalizing against the ß-galactosidase activity.

Figure 7 is a diagram showing functional domains of intracellular receptors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The effects of progestins and antiprogestins on PR and mutant PR have shed light on how a cell recognizes and responds to ligand activated PR or mutant PR. Deletion of 54 amino acids from the C-terminus of PR (PR-UP-1) greatly reduced progesterone (i. e. a PR agonist) binding but had a minimal effect on the receptor's affinity for RU486 (i. e. a PR

antagonist) (6). RU486 could function as an agonist on the mutant receptor PR-UP-1. Mutation of glycine 722 to cysteine within hPR reduced its affinity for RU486 while having no effect on progesterone binding (7). A monoclonal antibody to the C-terminal tail of hPR blocked binding of progesterone but not RU486 (8).

Without being bound by any theory, Applicant proposes that agonists and antagonists interact with overlapping though distinct regions within the PR ligand binding domain (LBD). The C-terminal tail of the receptor functions as a transcriptional repressor whose activity is disrupted by agonists alone. In addition, Applicant proposes that the ability to interact with one of the specific regions would be predictive of the biological activity of a ligand. A prediction of this hypothesis is that compounds which do not require the C-terminal 54 amino acids of hPR for high affinity binding, and therefore interact with PR-UP-1, would be unable to overcome the repressive effects of the carboxyl tail and would manifest antagonist activity on the full-length hPR receptor.

Example 1 : Identifying three distinct classes of PR-ligands by in vitro analysis of PR-transcriptional activity.

Applicant tested a number of structurally diverse PR ligands to see if they could be classified as agonists or antagonists based on their abilities to differentially activate PR-WT or the C-terminally truncated PR-UP-1 mutant.

This analysis was accomplished by assaying The transcriptional activity of PR-WT (A) and PR-UP-1 (B) in the presence of a series of a series of PR-ligands were assessed using a reconstituted hormone responsive transcription system in Saccharomyces cerevisiae (6). The results of this analysis are shown in Table 1 and Figure 1.

Table 1 illustrates the structure-activity relationships among PR ligands. Each ligand was tested for its ability to induce transcriptional activity of either PR-WT or PR-UP-1 using a reconstituted ligand responsive transcription system in Saccharomyces cerevisiae. The designation (+) indicates which receptor, PR-WT or PR-UP-1, the compound was most active on. Specifically, the compounds tested were: 1) RTI 3021-002,2) RTI 3021-003,3) RTI 3021-012, 4) RTI 3021-023,5) RTI 3021-020,6) RTI 3021-021, 7) RTI 3021-022,8) RTI 2207-213,9) RTI 2207-222, 10) 2207-225,11) RTI 2207-226.

Each compound was assessed multiple times with similar results.

As expected, those ligands with structures similar to the antiprogestin RU486 (RTI 3021-002,3021-003,3021-012 and 3021-023) (i. e. containing a dimethylaminophenyl substitution at the lip-position) functioned as agonists when assayed on PR-UP-1 but had no effect on the transcriptional activity of PR-WT. These compounds would likely function as PR antagonists.

Compounds with structures similar to progesterone (RTI 2207-213,2207-222,2207-225, and 2207-226) activated the transcriptional activity of PR-WT but were not active when analyzed on PR-UP-1 and therefore would likely function as agonists.

Unexpectedly, three ligands (RTI 3021-020,3021-021 and 3021-022) were found to activate both PR-WT and PR-UP-1 equally well, suggesting that there may be more than two classes of PR ligands.

To complete this analysis, a full dose response curve was performed using one ligand from each group.

Specifically, RTI 3021-023 (active on PR-UP-1 alone), RTI 2207-226 (active on PR-WT alone) and RTI 3021-020 (active on both receptors) were selected (Figure 1A & 1B). The results of this analysis confirm that PR-ligands can be separated into three mechanistically distinct groups representing agonists, antagonists and a third class, i. e. mixed agonists.

Example 2: Identifying three classes of PR-ligands: agonists, antagonists and mixed agonists.

To determine whether or not the three mechanistically distinct ligands would manifest unique activities when assayed in a bona fide progesterone target cell, Applicant assayed the agonist and antagonist activities of representative members of the three classes of ligand in the PR positive T47D breast cancer cell line. In addition to hPR-A and hPR-B, T47D cells express a tissue non-specific alkaline phosphatase, which has been shown to be positively regulated by PR (13). Thus, Applicant monitored alkaline

phosphatase activities in this cell line following administration of the test compound alone or in the presence of progesterone.

RTI 2207-226, which is predicted from the yeast data to be an agonist, induces alkaline phosphatase activity in a manner similar to progesterone (Figure 2A) and does not antagonize progesterone induced transcriptional activity (Figure 2B).

RTI 3021-023, which is predicted from the yeast data to be an antagonist, functions as an antagonist as shown by its ability to inhibit progesterone induced alkaline phosphatase activity (Figure 2B).

RTI 3021-020, which is predicted from the yeast data to belong to a third class of PR-ligand, functions as a partial agonist (Figure 2A) and antagonizes the alkaline phosphatase activity induced by progesterone to a level equivalent to the maximal agonist activity of RTI 3021-020 by itself (Figure 2B).

These results confirm that PR-ligands can be separated into at least three distinct classes and support the hypothesis that the biological activity of a compound can be predicted based upon its ability to interact with specific regions within the LBD of PR.

Example 3: Mixed agonist induces a unique conformation change with PR.

Applicant turned next to address possible mechanism (s) responsible for the mixed agonist activity RTI 3021-020. One explanation was that RTI 3021-020 may

impair PR-DNA binding activity, thus reducing the transcriptional efficacy of this ligand/receptor complex.

However, electromobility shift analysis (EMSA) demonstrated that the PR-DNA complex formed in the presence of RTI 3021-020 was qualitatively and quantitatively similar to that induced by the antagonist RU486, indicating that the PR-DNA complex formed in the presence of RTI 3021-020 is similar to that formed in the presence of RU486 and RTI 3021-020 manifests its unique activity by affecting a different step (s).

Applicant used a limited protease digestion assay to study conformation changes in PR when it interacts with a PR- ligand. Radiolabelled (35S) PR was produced in vitro using a coupled transcription/translation system. The labeled receptor was subsequently incubated in the absence or presence of ligand and then exposed to increasing concentrations of trypsin (Figure 3). The resultant proteolytic fragments were analyzed following separation on a denaturing gel as shown in Figure 3. The unliganded receptor (lanes 1-4) was totally degraded upon incubation with increasing concentrations of trypsin.

Incubation with progesterone or RTI 2207-226 (lanes 5-7) prior to trypsinization results in the protection of a predominant 30-kDa fragment.

When RU486 or the antagonist RTI 3021-023 was used (lanes 11-13), two major species of 27-kDa and 21-kDa fragments are observed.

Incubation with the mixed agonist RTI 3021-020 resulted in a pattern which was distinct from that produced by progesterone or RU486 in that only a 27-kDa band is protected

(lanes 8-10). Thus, the mixed agonist induces a conformation change within PR which is different from that induced by either agonists or antagonists.

Example 4: Mixed agonist is a full agonist of a mutant PR whose"antagonist binding pocket"is disrupted.

The mixed agonist RTI 3021-020 and the antagonist RU486 both contain a dimethylaminophenyl substitution at the lip-position (Table 1). This, coupled with the fact that both of these compounds bind and transcriptionally activate PR-UP-1, suggests that they may interact with PR through the "11a-pocket" ; a region whose integrity is required for the binding of lip-substituted antagonists (7).

To test this possibility, Applicant assayed in CV-1 cells the ability of these compounds to activate the transcriptional activity of either PR-WT or a receptor mutant PR-722, in which the"llp-pocket"had been mutated by substituting glycine 722 with cysteine.

The point mutation within PR-722 had no effect on the ability of progesterone to function as an agonist (Figure 4A), but diminished the ability of RU486 to antagonize progesterone activation (Figure 4B).

Surprisingly, RTI 3021-020 was converted from a mixed agonist into a full agonist when tested on PR-722 (Figure 4C), given that RTI 3021-020 does not appear to activate PR-WT in this assay. That the agonist activity of the mixed agonist has increased with the point mutation suggests that RTI 3021-020 does interact with the

"llp-pocket."Additionally, Applicant found that RTI 3020-020 showed some binding to PR-722, whereas RU486 and other antagonists such as RTI 3021-023 barely bind PR-722, if at all.

Example 5: Mixed agonist interacts with PR-891 and PR-WT in a way different from an agonist or antagonist.

Whole cell binding studies support that the mixed agonist RTI 3021-020 interacts with PR in a manner which is different from that of agonists and antagonists. The ability of representative members of each class of compounds to bind to either PR-WT or PR-891 (in which agonist binding has been destroyed) was examined by the ability of each compound to competitively inhibit binding of [3H] RU486.

The results of this analysis reveal that whereas RU486 binds equally well to PR-WT and the PR-891, the synthetic progestin R5020 fails to bind PR-891. However, unlike the pure antagonist RU486, the mixed agonist RTI 3021-020 demonstrates a lower binding affinity for PR-891 than for PR-WT. This suggests that in the context of the full length receptor, the mixed agonist does require sequences within the C-terminus for high affinity binding. However, unlike progesterone and other agonists, which have a very low affinity for PR-891 (Figure 5A), deletion of the C-terminus does not totally abolish RTI 3021-020 binding.

The behavior of RTI 3021-020 on PR-WT, PR-891 and PR 722 in these binding assays confirm that this compound belongs to a unique class of PR ligands which interact with the

receptor in a manner which is different form either PR agonists or antagonists.

Example 6: Mixed agonist has agonistic activity or antagonistic activity based on cell context.

T47-D breast cancer cells were transfected with an MMTV-LUC reporter plasmid and a CMV--galactosidase expression vector to control for transfection efficiency. In Fig. 6A, the transfected cells were incubated with no ligand or various concentrations of either progesterone or RTI 3021-020. In Fig. 6B, the transfected cells were incubated with no ligand or 10-7M progesterone along with various concentrations of either RU486 or RTI 3021-020. Although progesterone activates MMTV promoter, mixed agonist RTI 3021-020 only has antagonist activity and does not have any agonist activity on MMTV promoter. Comparing this result with the agonist activity of RTI 3021-020 on alkaline phosphatase promoter, it shows that mixed agonist has agonistic activity or antagonistic activity based on cell context.

Example 7: Converting an antagonist into a mixed agonist Pure antagonists such as RU486 have a dimethylaminophenyl substitution at the 11-ß position which facilitates a unique interaction with a region of the PR-LBD termed the"11--pocket" (6,7). Mixed agonists such as RTI 3021-020 have not only a dimethylaminophenyl substitution at

the il-P position but also an ethyl or other alkyl side-chain substituent at the 16a position.

Without being bound by any theory, Applicant hypothesizes that the 16a substitution within the RTI 3021-020 allows this class of lip-substituted compounds to interact with a more C-terminal portion of the LBD than compounds which lack this substitution (i. e., RU486); and this distinct mode of interaction is responsible for the unique alteration in the conformation of the progesterone receptor induced by RTI 3021-020.

Applicant further proposes the following model to explain how the cell distinguishes between hPR agonists and antagonists, and a model to explain the biological activity of PR-agonists, antagonists and mixed agonists. In the unliganded state, hPR is in a transcriptionally inactive conformation and consequently is associated with a putative repressor protein. Agonists bind to the more C-terminal portion of the receptor and hereby induce a structural change in the receptor which results in a transcriptionally active receptor, possibly due to the removal of the repressor protein and/or by inducing a structural change in the activation domains producing a more productive conformation for transactivation.

Antagonists, which tend bind to the more N-terminal part of the HBD induce a different conformation in the receptor, in which the C-terminus is not pulled in towards the receptor. The conformational change induced by antagonists may be insufficient to cause the removal of the repressor or allow activation by either AF1 or AF2.

Mixed agonists can bind to both the C-terminal and N-terminal regions of the HBD inducing a new conformation that is different from those induced by either agonists or antagonists. The mixed agonist conformation allows some partial activity, but does not allow full agonistic activity.

This may be the result of continued but less stable association with the repressor.

In the absence of hormone, the progesterone receptor resides in a latent form within target cells. Interaction with any high affinity ligand induces a conformational change in the receptor and causes the dissociation of the heat shock proteins. The binding of an agonist induces a specific conformational change, overcoming the repressive effect of the carboxyl tail, and permitting a productive association of the receptor with the transcription apparatus.

Occupancy of PR with an antagonist in a manner distinct from agonists also induces a conformation change.

However, this change is not sufficient to overcome the repressive activity of the PR-tail, and the resultant complex is transcriptionally inactive.

The mixed agonists, like RTI 3021-020, interact with PR in a distinct manner requiring sequences within the agonist and antagonist"pockets"of PR for high affinity binding.

Interaction with a member of this class of compounds results in a partial reversal of the inhibitory activity of the PR-tail and subsequent partial agonist activity. In the ways described above, receptor structure, coupled with the cell and promoter context in which it operates, determines the ultimate biological effect of the bound ligand.

Uses of Mixed Agonists The steroid hormone progesterone is a key modulator of the cellular processes required for the development and maintenance of reproductive function. Produced primarily by ovarian granulosa cells, it mediates its biological activity throughout the body by interacting with specific high affinity nuclear receptors located within target cell nuclei. Actions outside of the reproductive system have been implied by the localization of specific receptors for progesterone in"non endocrine"targets such as bone. These progesterone receptors are latent transcription factors which upon binding progesterone are capable of interacting with specific recognition sequences within target gene promoters. The consequence of these interactions are determined by the cell and promoter context of the DNA bound receptor.

From a clinical perspective regulation of progesterone receptor transcriptional activity is of extreme importance. There exists several pharmaceutical agents which are used in the clinic to mimic (agonists) or oppose (antagonists) the actions of progesterone. Below is a description of the current use of these compounds, their side effects and the advantages of a mixed agonist.

Oral contraceptives Progesterone agonist formulations have been used as single agents (Depo-provera or Norplant), or in combination with estrogens, to regulate ovulation and implantation. In addition to fertility control, these formulations are also used to treat abnormalities in the periodicity of the

menstrual cycle. Currently these drugs are delivered as pills, implants, injectables or as topicals (patch). Agonist containing vaginal rings and intrauterine devices are also being considered. Unfortunately, because the known progesterone agonists do not display any tissue selectivity, they activate the progesterone receptor in tissues other than those required to regulate reproductive function. The lack of selectivity may be responsible for weight gain, dysfunctional bleeding and mood disorders associated with current progestins. A mixed agonist which displays tissue (or processes) specific activities may be useful for these applications.

Hormone replacement therapy One of the problems associates with estrogen replacement therapy is that in post-menopausal women there is insufficient progesterone produced to counteract the proliferative activities of estrogen in the uterus. This unopposed action of estrogen is a risk factor for endometrial cancer. Consequently, it is becoming increasingly popular for physicians to use formulations containing progesterone for this activity. However, as the current progestins are not tissue selective there is concern that these agents in addition to counteracting the actions of estrogen in the endometrium (a desired activity) may also have the undesired side effect of reducing the efficacy of estrogen in the cardiovascular system, bone and in the central nervous system.

A mixed agonist may provide the desired selectivity.

Progesterone receptor antagonists Although progesterone agonists have been used clinically for quite some time, it is only in the last few years that progesterone receptor antagonists have been used.

Antiprogestins (like RU486) bind to the receptor and oppose the natural actions of progesterone. These compounds have been shown to be effective in the treatment of uterine fibroids, endometriosis, breast cancer, and brain meningiomas.

In addition to these activities, antiprogestins have also been shown to be effective as contraceptives, missed menses inducers, and as agents to facilitate termination of early stage pregnancies. For acute applications it appears as if the current antiprogestins are very good. However, for chronic treatments the current generation of antiprogestins have selectivity problems. Not only do they manifest antiprogestional activity in all tissues where a progesterone receptor is present but it has now been observed that these compounds bind also to the receptor for glucocorticoids.

Consequently, there is clinical demand for a new class of PR- antagonists which would exhibit more selectivity. The mixed agonists may provide these benefits.

Materials and Methods Yeast Transformation and -Galactosidase Assays.

All assays were performed in the yeast strain YPH500 (Mat a ura 3-25, lys 3-810a, ade 2-101o, trp 1-A63, his 3-A200, leu 2-A1) (9). Yeast cell transformations were performed using a modified lithium acetate protocol (10). Specifically YPH500 was transformed with the -galactosidase reporter

YrpG2. Leu (a gift from A. Nawaz, Baylor College of Medicine) and either the copper inducible PR-WT expression plasmid YEphPR-B (11) or the PR-UP-1 expression vector YEphPR-UP-1 (6). Transformed yeast were grown to exponential phase and then incubated with 100 AM copper sulfate and ligand for 4 hrs at 30°C. The cells were then lysed and assayed for P-galactosidase activity as described previously (12).

Alkaline Phosphatase Assay.

T47D cells were seeded in 96-well plates at 10,000 cells/well in RPMI media with 10% fetal calf serum (FSC).

Cells were washed with phosphate buffered saline (PBS) and fresh medium containing 2% FCS and ligand (10-6-10-9M) was added. The treated cells were incubated with ligand for 48 hrs, washed with PBS and fixed with 5k formalin at room temperature for 30 min. Cells were subsequently washed with PBS and assayed for alkaline phosphatase activity as described previously (13).

Limited Trypsin Digestion Assay.

Radiolabelled (35S) PR-WT was produced in vitro using the TNT coupled transcription/translation system (Promega).

For these reactions, the plasmid pBSII-PRB, which contains the cDNA for hPR-B, was used as a template. Labeled receptor was incubated with ligand at RT for 10 min, and then digested with trypsin as described by Allan et al. (14).

Mammalian Transfections and Luciferase Assays.

CV-1 cells were plated in 24 well plates at a density of 6x104 cells/ml and grown overnight in modified Eagle's media plus 10% FCS. Cells were transfected with 1 Ag of the PR expression plasmid pBK-CMV-PRB (15) or pBK-CMV-PR722 (pBK-CMV-PRB in which glycine 722 was converted to cysteine; a gift from Dawn Wen, Ligand Pharmaceuticals), 0.5 Ag pCMV-Gal (16) and 1.5 Ag MMTV-LUC using Lipofectin as described previously (17). All transfections were performed in triplicate. Following a 2 hr transfection, the cells were washed and fresh medium containing the test compound was added. Following a 48 hr incubation, assays of luciferase and P-galactosidase were performed as described previously (17).

Whole Cell Steroid Binding Assays.

COS-1 cells were plated in DMEM with 10% FCS at a density of 2 x 105 cells/well in a six well plate. After 24 hrs, the cells were transfected using an adenovirus system (V.

Allgood and N. L. Weigel personal communication) with 100 ng/well of the PR-WT expression plasmid pSV-PRA, PR-891 (pSV-PRA which lacks the C-terminal 42 amino acids) or PR-722 (pBK-CMV-PR722-CMV-PR722-CMV-PR722). At 48 hrs post-transfection, the medium was replaced with fresh DMEM containing 1 nM [3H] RU486 (a gift from R. Daraedt, Roussel Uclaf, Romanville, France) and varying concentrations of unlabeled ligands. Cells were incubated for 4 hr at 37°C, washed with ice cold PBS, and [3H] RU486 was extracted with ethanol and quantitated by liquid scintillation counting.

Parallel transfected cells were lysed and assayed for protein

concentration. receptor expression levels were normalized to protein and calculated as pmoles of steroid binding/mg protein. Values for competitive binding of unlabeled ligands are averages of duplicate determinations and were calculated as a W of total [3H] RU486 binding; set as 100%-ion the absence of competition. Non specific binding was determined by parallel incubations of mock transfected COS-1 cells and was between 10-150 of the total.

Pharmaceutical Formulations and Modes of Administration The particular compound that affects the disorders or conditions of interest can be administered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient (s). In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of a agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.

The compounds also can be prepared as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate and quinate. (See e. g., PCT/US92/03736). Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic

acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.

Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of the compound is first dissolved in a suitable solvent such as an aqueous or aqueous-alcohol solution, containing the appropriate acid. The salt is then isolated by evaporating the solution. In another example, the salt is prepared by reacting the free base and acid in an organic solvent.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e. g., for determining the LD50 (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population).

The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDgo/EDgo. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED, o with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Levels in plasma may be measured, for example, by HPLC.

The present invention also encompasses pharmaceu- tical compositions prepared for storage and subsequent administration, which have a pharmaceutically effective amount of the products disclosed above in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R.

Gennaro edit. 1985). Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid may be added as preservatives.

Id. at 1449. In addition, antioxidants and suspending agents may be used. Id.

The compositions of the present invention may be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions, suspensions for injectable administration; and the like. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (e. g., liposomes) may be utilized.

The pharmaceutically effective amount of the composition required as a dose will depend on the route of administration, the type of animal being treated, and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.

In practicing the methods of the invention, the products or compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, ordinarily in a mammal, preferably in a human, or in vitro.

In employing them in vivo, the products or compositions can be administered to the mammal in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperitoneally, employing a variety of dosage forms.

As will be readily apparent to one skilled in the art, the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and mammalian species treated, the particular compounds employed, and the specific use for which these compounds are employed. The determination of effective dosage levels, that is the dosage levels necessary to achieve the desired result, will be within the ambit of one skilled in the art. Typically, human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved. In non-human

animal studies, applications of products are commenced at higher dosage levels, with dosage being decreased until the desired effect is no longer achieved or adverse side effects disappear.

The dosage for the products of the present invention can range broadly depending upon the desired affects and the therapeutic indication. Typically, dosages may be between about 10 ßg/kg and 100 mg/kg body weight, preferably between about 100 ßg/kg and 10 mg/kg body weight. Administration is preferably oral on a daily basis.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e. g. Fingl et al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1 p. 1). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.

For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the

invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharma- ceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceuti- cally. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or

solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e. g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

Optionally, the suspension may also contain suitable stabiliz- ers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone

(PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings.

For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

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All publications referenced are incorporated by reference herein, including the nucleic acid sequences and amino acid sequences listed in each publication. All the compounds disclosed and referred to in the publications mentioned above are incorporated by reference herein, including those compounds disclosed and referred to in articles cited by the publications mentioned above.

This application is converted from provisional application serial no. 60/023,206, which is incorporated by reference herein in its entirety, including its figures, formulas and amino acid sequences and nucleic acid sequences described or disclosed therein.

Other embodiments of this invention are disclosed in the following claims.