WO2013071177A1 - Procédés et compositions pour l'inhibition de l'activité du récepteur des androgènes - Google Patents

Procédés et compositions pour l'inhibition de l'activité du récepteur des androgènes Download PDF

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Publication number
WO2013071177A1
WO2013071177A1 PCT/US2012/064538 US2012064538W WO2013071177A1 WO 2013071177 A1 WO2013071177 A1 WO 2013071177A1 US 2012064538 W US2012064538 W US 2012064538W WO 2013071177 A1 WO2013071177 A1 WO 2013071177A1
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WIPO (PCT)
Prior art keywords
compound
cell
heteroarylsteroid
cells
abiraterone
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PCT/US2012/064538
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English (en)
Inventor
Cy A. Stein
Harris Soifer
Bekir Cinar
Scott Chappel
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Tokai Pharmaceuticals, Inc.
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Publication of WO2013071177A1 publication Critical patent/WO2013071177A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones

Definitions

  • the androgen receptor (AR) is a ligand-dependent transcription factor responsible for the normal development of prostate tissue by regulating androgen-responstve gene expression during adolescence ( 1).
  • AR androgen receptor
  • anti-androgens such as bicalutamide and flutamide were developed to: a) block the binding of testosterone and dihydrotestosterone (DHT) to AR, b) cause the assembly of a transcriptionally inactive receptor, possibly by inducing a conformational change in the AR that either prevents receptor dimerization, diminishes double-stranded D A binding, or c) increase the recruitment of nuclear co- repressor complexes into holo-AR transcriptional complexes (3-5).
  • DHT dihydrotestosterone
  • AR gene amplification (10, 1 1)
  • AR mutation (12, 13)
  • AR-associated co-regulators 14
  • the transcriptional activity of the AR may also become entirely ligand-independent (17).
  • disruption of the AR can inhibit the proliferation of ostensibly androgen-refractory ceils (38, 19).
  • a reasonable therapeutic strategy would be to drastically reduce the levels AR protein in prostate cancer cells, by targeting its stability, degradation, expression and/or activity ( 18, 20-21).
  • oligonucleotides have been employed to down-regulate AR expression.
  • Molecules that have been shown to decrease the steady-state level of AR protein include: quercetin (22); the non-steroidal antiinflammatory flufenamic acid (23); resveratrol (24); the fiavone luteolin (25); docetaxel (which may be one of its major mechanisms of action clinically; [26]); phytocompounds from the oriental herbal medicine Wedelia chinensis (27); siRNAs (28); morpholino antisense oligonucleotides (oligos, [29]); antisense phosphorothioate oligos delivered by electroporation (30), and antisense locked nucleic acid (LNA [31 ]) and FANA (32) oligonucleotides delivered gymnotically (33).
  • quercetin 22
  • the non-steroidal antiinflammatory flufenamic acid 23
  • resveratrol 24
  • the invention provides a method of reducing the expression of androgen receptor protein in a cell by contacting the cell with a 17-heteroarylsteroid compound.
  • the compound is Compound 1.
  • the level of androgen receptor protein is reduced following the step of contacting the cell with the 17-heteroarylsteroid compound.
  • the level of androgen receptor mRNA is reduced.
  • the cell is a LNCaP or LAPC4 cell.
  • quantitation of androgen receptor mRNA is ascertained by qPCR.
  • the invention further provides a method of reducing nuclear translocation of androgen receptor in a cell, comprising contacting the cell with a 17-heteroarylsteroid compound.
  • the 17- heteroarylsteroid compound is Compound 1.
  • the ceil is a LNCaP or LAPC4 cell.
  • the invention also provides a method for reducing cap-dependent translation initiation in a cell, comprising contacting the cell with a 17-heteroarylsteroid compound.
  • the 17- heteroarylsteroid compound is Compound 1
  • the cell is a LNCaP or LAPC4 cell.
  • the 17-heteroarylsteroid compound reduces assembly of the eIF4F complex to the mRNA 5' cap.
  • the 17-heteroarylsteroid compound reduces phosphorylation of 4EBP 1.
  • the reduction of cap-dependent translation initiation is determined by an assay substantially as described in Example 9.
  • the invention additionally provides a method of suppressing tumor growth in a mammal having at least one prostate tumor, comprising administering to the mammal an effective amount of at least one 17-heteroarylsteroid compound, wherein the 17-heteroarylsteroid reduces cap-dependent translation initiation in the mammal.
  • the mammal is a human.
  • the 17- heteroaryl steroid compound is not Compound 1 or abiraterone.
  • a method of identifying a 17-heteroarylsteroid compound capable of suppressing tumor growth in a mammal having at least one prostate tumor comprising: a) providing at least one 17-heteroary steroid compound; b) determining the ability of the at least one 17- heteroarylsteroid compound to reduce cap-dependent translation initiation in a cell-based assay; c) selecting the compound(s) capable of reducing cap-dependent translation initiation in the cell-based assay.
  • the cell-based assay is conducted as described in Example 9.
  • Figure 1 shows the effect of 17-HASs on the levels of androgen receptor (AR) protein and AR mRNA.
  • AR protein from LNCaP cells cultured in charcoal-stripped serum (CSS) and treated with the indicated concentrations of Compound 1 (5-15 ⁇ ).
  • Control (C) cells were treated with an equal volume of DMSO. A representative Western blot of whole cell extracts collected on day 1 or day 3 post- treatment. The levels of a-tubulin were also determined as a loading control.
  • B Western blot analysis for AR protein from LNCaP cells cultured in CSS and treated for 3 days with the indicated concentrations of abiraterone alcohol (5-15 ⁇ ). Control (C) cells were treated with an equal volume of ethanoi. The levels of a-tubulin were also determined as a loading control.
  • C Western blot analysis for intracellular PSA protein levels in LNCaP cells cultured in FBS and treated for 3 days with the indicated
  • Control (C) cells were treated with an equal volume of either DMSO or ethanoi. The levels of a-tubulin were also determined as a loading control.
  • D Western blot analysis of AR steady-state protein levels in LNCaP cells cultured in CSS and treated with DMSO (D) or 5 ⁇ Compound 1 (T) for 5 days (left panel) and then sub-cultured into fresh media without drug (No T) and incubated for an additional 5 days. The levels of a-tubulin were also determined as a loading control.
  • Figure 2 shows a competitive binding assay of Compound 1 and abiraterone alcohol for the AR.
  • A Competitive binding curve of Compound 1 and abiraterone alcohol for the T877A mutant AR in LNCaP cells.
  • B-F Competitive binding curve of Compound 1 for the wild type AR (LAPC-4 (B)), mutant AR (LNCaP, T877A) (C)),
  • D-F AR expressed in PC3 cells: wild type AR (PC3 AR WT (D)), mutant W741C AR (PC3-AR-741C (E)), mutant W741L (PC3-AR-W741L (F)).
  • PC3 ceils the WT AR and mutant AR proteins were expressed by transient transfection, as described.
  • FIG. 3 shows suppression of androgen receptor trans-activation by 17-HASs.
  • 0.8 ⁇ .
  • PC3 clones that stably express the wild ty pe (black), W741C (white), or W741L (grey ) AR proteins were transiently transfected with the reporter vector pARE-4X-Luciferase and then stimulated with R1881 (1 nM). Ceils were co-treated with the indicated concentrations of Compound 1. The amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU).
  • (C) PC3 clones that stably express the WT, W741C, or W741L AR proteins were transiently transfected with the reporter vector pARE-4-Luciferase.
  • the cells were untreated (mock or control), treated with 1 nM Rl 881 , or treated with 10 ⁇ Casodex or 10 ⁇ Compound 1 in the absence of R1881.
  • the amount of transcriptional activity was normalized to Renilla luciferase and is expressed as normalized relative light units (RLU).
  • (D) LNCaP cells were either pre-treated with DMSO (CPD1 -) or pre-treated with 20 ⁇ Compound 1 (CPD1+) for 1 hour.
  • Figure 4 shows the effect of Compound 1 and abiraterone on the rate of AR degradation.
  • A, B A representative AR degradation time-course experiment in LNCaP cells is shown.
  • AR protein was measured at 4 h intervals from LNCaP cells cultured in androgen-free media and co-treated with cycloheximide (CHX, 100 ⁇ ) and 20 ⁇ Compound 1 (CPDl-20) or 20 ⁇ abiraterone alcohol (abiraterone).
  • CHX cycloheximide
  • CPDl-20 Compound 1
  • abiraterone alcohol abiraterone
  • FIG. 1 shows the effect of 17-HASs on AR translational machinery.
  • A, B Luciferase reporter assay, in which translation of the luciferase mRNA is under the control of AR 5' UTR (test) or IRE S (control).
  • 17-heteroarylsterotd compounds include 3 ⁇ -hydroxy- 17-(1 H-benztmtdazole- l -yl)androsta- 5.16-diene, also known as VN/124-1 ; herein “Compound ⁇ ” or “Cpdl "; and abtraterone alcohol, an active pharmaceutical ingredient and plasma enzymatic cleavage product of abiraterone acetate [35, 36].
  • Compound 1 modulate the activity of steroid hormone nuclear receptors and, as such, are useful for treating androgen receptor mediated diseases or conditions.
  • Compound 1 or 3-p-HydiOxy-17-(7//-benzimidazol-I -yl)androsta-5,16-diene) may be synthesized using standard synthetic techniques known to those of skill in the art or using methods known in the art in combination with methods described herein. In additions, solvents, temperatures and other reaction conditions presented herein may vary according to the practice and knowledge of those of skill in the art.
  • the starting material used for the synthesis of the Compound 1 can be obtained irom commercial sources, such as Aldrich Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St. Louis, Mo.), or the starting materials can be synthesized.
  • Compound 1 can be prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of the compound with a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of the compound with a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of the compound with a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically acceptable salt) by reacting the free base form of the compound with a
  • inorganic or organic acid including, but not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid metaphosphoric acid, and the like; and organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, p-toluenesulfonic acid, tartaric acid, trifluoroacetic acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, arylsulfonic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid,
  • a reference to a pharmaceutically acceptable salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs.
  • Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of Compound 1 can be conveniently prepared or formed during the processes described herein.
  • hydrates of Compound 1 can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents including, but not limited to, dioxane, tetrahvdrofuran or methanol.
  • organic solvents including, but not limited to, dioxane, tetrahvdrofuran or methanol.
  • the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.
  • Compound 1 includes crystalline forms, also known as polymorphs.
  • Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.
  • Compound 1 can be prepared as a prodrug.
  • Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some process, such as conversion by a metabolic pathway .
  • Some prodrugs have a chemical group present on the prodrug that renders it less active and/or confers solubility or some other property to the drug. Once the chemical group lias been cleaved and/or modified from the prodrug the active drug is generated.
  • Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. Prodrugs may, for instance, be bioavailable by oral administration whereas the parent is not.
  • the prodrug may also have improved solubility in pharmaceutical
  • compositions over the parent drug over the parent drug.
  • a prodrug would be a derivative which is administered as an ester (the "prodrug") to facilitate absorption in the gastrointestinal tract where improved water solubility is beneficial, but which then is metabolicaily hydrolyzed to a carboxylic acid and the active entity.
  • Compound 1 A further example of a prodrug might be a short peptide
  • Prodrugs may be designed as reversible drug derivatives, for use as modifiers to enhance drug transport to site-specific tissues.
  • the design of prodrugs to date has been to increase the effective water solubility of the therapeutic compound for targeting to regions where water is the principal solvent. See, e.g., Fedorak et al., Am. J. Physiol. , 269:G210-218 ( 1995); McLoed et al., Gastroenterol 106:405-413 (1994); Hochhaus et al., Biomed. Chrom., 6:283-286 (1992); J. Larsen and H. Bundgaard, Int. J.
  • prodrug derivatives of Compound 1 can be prepared by methods known to those of ordinary skill in the art (e.g., for further details see Sauinier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985). Prodrug forms of the herein described compounds, wherein the prodrug is metabolized in vivo to produce a derivative as set forth herein are included within the scope of the claims. Indeed, some of the herein-described compounds may be a prodrug for another derivative or active compound.
  • Sites on the aromatic ring portion of Compound 1 can be susceptible to various metabolic reactions, therefore incorporation of appropriate substituents on the aromatic ring structures, such as, by way of example only, halogens can reduce, minimize or eliminate this metabolic pathway.
  • Various methods of making Compound 1 are contemplated.
  • one or more of the following chemical reactions is performed in an inert atmosphere, for example, nitrogen or argon.
  • the temperature of the reaction is monitored.
  • the reaction is monitored by HPLC or TLC.
  • the pH of the reaction is monitored.
  • the temperature of the reaction is controlled.
  • the purity of the product is determined by HPLC.
  • the experiments are run on small scale, medium scale, large scale, analytical scale, or manufacturing scale.
  • the product is clarified by filtration through a pad comprising one or more of silica gel and celite.
  • the synthesis is performed on large scale.
  • large scale comprises a scale of about 1 to about 10 kg.
  • the synthesis is performed on manufacturing scale.
  • manufacturing scale comprises a scale of greater than about 10 kg.
  • manufacturing scale comprises a scale of about 10 to about 1 ,000 kg.
  • manufacturing scale comprises a scale of about 10 to about 100 kg.
  • manufacturing scale comprises a scale of about 10 to about 50 kg.
  • manufacturing scale comprises a scale of about 33.4 kg.
  • an experiment is performed on a smaller scale to gather information to be used to plan or perform synthesis on a manufacturing scale.
  • the results obtained on the smaller scales are expected to be reproducible on manufacturing scale.
  • the results obtained on smaller scales are not expected to be reproducible on manufacturing scale.
  • the yields obtained on manufacturing scale are greater than the yields obtained on smaller scales. In some embodiments, the yields obtained on manufacturing scale are lesser than the yields
  • a solution of a compound of Formula i in a solvent is prepared.
  • a compound of Formula ii is then contacted to the solution, and the resultant mixture is heated in the presence of a base for a period of time sufficient to provide a compound of Formula iii.
  • the period of time is about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, or about 24 hours.
  • the time is from about 1 hour to about 24 hours.
  • the base comprises lithium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, a sodium phosphate, or a potassium phosphate.
  • the solvent comprises DMF.
  • the temperature is about 50 °C, about 70 °C, about 100 °C, about 150°C, or a temperature effective to sustain reflux conditions. In some embodiments, the temperature is from about 50 °C to about 200 °C.
  • the compound of Formula iii can be isolated from the reaction mixture and purified by any method known to one of skill in the art. Such methods include, but are not limited to, pouring an aqueous mixture into the reaction mixture, thereby effecting the precipitation of compound iii as a solid.
  • the isolated compound of Formula iii may optionally be purified by any method known to one of skill in the art. Such methods include, but are not limited to, trituration with water.
  • a solution of a compound of Formula iii in a solvent is prepared, and the solution is contacted with a catalyst for a period of time sufficient to provide a compound of Formula i .
  • the period of time is about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, or about 24 hours. In some embodiments, the time is from about 1 hour to about 24 hours.
  • the catalyst comprises palladium on carbon, platinum on carbon, a transition metal salt, or a transition metal complex.
  • the solvent comprises N- methylpyrroiidone.
  • the temperature is about 50 °C, about 70 °C, about 100 °C, about 150 °C, about 190 °C, about 200 °C or a temperature effective to sustain reflux conditions. In some embodiments, the temperature is from about 50 °C to about 250°C.
  • the compound of Formula iv can be isolated from the reaction mixture and purified by any method known to one of skill in the art. Such methods include, but are not limited to, in-line filtration. The isolated compound of Formula iii may be isolated from the reaction mixture and purified by any method known to one of skill in the art. Such methods include, but are not limited to, in-line filtration. The isolated compound of Formula iii may
  • a solution of a compound of Formula iv in a solvent is prepared, and the solution is contacted with a base for a period of time sufficient to provide a compound of Formula v (i.e., Compound 1 ).
  • the period of time is about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, or about 24 hours. In some embodiments, the time is from about i hour to about 24 hours.
  • the base comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, lithium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, a sodium phosphate, or a potassium phosphate.
  • the solvent comprises water, methanol, ethanol, 2-propanol, t-butanol, or mixtures thereof. In some embodiments, the solvent comprises methanol and the base comprises sodium methoxide.
  • the temperature is about 35 °C, about 50 °C, about 70 °C, about 100 °C, or a temperature effective to sustain reflux conditions.
  • the temperature is from about 25 °C to about i 00 °C.
  • the compound of Formula v can be isolated from the reaction mixture and purified by any method known to one of skill in the art. Such methods include, but are not limited to, extraction.
  • the isolated compound of Formula iii may optionally be purified by any method known to one of skill in the art. Such methods include, but are not limited to, trituration.
  • a pharmaceutical composition refers to a mixture of Compound i with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism.
  • Pharmaceutical composition containing Compound 1 can be administered in therapeutically effective amounts as pharmaceutical compositions by any conventional form and route known in the art including, but not limited to: intravenous, oral, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, otic, nasal, and topical administration.
  • composition containing Compound 1 in a targeted drug deliver system, for example, in a liposome coated with organ-specific antibody.
  • the liposomes will be targeted to and taken up selectively by the organ.
  • the pharmaceutical composition containing Compound 1 may be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.
  • Compound 1 can be formulated readil by combining the active compounds with pharmaceutically acceptable carriers or excipients well known in the art.
  • Such carriers enable the compounds described herein to be formulated as tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Dragee cores are provided with suitable coatings.
  • concentrated sugar solutions may be used, which may optionall contain gum arabic, talc, polyvinylpyrrolidone, 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.
  • compositions 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 capsule comprises a hard gelatin capsule comprising one or more of pharmaceutical, bovine, and plant gelatins.
  • a gelatin is alkaline processed.
  • 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.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets, lozenges, or gels formulated in conventional manner.
  • Parental injections may involve for bolus injection or continuous infusion.
  • the pharmaceutical composition of Compound 1 may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • 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 fatt 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.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. , sterile pyrogen -free water, before use.
  • Compound 1 can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments.
  • Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • Formulations suitable for transdermal administration of compounds of the invention may employ transdermal delivery devices and transdermal delivery patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Such patches may be constructed for continuous, pulsatile, or on demand deliver ⁇ ' of pharmaceutical agents. Still further, transdermal delivery of Compound 1 can be accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches can provide controlled delivery of Compound 1 . The rate of absorption can be slowed by using rate-controlling membranes or by trapping the compound within a poly mer matrix or gel. Conversely, absorption enhancers can be used to increase absorption.
  • An absorption enhancer or carrier can include absorbable pharmaceutically acceptable solvents to assist passage through the skin.
  • transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionall with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
  • Compound 1 may be in a form as an aerosol, a mist or a powder.
  • Pharmaceutical compositions of the invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, such as, b way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a pow der mix of the compound and a suitable powder base such as lactose or starch.
  • Compound I may also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like, in suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted.
  • rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas
  • conventional suppository bases such as cocoa butter or other glycerides
  • synthetic polymers such as polyvinylpyrrolidone, PEG, and the like
  • therapeutically effective amounts of Compound 1 provided herein are administered in a pharmaceutical composition to a mammal having a disease or condition to be treated.
  • the mammal is a human.
  • a therapeutically effective amount can vary widely depending on the sev erity of the disease, the age and relative health of the subject, the potency of the compound used and other factors.
  • the compounds can be used singly or in combination with one or more therapeutic agents as components of mixtures.
  • compositions may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art.
  • Pharmaceutical compositions comprising a compound of the invention may be manufactured in a conventional manner, such as, by w ay of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • the pharmaceutical compositions will include at least one pharmaceutically acceptable earner, diluent or excipient and a compound of the invention described herein as an active ingredient in free-base form, or in a pharmaceutically acceptable salt form.
  • the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type of activity.
  • compositions comprising the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid.
  • Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories.
  • Liquid compositions include solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, or nanoparticles comprising a compound as disclosed herein.
  • Semi-solid compositions include, but are not limited to, gels, suspensions and creams.
  • compositions may be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions may also contain minor amounts of nontoxic, auxil obviouslyy substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.
  • Compound 1 can be used in the preparation of medicaments for the treatment of diseases or conditions in which steroid hormone nuclear receptor activity contributes to the pathology and/or symptoms of the disease.
  • a method for treating any of the diseases or conditions described herein in a subject in need of such treatment involves administration of pharmaceutical compositions containing at least one compound of the invention, or a pharmaceutically acceptable salt,
  • compositions containing the compound(s) described herein can be administered for prophylactic and/or therapeutic treatments.
  • the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, or ameliorate the condition itself.
  • Amounts effective for this use will depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. It is considered well within the skill of the art for one to determine such therapeutically effective amounts by routine experimentation (including, but not limited to, a dose escalation clinical trial).
  • compositions containing the compound(s) described herein can be used to treat a steroid hormone disorder, disease or condition selected from: primary and secondary hyperaldosteronism, increased sodium retention, increased magnesium and potassium excretion (diuresis), increased water retention, hypertension (isolated systolic and combined systolic diastolic), inflammation, malignancies such as leukemias and lymphomas, Cushing's syndrome, congenital adrenal hyperplasia, polycystic ovarian syndrome, endometrial cancer, cervical cancer, hypocalcaemia, hyperglycemia, chronic primary adrenal insufficiency, secondary adrenal insufficiency, alopecia, prostate cancer, benign prostatic hyperplasia, alopecia, anorexia nervosa, breast cancer, AIDS, cachexia, for hormone replacement therapy (HRT), employed in male contraception, for male reproductive conditions, primary or secondary male hypogonadism, testicular cancer, ovarian cancer, lung cancer,
  • HRT
  • the method comprising administering to the patient an effective amount of a compound described herein, or a tautomer, prodrug, solvate, or salt thereof.
  • the compositions containing the compound(s) described herein can be used to treat a steroid hormone mediated cancer.
  • steroid hormone mediated cancer is prostate cancer.
  • the compositions containing the compound(s) described herein can be used to treat castration resistant prostate cancer.
  • steroid hormone mediated cancer is breast cancer.
  • steroid hormone mediated cancer is endometrial cancer.
  • steroid hormone mediated cancer is ovarian cancer.
  • steroid hormone mediated cancer is prostate cancer.
  • steroid hormone mediated cancer is cervical cancer.
  • the compositions containing the compound(s) described herein can be used to treat congenital adrenal hyperplasia. In some embodiments, the compositions containing the compound(s) described herein can be used to treat polycystic ovarian syndrome. In some embodiments, the compositions containing the compound(s) described herein can be used to treat conditions associated with excessive androgens. In certain embodiments, conditions associated with excessive androgens include, but are not limited to, hirsutism, oligomenorrhea, amenorrhea, anovulation, androgenic alopecia, hypergonadism, excessive acne, and/or virilization. In certain embodiments, conditions associated with excessive androgens are metabolic diseases or conditions that include, but are not limited to type 2 diabetes, dyslipidemia, and coronary artery disease.
  • the administration of the compounds may be administered chronically , that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.
  • the administration of the compounds may be given continuously or temporarily suspended for a certain length of time (i.e., a "drug holiday").
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease or condition is retained. Patients can, however, require intermittent treatment on a long-term basts upon any recurrence of symptoms.
  • the compounds described herein may be administered therapeutically effective amounts of at least one of the compounds described herein (or a pharmaceutically acceptable salts, pharmaceutically acceptable N-oxides, pharmaceutically active metabolites, pharmaceutically acceptable prodrugs, and pharmaceutically acceptable solvates thereof) in combination with another therapeutic agent.
  • a pharmaceutically acceptable salts, pharmaceutically acceptable N-oxides, pharmaceutically active metabolites, pharmaceutically acceptable prodrugs, and pharmaceutically acceptable solvates thereof in combination with another therapeutic agent.
  • an anti-inflammatory agent in combination with the initial therapeutic agent.
  • the therapeutic effectiveness of one of the compounds described herein may be enhanced by administration of an adjuvant ⁇ i.e., by itself the adjuvant ma only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced).
  • the benefit of experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit.
  • the overall benefit experienced by the patient may simply be additive of the two therapeutic agents or the patient may experience a synergistic benefit.
  • dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the disease or condition being treated and so forth.
  • the compound provided herein may be administered either simultaneously with the biologically active agent(s), or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein in combination with the biologically active agent(s).
  • the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents. Multiple therapeutic combinations are envisioned.
  • Compound 1 may also be used in combination with procedures that may provide additional or synergistic benefit to the patient.
  • patients are expected to find therapeutic and/or prophylactic benefit in the methods described herein, wherein pharmaceutical composition of the invention and /or combinations with other therapeutics are combined with genetic testing to determine whether that individual is a carrier of a mutant gene that is known to be correlated with certain diseases or conditions.
  • Compound I and combination therapies can be administered before, during or after the occurrence of a disease or condition, and the timing of administering the composition containing a compound can vary.
  • the compounds can be used as a prophylactic and can be administered continuously to subjects with a propensity to conditions or diseases in order to prevent the occurrence of the disease or condition.
  • the compounds and compositions can be administered to a subject during or as soon as possible after the onset of the symptoms.
  • the administration of the compounds can be initiated within the first 48 hours of the onset of the symptoms, preferably within the first 48 hours of the onset of the symptoms, more preferably within the first 6 hours of the onset of the symptoms, and most preferably within 3 hours of the onset of the symptoms.
  • the initial administration can be via any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof.
  • a compound is preferably administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary' for the treatment of the disease, such as, for example, from about 1 month to about 3 months.
  • the length of treatment can vary for each subject, and the length can be determined using the known criteria.
  • the compound or a formulation containing the compound can be administered for at least 2 weeks, preferably about 1 month to about 3 years, and in some embodiments from about 1 month to about 10 years.
  • the pharmaceutical composition described herein may be in unit dosage forms suitable for single administration of precise dosages.
  • the formulation is divided into unit doses containing appropriate quantities of one or more compound.
  • the unit dosage may be in the form of a package containing discrete quantities of the formulation.
  • Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • Aqueous suspension compositions can be packaged in single-dose non-reclosable containers.
  • multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition.
  • formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative.
  • the daily dosages appropriate for Compound 1 described herein are from about 0.03 to 60 mg/kg per body weight.
  • An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 1 mg to about 4000 mg, conveniently administered in divided doses, including, but not limited to, up to four times a day or in retard form.
  • Suitable unit dosage forms for oral administration comprise from about 1 mg to about 4000 mg active ingredient.
  • a single dose of compounds of the invention is w ithin the range of about 50 mg to about 2,000 mg.
  • a single dose of compounds of the invention is about 90 mg, about 200 mg, about 250 mg, about 325 mg, about 650 mg, about 975 mg, about 1300 mg, about 1625 mg, or about 1950 mg.
  • an administration of compounds of the invention of about 90 mg, about 325 mg, about 650 mg, about 975 mg, about 1300 mg, about 1625 mg, or about 1950 mg is given as multiple doses.
  • Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, for determining the LD 50 (the dose lethal to 50% of the population) and the ED 5 o (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD S0 and ED 50 .
  • Compounds exhibiting high therapeutic indices are preferred.
  • the data obtained from 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 50 with minimal toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • Suitable compounds that may be used, in addition to Compound 1 , as agents for treating steroid hormone mediated disorders include, but are not limited to, hormone ablation agents, anti-androgen agents/anti-androgens, anti-estrogen agents/anti- estrogens, differentiating agents, anti -neoplastic agents, kinase inhibitors, anti-metabolite agents, alkylating agents, antibiotic agents, immunological agents, interferon-type agents, intercalating agents, growth factor inhibitors, cell-cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, mitotic inhibitors, matrix metalloprotease inhibitors, and genetic therapeutics.
  • the amount of the additional agents for treating steroid hormone mediated disorders administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the cancer, whether administered alone or in combination with Compound 1.
  • Examples of some of the above classes of agents for treating steroid hormone mediated disorders are listed below for purposes of illustration and not for purposes of limitation, as these examples are not all- inclusive. Many of the examples belo could be listed in multiple classes of agents for treating steroid hormone mediated disorders and are not restricted in any way to the class in which they are listed.
  • Compound 1 may be administered with an hormonal ablation agent, such as deslorelin, leuprolide, goserelin or triptorelin.
  • an hormonal ablation agent such as deslorelin, leuprolide, goserelin or triptorelin.
  • the amount of the hormonal ablation agent administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the steroid hormone mediated disorder, whether administered alone or in combination with Compound 1.
  • Suitable anti-androgen agents include but are not limited to bicalutamide, flutamide and nilutamide.
  • the amount of the anti-androgen agent administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the steroid homione mediated disorder, whether administered alone or in combination with Compound 1.
  • Suitable anti-estrogen agents include but are not limited to tamoxifen, raloxifene, 4- hydroxy tamoxifen (afimoxifene), ciomifene, arzoxifene, avalanose, ormeloxifene, and toremifene.
  • the amount of the anti-estrogen agent administered to a mammal having a steroid homione mediated disorder is an amount that is sufficient to treat the steroid hormone mediated disorder, whether administered alone or in combination with Compound 1.
  • Compound 1 may be administered with a differentiating agent.
  • suitable differentiating agents include, but are not limited to, polyamine inhibitors; vitamin D and its analogs, such as caicitriol, doxercalciferoi and seocalcitol; metabolites of vitamin A, such as ATRA; retinoic acid; retinoids; short-chain fatty acids; phenylbutyrate; and nonsteroidal anti-inflammatory agents.
  • the amount of the differentiating agent administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the steroid hormone mediated disorder, whether administered alone or in combination with Compound I .
  • Compound 1 may be administered with an anti-neoplastic agent, including, but not limited to, tubulin interacting agents, topoisomerase inhibitors and agents, acitretin, alstonine, amonafide, amphethinile, amsacrine, ankinomycin, anti-neoplaston, aphidicolin glycinate, asparaginase, baccharin, batracylin, benfluron, benzotript, bromofosfamide, caracemide, carmethizole hydrochloride, chlorsulfaquinoxaione, clanfenur, claviridenone, crisnatol, curaderm, cytarabine, cytocytin, dacarbazine, datelliptinium, dihaematoporphyrin ether, dihydrolenperone, dinaline, distamycin, docetaxel, elliprabin,
  • Compound 1 may also be used with a kinase inhibitor, including p38 inhibitors and CDK inhibitors, TNF inhibitors, metallomatrix proteases (MMP) inhibitors; COX-2 inhibitors, including celecoxib, rofecoxtb, parecoxib, valdecoxib, and etoricoxib; SOD mtmics; or a v ,p 3 -inhibitors.
  • the amount of the kinase inhibitor administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the steroid hormone mediated disorder, whether administered alone or in combination w ith Compound 1.
  • Compound 1 may be administered with an anti-metabolite agent.
  • Suitable anti-metabolite agents may be selected from, but are not limited to, 5-FlJ-fibrinogen, acanthifolie acid, aminothiadiazole, brequtnar sodium, carmofur, cyclopentyl cytosine, cytarabine phosphate stearate, cytarabine conjugates, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, doxifluridine, camrabine, fioxuridine, fludarabine phosphate, 5-fluorouracil, N-(2'-furanidyl)-5-ftuorouracil, isopropyl pyrrolizine, methobenzaprim, methotrexate, norspermidine, pentostatin, piritrexim, plicamycin, thioguanine, tiazofurin, trimetrexate, tyrosine kinase inhibitors, and uricyt
  • Compound 1 may be administered with an alkylating agent, that may be selected from, but not limited to, aldo-phosphamide analogues, altretamine, anaxirone, bestrabucil, budotitane, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyplatate,
  • alkylating agent may be selected from, but not limited to, aldo-phosphamide analogues, altretamine, anaxirone, bestrabucil, budotitane, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyplatate,
  • the amount of the alkylating agent administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the steroid hormone mediated disorder, whether administered alone or in combination with Compound 1.
  • Compound 1 may be administered with an antibiotic agent.
  • Suitable antibiotic agents may be selected from, but are not limited to, aclarubicin, actinomycin D, actinoplanone, adriamycin, aeroplysinin derivative, amrubicin, anthracycline, azinomycin-A, bisucaberin, bleomycin sulfate, bryostatin-1 , calichemycin, chromoximycin, dactinomycin, daunorubicin, ditrisarubicin B, dexamethasone, doxorubicin, doxorubicin-fibrinogen, elsamicin-A, epirubicin, erbstatin, esorubicin, esperamicin-Al, esperamicin-Alb, fostriecin, giidobactin, gregatin-A, grincamycin, herbimycin, corticoster
  • Compound 1 may also be used with other anti-cancer agents, including but not limited to, acemannan, aclarubicin, aldesleukin, alemtuzumab, alitretinoin, altretamine, amifostine, amsacrine, anagrelide, anastrozole, ancestim, bexarotene, broxuridine, capecitabine, celmoleuktn, cetrorelix, cladribine, clotrimazole, daclizumab, dexrazoxane, dilazep, docosanoi, doxifluridine, bromocriptine, carmustine, cytarabine, diclofenac, edelfosine, edrecolomab, eflornithine, emitefur, exemestane, exisulind, fadrozole, filgrastim, finasteride, fludarabine
  • Compound 1 may also be administered or combined with steroids, such as corticosteroids or glucocorticoids, non-limiting examples of such suitable steroids including hydrocortisone, prednisone, and dexamethasone.
  • steroids such as corticosteroids or glucocorticoids, non-limiting examples of such suitable steroids including hydrocortisone, prednisone, and dexamethasone.
  • Compound 1 and the steroid may be administered in the same or in different compositions, with the amount of the steroid administered to a mammal having a steroid hormone mediated disorder is an amount that is sufficient to treat the steroid hormone mediated disorder, whether administered alone or in combination with Compound 1.
  • compositions comprising both Compound 3 and a steroid, particularly a corticosteroid, or more particularly, a glucocorticoid.
  • Steroids within the scope of the disclosure include, but are not limited to, (1) hydrocorti sone (Cortisol; cyprionate oral;
  • prednisolone e.g., DELTA -CORTEF® prednisolone sodium succinate, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate
  • prednisone and combinations thereof See, e.g., Goodman & Oilman's The Pharmacological Basis of Therapeutics, 10* sup. Edition 2001.
  • single unit solid oral dosage forms that comprise an amount from about 20 mg to about 500 mg of Compound 1 and from about 0.5 mg to about 3.0 mg of a steroid, e.g., a glucocorticoid, in a single composition, optionally with one or more excipients, carriers, diluents, etc.
  • the single unit dosage form may comprise about 250 mg of Compound 1 and about 1.0 mg, 1.25 mg, 1.5 mg, or 2.0 mg of a steroid, such as, but not limited to, corticosteroids or glucocorticoids.
  • Pharmacokinetic and pharmacodynamic data can be obtained by known techniques in the art. Due to the inherent variation in pharmacokinetic and pharmacodynamic parameters of drug metabolism in human subjects, appropriate pharmacokinetic and pharmacodynamic profile components describing a particular composition can vary. Typically, pharmacokinetic and pharmacodynamic profiles are based on the determination of the "mean" parameters of a group of subjects. The group of subjects includes any- reasonable number of subjects suitable for determining a representative mean, for example, 5 subjects, 10 subjects, 16 subjects, 20 subjects, 25 subjects, 30 subjects, 35 subjects, or more. The "mean" is determined by calculating the average of all subject's measurements for each parameter measured.
  • the pharmacokinetic parameters can be any parameters suitable for describing the present composition.
  • the C mai can be not less than about 500 ng/ml; not less than about 550 ng/ml; not less than about 600 ng/ml; not less than about 700 ng/ml; not less than about 800 ng/ml; not less than about 880 ng ml, not less than about 900 ng/ml; not less than about 100 ng/ml; not less than about 1250 ng/ml; not less than about 1500 ng/ml, not less than about 1700 ng/ml, or any other C ma appropriate for describing a pharmacokinetic profile of Compound 1.
  • the C TOax can be not less than about 500 pg/ml; not less than about 550 pg/ml; not less than about 600 pg/ml; not less than about 700 pg/ml; not less than about 800 pg/ml; not less than about 880 pg/ml, not less than about 900 pg/ml; not less than about 1000 pg/ml; not less than about 1250 pg/ml; not less than about 1500 pg/ml, not less than about 1700 pg/ml, or any other C max appropriate for describing a pharmacokinetic profile of a compound formed in vivo alter administration of Compound 1 to a subject.
  • the T max can be, for example, not greater than about 0.5 hours, not greater than about 1.0 hours, not greater than about 1.5 hours, not greater than about 2.0 hours, not greater than about 2.5 hours, or not greater than about 3.0 hours, or any other T mai appropriate for describing a pharmacokinetic profile of Compound 1.
  • the AU o-i nt can be, for example, not less than about 590 ng*hr/mL, not less than about 1500 ng « hr/mL , not less than about 2000 ng « hr/mL , not less than about 3000 ng.
  • the AU o-mo can be, for example, not less than about 590 pg » hr/mL, not less than about 1500 pg » hr/mL, not less than about 2000 pg*hr/mL, not less than about 3000 pg*hr/mL, not less than about 3500 pg*hr/mL, not less than about 4000 pg*hr/mL, not less than about 5000 pg'hr/niL, not less than about 6000 pg « hr/niL, not less than about 7000 pg*hr/mL, not less than about 8000 pg « hr/mL, not less than about 9000 pg « hr/mL, or any other AUC o-in appropriate for describing a pharmacokinetic profile of a compound formed in vivo after administration
  • the plasma concentration of Compound 1 about one hour after administration can be, for example, not less than about 140 ng/ml, not less than about 425 ng/ml, not less than about 550 ng/ml, not less than about 640 ng/ml, not less than about 720 ng/ml, not less than about 750 ng/ml, not less than about 800 ng/ml, not less than about 900 ng/ml, not less than about 1000 ng/ml, not less than about 1200 ng/ml, or any other plasma concentration of Compound 1.
  • the pharmacodynamic parameters can be any parameters suitable for describing the present composition.
  • the pharmacodynamic profile can exhibit decreases in AR protein or endogenous androgens for, by way of example only, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours or at least about 24 hours.
  • the pharmacodynamic profile can exhibit an inhibition of androgen sy nthesizing enzymes, including CYP17, for, by way of example only, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours or at least about 24 hours.
  • the pharmacodynamic profile can exhibit reduction of androgen signaling, for, by way of example on!y, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours or at least about 24 hours.
  • PC3 (CRL-1435) and LNCaP (CRL-1740) cells were maintained in RMPI media supplement with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin G sodium/100 mg/mi streptomycin sulfate, sodium pyruvate, and non-essential amino acids at 37°C in a humidified 5% C02 incubator.
  • LAPC-4 cells a generous gift of Dr. R. Reiter (UCLA, Los Angeles, CA), were maintained similarly , but in IMDM media supplemented with 5% heat inactivated fetal bovine serum.
  • Cells expressing either the wild type (WT) or AR mutant proteins were created by stable transfection of PC3 (AR null) cells with pCIneo-hAR (WT), pCIneo-hAR-W741 C, or pCIneo-hAR-W741L (generous gifts of Dr. S.P. Balk, Beth Israel Medical Center, Boston, MA). Where indicated, cells were cultured in phenol red-free, steroid-free media, consisting of basal media supplemented with 5-10% dextran-coated, charcoal-stripped FBS. Compound 1 , previously known as VN/124-1 , was prepared as described.
  • Compound 1 was dissolved in DMSO prior to use.
  • Bicalutamide (Casodex®) was dissolved in DMSO prior to use and was a generous gift of V. Njar (Jefferson Medical University, Philadelphia, PA).
  • R1881 was obtained from Perkin Elmer and dissolved in DMSO.
  • Cycioheximide was obtained from Sigma and dissolved in ethanol.
  • MG 132 was dissolved in DMSO and was obtained from EMS Scientific (San Diego, CA).
  • 7-methyl-GTP (7mG) sepharose beads were obtained from Amersham Biosciences/GE Healthcare (Piscataway, NJ).
  • anti-androgen receptor clone F39.4.1, BioGenenix, San Ramon, CA, 1 :400
  • anti-a-tubulin clone B-5-1 -2, Sigma, St.
  • a dissociation step was also performed to confirm the amplification of a single product.
  • the relative standard curve method was used to quantify the amount of AR and RPLPO mRNA in each sample.
  • K c the "competition constant", as it henceforth will be in this work).
  • PC-3 cells were first transiently transfected with pCIneo-AR-WT, pCIneo-AR-W741C, or pClneo-AR-W741 L.
  • Ceils treated for the indicated time periods were collected by brief trypsinization, resuspended in media containing 10% FBS and returned to the humidified 5% CO? incubator for 30-45 minutes.
  • Apoptotic cells were measured with the Annexin V-F1TC Apoptosis Detection Kit (BD Biosciences, San Jose, CA). Propidium iodide (1 mg/mL) was added just prior to flow cytometric analysis (Becton Dickinson FACScan). Additional samples were prepared from vehicle-treated cells and were unstained, stained with PI alone, or stained with Annexin V-FITC aione. Ten thousand cells per sample were analyzed. Apoptosis studies were repeated a minimum of two times.
  • LNCaP or LAPC-4 cells were plated onto 6-well plates in Phenol red-free media containing charcoal-stripped FBS and treated with cyeloheximide ( 100 fM) along with vehicle (DMSO or Ethanol) or Compound I or abiraterone alcohol.
  • the amount of AR protein as a function of time was determined by Western blot analysis as described above. Differences in the rate of AR degradation between test compound and control were determined by Mann- Whitney statistical analysis.
  • pARE4-luciferase contains four Androgen Response Elements (AREs) cloned in tandem into pGL3 (Promega, Madison, WT).
  • pRL-CMV-Renilla is a cytomegalovirus (CMV) promoter- driven Renilla luciferase control plasmid.
  • PC3 cells stably expressing WT or mutant AR proteins were seeded into poly-lysine-coated plates using phenol red-free, steroid-free RMPI complete media without antibiotics and transfected 24 h later with 100 g pARE4-Luciferase and 100 pg pRL-CMV-Renilla using Lipofectamme 2000 (Invitrogen, Carlsbad, CA). 24 h post-transfection, the medium was changed to fresh phenol red-free, steroid-free RMPI complete media and hormones or drugs were added at the indicated concentrations. Firefly and Renilla luciferase activities were determined 18 h later using the Dual Luciferase Kit (Promega, Madison, WI).
  • Data shown represent the mean and standard deviation of three independent experiments performed in triplicate and is expressed as relative light units (RLU, Firefly luciferase/Reniila luciferase).
  • RLU relative light units
  • IC 50 values dose-response data were analyzed by non-linear regression to fit the data to the log (inhibitor) vs. response with variable slope using Graphpad Prism software.
  • LNCaP cells were seeded in 24-well plates at 70% confluence a day before transfections. Cells then were trans ected with plasmid DNA (0.5 tg/well) carrying pIR-AR 5'UTR-Luc (5'UTR; test) or pIRES-Luc (IRES, control) for 6 h in serum-free and antibiotic-free conditions. Transcription of both luciferase reporter genes is under the control of the CMV promoter. Constructions of the plasmid DNA were described elsewhere (see reference 49 for details). Lipofectamine 2000 reagent as used in ail transfections according to the manufacturer's instructions (Invitrogen).
  • Luciferase reporter gene activities were measured using Luciferase Assa System from Promega (Madison, WI) at 36 h post treatment using a BMG Labtech microplate reader (Car ⁇ , NC). Relative luciferase units were normalized to total protein and then normalized to vector control (pIR-AR 5'UTR-Luc) and the result was presented as luciferase activity.
  • LNCaP cells in 96-well plate were seeded 24 h prior to drug treatment and then treated with control (mock).
  • LNCaP cells were seeded in 6-well plates 24h prior to drug exposures. Cells were then exposed to doses of Compound 1 or abiraterone alcohol (0, 10, and 20 ⁇ ) in 5%FBS -Medium for 36 hours. Cells were washed with ice cold PBS and total cell lysates were prepared in cap binding buffer containing 150 mM NaCI, 50 mM Tris, pH 7.5, 50 niM NaF, 10 mM Na pyrophosphate, 1 mM EDTA, 2.5 mM Na orthovanadate supplemented with cocktail of protease inhibitor (Calbiochem ) from indicated conditions.
  • cap binding assay 25 ⁇ of pre-washed 7-methy -GTP (7mG) sepharose beads slurry was added to 150 ⁇ g total proteins and incubated at room temperature for 1 hour. Samples were then washed with cap binding buffer and quenched with sample buffer. Samples were boiled and equal amount of elute and 10 ⁇ g of total lysates were resolved by 7.5% (for the detections of eIF4G and AR proteins) or by 12% (for the detection of eIF4E, 4EBP1, phospho-4EBP 1 and AR proteins) SDS-PAGE and then transferred onto nitrocellulose membranes.
  • Example 10 Reduction of AR protein levels by Compound 1 and abiraterone alcohol.
  • LNCaP and LAPC-4 The effects of treatment with these two compounds on AR expression was measured in two prostate cancer cell lines, LNCaP and LAPC-4, that express AR and respond to androgen signaling.
  • LNCaP cells were cultured in medium supplemented with charcoal-stripped serum (CSS, T ⁇ 1 nM) followed by treatment with increasing concentrations of Compound 1 , the steady-state levels of AR protein were markedly decreased (up to 84%, 15 ⁇ Compound 1 , Day 3, Fig. 1 A).
  • Abiraterone alcohol, another potent Cypl 7 inhibitor produced a similar dose-dependent decrease in AR protein levels in LNCaP cells (Fig. IB).
  • LAPC-4 cells which over-express the WT AR, the concentration of 17-LTAS required for maximum silencing of the AR is -5-fold lower than in LNCaP cells (1 .0 ⁇ vs. 5.0 ⁇ ) (Fig. I E).
  • LAPC-4 ceils abiraterone alcohol reduced AR expression to a greater extent than
  • Compound 1 at concentrations greater than or equal to 1 ⁇ .
  • AR mRNA levels were reduced by 38%.
  • no reduction in AR mRNA was observed in LNCaP cells treated with 10 ⁇ Compound 1 (Fig. IF).
  • Example 11 Binding to wild type and mutant AR by Compound I and abiraterone alcohol.
  • the K t can be estimated to be greater than 3 ⁇ .
  • Example 12 Antagonism of AR transcriptional activity by Compound 1 and ahiraterone alcohol. f 00101]
  • the disparate binding of these 17-HASs to the WT and mutant AR proteins suggests that abiraterone alcohol and Compound 1 might also exhibit different abilities to antagonize AR activity.
  • AR-dependent promoter reporter gene activation pARE4-Luc was measured in following transient transfection of PC3 cells that were co-transfected with WT AR. These cells were then treated by Rl 881 and either Compound 1 , abiraterone alcohol or bicalutamide.
  • Compound 1 exhibited a significantly greater ability to block Rl 881 -induced AR transcriptional activation compared with abiraterone alcohol (IC 50 [c ⁇ >rapoand ij 4.7 ⁇ ) (Fig. 3A).
  • IC 50 [c ⁇ >rapoand ij 4.7 ⁇ )
  • Fig. 3A The ability of the 17-HASs to block ligand-dependent AR transactivation against that of bicalutamide as compared.
  • the AR is rapidly translocated to the nucleus upon androgen binding.
  • AR levels were measured in the cytoplasmic and nuclear fractions following pre-treatment of LNCaP cells with Compound 1 prior to exposure to the synthetic androgen R1881 (Fig. 3D).
  • CPD1-/R1881 + In cells pre-treated with vehicle and then exposed to androgen (CPD1-/R1881 +), AR levels in the ucleus were approximately 5-fold higher than in untreated cells (Compound 1 -/R1881 -). While pre-treatment of LNCaP cells with Compound 1 had little effect on AR levels in the cytoplasm, AR levels in the nucleus were reduced approximately 3 -fold in cells pre-treated with Compound 1 (Compound 1+/R1881 -) relative to untreated cells.
  • Example 13 Compound 1 and abiraterone alcohol do not affect the rate ofAR degradation.
  • CHX protein synthesis inhibitor cycioheximide
  • Fig. 4A abiraterone
  • Compound 1 and abiraterone alcohol target translationai machinery to reduce AR protein levels.
  • AR expression can also be regulated at the translationai level through alterations in the cap-dependent translationai machinery (49).
  • AR 5' IJTR- directed luciferase (pIR-AR 5'-UTR-Luc) gene expression was first measured at the translationai level, along with control pIRES-Luc (49, 50). The inhibition of pIRES-Luc and pIR-AR 5 -UTR-Luc by Compound 1 are individually shown in Fig.
  • Fig. 5C demonstrated that Compound 1 , in a dose-dependent manner, reduced the binding of e!F4E and eIF4G to 7 m G-sepharose b 50% compared to untreated control cells (DMSO); this closely correlated w ith reduced levels of AR protein as demonstrated by western blot (Fig. 5C, input). Similar results were obtained with abiraterone alcohol (Fig. 5D).
  • Reid AH Attard G, Danila DC, Oommen NB, Olmos D, Fong PC, Molife LR, Hunt J, Messiou C, Parker C, Dearnaley D, Swennenhuts JF, Terstappen LW, Lee G, Kheoh T, Molina A, Ryan CJ, Small E, Scher HI, de Bono .IS. (2010) J. Clin. Oncol. 28, 1489-1495

Abstract

La présente invention concerne des méthodes de traitement pour le traitement du cancer de la prostate. Dans un aspect, l'invention concerne un procédé de réduction de l'expression d'une protéine de récepteur des androgènes dans une cellule par la mise en contact de la cellule avec un composé 17-hétéroarylstéroïde. Dans certains modes de réalisation, le composé est le Composé 1. Dans certains modes de réalisation, le niveau de protéine du récepteur des androgènes est réduit suivant l'étape de mise en contact de la cellule avec le composé 17-hétéroarylstéroïde. Par exemple, le niveau d'ARNm du récepteur des androgènes est réduit. Dans certains modes de réalisation, la cellule est une cellule LNCaP ou LAPC4.
PCT/US2012/064538 2011-11-10 2012-11-09 Procédés et compositions pour l'inhibition de l'activité du récepteur des androgènes WO2013071177A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014111815A2 (fr) * 2013-01-18 2014-07-24 Cortendo Ab (Publ) Abiratérone et ses analogues pour le traitement de maladies associées à une surproduction de cortisol
WO2014111815A3 (fr) * 2013-01-18 2014-11-06 Cortendo Ab (Publ) Abiratérone et ses analogues pour le traitement de maladies associées à une surproduction de cortisol
CN104017045A (zh) * 2014-06-23 2014-09-03 广州艾格生物科技有限公司 甾体cyp17抑制剂的新型药物前体及其应用、制备方法
CN104017045B (zh) * 2014-06-23 2016-01-13 广州艾格生物科技有限公司 甾体cyp17抑制剂的新型药物前体及其应用、制备方法
US10722527B2 (en) 2015-04-10 2020-07-28 Capsugel Belgium Nv Abiraterone acetate lipid formulations

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