Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/365—Lactones
  • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
  • A61K31/37—Coumarins, e.g. psoralen
• • 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
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
• • 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
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
  • A61K31/569—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone substituted in position 17 alpha, e.g. ethisterone
• • 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
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
  • C07J1/0003—Androstane derivatives
  • C07J1/0014—Androstane derivatives substituted in position 17 alfa, not substituted in position 17 beta

Definitions

• the present invention relates to novel steroid derivatives, which are useful as medicaments.
• the invention also relates to the use of steroid derivatives in the manufacture of a medicament e.g. for the treatment of a benign and/or malignant tumour, such pharmaceutical com- positions, as well as method for treating benign and malignant tumours.
• US patent no. 5,912,240 (Loria) describes the steroid androstene-3 ⁇ ,17 ⁇ -diol (17 ⁇ -AED) and antitumoural effects through inhibiting growth and inducing apoptosis in all neoplastic cell lines. Apoptosis is demonstrated therein in vitro in three neoplastic myeloid cell lineages. In two breast cancer cell lines growth-inhibition is demonstrated. However, no apoptosis was demonstrated in the two breast cancer cell lines.
• Nuclear receptor PPAR ⁇ is a transcription factor belonging to the steroid hormone receptor superfamily. Nuclear receptors link extracellular hormone signals to a transcriptional response. This is done through binding of the receptor to response elements located within promoter regions of target genes. Some nuclear receptors of this family exert only ligand- dependent effects, while others function in the absence of ligands.
• steroid hormone receptor superfamily includes glucocorticoid receptor and receptors for estrogens, androgens, progestins, thyroid hormone, retinoic acid, 9-cis-retinoic acid, peroxisome proliferators, vitamin D and ecdysone.
• the receptor consists of six domains. Counting from the N-terminal A-F, where ligand- independent function (AF-1) resides in A/B and ligand-dependent function AF-2) in E.
• the C- domain is the DNA-binding domain (DBD).
• Waxman (Role of metabolism in the activation of dehydro-epiandrosterone as a peroxisome proliferator, DJ Waxman, J of Endocrinology, vol 150, suppl. Sept 1996) indirectly demonstrated ligand activity of 3 ⁇ -sulfate of 5-androstene-3 ⁇ ,17 ⁇ -diol to PPAR .
• One object of the invention is to provide a novel compound, which is capable of controlling cell cycle arrest and/or an angiostatic effect, which compound is useful as a medicament.
• This and other objects of the invention are achieved as described by the appended claims. More specifically, the invention- relates to administrating a therapeutic dose of such a steroid to cause a down-regulation of cyclin DI or ⁇ -catenin in instances where these factors are overexpressed and present a hindrance to therapy, to remove the cell-cycle block or to down- regulate pathologic vascularization.
• Examples of such instances are colorectal carcinomas which are mutated in the APC-gene in a majority of cases, which causes an upregulation of ⁇ - catenin, a minority of prostate cancers with increased expression of cyclin DI or ⁇ -catenin as well as some mammary cancers with increased expression of ⁇ -catenin of phenotypic reasons.
• the mantle-cell lymphomas known to be resistant to virtually all known treatment, which are characterized by their expression of cyclin DI.
• the effects obtained according to the present invention can be used by them or combined with traditional cytostatic therapy or irradiation.
• it can be useful or necessary to combine the novel steroid according to the invention with ligands to nuclear receptors such as androgens, anti-androgens, estrogens, anti-estrogens, retinoic acid derivatives, del- tanoids, levaxin etc.
• the invention also deals with a novel PPAR ⁇ -ligand and PPAR ⁇ -ligand activity of 3 ⁇ -steroid sulfates, how to promote or to block such activity, and advantageous applications thereof.
• the invention also encompasses the new steroids as such and their effects.
• Figure 1 is a picture of undifferentiated control and tumour treated with 17 +17 ⁇ -AED.
• Figure 2 shows Western blots as discussed in relation to Table IV.
• Figure 3 illustrates effects on VEGF of 17 ⁇ -AED, 17 ⁇ -AED, 17 ⁇ OH-pregnenolone and 5- androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol.
• Figure 4 shows Western blots, illustrating effects of 17 -AED, 17 ⁇ OH-pregnenolone and 17 ⁇ - AED on expression of ⁇ -catenin, cyclin DI and COX-2 in Dunning AT-1, rat prostate tumours.
• Figure 5 shows a comparison of proportion of cells in Gl, S-phase in untreated controls (CA- CD) and after treatment with 17 -AED (alfaA-D) illustrated by representative examples.
• Figure 6 illustrates how 17 ⁇ -AED,170H-pregnenolone, 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ and their respective 3 ⁇ -sulfates were investigated for PPAR ⁇ -ligand activity in a ligand-induced coactiva- tor assay.
• the present invention relates to a novel steroid derivative for use as a medicament.
• the derivative according to the invention is described by either one of the general formulas (I) and (II) below, the only difference between said formulas being the nature of the bond between carbon atoms nos. 5 and 6, which is a double bond in formula (I), as shown below:
• steroids have RnO-substituents in 3 ⁇ -position and R 3 -substituents in 17 ⁇ -position and optional substituents in 7 and 17 ⁇ -position.
• Ri on oxygen at position 3 can be: (i) a hydrogen atom, (ii) an N0 2 , an S03H, an -OP(OH)3, an acyl group, or any other group forming an ester with an inorganic or organic acid, (iii) a protecting group, such as CH 3 , CH 2 OMe, CH 2 0-alkyl (iv) any other aliphatic chain which can be straight or branched, saturated or unsaturated, substituted or unsubstituted, cyclic, including mixed cyclic and aliphatic substituents, saturated or aromatic or heterocyclic substituents containing up to 20 carbon atoms.
• Substituents may be selected from OH, halogen (F, Cl, Br, I), amino, aikylamino or dialkylamino.
• R can form ethers or esters with the steroid.
• R 2 can be hydrogen in formula (II) and can be hydrogen in formula (I) provided R ⁇ s not H, or can be R 'O in or ⁇ -position of the carbon number 7, where R' independently of R t may be any substituent acceptable for R x .
• R 3 is always in 17 ⁇ -position and may be an hydroxyl-group, an acyl-group or may be R ' 'O, where R ' ' may be any other group forming an ether or an ester as described for Ri or any other substituent acceptable for, but independent of R ⁇
• R 4 Js always in 17 ⁇ -position and can be a hydrogen atom, an alkyl group, an acyl-group, an alkoxy group, the latter of the formula R ' ' 'O, wherein R " ' may be any other group forming an ether or an ester or any other substituent acceptable for, but independent of R x .
• R 4 can be an acyl group, in which hydrogen or an alkoxy or alkyl group may be attached to the keto group.
• R 4 can be acetyl (CH 3 CO), as in 170H-pregnenolone, where a methyl is attached to the keto-group.
• This keto-carbon numbered 20 could have any alkyl, alkenyl, aryl, including branched side chains or mixed aromatic and aliphatic side chains, including cyclic saturated hydrocarbons as well as heterocyclic rings or heteroaliphatic chains containing e.g. N, P, O, Si, F, S, Se, CN, halogens and containing up to 20 carbons.
• said steroid is, 17-hydroxy-pregnenolone (17 ⁇ -OH), ⁇ -5-androstene- 3 ⁇ ,17 -diol, ⁇ -5-androstene-7-oxo-3 ⁇ , 17 ⁇ -diol and/or 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol.
• said steroid is the corresponding pregnane- and/or androstane- derivatives.
• the above mentioned effects are independent of any direct apop- totic effect on the cells of said tumour.
• novel steroids according to the invention are useful in medicaments, which when administered to a patient in need of therapy is capable of providing one or more antitumoural effects through interference with the Wnt signaling pathway.
• medicaments are especially advantageous for the treatment of tumours where an overexpression of factors from this pathway due to a mutation in factors regulating this pathway or where a phenotypic overexpression occurs, where tumours have been shown to be resistent to some forms of conventional treatment.
• a medicament which comprises one or more steroids according to the invention is for the treatment and/or prevention of a medical condition selected from the group consisting of colon malignancies with a genetic overexpression, cancers caused by a phenotypic upregulation of cyclin DI and/or ⁇ -catenin (estrogen receptor- negative breast cancers), lung cancers, melanomas, mantle cell lymphomas and other B-cell lymphomas characterized by over-expression of cyclin DI, parathyroid adenomas and can- cers, head and neck tumours of squamous cell origin, oesophagal tumours and tumours and other pathologic conditions dominated by a destructive neovascularisation (diabetic retinopa- thy, exsudative forms of macular degeneration, corneal neovascularisation, vascular tumours as hemangiomas, malignant vascular tumours, midline granulomas and uncontrolled growth of scars as in
• the present invention relates to the use of a steroid derivative of 5- androstene-, 5-pregnenolone or corresponding saturated derivatives (androstane- or pregnane-) in the manufacture of a medicament for the treatment and/or prevention of a benign and/or malignant tumour, which medicament is capable of interrupting disturbances in Wnt- signaling, such as cell-cycle arrest in Gl-phase, and/or providing an angiostatic effect.
• said steroid derivate is described by formula (I) or (II) as shown and defined above.
• R R' and/or R" of said formulas form one or more ether(s) and/or ester(s) with the steroid.
• R 4 can be an acyl group, in which a hydrogen, or an alkoxy or alkyl group, is attached to the keto group.
• R 4 is acetyl (CH CO), where a methyl is attached to the keto group, and this keto carbon in position 20 has an alkyl, alkenyl, aryl, including branched, side chain or a mixed aromatic and aliphatic side chain, including cyclic saturated hydrocarbons as well as heterocyclic rings or heteroaliphatic chains, such as those comprising N, P, O, Si, S, Se, CN, or one or more halogen and comprises up to 20 carbons.
• CH CO acetyl
• the steroid is selected from the group consisting of 17- hydroxy-pregnenolone (17 ⁇ -OH), ⁇ -5-androstene-3 ⁇ ,17 ⁇ -diol, ⁇ -5-androstene-3 ⁇ ,17 ⁇ - diol-7-oxo, 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol, 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol and 5-androstene- 3 ⁇ ,17 ⁇ -dioI-7-one, 5-androstane-3 ⁇ ,7 ⁇ ,17 ⁇ -triol, 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol, 5- androstane-3 ⁇ ,7 ⁇ ,17 ⁇ -trioI, and 5-androstane-3 ⁇ ,17 ⁇ -diol.
• One or more pregnane- and/or androstane-derivative corresponding to the steroid can be used in the manufacture of the medicament according to the invention.
• the medicament produced according to this aspect of the invention useful is for the treatment and/or prevention of one or more medical conditions selected from the group that consists of colon malignancies and other malignancies with a genotypic or phenotypic overex- pression of factors belonging to the Wnt-signaling pathway, such as lung cancers, melanomas, breast cancers, mantle cell lymphomas and other lymphomas as well as a fraction of prostate cancers, characterized by an up-regulation of said factors, head and neck cancers of squamous cell origin, oesophagal cancers, parathyroid cancers or adenomas or other tumours characterized by a disturbance in Wnt-signaling; and conditions dominated by pathologic ne- ovascularisation, such as diabetic retinopathy, exsudative forms of macular degeneration, corneal neovascularisation, and vascular tumours.
• a deregulation of Wnt-signaling exists in the context of prostate cancer a deregulation of W
• In androgen-refractory progression AR is activated also by ligand-independent factors such as epidermal growth factor (EGF) and IL-6. EGF-receptor-activation in turn up-regulates expression of cyclin DI (see references in discussion).
• EGF epidermal growth factor
• IL-6 ligand-independent factors
• EGF-receptor-activation in turn up-regulates expression of cyclin DI (see references in discussion).
• the above described two compounds S4 and S8 are thus of potential value only in a small subfraction of human prostate cancers, express- ing aberrant Wnt-signaling and their usefulness is further limited by their androgenic activity and their metabolisation to other androgens.
• such a process may be limited or stopped through treatment with a steroid according to the invention alone or as pre-surgical or pre- radiological treatment or in combination with cytotoxic drugs, interferons, cytokines or steroid hormones.
• the present invention relates to a method of producing a medicament for the treatment of a benign and/or malignant tumour, comprising the steps of
• the active ingredient of said medicament may be the corresponding androstene (or androstane) derivative or an ester thereof with organic or inorganic acid.
• organic acid it can be comprised of up to 25 carbon atoms (S4 and S8).
• the effect of the medicament can be magnified or prolonged by simultaneously administrated sulphatase inhibitor, such as Coumate®.
• the corresponding androstane derivative (S8, formula shown above) is produced from 17 ⁇ - or 17 ⁇ -AED (inversion of 17 ⁇ - to 17 ⁇ through Mitsunobu reaction, described in M&M) through protection of hydroxy groups with acetate and then reducing the double bound with Raney catalyst.
• said enzyme is DHEA-sulfotransferase or a phenolsul- photransferase.
• the present medicament is a medicament, which enhances the effect of an estrogen receptor- ⁇ (ER- ⁇ ) blockade.
• the invention also includes the medicaments produced according to the method described above per se, as will be exemplified below.
• the medicament produced according to the present method is useful for the treatment and/or prevention of a condition selected from the group consisting of urothelial cancers, gastric cancers, cancers of the smaller intestine, pancreatic cancers, tumours derived from endothe- lial cells, leiomyosarcomas, cancer of the colon, chorioncarcinomas, adenocarcinomas of the lung and liposarcomas, and pathology of the eye tissues, such as cells of the macula and glaucoma
• the invention relates to the use of 5-androstene-17 ⁇ -oI-3 ⁇ -sulfate (17 - AEDS) and/or androstane-17 ⁇ -ol-3 ⁇ -sulfate in the manufacture of an immunomodulating medicament, e.g. for the treatment and/or prevention of an inflammatory disease, such as rheumatoid arthritis, arthrosis, or inflammatory bowel disease, or a disease caused by an exaggerated or persisting T-helper-1 response, such as multiple sclerosis or Guillain Barres syndrome.
• the invention also relates to 5-androstene-17 -ol-3 ⁇ -sulfate (17 ⁇ -AEDS) and/or androstane-17 -ol-3 ⁇ -sulfate for use as medicaments.
• the present medicaments are suitable for administration in either their native form or in the form of a hydrolysable prodrug, i.e. an inactive form of the drug which is easily converted into active drug through hydrolysis in the environment of choice.
• a prodrug may be an ether or an ester of said medicament.
• Such a drug or prodrug is suitably administrated by topical injection, by intratumoural injection, by parenteral administration or intra-arterially, through selective Catheterization of an artery supporting a tumour or a site of pathologic neovascularisation, in the form of a pharmacologically acceptable sterile solution or suspension.
• a simultaneous injection of soluble starch particles of calibrated size (Spherex®) may enhance and prolong the effect of the drug and will also counteract the stimulus to neovascu- larisation and regrowth of the tumour produced by the resulting hypoxia.
• the drug may be used as topical in the form of a pharmacologically acceptable solution, cream or jelly with for instance cyclodextrin applicated to the eye, to the mucosa of mouth, nose, vagina or rectum.
• a compress soaked in drug solution may be used for in- stance in preventing excessive scar-formation.
• the drug may also be taken orally, as rectal or vaginal suppositories, creams or enemas.
• the drug may be taken as an en- tero-capsule, either in the form of suitable sulfated form or as native steroid (S4 or S8) as DHEA-sulphotransferase is present in the smaller intestine.
• the sulfates of drugs S4 or S8 may be given intravesically to the urine bladder through a catheter a demure of a sterile solution in the case of superficial bladder cancer.
• cytotoxic drugs such as anthracyclines, such as doxorubicin, daunorubicin, epirubicin, idarubicin or mitoxantron, vincaalkaloids such as vinblastin, vincristin, vindesin or vinorelbin, taxanes such as docetaxel or paclitaxel, alkylating drugs such as ifosfamid, cyclo- fosfamid, busulfan, thiotepa, nitrosoureas such as lomustine, chlorambucil, dacarbazine, cis- platin, paraplatin or oxaliplatin, topoisomerasell-inhibitors such as etoposid or teniposid, to- poisomerase-I-inhibitors such as topotecan or irinotecan, antimetabolites such as meth-
• cytotoxic drugs such as anthracyclines, such as doxorubicin, daun
• the effect of the novel compounds according to the invention may also be attenuated through the use of corticosteroids, retinoids, deltanoids, thyroid hormones, sex steroids and other nuclear receptor ligands.
• Figure 1 is a picture of undifferentiated control and tumour treated with 17 +17 ⁇ -AED.
• Figure 2 shows Western blots as discussed in relation to Table IV.
• ⁇ rat tumour exposed to 17 ⁇ -AED (which is represented to the left of control to point out the difference in exposure time compared to the other treatments). The drug was allowed to act for 96 hours.
• ⁇ tumour treated with 17 ⁇ -AED, with an 8 times higher dose acting for 456 hours.
• + ⁇ shows sequential treatment with ⁇ for 96 hours followed by ⁇ for 360 hours.
• Figure 3 illustrates effects on expression of VEGF of ⁇ -AED, ⁇ -AED, 5-androstene 3 ⁇ ,7 ⁇ ,17 ⁇ - triol and 17 ⁇ OH-pregnenolone.
• A Untreated controls. Stained section to the left followed by negative control of same tumour.
• B 17alpha-AED; Stained section to the left followed by negative control of same tumour.
• C 17beta-AED; D: From left to right 1-3 tumour samples treated with androstene-3beta,7beta,17alpha-triolstained for VEGF, 4 negative control, same treatment.
• Figure 4 shows protein blots of samples of Dunning AT-1 rat tumours treated with ⁇ -AED, 17 ⁇ OH-pregnenolone and ⁇ -AED as well as untreated samples (C1-C3). Influence of treatment on expression of ⁇ -catenin, cyclin DI and COX-2 is demonstrated by representative examples.
• Figure 5 shows representative examples of the effects on the cell cycle of Dunning AT-1, rat prostate cancer after treatment with 17 ⁇ -AED (alpha A-D) compared to untreated control tumours (CA-CD).
• the untreated tumours show a strong expression of cyclin DI in accordance with a large proportion of the cells in Gl.
• tumours treated with 17 ⁇ -AED there is a decrease in Gl and an increase of cells in S-phase, paralleled by a decrease in cyclin DI.
• the pattern combined with the results from protein blotting and demonstrated lack of apoptosis indicates cell-death of non-apoptotic nature, in S or G2.
• Figure 6 illustrates how ⁇ -AED, 170H-pregnenolone and 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol and their corresponding 3 ⁇ -sulfates were investigated for PPAR ⁇ -ligand activity in a ligand-induced coactivator assay.
• SRC-1 positive control
• SRC-1 negative control rat liver cyto- sol from a male rat.
• the only sample demonstrating PPAR ⁇ -ligand activity is the 5-androstene-17 ⁇ -ol-3 ⁇ -sulfate.
• reaction mixture was warmed slowly to 0°C and stirred for 30 minutes.
• Aqueous NaOH (2M, 6 ml) was added carefully and the mixture extracted three times with diethylether. The combined extracts were dried over MgS0 4 and the solvent evaporated.
• Group 1 a single dose of 80mg ⁇ 5-androstene-3 ⁇ ,17 ⁇ -diol (Sigma Chemicals) s.c.
• Group 2 and 3 a single dose of 10 mg of ⁇ 5-androstene-3 ⁇ ,17 ⁇ -diol, (Steraloid Inc.).
• PEG 400 (Sigma Chemicals) and ethanol, 0,5 ml was used as vehicle and injected s.c. adja- cent to the tumour site.
• group 3 previously treated with 10 mg of ⁇ 5-androstene-3 ⁇ ,17 ⁇ -diol, received a single injection of 80 mg of ⁇ 5-androstene-3 ⁇ ,17 ⁇ -diol in the same way as groupl.
• Immunostaining was done on formalin fixed sections against VEGF, clone C-l, IgG2a, sc- 7269, Santa Cruz. Working dilution 1: 100.
• As negative control antibody a mouse IgG, clone DAK-GOl, kappa, x 931,Dako, was used with a working dilution of 1:50. Tumours were investigated for increased apoptosis with the Apop-Tag in situ apoptosis detection kit (Oncor, Gaithersburg, MD).
• Group 1 received a single dose of 10 mg 17 ⁇ -AED sc. near tumour.
• Group 2 received a single dose of 80 mg 17 ⁇ -AED in the same way.
• Group 3 received a single dose of 25 mg 17 ⁇ OH-pregnenolone in the same way.
• Group 4 received a single dose of 7,5 mg 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol.
• a fifth group served as tumour bearing controls.
• DU-145 Human prostatic cancer cell lines, PC-3 and DU-145, were used. Frozen cell lines were established in culture. DU-145, which was received as a tumour piece, was first disintegrated in a sterile Petri dish, using a pair of scissors. The disintegrated tumour was transferred to a 75 cm 3 cell-culture flask containing medium. For medium, Ham 's F 10 was used for PC-3 and RPMI 1640 for DU-145.
• FBS 10%, 0.2 % NaHC03 (7.5 %), 2mM L-glutamine (200 mM) and 0.005 mg/ml gentamycin (5mg/ml) were added to culturing media for both cell lines.
• Cells were grown in 75 cm 3 cell culture flasks until nearly confluent and culture medium was changed twice weekly.
• the concentration of AED in each culture flask was 100 nM. Controls were exposed to the same concentration of DMSO and ethanol in culture medium.
• a control was prepared, containing no cell lysate but the same amounts of protease assay buffer, lysis buffer and Ac-DEVD-AMC substrate. Fluorescence was measured using a spectrofluorometer (RF 540,Shimadzu Data Recorder DR3, Instrument AB, Lambda) at 415-450 nm with an excitation wavelength of 380 nm.
• Trypan blue exclusion trial lOO ⁇ L aliquots of the initial cell suspension in PBS were used to determine the viability of cells collected by means of the trypan blue exclusion assay. 10 ⁇ L of the cell suspensions were mixed with 10 ⁇ L trypan blue solution (Sigma Chemical Co. # T8154) and left to sit for 3 minutes. The numbers of viable and dead cells were counted in a hemocytometer.
• her-2 antibodies As no preservative was added to her-2 antibodies, they were added at 2 occasions, 24 hours apart during 72 hours of incubation, (taking the limited half-life of the antibody into consideration). Representations of each cell line containing no steroid were also treated with herceptin as above. For each cell line and variation of treatment 3 different culture flasks were used.
• rat liver cytosol a mixture of 100 ⁇ L rat liver cytosol and 20 ⁇ L of 3 '-phosphoadenosine-5 '- phosphosulphate (PAPS), corresponding to approximately 0,16 mg of PAPS was shared be- tween and added to cultures containing 17 ⁇ -AED.
• PAPS 3 '-phosphoadenosine-5 '- phosphosulphate
• estrogen receptor ⁇ is expressed in both cell lines and estrogen receptor ⁇ in PC-3 cells only.
• ICI 172,780 in a 50nm concentration was used and added at two occasions 36 hours apart.
• the estrogen receptor blocked cultures were then treated with +/-HER2 antibodies and +/- 17 ⁇ -AED-3 ⁇ - sulfate in the concentrations and manner as in experiment with PC-3 and DU145 with the exception that estrogen receptors were blocked as described.
• 3T3L1 fibroblasts from ATCC (embryonic muscle cell-line) batch F-12732 (batch-date 930301) were grown in 500 ml of DMEM, 50 ml of fetal bovine serum (FBS) and 11 ml of PEST. Medium was changed every second day. Cells were passaged when they were 80% confluent.
• ATCC embryonic muscle cell-line
• FBS fetal bovine serum
• fibroblasts were incubated in DMEM+ 10% FCS 4- 5 ⁇ g/ml of insulin. 0.1 mM of IBMX and 0.25 ⁇ M of dexametason was added. After 2 days cells were transferred to DMEM+10% FCS + 5 ⁇ g/ml insulin. Medium was then changed every second day and consisted of DMEM + 10% FCS. Ethanol in distilled water was added, keeping the ethanol concentration below 0.1%. This served as positive control for differentiation.
• Fibroblasts were also incubated with the same solutions except that IBMX and dexa- methasone were exchanged with solutions of 17 ⁇ -AED in ethanol and water, making the con- centration of 17 ⁇ -AED 100 or 200 nM in the cultures. Cultures were kept in incubator until confluent.
• Thawed rat tumours from both experiments above were homogenized in ice cold lysis-buffer. (160 mM NaCl, 10 mM HEPES, 2 mM CaCl2, 5% SDS, 0,5% Triton X-100, 100 ⁇ g/ml phenyl- metylsulfonyl fluoride, 1 ⁇ g/ml leupeptin and 2 ⁇ g/ml aprotinin), placed on ice for 15 minutes and following clarification centrifuged for 10 minutes at 13,000 g. Protein content of homoge- nates were determined by Lowry assay.
• Protein in lysates (90 ⁇ g) were separated by electrophoresis using 8% SDS-PAGE and sepa- rated proteins were transferred onto a nitrocellulose membrane (Amersham) in trans-blot electrophoretic transfer cell (Bio-Rad Laboratories).
• TTBS lx TTBS: 20mM Tris, 150mM NaCl, pH 7.5, 0,1% Tween-20
• blots were probed with mouse monoclonal IgGl PPAR ⁇ anti- body (Anti-PPAR-gamma (E8) Santa Cruz Biotechnology) diluted 1:400 in TTBS containing 3% (w/v) non-fat dried milk.
• the blots were striped and rehybridized with antibodies against ⁇ -catenin (C-18, sc. 1496) and COX-2 (C-20, sc 1745) antibodies.
• tumours were investigated for expression of ⁇ -catenin, COX-2 and cyclin DI (A- 12, sc. 8396).
• Protein blotting was repeated for DU-145 and PC-3 cell lines, which were also investigated for expression of PPAR ⁇ through antibody (H-74, sc 7197).
• ApcDNA3-SRC-l construct was used as template to prepare ( 35 S -methionine)-SRC-l by in vitro transcription and translation using the TNT® Coupled Reticulocyte Lysate System (Promega).
• the bacteria were lysed by sonication in TEDG buffer (50mM Tris (pH 7.4), 1.5 mM EDTA, 10% glycerol, 0.4 M NaCl, 0.1 mM DTT) contain- ing the following protease inhibitors: 0.5 mM PMSF, 1 mM benzamidine, lO ⁇ g/ml leupeptin, lO ⁇ g/ml antipain and lO ⁇ g/ml aprotinin.
• the lysates were centrifuged at 100,000 x g for 60 min in a SW 41 rotor using a Beckman L8-70 M ultracentrifuge.
• the fusion protein was immobilized on GSH Sepharose beads (Pharmacia) and then incubated with potential ligands in (Fig.5) lOmM Tris (pH7.4), 0.12 M KCI, 8% glycerol, 4mM DTT, and 0.5% CHAPS (buffer AA) for 30 min at room temperature. Thereafter, ( 35 S methionine) SRC-1 (ca 0.1 ⁇ Ci) was added, and the beads were incubated for another hour at 4°C and then washed extensively with buffer A. SDS sample buffer was added to the beads, and the samples boiled prior to separation on 7.5% SDS-PAGE. Radioactivity was detected by autoradiography.
• Lioand-induced coactivator interaction assay ⁇ - and ⁇ -AED were investigated in above assay for PPAR ⁇ ligand activity in 10 and 100 ⁇ M solutions in ethanol 10%, in distilled water. lO ⁇ L aliquots of ⁇ -AED-solution or ⁇ -AED-solution were also added to a preparation of a male rat liver cytosolic preparation prepared as follows:
• a homogenate of a rat liver was prepared using a food processor.
• the liver homogenate was protected from serine-proteases by the addition of 2mmol of PMSF, lmmol of EDTA and 10 mmol Tris-HCI buffered to a pH of 7,40.
• the homogenate was prepared at 4°C and then centrifuged at 15000g for 10 minutes, followed by centrifugation at lOOOOOg for an hour at 4°C.
• the supernatant was divided into test tubes containing one of the following steroids: 17 ⁇ -AED or 17 ⁇ -AED in 5 ⁇ M concentration together with 100 ⁇ L of liver cytosol.
• One test tube served as positive control and contained SRC-1.
• 17 ⁇ -AED was treated in the method described by Arnostova, Libuse M.; Pouzar, Vladimir; Drasar, Pavel. Inst. Org. Chem. Biochem., Czech. Acad. Sci., Prague, Czech. Synth. Commun. (1990), 20(10), 1521-9, where acetate is used as protection group and pyridine -S0 3 complex as the sulfating agent.
• Deprotection with an excess of 0,8M NaOH in MeOH-water provides the desired hydroxy sulfate, which is investigated, for PPAR ⁇ -activity in the ligand- activated-coactivator receptor assay described above. This shows PPAR ⁇ -activity for the 17 ⁇ - AED-3 ⁇ sulfate.
• rat prostatic tumour changed tumour appearance from anaplastic pattern to a tumour showing glandular differentiation.
• human prostatic carcinomas express PPAR ⁇ and since activation of this receptor can lead to differentiation in other types of tissue expressing PPAR ⁇ , protein blotting with antibodies against PPAR- ⁇ was performed in order to see if an attenuation of the expression of this nuclear receptor could explain the differentiation.
• 3T3-L1 mouse fibroblasts
• a pre-confluent culture of 3T3L1 mouse fibroblast culture was exposed to 100 and 200 nmo- lar 17 ⁇ - or ⁇ -AED to see if a differentiation into adipocytic phenotype would take place.
• caspase- 3 or caspase-7 lacks caspase 3 activity during apoptosis. Instead it has caspase-7 which is less effective in mediating apoptosis than cas- pase-3. Both caspases, however, recognize the same amino acid motif and the substrate used is thus suitable for detecting both) showed a significant decline when DU-145 was grown in the presence of 17 ⁇ -AED compared to growth in 17 ⁇ -AED or controls. Interestingly a significant decrease in total number of cells was noted after treatment with 17 ⁇ -AED in DU- 145 cells. This suggests a growth inhibitory effect of both steroids in DU-145 cells.
• Caspase 3-mediated fluorescence in PC-3 cells treated with 17 ⁇ -AED shows an essentially unaltered intensity compared to untreated controls.
• intensity is corrected for cell numbers there is however a decrease, corresponding to a decrease in apoptosis, which is proba- bly significant.
• For 17 ⁇ -AED approximately a doubling of fluorescence, corresponding to an increase in apoptosis was seen. Looking at total number of cells in the PC-3 cultures reveals an increase in total number of cells with 59% in cultures treated with 17 ⁇ -AED compared to controls.
• table I shows PC-3 and DU-145 cells treated with 17 ⁇ -AED,17 ⁇ -AED or both and num- ber of dead and viable cells compared to untreated controls. Estimations for each group are based on two different measurements from three different culture flasks.
• table II shows the mean fluorescence in DU-145 and PC-3 samples estimated with spectrofluorometry in caspase-3, apoptosis assay.
• Table III shows estimations of viability and apoptosis in HER-2/neu expressing DU-145 and PC-3 androgen-refractory human prostate cancer cell lines and human mammary cancer cell lines MCF-7 (estrogen receptor positive) and SKBR-3 (estrogen receptor negative) and the influence of HER-2/neu antibody herceptin, 3 ⁇ -sulfate of 17 ⁇ -AED and their combination.
• CFA chlorofluoresceindiacetate
• PI propidiumiodide
• SKBR-3 a decrease in viable cells and an increase in dead cells is seen in 17 ⁇ -AED - 3 ⁇ sulfate treated cultures.
• Treatment of cell cultures with HER-2 antibodies shows no signifi- cant effects on the proportion between surviving and dead cells.
• the combination of HER-2 antibodies and 17 ⁇ -AED-3 ⁇ sulfate seems to protect SKBR-3 cells where a significantly increased proportion of cells survives. There is a weak but similar tendency in the other cell types as well, except for DU-145 where this combination gives a reduction in surviving cells.
• Exposure to steroid was 80 hours in all arms (contrary to the previous experiment where tumours were exposed for 96 hrs ( ⁇ -AED) or 456 hours ( ⁇ -AED and ⁇ + ⁇ -AED)).
• tumours treated with ⁇ -AED showed devitalized tumour tissue in circumscribed areas, giving the tumours a patchy appearance on microscopy. Onset of anti- tumoural effect was immediate. Considering the delayed onset of effect in ⁇ -AED treated tumours, and the very slow onset reported using troglitazone in vitro on human prostate cancer cell lines, another mechanism than increase of apoptosis through activation of PPAR ⁇ must be suspected.
• VEGF Vascular Endothelial Growth Factor
• Results can be seen in fig.3 showing 5 ⁇ sections of whole tumours where A. are tumour sections from untreated controls. Every second section is a negative control.
• C. Are tumours treated with 17 ⁇ -AED. Every second sample is a negative control.
• D. Are 5-androstene-3 ⁇ ,7 ⁇ ,17 ⁇ -triol with three treated samples to the left followed by a negative control. To the right three samples treated with 170H-pregnenolone, followed by a negative control.
• Proportion of cells in Gl was 59% in control tumours (55,52,65,67) and 35% (30,41,43) in treated tumours. Comparison of cells in G2 phase was difficult to estimate due to wide distri- bution of values.
• Fig. 4 shows cell-cycle analysis for four different tumours treated with 17 ⁇ -AED compared to four different tumours of untreated controls.
• Cell-cycle analysis is complicated by a considerable part of the cells being necrotic, causing a disturbance to interpretation of cells in G2.
• PPAR ⁇ -ligand activation assay ⁇ - and ⁇ -androstenediols were investigated for PPAR ⁇ -ligand activity in a ligand-induced coac- tivator assay. No sign of transcription pointing to a ligand activity in the investigated andros- tenediols per se was seen.
• Liver-cytosol incubated for two hours with ⁇ - or ⁇ -AED was investigated in ligand-induced coactivator assay. Weak bands with position corresponding to the positive control were present in liver cytosol. An identical result was seen when 17 ⁇ -AED was added to the liver cytosol.
• the experiment is repeated with 17 ⁇ -AED, 17 ⁇ -AED+/- PAPS and liver cytosol +/- heat inactivation by heating liver cytosol to 45°C for 15 minutes to inactivate DHEA-suIfotransferase.
• Fig. 5 shows positive control (SRC-1) to the left, followed by negative control and then native steroids 17 ⁇ -AED, 170H-pregnenolone and 3 ⁇ ,7 ⁇ ,17 ⁇ -androstenetriol + liver cytosol followed by steroids + liver cytosol + PAPS.
• SRC-1 positive control
• FIG. 5 shows positive control (SRC-1) to the left, followed by negative control and then native steroids 17 ⁇ -AED, 170H-pregnenolone and 3 ⁇ ,7 ⁇ ,17 ⁇ -androstenetriol + liver cytosol followed by steroids + liver cytosol + PAPS.
• a significant difference in signal is seen for the combination of 17 ⁇ -AED + liver cytosol + PAPS. All other combinations, except positive control give insignificant responses.
• Loria describes growth inhibition and apoptosis in monocytic tumour cell lines and growth inhibition in mammary cancer cell lines "independent of estrogen- or androgen receptors".
• the mechanism there is no suggestion as to the mechanism, and therefore, the practical applicability of said teachings of Loria is limited.
• Estrogen-receptor ⁇ (ER ⁇ ) on the other hand is expressed in both cell lines. Estrogen-receptor ⁇ (ER ⁇ ) only in PC-3. Treatment of both cell lines with ICI 172,780, which blocks both receptors gives a considerable growth inhibition and cell-death by itself in PC-3 cells.
• PAI-1 is down-regulated and PA thus up-regulated, speaking in favor of an angiogenic rather than angiostatic effect (Thiazolidinediones down-regulate PAI-1 expression in HUVEC: A possible role for PPAR ⁇ in endothelial function: Kato K et al: Biochem Biophys. Res. Commun.1999 May 10; 258(2) : 431-5).
• ⁇ -catenin is part of Wnt-signaling pathway and has been shown to influence cell cycle regulation and entry (Wnt-5a signaling in human mammary cells: Implications for the development of Breast Cancer: Marzieh J ⁇ nsson. Doctoral dissertation, Lund, Sept. 2000).
• tumours treated with 17 ⁇ -AED a growth-stimulatory effect of this compound in tumour was observed for more than a week, before this was interrupted by an immunological antitu- moural response. This effect is independent of estrogen- and androgen-receptor activation, since the used AT-1-tumour completely lacks such receptors. Since the first investigations of ⁇ -catenin and COX-2 expression in tumours treated with 17 ⁇ -AED were not fully reliable, due to the fact that they were based on tumour investigation after 19 days of treatment, whereas the tumours treated with 17 ⁇ -AED where treated only for 96 hours, the experiment was re- peated with 17 ⁇ -AED,17 ⁇ -AED,
• 17 ⁇ -AED which is converted into epitestosterone, dehydro-epiandrosterone, androstenedione or estradiol.
• ⁇ -catenin when up-regulated will enhance the effect of potential ligands on AR. Andros- tenedione, androstenediols, dehydro-epiandrosterone but also estradiol (all possible metabolites of 17 ⁇ -AED) and the common androgen-receptor blockers as for instance bicalu- tamide have been demonstrated to increase transcriptional activity in AR, resulting in disease progression ( ⁇ -catenin affects androgen-receptor activity and ligand specificity: Tru- ica CI et al; Cancer Research. 60(17) : 4709-13,2000 Sepl.).
• C) 17 ⁇ -AED-3 ⁇ -suIfate will be formed with increasing efficiency if the organism is deprived of other potential stimulators of androgen-receptor transcription.
• the mentioned AR- ligands will compete with the sulfate, which is a PPAR ⁇ -ligand for co-factors necessary for receptor-activation, among them SRC-1 and ARA-70.
• Overexpression of cyclin DI is es- timated to 4.2% of prostate cancers according to one source, but is probably more common than so, as androgen-independent progression through EGF-receptor stimulation, which is a common mechanism in disease progression, causes up-regulation of cyclin DI.
• the down-regulation of COX-2 seen when 17 ⁇ -AED and 17 ⁇ -AED are combined can either be a result of combined PPAR ⁇ and ⁇ -activation or a result of PPAR ⁇ -activation.
• Presence of testosterone, dihydrotestosterone and other ligands to AR, such as 17 ⁇ -AED and possibly 17 ⁇ -AED, or at least its metabolite, epitestosterone are likely to counteract the effects of PPAR ⁇ -ligands.
• Flutamide and bicalutamide as means to block AR-activation are doubtful or at least incomplete as androgenic properties are activated in these substances in the presence of ARA-70 as well as in cases of up-regulated ⁇ -catenin.
• Androgen receptors are expressed in many organs. What is known of the down-regulation of the receptor is that it is partly influenced by ER ⁇ , which has a down-regulatory effect. It is of course influenced by diminished access to ligands, such as testosterone or dihydrotestoster- one. It is also downregulated by resveratrol and by activation of the Arylhydrocarbon- receptor.
• IL-4 a cytokine resulting in a T-helper 2 type response, the very opposite to what is useful in tumour immunotherapy.
• the concept to use sequential treatment with first 17 ⁇ -AED followed by 17 ⁇ -AED is thus not useful at all to achieve an antitumoural effect.
• this combination also lacked antitumoural effects, but even stimu- lated tumour-growth.
• ER ⁇ and ER ⁇ calls for an effective receptor blockade. Tamoxifen or raloxifen can be used for this purpose. From what is known today about the controlling function of ER ⁇ there is no need to control the activity of this receptor as this is the receptor that suppresses ER ⁇ -activity through its ligand- 3 ⁇ ,17 ⁇ -androstanediol. In a tumour system, this does not necessarily hold true, as cell-signaling might be defect.
• PPAR ⁇ agonists increase VEGF expression in human vascular smooth muscle cells: Ya- makawa K et al, Biochem. Biophys. Res. Commun. 2000 Mayl9, 271(3) :571-4 ). Decrease in MMP-9 was also observed after treatment with PPAR ⁇ -ligands. Publications mainly report an upregulation of VEGF through PPAR ⁇ . A strong upregulation of VEGF in p53-mutated tumours is also reported.
• the present invention provides evidence to different mechanisms behind the growth-inhibition observed in some neoplastic cell lines and the antitumoural effects seen in vivo in Dunning AT-1 rat prostatic cancer.
• Effects of this type are mainly dependent on sulfotransferase activity and preferably DHEA- sulfotransferase, which is present in the liver, in adrenals, in testes and in small intestine. Its presence in placenta is not yet documented, but the related pregnenolone-sulfotransferase is present. It is likely that a sulfotransferation with much lower substrate specificity occurs also elsewhere, especially with pregnenolone-sulfotransferase and estrogen-sulfotransferase, the latter being present in many tissues.
• the present inventors have demonstrated growth inhibition in 3T3L1 fibroblasts and in ER- negative breast cancer cell line SKBR-3 treated with 17 ⁇ -AED-3 ⁇ -sulfate.
• human androgen-refractory prostate cancer cell lines PC-3 and DU-145 a considerable cell death in PC-3 cells was shown when estrogen-receptors were blocked. No such effect was however seen in DU-145. The latter is possibly dependent on very low expression of PPAR ⁇ in this cell line, which is instead dominated, by a strong expression of PPAR ⁇ .
• tumours known to express PPAR ⁇ and hence expected to respond to 3 ⁇ -sulfate of 17 ⁇ - AED are urothelial cancers, gastric cancers, malignancies derived from endothelial cells, smooth muscle cells, cancer of the colon, chorioncarcinomas, adenocarcinomas of the lung, gastric cancers and liposarcomas as well as several aspects of pathology of the eye tissues, such as cells of the macula and glaucoma which are all influenced by therapy with PPAR ⁇ - ligands.
• Monocytes and lymphocytes are downregulated in their production of proinflamma- tory cytokines (IL-1, TNF- ⁇ and IL-6) as well as cytokines of Thelperl-profile in T-cells (IFN- ⁇ ,
• TNF- ⁇ and IL-2 are promoted consistent with a stimulation of a T- helper 2-profile.
• This also includes inflammatory bowel diseases such as Crohn ' s disease, diseases of the placental tissue, autoimmune, inflammatory diseases such as rheumatoid arthritis, neurodegenerative diseases such as multiple sclerosis and Guillain-Barres syndrome and many others.
• the second mechanism of action is cell cycle regulatory and antiangiogenic and not mediated through PPAR ⁇ , as demonstrated by an even stronger antitumoural effect from 170H- pregnenolone than 17 ⁇ -AED.
• the present invention further encompasses prodrugs of the compounds of the invention, whereby such prodrugs encompass esters as well as other prodrugs encompassing protecting groups on the hydroxy-groups, which protecting groups are cleaved off during metabolism.
• the present compounds are administered in therapeutically effective amounts, and preferably in such amounts as to reach a blood serum concentration of 50 to 500 nM.
• compositions of the invention are prepared in the form of granules, tablets, or injectable solution, containing the active compound together with one or more therapeutically inert excipients.
• the compositions may comprise from 0.5 to 99.5 % by weight of the active drug.
• the compounds may be administered in combination with cationic dextranes, as well as they can be administered in the form of amino substituted compounds, wherein one or more of carbon numbers 7, 11, and 16 may be substituted.

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