US 2004O110733A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0110733 A1 Borlak et al. (43) Pub. Date: Jun. 10, 2004

(54) METHODS AND AGENTS FOR TREATING wherein Said method and agent consist in increasing test CARDOWASCULAR DISEASES osterone concentration in pathological tissues to normal levels or inhibiting and/or eliminating metabolites from (75) Inventors: Jurgen Borlak, Lehrte (DE); Thomas metabolism. Thum, Hannover (DE) The testosterone concentration in pathological tissues can be Correspondence Address: increased to normal levels by administering at least one KURT BRISCOE Substance from the following groups: NORRIS, MCLAUGHLIN & MARCUS, PA. 220 EAST 42ND STREET, 30TH FLOOR testosterone, NEW YORK, NY 10017 (US) Substances with effects similar to those of testosterone; (73) Assignee: Fraunhofer-Gesellschaft ZL Forderung der angewandten Fors testosterone mimetics, chung e.V., Munchen (DE) Substances that enhance testosterone Synthesis, (21) Appl. No.: 10/309,562 Substances that inhibit testosterone metabolism. (22) Filed: Dec. 4, 2002 Metabolites from testosterone metabolism can be inhibited and/or eliminated by administering at least one Substance (30) Foreign Application Priority Data from the following groups: Sep. 4, 2002 (EP)...... EP O209031.5 Substances that bind to the receptor, causing the receptor levels to be regulated and thus normal Publication Classification ized; Substances that bind to the , regulat (51) Int. Cl...... A61K 31/57 ing the androgen receptor-mediated gene expression (52) U.S. Cl...... 514/171 by inhibiting it, as is observed in cardiac hypertro (57) ABSTRACT phy. This invention relates to a method and an agent for treating Areas of application are medicine and pharmaceuticals cardiovascular diseases, especially cardiac hypertrophy, industry. Patent Application Publication Jun. 10, 2004 Sheet 1 of 10 US 2004/0110733 A1

Jun. 10, 2004 Sheet 2 of 10 US 2004/0110733 A1

O

controlLW LVAD

No.control V LVAD LV hypertroph

Patent Application Publication Jun. 10, 2004 Sheet 3 of 10 US 2004/0110733 A1

Figure 3

Androstenedione O.5 O LVAD LW normal LVH LVH + CO LVH, boilt microsomes

0.35 E O.3 St 0.25 O2 0,15 DH 0,1 O,05"6 8 LVAD LW northa VH LVH - LVH, boilt Firasteride microsomes

s E S. LVAD ss LW normal LVH

is

s E 3. 5 normotensive V s SHRW

d

:

Patent Application Publication Jun. 10, 2004 Sheet 4 of 10 US 2004/0110733 A1

Figure 4

Testosterone

Androstered one

1a-HT no.

2a-Frr -e- i6a-HT -CO Gb-HT --> 7a-HT -->

f T Patent Application Publication Jun. 10, 2004 Sheet 5 of 10 US 2004/0110733 A1

Figure 5 A

w is 2000 9. 1800 9 1600 is 1400 1200 -

5 EL 1000 Androgen rec A as 800

s as 600 400 s 200 2 s s control V WAD LV hypertroph

3500

3000 2SOO 2000 Androgen rec 500 1000 500

normo tensive SHR LV W

s 80

o 5 40

20 O control treated (100M testosterone) Patent Application Publication Jun. 10, 2004 Sheet 6 of 10 US 2004/0110733 A1

Figure 6

2000

1 7

150 O

1250 nor Totensively 100O SHRLV

750

2

CYP21 CYP23 Patent Application Publication Jun. 10, 2004 Sheet 7 of 10 US 2004/0110733 A1

Figure 7 A

1 4 0 120 100 80 alpha MHC 60 40 20

control LV WAD LV hypertroph

160 140 120 100 80 alpha MHC 60 40 20

normotensive SHRLV

V

160 1 4 0 120 1 OO alpha MHC 80 60

40

control treated (100 M tes to sterone) Patent Application Publication Jun. 10, 2004 Sheet 8 of 10 US 2004/0110733 A1

Patent Application Publication Jun. 10, 2004 Sheet 9 of 10 US 2004/0110733 A1

Figure 9

Matrix design Reference AGAACANNNTGTTCT AR consensus sequence obtained from Transfac databank AGTACGTGATGT CT Karvonen et al., J Biol Chem, 1997 GGTTCTTGGAGTACT Claessens et al., J Biochem Mol Biol, 2001 TGGTCAGCCAGTTCT Claessens et al., J Steroid Biochem Mol Biol, 2001 GGCTCTTTCAGTTCT Claessens et al., J Steroid Biochem Mol Biol, 2001 AGAACAGCAAGTGCT Cleutjens et al., J Biol Chem, 1996 used matrix for search of potential ARE binding sites

Z20656

Patent Application Publication Jun. 10, 2004 Sheet 10 of 10 US 2004/0110733 A1

Figure 10

human i 450 400 400 350 ss 350 a 300 5 250300 isa S 2so $200 - 5 alphe reductase O 200 5alpharoductase x - 150 g 1so 100 100 50 so O o 2 control LV LWAD LV nofrnotonsvg Shirlw hypertroph W

250 ado is 700 isis a 200 is ae 600 is 150 3 500 a 400 tibiSof 5 : 100 17thsot : 5 soo e 200 50 - aSp tooO 2 for Totesye SHRLW O control LW WAO LW hypertroph e LW

200 s 180 2 a 160 t 120140 is a a 100 80 60

20 O e controllW LWA) LW hypertroph e nortotons welW SHRLW US 2004/0110733 A1 Jun. 10, 2004

METHODS AND AGENTS FOR TREATING Secretion. In other words, administration of to men CARDIOVASCULAR DISEASES has an effect Similar to that of Surgical castration, only that it is caused by hormones. DESCRIPTION 0006. In order to avoid the effects of , chemi 0001) 1. Field of the Invention cally Synthesized are used as receptor blocker. Due to their structure, which is similar to that of testoster 0002 This invention relates to methods and agents for one, they can bind to the androgen receptor, inhibiting the treating cardiovascular diseases, especially cardiac hyper Signal transduction mediated by the androgen receptor. trophy. Areas of application are medicine and pharmaceu Known blockers are cyproteron and cyproteron . ticals industry. Under the influence of antiandrogens, development of the Seminal duct and its accessory glands as well as the penis is 0003 2. Background of the Invention inhibited. In addition, differentiation of the normal pheno 0004 Testosterone is an androgen that is primarily pro type into the male phenotype in the hypothalamus is duced in the Leydig cells of the testis. It can either act blocked. In adult animals, antiandrogens inhibit Spermato directly or, after modification, via enzymes, e.g., after reduc genesis as well as Seminal vesicle and cause prostate and oil tion at position 5 of the Steroid skeleton. Androgenic Sig gland atrophy. Furthermore, antiandrogens increase Secre naling (hormone effect) is responsible for the development tion of FSH- and LH-releasing hormones and thus also of male SeX characteristics and affects metabolic processes release of FSHS and LHS. of the oil glands and accessory reproductive glands (e.g., 0007 An endocrine disorder of the testes caused by prostate, Seminal vesicles). These effects are primarily medi adenomas of the Leydig cells is Seldom and leads to Pubertas ated by 5-alpha- (5-alpha-DHT) (Rosen praecox in adolescents, i.e., an abnormally early puberty. field et al., 1998). Testosterone and numerous derivatives regulate Spermatogenesis, recruit the Substances required for 0008 Indications for androgen therapy include general Spermatogenesis, including fructose, increase protein Syn androgen deficiency, e.g., after castration, male climacteric, thesis, promote bone growth and enhance (O’Donnell impotentia generandi due to oligoZoospermia and impoten et al., 1999; Mason and Morris, 1997). Inside the prostate, tia coeundi. Administration of anabolics (Such as testoster 5-alpha-DHT may induce hypertrophy (Schroder, 1994); one and dihydrotestosterone) causes increased muscle 5-alpha steroid reductase inhibitors are thus used for the building (Sinha-Hikim et al., 2002). Most of the molecular treatment of prostate tumor patients to bring about reduction causes, however, remain unknown. in size of the hypertrophic gland tissue (Schulman, 2001). At 0009 Multicentrical studies currently investigate the present, 5-alpha Steroid reductase inhibitors are used to treat therapeutic benefit of testosterone Substitution during "male hormone-responsive alopecia, prostate hyperplasia and menopause”. Interim analyses Support an increase in bone prostate cancer (Schulman, 2001). Testosterone metabolism and muscle mass and a decrease in body fat after testoster is regulated Via different enzymatic pathways in the body, one replacement. In contrast, administration of 5-alpha including the cytochrome P450 -monooxygenase System, dihydrotestosterone caused a decrease of the testosterone Several 17-beta-hydroxy-Steroid dehydrogenases, 3-beta-hy plasma level as well as the LH and FSH levels. droxy-Steroid dehydrogenase, aromatase and 5-alpha Steroid reductase (FIG. 1). In most of the target organs, testosterone 0010. The biosynthesis of testosterone and dihydrotest is thus reduced to 5-alpha DHT, a biologically highly active osterone is Similar to that of adrenocortical , i.e., via androgen, and, bound to cytoplasmatic receptors, transferred delta-5-pregnenolone from the cholesterol pathway. In into the nuclei, where a hormone-receptor-chromatin com humans, Subsequent Synthesis is predominantly carried out plex is formed (Porto et al., 1995). This initiates an activa via hydroxylation (hydroxypregnenolone) and additional tion of Specific genes, which in turn, induce mRNA synthe metabolism to become dihydroepiandrosterone (DHEA). In sis and the ribosomal translation and, finally, leads to a de most of the target organs, testosterone is reduced to dihy novo protein Synthesis. drotestosterone and Stereo- and regio-Selectively oxidized or hydroxilated via complex oxidation products. Studies by 0005 Both spermatogenesis and androgen secretion are Thum and Borlak (2000) on isolated heart muscle cells of controlled by hormones of the anterior lobe of the pituitary the rat and Subcellular fractions (microSomes) of the human gland; together with androgens follicle-stimulating hor heart have shown that testosterone is metabolized to differ mones (FSHS) promote spermatogenesis by Supporting the ent products (including oxidation, reduction, isomerization). function of Sertoli cells. Luteinizing hormones (LHS) affect Some of these metabolites, Such as dihydrotestosterone, are the Leydig cells and Stimulates androgen release. These capable of binding to androgen receptors. After interaction Secreted androgens have an inhibiting effect both on the between ligand and receptor, the androgen receptor is trans release of FSHs and on that of LHS. The relatively slight located into the nucleus and, after relevant modifications, a inhibiting effect of these androgens is Supported by , non-covalent bond is established between the receptor and which is Synthesized in the testis and converted from test responsive elements of numerous target genes (Porto et al., osterone in the peripheral tissues. Injections of testosterone 1995). Only little is known, however, about the interaction into the hypothalamus cause testicular atrophy and a between different transcription factors including hierarchy decrease in Secretion of a joint releasing hormone for FSH and network as well as protein-protein interactions and the and LH. Androgen release is strongly inhibited by estrogens androgen receptor in transcriptional activation of androgen and, to a lesser extent, by . Via a feedback receptor-responsive genes. There are indications that a tran mechanism, they inhibit the release of gonadotropic hor Sient therapy of preterm and newly born infants with glu mones. Lack of FSHs thus leads to an interruption of cocorticoid dexametasone causes cardiac hypertrophy (Skel Spermatogenesis, and deficiency of LHS Stops androgens ton et al., 1998). In addition, it is known that premenopausal US 2004/0110733 A1 Jun. 10, 2004

Women have a lower risk of cardiovascular disease. droxylation products were observed. In a comparison of might possibly have a cardio-protective effect, because this normal with hypertrophic heart muscle tissue, the Studies protection decreases after the menopause and is accompa show that the expression of genes that code isoforms for nied by a decrease in estrogen level. The to date worldwide different P450 mono-OXygenases, Such S largest study (Women Health Study) with more than 16000 CYP2A%.CYP4A11, P450 aromatase, and also renin, is postmenopausal women investigated the benefit of a hor upregulated (FIG.2). The highly significant induction of the mone combined Substitution therapy (estrogen plus proges P450 mono-oxygenase expression leads to an increased terone). Surprisingly, this therapy led to an increase in the concentration of the translated P450 proteins, which can be risk of cardiac diseases, apoplexy, and invasive breast cancer made responsible for the altered testosterone metabolism. The investigations provide evidence for a dysfunction / by 20% and of thrombosis by 50% (JAMA, 2002). excessive production of 5-alpha-dihydrotestosterone, 0011. It has already been described earlier on that test , and additional Stereo- and regio-Selective osterone is metabolized in Several organs and plays an hydroxy-derivates in human hypertrophic heart tissue, important role in the differentiation of the male phenotype. which play a causal role in the biological Signaling of Metabolites of testosterone include dihydrotestosterone, disease development (either receptor-mediated or -indepen which can establish an effective bound to the androgen dent, FIGS. 3 and 4). By inhibition of 5-alpha reductase receptor, thus inducing its activation. Furthermore, it is with , production of dihydrotestosterone in the known that the androgen receptor controls certain genes in heart tissue is reduced and normalized (FIG. 3). The the transcriptional regulation and thus directly influences the increased testosterone metabolism causes a marked decrease transcriptional activation of these genes. Also, as aforemen in the testosterone Serum level and a Statistically highly tioned, cytochrome P450 -dependent mono-oxygenases Significant (>12-fold) induction of the androgen receptor have crucial functions in the Steroid metabolism and are expression (see FIG. 5). The induction of the androgen responsible for regio- and Stereoselective hydroxylation. receptor is a biological response to the continuously decreas Here, tissue-specific metabolism via cytochrome P450 ing testosterone Serum level in hypertrophic hearts. mono-oxygenases takes on a special role, especially as the different isoforms are not expressed ubiquitously So that 0015 Additional experiments on spontaneously hyper metabolism, and thus also the metabolite profile, varies tensive rats (SHR), an established model of pathological between different tissue types and organs. The effects of hypertrophy, Support the findings obtained with human these metabolites therefore depend on the individual cell hypertrophic hearts (FIGS. 3, 5 and 6). The heart tissue of types and tissues, whereby the androgen receptor plays a the SHR strain also showed a markedly altered testosterone Special role. Consequently, the presence of the androgen metabolism, and a highly significant increase in Stereo- and receptor and the receptor levels are of crucial importance for regio-selective testosterone metabolites was observed (FIG. the biological Signal transfer mediated by androgen and 3). When compared to normotensive rats, a nearly 30-fold androgen-receptor ligands. induction of the androgen receptor can be observed in the hypertrophic heart tissue of the pathological rats (FIG. 5). 0012 Onset and development of cardiac hypertrophy is Similarly, the intensified testosterone metabolism also considered to be influenced by multiple factors, and different caused a decrease in testosterone Serum levels in the heart hypotheses are currently being discussed. On the one hand, muscle, and this lack of testosterone in the tissue induced an overcompensation of heart muscle cells activates different intensified expression of the androgen receptor. A lack of receptors, Such as G-protein-coupled and tyrosine kinase testosterone in tissue causes induction of the androgen dependent receptors, which then cause an intensified Syn receptor in the heart. thesis of proteins that damage the heart muscle. Further more, the production of growth factors increases, Such as an 0016 Further studies provide evidence for a strong increased Secretion of fibroblastic growth factor and trans reduction of the expression of alpha-MHC in pathological/ forming growth factor beta, which in turn causes cellular hypertrophic heart tissue (FIG. 7). Alpha-MHC is respon hypertrophy of the heart muscle cells. The role of hormones Sible for coding a structure protein in the heart muscle and in the development of cardiac hypertrophy, however, is thus already being used as a marker of diseases in remains unknown. differential diagnosis of cardiac hypertrophy in university hospitals. Bioinformatic analyses of the promotor region of 0013 The invention was based on the general aim of the alpha-MHC-coded gene yield proof for the presence of finding novel methods for treating cardiovascular diseases, several androgen receptor binding sites (FIG. 9). This especially cardiac hypertrophy. The objective of the inven indicates that testosterone itself, and also ligands activating tion is to provide agents with which cardiac hypertrophy can the androgen receptor, control alpha-MHC gene expression. be effectively treated. Furthermore, these agents are to be A decrease in testosterone Serum level, characteristic of indicated for treating other cardiovascular diseases. The cardiac hypertrophy, however, leads to a weaker expression invention is based on numerous gene and protein expression of alpha-MHC. Studies. Comparative Studies on explanted hearts of patients with differentially diagnosed cardiac hypertrophy and 0017 Based on these study results, the method and healthy hearts that had not been released for transplantation agents of the present invention for treating cardiovascular were conducted (eight patients with dilated cardiomyopathy diseases, especially cardiac hypertrophy, are disclosed. (DCM); n=5 had a left ventricular assist device (LVAD) implanted for more than six months and n=3 patients had 0018. The invention is employed by the methods DCM combined with cardiac hypertrophy). described in the claims. 0.014 Quite unexpected and surprisingly, these studies 0019. The method for treating cardiovascular diseases, confirm a markedly altered metabolism of testosterone in especially cardiac hypertrophy, consists in an increase in hypertrophic heart tissue. A highly significant increase in testosterone concentration in pathological tissues to normal production of 5-alpha-dihydrotestosterone and additional levels or in inhibiting and/or eliminating metabolites from P450 -catalyzed stereo- and regio-Selective oxidation/hy testosterone metabolism. US 2004/0110733 A1 Jun. 10, 2004

0020 Testosterone concentration in pathological tissues 0040 e) 3-beta-hydroxy-Steroid-dehydrogenase can be increased to normal levels by administering at least inhibitors; and/or one Substance from the following groups: 0041 f) aromatase inhibitors. 0021 testosterone; 0042 AS Synthetic analogue gonadoliberines are prefer 0022 substance with effects similar to those of ably used: BuSerelin, GoSerelin, , Navarelin, teStoSterOne Triptorelin; as antagonists of hormones inhibiting gonadot 0023 testosterone mimetics; ropine-releasing hormones are preferably used Abarelix, Antarelix, , Ganirelix Acetat, Iturelix; and as 0024 substances that enhance testosterone synthesis antagonists of hormones inhibiting lutenizing hormone releasing hormones (LHRH) are preferably used teverelix, a 0025 substances that inhibit testosterone metabo Synthetic decapeptide that contains five aminoacids in lism. D-configuration with the sequence Ac-D-Nal-D-Cpa-D-Pal 0.026 Metabolites of testosterone metabolism can be Ser--Tyr-D-Hci-Leu-Lys-(iPr)-Pro-D-Ala-NH2 inhibited and/or eliminated by administering at least one (C47H10OCIN15O14). Substance from the following groups: 0.043) Substances to be used as cytochrome P450(CYP) 0027 substances that bind to the androgen receptor, mono-oxygenase inhibitors include: amiodarone, cimeti causing the receptor levels to be regulated and thus dine, fluoroquinolone, fluvoxamine, furafylline, methoX normalized; Salen, mibefradile, ticlopidine, thiotepa, , felbam 0028 substances that bind to the androgen receptor, ate, fluoxetine, fluvoxamine, indomethacine, , regulating the androgen receptor-mediated gene lansoprazole, modafinil, omeprazole, paroxetine, topira mate, fluconazole, fluvastatin, isoniazide, lovastatine, par expression by inhibiting it, as is observed in cardiac oxetine, phenylbutaZone, probenecid, Sertraline, Sul hypertrophy. famethoxazole, Sulfaphenazole, teniposide, trimethoprime, 0029. This invention comprises agents for treating car Zafirlukast, clecoxib, , chlorpheniramine, diovascular diseases, especially cardiac hypertrophy, clomipramine, cocaine, doxorubicine, halofantrine, red-ha wherein Said agents contain Substances that increase test loperidol, levomepromazine, , methadone, osterone concentration in pathological tissues to normal mibefradil, moclobemide, quinidine, ranitidine, ritonavir, levels or inhibit and/or eliminate metabolites from testoster terbinafine, diethyl dithiocarbamate, disulfiram, delavirdine, one metabolism. indinavir, nelfinavir, ritonavir, Saquinavir, ciprofloxacin, clarithromycin, diltiazem, erythromycin, , grape 0030 The agents contain at least one substance from the fruit juice, itraconazole, , nefazodone, norfloxa following groups, which increase testosterone concentration cin, norfluoxetine, or troleandomycin. In addition, the inven in pathological tissue to normal levels: tion can be realized with Substances that inhibit the 0031 testosterone, preferably as testosterone esters, expression of genes coding for cytochrome P450(CYP) 19-nortestosterone, , test mono-oxygenase isoforms, such as CYP2A%CYP4A11, osterone undecanoate, , fluoxymester CYP2J2, or P450 aromatase. One, 0044) 5-alpha steroid reductase inhibitors, especially fin 0032 Substances with effects similar to those of asteride, are especially Suitable as Steroid reductase inhibi testosterone, preferably decanoat, closte torS. bole acetate, methenolone acetate, androstendione, ; 0045 acetate is preferably used as isomerase inhibitor. 0033) testosterone mimetics, 0046) Agents preferred as 17-beta-hydroxy-steroid-dehy 0034) substances that enhance testosterone synthe drogenase inhibitors include 3beta-peptido-3alpha-hydroxy sis, preferably Synthetic analogue gonadoliberine 5alpha--17-on-derivates, Substances with a and additional non-Steroid Substances that promote 17-spiro-gamma-lactone group, and 3beta-(N-adamantylm an increased testosterone release as well as antago ethyl-N-butanoyl)aminomethyl-3 alpha-hydroxy-5-alpha nists of hormones inhibiting gonadotropine-releas androstane-17on. ing hormones and lutenizing hormone-releasing hor 0047 is preferably used as 3-beta mones (LHRH); hydroxy-Steroid-dehydrogenase inhibitor, and , 0035 substances that inhibit testosterone metabo fadrozole, , , , and lism, especially Synthesis of dihydrotestosterone; 1,4,5-androstene-3,17-dione are preferably used as aro Substances preferred include: matase inhibitor. 0036) a) cytochrome P450(CYP) mono-oxygenase 0048. In addition, the agents of this invention may con inhibitors; and/or tain one Substance of the following groups, which inhibit and/or eliminate the metabolites of the testosterone metabo 0037 b) steroid reductase inhibitors; and/or lism and their effects: 0038 c) isomerase inhibitors; and/or 0049 Substances that bind to the androgen receptor, 0039 d) 17-beta-hydroxy-steroid-dehydrogenase causing the receptor levels to be regulated and thus inhibitors; and/or normalized; US 2004/0110733 A1 Jun. 10, 2004

0050 substances that bind to the androgen receptor, 0062 Possible contamination with genomic DNA was regulating the androgen receptor-mediated gene prevented by direct addition of RNA extracts to the PCR expression by inhibiting it, as is observed in cardiac cocktail. The PCR reactions were made in the linear ampli hypertrophy. fication range. Amplificates were separated onto a 1.5% agarose gel, Stained with ethidium bromide and photo 0051. Here, the use of antagonists and/or ligands of the graphed on a transilluminator (Kodak Image Station 440). androgen receptor is preferred. Quantification was achieved using the program Kodak 1D 0.052 Preferred antagonist of the androgen receptor are 3.5 Network. Tables 1A and B depict the sequences of the and , and preferred ligands of the oligonucleotides used for amplifying the different genes. androgen receptor include chiral and non-chiral analogue 0063 Quantitative Real-Time RTPCR bicalutamides, chiral and non-chiral analogue bicalutamides with a trifluoride methyl group, and chiral and non-chiral 0064. The studies were conducted with a lightcyler analogue bicalutamides with a Sulfide group. (Roche Diagnostics, Mannheim, Germany). One ul of the cDNA diluted at a ratio of 1:10 was mixed with 0.5uM of the 0053. Furthermore, the agents of the invention may con specific primer, 4mm MgCl2, and 2ul Master-SYBR-Green tain alpha-MHC and/or Substances that increase the expres PCR Mix (Roche Diagnostics, Mannheim, Germany) and sion of alpha-MHC. adapted to a final volume of 20l. After initial denaturation at 95 C. for 30s, PCR reaction was started at 55 C.-58 C. 0054. In the following, the invention is further illustrated (annealing) for 7s, followed by an extension phase of 12-20s by reference to examples and figures. at 72 C. and a subsequent denaturation phase of 1s at 95 C. Table 1 shows the oligonucleotide Sequences. By the end Study Description of each extension phase, fluorescence was measured to be used for evaluation. To guarantee that no unspecific primer 0055 General dimers are measured, fluorescence detection was conducted 0056 Animal experiments above the temperature characteristic of primer dimers (as assessed in preliminary Studies). Amplification was followed 0057 Male normotensive Sprague-Dawley and sponta by a melting curve analysis, measuring fluorescence by neously hypertensive rats (SHR, weighting approx. 200g) by slowly increasing the temperature of the DNA synthesis Charles River (Sulzfeld, Germany) were used. Before Sur products from 65° C. to 95°C. (0.1° C./s). The exact amount gery, the animals were anesthetized with intreaperitoneal of the obtained PCR product was determined by co-ampli administration of ketamin (anesthetic, 0.1ml/100g body fication of Standardized cardiac cDNA and according to the weight), xylazin-hydrochloride (muscle relaxant; 0.05ml/ manufactures instructions. 100g body weight) and heparin (2000 i.e. heparin). EXAMPLE 1. 0.058 Isolation of total RNA and reverse transcription 0065. In cell culture experiments with isolated heart into cDNA muscle cells from the rat, addition of testosterone to the 0059 Total RNA was isolated from heart tissue using the culture medium reduces and normalizes androgen receptor Total RNA. Isolation Kit (Macherey-Nagel, Germany) expression (FIG. 5c). according to the manufacturers instructions. The quality of EXAMPLE 2 the isolated RNA was assessed by evaluating the ribosomal bands on agarose gel (1%). Of each sample, 2ug RNA was 0066 Experiments on rat heart muscle cells provide used for reverse transcription. RNA and random primer evidence for the therapeutic effect of a testosterone Substi (Roche Diagnostics, Mannheim, Germany) were preheated tution, causing a normalization of alpha-MHC expression at 70° C. for 10 min. and subsequently incubated with (FIGS. 7 and 8). 5xRT-AMV buffer (Roche Diagnostics, Mannheim, Ger EXAMPLE 3 many), dNTPs (1 mM, Roche Diagnostics, Mannheim, Ger many), 40U RNAse inhibitor (Stratagene, Amsterdam, 0067. An established cardiac hypertrophy starts to Netherlands) and 20UAMV-RT (Promega, Mannheim, Ger regreSS after, e.g., implantation of a left Ventricular assist many) at a total volume of 20ul for 60 min. at 42° C. Next, device (LVAD). Studies with LVAD implantation tissue are the added enzymes were inactivated by denaturation for 5 especially valuable because the patients are first differen minutes at 95 C. tially diagnosed with cardiac hypertrophy which, due to the assist device, regreSSes after considerable time has passed, 0060. Thermocycler RT Polymerase Chain Reaction normalizing the heart's function. Therefore, testosterone (PCR) metabolism in LVAD-Supported hearts and in patients under conventional treatment was investigated. When comparing 0061 PCR reactions were carried out in a thermocycler healthy patients with those with LVAD implants, metabo (T3, Biometra, Germany) under the following conditions: lism of testosterone into different metabolites (5-alpha-DHT, Denaturation at 94 C. for 30s, annealing at 57 C. for 60s different hydroxylation products) in hypertrophic heart was and extension at 72 C. for 60s to amplify the cytochrome markedly increased (FIG.3). We therefore provide evidence P450 isoforms, alpha-MHC (33 cycles each) and cyclophilin for a causal relationship between cardiac hypertrophy and an (26 cycles). Annealing temperature for detecting the andro altered Steroid metabolism of testosterone. gen receptor, 17beta-hydroxy-Steroid-dehydrogenase 1-4, 3-beta-hydroxy-steroid-dehydrogenase 1, P450 aromatase, EXAMPLE 4 renin, c-jun and 5-alpha reductase was 55 C. for 60s, and 0068. It is shown that decreased testosterone metabolism 30-35 PCR cycles were carried out. in healthy and LVAD-treated heart tissue is coupled with a US 2004/0110733 A1 Jun. 10, 2004

decreased expression of different cytochrome P450 mono clinical relevance. Studies on isolated heart muscle cells oxygenases (CYP2A%CYP2J2, CYP4A11) (FIG. 2). provide clear evidence for a therapeutic effect of testoster one itself, as has been shown with the example of the 0069. In addition, when compared to hypertrophic hearts, alpha-MHC-coded gene. The repression of the alpha-MHC the genes c-jun, renin, 5-alpha reductase and the expression gene expression observed during the clinical Study corre of the androgen receptor are significantly repressed in lated well with cardiac hypertrophy and has thus been LVAD-supported hearts (FIGS. 2 and 10), while expression accepted as biological marker. Further Studies on explanted of alpha-MHC in healthy and LVAD-supported heart tissue, heart and experimental investigations in the rat model pro which leads to reduced intraventricular pressure, is greater vide evidence for a deregulation of the androgen receptor in (FIG. 7). This proofs that the dysfunctioning steroid the hypertrophic heart. The induction of the androgen recep metabolism in cardiac hypertrophy can be normalized after tor observed in the hypertrophic heart is a biological LVAD implantation and that a regreSS of cardiac hypertro response/adaptation to the markedly decreased testosterone phy after LVAD implantation can also be clinically diag level in the heart tissue. Testosterone and 5-alpha-dihy nosed using echocardiography (Zafeiridis et al., 1998). drotestosterone activate the androgen receptor differently, 0070. In Summary, steroid metabolism in human hyper regulating different androgen receptor-responsive genes. trophic hearts and, in addition, also in pathological rat Thus, due to the different ligands of the receptor (testoster models is significantly altered, a finding that came unex one versus dihydrotestosterone), different target genes are pectedly and Surprisingly. The experiments that have led to activated. In the case of cardiac hypertrophy, leSS receptor is the invention document a pathological induction of CYP present to bind to the responsive elements of the alpha-MHC mono-oxygenases and enzymes involved in testosterone gene's promoter, thus deregulating alpha-MHC. A decreased metabolism and Studies with tissues and Subcellular frac expression of alpha-MHC, however, leads to negative inot tions from biopsy materials of hypertrophic and LVAD ropism and lower cardiac output, as is clinically observed in implanted heart tissues show that the investigations are of advanced cardiac hypertrophy and cardiac insufficiency.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 &2 11s LENGTH 10 &212> TYPE PRT <213> ORGANISM: Artificial &22O > FEATURE <223> OTHER INFORMATION: LHRH analogue. &22O > FEATURE <221 NAME/KEY: Modified <222> LOCATION: (1) . . (1) <223> OTHER INFORMATION: The alanine at position 1 is N-acetyl-D-3- (2-naphthyl)-Ala. &22O > FEATURE <221 NAME/KEY: Modified <222> LOCATION: (2) ... (2) <223> OTHER INFORMATION: The phenylalanine at position 2 is D-3- (4-chlorophenyl)-Ala. &22O > FEATURE <221 NAME/KEY: Modified <222> LOCATION: (6) . . (6) <223> OTHER INFORMATION: The lysine at position 6 is D-6-carbamoyl lysine. &22O > FEATURE <221 NAME/KEY: Modified <222> LOCATION: (3) . . (3) <223> OTHER INFORMATION: The alanine at position 3 is D-3 (3-pyridyl)- Ala. &22O > FEATURE <221 NAME/KEY: Modified <222> LOCATION: (8) . . (8) <223> OTHER INFORMATION: N-epsilon-isopropyl-Lys. &22O > FEATURE <221 NAME/KEY: Modified <222> LOCATION: (10) . . (10) <223> OTHER INFORMATION: The amino acid at position 10 is D-Ala-NH2. <400 SEQUENCE: 1 Ala Phe Ala Ser Tyr Lys Lieu Lys Pro Ala 1 5 10 US 2004/0110733 A1 Jun. 10, 2004

-continued <210> SEQ ID NO 2 &2 11s LENGTH 15 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE THER INFORMATION: Matrix derived using GEMS Launcher Release 3.0 Software. &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (1) . . (1) <223> OTHER INFORMATION: d may be either a g or t. &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (3) . . (3) <223> OTHER INFORMATION: n may be either a g c or t. &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (4) ... (4) <223> OTHER INFORMATION: w may be either a or t. <400 SEQUENCE: 2 dignwogggga gttct 15

What is claimed: 5. Agents of claim 4, wherein 1. A method for treating cardiovascular diseases, espe Said agents contain at least one Substance from the fol cially cardiac hypertrophy, wherein lowing groups, which increase testosterone concentra testosterone concentration in pathological tissues is tion in pathological tissue to normal levels: increased to normal levels or metabolites from test testosterone, osterone metabolism are inhibited and/or eliminated. 2. The method of claim 1, wherein Substances with effects similar to those of testosterone; testosterone mimetics, testosterone concentration in pathological tissues is increased to normal level Substances that enhance testosterone Synthesis, by administering at least one Substance from the follow Substances that inhibit testosterone metabolism. ing groups: 6. Agents of claims 4-5, wherein Said agents preferably contain testosterone in the form of testosterone, testosterone esters, 19-nortestosterone, testosterone Substances with effects Similar to those of testosterone propionate, , mesterolone, and . testosterone mimetics, 7. Agent of claims 4-5, wherein Substances that enhance testosterone Synthesis Said agents contain Substances with effects Similar to those of testosterone, preferably nandrolone decanoat, Substances that inhibit testosterone metabolism. clostebole acetate, acetate, androstenedi 3. The method of claim 1, wherein one, dehydroepiandrosterone. metabolites of testosterone metabolism can be inhibited 8. Agent of claims 4-5, wherein and/or eliminated by administering at least one Sub Said agents contain Substances that enhance testosterone stance from the following groups: Synthesis, preferably Synthetic analogue gonado liberines and additional non-Steroid Substances that Substances that bind to the androgen receptor, causing promote increased testosterone release as well as the receptor levels to be regulated and thus normal antagonists of hormones inhibiting gonadotropine-re ized; leasing hormones and lutenizing hormone-releasing Substances that bind to the androgen receptor, regulat hormones (LHRH). ing the androgen receptor-mediated gene expression 9. Agents of claims 4-5 and 8, wherein by inhibiting it, as is observed in cardiac hypertrophy Buserelin, Goserelin, Leuprorelin, Navarelin, Triptorelin 4. Agents for treating cardiovascular diseases, especially are preferably used as Synthetic analogue gonado cardiac hypertrophy, wherein liberines. Said agents contain Substances that increase testosterone 10. Agents of claims 4-5 and 8, wherein concentration in pathological tissues to normal levels or Abarelix, Antarelix, Cetrorelix, Ganirelix Acetat, Iturelix inhibit and/or eliminate metabolites from testosterone are preferably used as antagonists of hormones inhib metabolism. iting gonadotropine-releasing hormones. US 2004/0110733 A1 Jun. 10, 2004

11. Agents of claims 4-5 and 8, wherein 15. Agents of claims 4-5 and 12, wherein teverelix, a Synthetic decapeptide that contains five ami cyproteron acetate is preferably used as iSomerase inhibi noacids in D-configuration with the Sequence Ac-D- tor. Nal-D-Cpa-D-Pal-Ser--Tyr-D-Hci-Leu- Lys-(iPr)- 16. Agents of claims 4-5 and 12, wherein Pro-D-Ala-NH2 (C47H1 OOCIN 15014) are 3beta-peptido-3alpha-hydroxy-5-alpha-androstan-17-on preferably used as antagonists of hormones inhibiting derivates, Substances with a 17-spiro-gamma-lactone lutenizing hormone-releasing hormones (LHRH). group, 3beta-(N-adamantylmethyl-N-butanoyl)ami 12. Agents of claims 4-5, wherein nomethyl-3 alpha-hydroxy-5-alpha-androstane-17-on Said agents contain the following Substances that inhibit are preferably used as 17-beta-hydroxy-steroid-dehy testosterone metabolism, especially Synthesis of dihy drogenase inhibitor. drotestosterone, preferably: 17. Agents of claims 4-5 and 12, wherein a) cytochrome P450(CYP) mono-oxygenase inhibitor; cyproterone acetate is preferably used as 3-beta-hydroxy and/or Steroid-dehydrogenase inhibitor. b) steroid reductase inhibitor; and/or 18. Agents of claims 4-5 and 12, wherein tamoxifen, fadrozole, aminoglutethimide, formeStane, c) isomerase inhibitor; and/or testolactone, 1,4,5- androsatriene-3,17-dione are pref d) 1 7-beta-hydroxy-steroid-dehydrogenase inhibitor; erably used as aromatase inhibitor. and/or 19. Agents of claim 4, wherein e) 3-beta-hydroxy-steroid-dehydrogenasen inhibitor; and/ Said agents contain at least one Substance from the fol O lowing groups that inhibit and/or eliminate metabolites f) aromatase inhibitor. from testosterone metabolism and their effects: 13. Agents of claims 4-5 and 12, wherein Substances that bind to the androgen receptor, causing agents preferably used as cytochrome P450(CYP) mono the receptor levels to be regulated and thus normal oxygenase inhibitor include amiodarone, cimetidine, ized; fluoroquinolone, fluvoxamine, furafylline, methoX Substances that bind to the androgen receptor, regulat Salen, mibefradile, ticlopidine, thiotepa, cimetidine, ing the androgen receptor-mediated gene expression felbamate, fluoxetine, fluvoxamine, indomethacine, by inhibiting it, as is observed in cardiac hypertro ketoconazole, lanSoprazole, modafinil, omeprazole, phy. paroxetine, topiramate, fluconazole, fluvastatin, iso 20. Agents of claims 4 and 16, wherein niazide, lovastatine, paroxetine, phenylbutaZone, Said agents contain probenecid, Sertraline, Sulfamethoxazole, Sul faphenazole, teniposide, trimethoprime, Zafirlukast, antagonists of the androgen receptor, and/or clecoxib, chlorpromazine, chlorpheniramine, clomi ligands of the androgen receptor. pramine, cocaine, doxorubicine, halofantrine, red-ha 21. Agents of claims 4 and 16-17, wherein loperidol, levomepromazine, metoclopramide, metha done, mibefradile, moclobemide, quinidine, ranitidine, flutamide and bicalutamide are preferably used as antago ritonavir, terbinafine, diethyl dithiocarbamate, disul nists of the androgen receptor. firam, delaVirdine, indinavir, nelfinavir, ritonavir, 22. Agents of claims 4 and 16-17, wherein Saquinavir, ciprofloxacin, clarithromycin, diltiazem, chiral and non-chiral analogue bicalutamides, chiral and erythromycin, gestodene, grapefruit juice, itraconazole, nion-chiral analogue bicalutamides with a trifluoride mifepristone, nefazodone, norfloxacin, norfluoxetine, methyl group, and chiral and non-chiral analogue troleandomycin and/or Substances that inhibit the bicalutamide with a Sulfide group are preferably used as expression of those genes coding cytochrome P450 ligands of the androgen receptor. (CYP) mono-oxygenase isoforms, Such as 23. Agents of claims 4 and 16-19, wherein CYP2A5/7CYP4A11, CYP2J2, or P450 aromatase. Said agents contain alpha-MHC and/or Substances that 14. Agents of claims 4-5 and 12, wherein enhance expression of alpha-MHC. 5-alpha-Steroid-reductase inhibitor, preferably finasteride, is preferably used as Steroid-reductase inhibitors. k k k k k