WO1998003183A1

WO1998003183A1 - Use of 5,6-dihydro-5-azacytidine in the treatment of prostate cancer

Info

Publication number: WO1998003183A1
Application number: PCT/US1997/013102
Authority: WO
Inventors: Daniel D. Von Hoff; Elzbieta Izbicka
Original assignee: Ilex Oncology, Inc.
Priority date: 1996-07-22
Filing date: 1997-07-22
Publication date: 1998-01-29
Also published as: AU4046197A

Abstract

A method for treating prostate cancer, comprising administering an effective amount of 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof, either alone, or in combination with hormonal therapy. The invention includes a method for increasing expression of the androgen receptor in a prostate cancer cell, a method of increasing E-cadherin expression in a prostate cancer cell, and a method of inducing apoptosis in a prostate cell.

Description

USE OF 5,6-DIHYDRO-5-AZACYTIDINE IN THE TREATMENT OF PROSTATE CANCER

Priority of Invention This application claims priority of invention under 35 U.S.C. § 1 19(e) from U.S. Provisional Application Number 60/022,042, filed 22 July 1996.

Background of the Invention

There are approximately 200,000 new cases of prostate cancer and approximately 38,000 deaths from prostate cancer in the U.S. annually according to CA-A Cancer Journal for Clinicians, 44(1), Jan/Feb 1994. Prostate cancer is the second most common malignancy among men and accounts for 13% of all male cancers. Recent studies suggest that the androgen receptor (AR) "has a critical biologic role in a subgroup of patients with advanced prostate cancer." (Wilson, J. D. (May, 1995), N. Engl. J. Med.. 322(21): 1440- 1441). However, the occurrence of AR-negative prostate cancer is not well documented.

Patients with metastatic prostate cancer are primarily treated using chemical or surgical hormone manipulation therapies. Unfortunately, most patients relapse as a result of the outgrowth of androgen-independent tumor cells. There is a need therefore for new therapies to overcome or avoid this characteristic of prostate cancer. Recent scientific findings suggest that the resistance of prostate cancer to hormone therapies may result from changes in androgen receptor gene expression caused by hypermethylation.

Transcriptional inactivation of some genes closely correlates with hypermethylation of the cytosine-rich region of gene promoters, known as CpG islands. Immortalized and malignant cells show increased cytosinc DNA methyltransferase (C-MT) activity, which is responsible for promoter hypermethylation. Falette and Watts found no relationship between estrogen receptor (ER) gene expression and ER gene methylation pattern, however, recent studies directly linked the methylation of the CpG islands in the ER gene promoter region with the loss of ER expression in cultured human breast cancer cell lines (Falette, N.S. et al. (1990), Cancer Res.. 50:3974-3978; Watts, C. K. W. et al. (1992), Mol. BioL 41 :529-536; Ottaviano, Y. L. et al. (1994), Cancer Res.. 54:2552-2555). Hypermethylation of the CpG islands is catalyzed by cytosine DNA methyltransferase (C-MT). C-MT expression and activity are significantly elevated in progressive stages of colon cancer (Issa, J. P. et al. (1993), J. Natl. Cancer Inst.. 85:1235-1240). Ferguson reverted ER(-) breast cancer cells to the ER(+) phenotype by demethylation with azacytidine derivatives, which inhibit C-MT activity (Ferguson, A. T. et al. (1994), Cancer Res.. 54:2279-2283).

Expression of E-cadherin (E-cad), the "invasion-suppressor" gene is silenced by hypermethylation in human carcinomas and in human breast and prostate carcinomas (Yoshiura, K. et al. (1995), Proc. Natl. Acad. Sci. USA. 92:7416- 7419;Graff, L. R. et al. (1995), Cancer Res.. 55:5195-5199). The latter study used azacytidine (azaC) to partially restore E-cad expression. These in vitro findings suggest that hypermethylation could account for transcriptional inactivation of a single gene and demethylation could reactivate the expression of those genes.

Unfortunately, there is still no known therapy that can convert hormone refractory prostate cancer to hormone sensitivity so that the tumors will respond to conventional hormonal manipulation. Such a therapy would satisfy a long felt need in the art.

Summary of the Invention The present invention seeks to overcome deficiencies in the art by providing a treatment of prostate cancer that can cause the reversion of a hormone refractory prostate cancer cell to a hormone sensitive cell. Without limiting the present invention to a any single theoretical basis, it is contemplated that androgen independence of prostate cancer cells is associated with a lack of expression of the androgen receptor, possibly due to methylation of a gene control region mediated by cytosine-DNA-methyltransf erase (C-MT). The methods of the present invention utilize 5,6-dihydro-5-azacytidine, an inhibitor of C-MT, to restore expression of the androgen receptor, among other effects, and to cause the reversion of the cell to hormone sensitivity, thereby rendering the cell treatable with luteinizing hormone-releasing hormone (LHRH) agonists, androgen antagonists or other hormone manipulation. An aspect of the present invention, therefore, is a combination therapy using DHAC with conventional hormone therapy in the treatment of hormone refractory prostate cancer.

In a certain broad aspect, the present invention may be described as a method of increasing expression of the androgen receptor in a prostate cancer cell comprising contacting the cell with 5,6-dihydro-5-azacytidine under conditions effective to increase expression of the androgen receptor. As shown herein, androgen receptor expression is increased in both androgen receptor negative and androgen receptor positive cells. Therefore, in light of this data, and in view of the other effects described herein, (increase in apoptosis and increased expression of E-cadherin), it is understood that treatment with DHAC may be beneficial for hormone refractory tumors and for hormone sensitive tumors as well.

Another aspect of the present invention is a method of increasing E- cadherin expression in a prostate cancer cell, comprising contacting the cell with 5,6-dihydro-5-azacytidine. An increase in E-cadherin levels is an indication that a prostate tumor is expected to be more defined and less aggressive (better behaved). Therefore, an increase in E-cadherin as described herein is a further and important benefit of contacting a prostate tumor or cell with DHAC. A surprising benefit of the disclosed methods is also an increase in apoptosis in prostate cancer cells upon contact with DHAC. Because prostate cancer represents, in some way, a failure of the apoptotic mechanism of the cell, an increase in apoptosis of the cancer cells is a promising benefit of the disclosed treatment methods.

In the methods described herein, the preferred cell to be contacted with DHAC is a human neoplastic prostate cell, and preferably the cell is contained in a subject and most preferably in a human prostate cancer patient. An effective amount is described herein as an in vitro concentration of from a detectable level to about 10 μM 5,6-dihydro-5-azacytidine. The in vitro level is that amount that is demonstrated to be effective in an in vitro embodiment, and is indicative of the level to be achieved at the site of the prostate cell in a subject. In certain broad aspects, the invention may be described as a method of treating a prostate tumor, including metathesis, in a subject comprising administering 5,6-dihydro-5-azacytidine to said subject in an amount effective to convert a hormone refractory tumor to a hormone sensitive tumor, or as a method of increasing E-cadherin expression in a prostate tumor in a subject, or even as a method of increasing apoptosis in prostate tumor cells in a subject.

The method comprises administering to a subject an effective amount of DHAC, wherein an effective amount is about 100 to about 2000 mg/m² per day. In certain preferred embodiments, this amount is administered for about 5 to 7 days, every three weeks to a month as determined by the practitioner. It is contemplated that the daily dosage may be higher in some cases, in particular when the duration of administering is shorter, even including up to 8 grams/m² in a single day. However, due to the side effects associated with DHAC, the preferred method is a lower dosage over a period of 5-7 days.

Brief Description of the Drawings

FIG. 1 shows the expression of the androgen receptor in LNCaP cells after seven days at various concentrations of DHAC.

FIG. 2 shows the expression of the androgen receptor in DU145 cells after seven days at various concentrations of DHAC. FIG. 3 shows the expression of the androgen receptor in MPC3 cells after seven days at various concentrations of DHAC.

FIG. 4 shows C-MT activity in LNCap cells after treatment at various concentrations of DHAC.

FIG. 5 shows C-MT activity in DU145 cells after treatment at various concentrations of DHAC. FIG. 6 shows the levels of apoptosis in DU145 cells (TdT assay) at 1, 3 and 5 days of culture after treatment with DHAC.

FIG. 7 shows the levels of apoptosis in LNCap cells (TdT assay) at 1 , 3 and 5 days of culture after treatment with DHAC. FIG. 8 shows cell growth in DU145 cells after treatment with DHAC.

FIG. 9 shows cell growth in LNCap cells after treatment with DHAC.

FIG. 10 shows upregulation of androgen receptor expression in all four tested lines of human prostate cells.

FIG. 1 1 shows the regulation of AR gene expression and C-MT activity by DHAC in TSUPrl prostate carcinoma.

Detailed Description The present invention may be described in a broad aspect as a method of treating prostate cancer by the administration of 5,6-dihydro-5-azacytidine. This treatment is shown herein to have multiple effects that are contemplated to be beneficial to a prostate tumor patient. One such effect is the upregulation of the androgen receptor protein. It is the down-regulation of androgen receptor that is thought to be the cause of hormone refractory tumors, which do not respond to hormonal manipulation. The increase in expression of the androgen receptor caused by contacting those cells with 5, 6-dihydro-5 -azacytidine is expected to result in a reversion of a hormone refractory prostate tumor to hormone sensitivity. Results obtained and disclosed herein with prostate cancer cell lines that are both androgen receptor positive and negative demonstrate the ability of this method to increase androgen receptor expression. Other broad aspects of the present invention include methods of increasing apoptosis in prostate cancer cells by contacting those cells with DHAC. This surprising result is an important embodiment of the present invention, particularly if those results are tumor cell specific.

An important embodiment of the present invention is a method of increasing expression of E-cadherin in a prostate cancer cell by contacting the cell with DHAC. E-cadherin (E-cad) is reported to suppress tumor cell invasion and metastasis in experimental tumor models and E-cad expression is low in poorly differentiated, advanced stage carcinomas (Graff, L. R. et al. (1995), Cancer Res.. 55:5195-5199). This loss of expression may be due to hypermethylation of the E-cad gene promoter region, and therefore, administration of DHAC may have the effect of hypomethylation of that region, resulting in increased expression of E-cad and a less aggressive tumor.

Without limiting the invention to any one mechanism, the benefits of the present methods are contemplated to be at least in part due to an effect on the activity of cytosine DNA methyltransferase (C-MT), as shown herein. Various genes are upregulated or down-regulated as part of the process of conversion of a normal cell to a cancer cell, and DHAC is shown herein to be effective in altering the expression of various cancer genes, possibly by affecting the activity of C-MT. A further aspect of the present invention is a method of causing the reversion of hormone insensitive prostate tumors and metastases to hormone sensitivity, thus allowing the tumor to be treated or controlled by conventional hormone manipulation therapy.

Hormone therapies useful for treating prostate cancer include, orchiectomy (castration) and treatment with agonists of lutenizing hormone- releasing hormone (LHRH) (e.g. leuprolide acetate or goserelin acetate). Such therapies may additionally be augmented by administration of an agent which inhibits the action of androgens (e.g. bicalutamide or flutamide). G. Alivizatos and G. Oosterhof, Anti-Cancer Drugs, 4, 1993, 301-309. Because 5,6-dihydro- 5-azacytidine and its salts convert androgen independent cancer cells to androgen dependent cells, which are amenable to hormone therapy, 5,6-dihydro- 5-azacytidine or its pharmaceutically acceptable salts may preferrably be administered in combination with hormone therapies. Accordingly the invention also provides a method for treating prostate cancer in a mammal (e.g. a human), comprising administering 5,6-dihydro-5-azacytidine or a pharmaceutically acceptable salt thereof, in combination with hormone therapy. The invention also provides a method for treating prostate cancer in a mammal (e.g. a human), comprising administering 5,6-dihydro-5-azacytidine or a pharmaceutically acceptable salt thereof, in combination with an agent that inhibits the action of androgens. It is to be understood that according to the invention, when 5,6- dihydro-5-azacytidine or a pharmaceutically acceptable salt thereof, is administered in combination with an LHRH agonist or in combination with an agent which inhibits the actions of androgens, or in combination with both such agents, the individual agents (including 5,6-dihydro-5-azacytidine or a pharmaceutically acceptable salt thereof) may be administered concurrently, alternately, or cyclically, or in any combination thereof, provided the combined administration of the agents produces a beneficial therapeutic effect. The present disclosure includes examples in which four human prostate carcinoma cell lines (LNCaP, DU145, MPC3, and TSUPrl) were incubated for a week with 5 μM azaC or 5 μM 5,6-dihydro-5-azacytidine (DHAC). Expression of the AR protein was determined in cell lysates by a ligand competition assay, while C-MT activity was measured by a method used by Issa and colleagues (Issa, J. P. et al. (1993), J. Natl. Cancer Inst.. 85: 1235-1240). The data suggest that when the prostate cancer cells were treated for a week with 5 μM DHAC, the expression of the AR gene increased in all groups, especially in the AR(+) cells (LNCaP) (FIG. 10). In TSUPrl, upregulation of the AR expression was clearly paralleled by an inhibition of C-MT activity (FIG. 1 1 ). Three of the human prostate cancer cell lines (MPC3, DU145, and

TSUprl) express low levels of the AR receptor, and one (LNCaP) expresses high levels of the receptor. The cells are treated in culture with two types of C-MT inhibitors: 5-azacytidine (azaC) and 5,6-dihydro-5-azacytidine (DHAC). To determine the optimal conditions for treatment, the cells are cultured in the presence of 10% fetal calf serum for 3, 5, and 7 days with a single dose of

DHAC or azaC (0.1, 0.5, 2.5, and 10 μM). In a pulse protocol, DHAC or azaC (0.1 , 0.5, 2.5, and 10 μM) is freshly added to the cells every 2 days for a total of 8 days. Control groups in each cell line have no inhibitor added. At the end of each time point, the cells are harvested and cell pellets are collected by centrifugation and several assays are performed. AR protein expression may be determined by a ligand competition assay. The pellet is solubilized and the cytosolic fraction is isolated by ultracentrifugation. The cytosol is incubated with ³H dihydrotestosterone (DHT) in the presence of excess unlabeled DHT to determine non-specific binding. The amount of specifically bound DHT is measured by liquid scintillation counting.

AR gene expression is determined by RT PCR and northern blots. Messenger RNA is extracted from the pellet using Fast Trac (Stratagene) reagent. Complementary DNA (cDNA) is synthesized form an mRNA template and the polymerase chain reaction (PCR^®) is done using human-specific oligonucleotide probes for the AR. Positive controls (β-actin) and negative controls are included. Northern blots are hybridized with ³²P labeled cDNA probes for the AR. Quantitation of the binding is done by video imaging.

Cytosine-DNA methyltransferase (C-MT activity is also assayed. The pellet is lysed in the presence of protease inhibitors and treated with RNase to deplete RNA, which inhibits C-MT activity. The C-MT activity is determined using poly(dI/dC) and ³H S-adenosyl methionine as the substrates, as described by Issa and colleagues (Issa, J. P. et al. (1993), J. Natl. Cancer Inst.. 85: 1235- 1240).

To examine whether DHAC and azaC affected the expression of other genes, DU145 and LNCaP cell lysates, which had previously tested positively for increased expression of the AR, were electrophoresed in polyacrylamide gels and transblotted to nitrocellulose. The blots were developed with rabbit anti-E- cadherin polyclonal antibody. The results showed that DHAC and azaC increased the expression of E-cadherin. This discovery indicates that the methods of using DHAC disclosed herein are not limited to a single mechanism, but DHAC may act as a multifunctional agent for the treatment of prostate cancer. This agent and other C-MT inhibitors like DHAC, in addition to increasing the expression of the AR gene, might also help in restoring the expression of "invasion-suppressor" genes such as E-cadherin. Therefore, the benefits of restored expression of the AR gene would be enhanced by the restoration of the cell's natural defense mechanisms. DHAC

DHAC or 4-amino-3,6-dihydro-l-β-D-ribofuranosyl-l,3,5-triazin-2(lH)- ine-monohydrochloride was developed as an alternative to 5-azacytidine, an antimetabolite with demonstrated antitumor activity in children and adults with acute non-lymphocytic leukemia. A method of preparing DHAC from 5- azacytidine by reduction of the 5,6-double bond of 5-azacytidine is described in U.S. Patent 4,058,602 (incorporated herein in its entirety by reference). DHAC demonstrated similar in vitro and in vivo activity to that of 5 -azacytidine although higher doses are required. The major dose limiting toxicity of 5- azacytidine given as a bolus dose is nausea and vomiting which can be overcome by a five day continuous infusion. 5-azacytidine is typically administered intravenously from 50 to 400 mg/m²/day for five days at intervals of 2 to 3 weeks (Remington's Pharmaceutical Sciences, 18th edition, 1990, Mack Publishing Company, Easton, PA, 18042 pi 158). However, 5-azacytidine is unstable in aqueous solution. Therefore, Beisler synthesized DHAC to overcome 5-azacytidine's water instability and to allow for continuous infusion without concern for drug hydrolysis in aqueous formulation (Beisler, J. A. et al. (1 77), J. Med. Chem.. 20:806-812).

5,6-Dihydro-5-azacytidine is normally available as a white lyophilized powder. Intact vials are stable for at least 4 years at room temperature or at 4°C. A 500 mg vial may be reconstituted with 9.6 ml of Sterile Water for Injection, USP. This results in a clear solution containing 50 mg of DHAC and 30 mg of mannitol at a pH of 3 to 5. The drug may be diluted in multiple types of intravenous infusion solutions. The reconstituted solutions are stable for at least 48 hours at room temperature. DHAC is also stable for 48 hours when diluted into 5% dextrose, 0.9% sodium chloride and Lactated Ringer's Injection, USP.

DHAC is not a prodrug of 5 -azacytidine, but is incorporated into nuclear RNA. It inhibits methylation of ribosomal and transfer RNA. It also inhibits transcription of ribosomal RNA and nuclear RNA. The overall effect is a decrease in the synthesis of methylated bases into RNA and a decrease in protein synthesis. DHAC can also substantially reduce DNA methylation levels. Accordingly, in addition to being useful for treating prostate cancer, DHAC may be useful for the treatment of other diseases wherein hypermethylation is implicated. Such diseases include but are not limited to thalassemia major and myodisplastic syndrom. The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

As shown herein, 5,6-dihydro-5-azacytidine (DHAC) upregulated the expression of the androgen receptor (AR) in LNCaP (FIG. 1) and DU145 (FIG. 2) cells. The expression of AR increased in DU145 cells from an undetectable level to 1.5 femtomoles/mg cytosol protein. The expression of AR in MPC3 cells was not significantly affected (FIG. 3). In addition, C-MT activity was strongly inhibited by DHAC in LNCaP cells (80% inhibition ) (FIG. 4) and was apparently increased in DU145 cells (FIG. 5). In comparison with untreated cells as a control, 5 μM DHAC stimulated apoptosis in DU145 cells at day 3 (>200%) (FIG. 6) and in LNCaP cells at day 5 (>300%) (FIG. 7). At the same time, the growth rate in DU145 were unaffected by DHAC (FIG. 8). In LNcCaP, DHAC decreased the growth by 44% (FIG. 9). Tissue Culture Methods

The study utilized four prostate cancer cell lines: DU-145, TSUprl, and MPC3, which have been reported to be androgen receptor (AR) negative, and the LNCaP cell line which is AR positive. The cells were cultured in RPMI 1640 medium (BRL Gibco) in the presence of 10% FBS and 1% L-glutamine at 37°C in 5% CO₂ atmosphere. Triplicate Tl 50 flasks for each drug concentration and for untreated controls were seeded with 5-7 x 10⁶ cells to yield confluent flasks at day 7. The cells were treated in culture with 5,6-dihydro-5-azacytidine hydrochloride (DHAC) (Ben Venue Laboratories, Inc., Bedford, Ohio), obtained through Ilex Oncology, San Antonio, Texas. DHAC was added to the cells to achieve a final concentration of 2.5, 5.0, and 10.0 μM. On day 7, the cells were detached with 0.25% trypsin in EDTA, washed with PBS, and pelleted. The pellets from each line were divided into fourths. One fourth was used in the CMT assay and the remaining three fourths were used in the androgen receptor assay. The pellets were stored at -70°C and thawed only once.

Androgen Receptor Assay

Pellets from prostate cells were homogenized on ice in chilled 10 mM Tris pll 8, 1.5 mM EDTA, 20 mM sodium molybdate, 10% glycerol, and 5 mM DTT (TEM₂₀DG) buffer in a glass-Teflon homogenizer with a motor-driven pestle at a proportion of 3 ml buffer per 0.1 ml packed cells. The homogenate was centrifuged at 40,000 rpm for 30 minutes, the supernatant containing cytosol proteins was incubated with pellet from a half volume of dextran-coated charcoal (DCC) for 10 minutes, and centrifuged 10 minutes at 2,000xg. Triplicate 200 μl aliquots of cytosol were incubated overnight with 2 x 10^~9 M (³H)-DHT (130 Ci/mMol). Parallel triplicates contained 2 x 10^'7 M unlabeled DHT to determine nonspecific binding. The reaction was stopped by adding 500 μl DCC suspension, the tubes were mixed every 5 minutes for 15 minutes and then centrifuged for 10 minutes at 800xg. The samples (500 μl) were counted with Ready Protein Liquid Scintillation Cocktail (Beckman) in a Beckman LS-5000 liquid scintillation counter for 10 minutes. Protein determination was done by the method of Lowry. The results were expressed as femtomoles of the AR per mg of cytosol protein. Cvtosine-DNA methvltransferase fC-MTI activity assay The cytosine-DNA methyltransferase (C-MT) activity assay was done according to the modified method described in Issa, J. P. et al. (1993), J. Natl. Cancer Inst.. 85:1235-1240). Human prostate cell pellets were homogenized with disposable plastic pestles in 1.5 ml Eppendorf tubes containing 500 μl of 50 mM Tris (pH 7.8), 1 mM EDTA, 0.1% sodium azide, 10% glycerol, 1% Tween 80, 1 mM dithiothreitol, 6 mg/ml phenylmethyl sulfonyl fluoride, and 100 μg/ l Rnase A (lysis buffer). The homogenate was passed several times through a 27 gauge needle and aliquots were removed for protein measurement by measuring the absorbance of the samples at 280 nm (A_2g0) against the lysis buffer. For DNA C-MTase activity determination, 5 μg of the cellular protein lysate was mixed with 0.5 μg of a synthetic DNA polymer, poly (d(I-C) (Pharmacia Biotech, Piscataway, NJ) and 3 μCi S-adenosyl methionine (80 Ci/mmol) in a total volume of 20 μl and incubated at 37°C for 2.5 hours. The reaction was stopped by adding 300 μl of 1% aminosalicylate, 5% butanol, 125 mM NaCl, 0.25 mg/ml salmon sperm DNA, and 1 mg/ml proteinase K. The samples were vortexed briefly and incubated another 30 minutes at 37°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation. Samples were placed at -70°C for 15 minutes and centrifuged at 12,000xg at 4°C for 5 minutes. The pellet was washed twice in 1 ml ice cold 70% ethanol, and air dried. RNA was removed by resuspension in 25 μl of 0.3 M NaOH and incubation at 37°C for 30 minutes. Twenty μl aliquots were spotted on Whatman GF/C filters, dried at 80°C for 5 minutes, washed in 1 ml ice cold 5% TCA, then 1 ml ice cold 70% ethanol, placed in 3 ml of EcoLume scintillation fluid, and counted in a Beckman scintillation counter for 2 minutes. All assays were done in triplicate. Terminal deoxynυcleotidyl transferase assay (TdT assay

The terminal deoxynucleotidyl transferase assay (TdT) detects apoptotic cells by direct fluorescence detection of digoxigenin-labeled genomic DNA. In the assay the addition of digoxigenin-11-dUTP and dATP to DNA is catalyzed by TdT. The heteropolymer product is detected with fluorescein-labeled anti- digoxigenin antibody. Upon excitation at 494 nm, an intense emission signal is generated at 523 nm. For each time point (day 1, 3, and 5) and for each DHAC concentration (0, 2.5, 5.0, and 10 μM), one T75 flask was seeded with 1.5 million cells. The drug was added on day 0. On the appropriate day, the cells were harvested, counted, then centrifuged and incubated for 10 minutes in 4% formalin. The cells were then spread on silanized slides and air dried. The slides were washed in PBS, and incubated with TdT and the digoxigenin-labeled substrates (Oncor-ApopTag kit) and incubated for 1 hour at 37°C. After incubation the cells were washed in a stop/wash buffer for 30 minutes and then antidigoxigenin-fluorescein was added for 30 minutes. The cells were counterstained with propidium iodide/Vectashield and viewed under a fluorescence microscope. A minimum of 500 cells were counted and the percentage of apoptotic cells was calculated.

Example 2 For the in vivo studies, nude mice are implanted with human prostate cancer cells that express low and intermediate [AR(-)], or high levels [AR(+)] of the androgen receptor gene. The animals are treated with variable doses of cytosine DNA methyltransferase (C-MT) inhibitors and varying schedules of administration and exposure to the agents, to determine the effects of C-MT inhibitors on the expression of AR in primary and metastatic tumor sites, tumor burden, number of metastases, body weight and animal survival. The preferred inhibitor is 5,6-dihydro-5-azacytidine (DHAC), which may be compared to the more toxic 5-azacytidine.

The drug's ability to increase expression of the androgen receptor in the Dunning Mat Ly-Lu rat prostate cancer line is tested. These cancer cells are androgen receptor negative and highly aggressive. The parent rat prostate cancer cell line, HI-F, which is androgen insensitive but has detectable levels of androgen receptors is also tested. When treatment with drug increases androgen receptor levels, the effect on animal survival is tested by hormone ablative therapy (surgical castration).

DHAC has been used in a mouse myeloma model at a dose of 200 mg/kg. However, the dosage in the present study is optimized as follows: Eighteen animals are inoculated with tumor and randomized into three groups of six to receive 100 mg/kg or 200 mg/kg IP in a single daily dose for five days. The third group receives no drug. Two animals from each group are sacrificed at 7 days, 14 days and 21 days. Tumors are harvested and androgen receptor levels are measured.

When the optimum dose has been established, 80 animals are injected with tumor cells into the hind limb and randomly assigned to 4 groups of 20 as follows: a) untreated animals b) untreated animals castrated on day 10 c) DHAC treated animals d) DHAC treated animals castrated on day 10.

Treated animals are given a daily dose of DHAC beginning on day zero to day four, for a total of five doses. Animals are closely monitored for tumor size, and if large volumes (>20 cc) occur, the animals are sacrificed. Animals are followed until all untreated animals are sacrificed. Tumors are harvested in all groups for receptor analysis. If treated, castrated animals show decrease in tumor size and prolonged survival, they are maintained until there is a statistical difference in survival curves over the other groups, at which time all surviving animals are sacrificed. The protocol is done first in the aggressive Mat Ly-Lu cell line, followed by an identical study in the Hi-F line.

Example 3 Clinical trials of DHAC in the treatment of prostate cancer are designed and conducted as follows.

Patients with AR(-) biopsy-proven prostate cancer which is refractory to hormonal treatment are treated with DHAC and then re-biopsied to determine the AR status of their tumors. If their tumors are converted to the AR(-) state, the patients are treated with hormonal manipulation therapy and observed for response to that therapy.

Two trials are conducted for a period of about 18 months with approximately 35 patients in each trial. The trial design is administration of DHAC, followed by assay of the androgen receptors and then treatment with an anti-androgen such as flutamide. The trial endpoints are patient response and conversion of tumor cells from AR-negative to AR-positive. It is contemplated that those tumors that are converted to AR-positive are then responsive to hormone therapy. DHAC may be administered on a monthly or three week schedule as a continuous infusion of 5 to 8 g/m² given in 24 hours, or infused over a period of up to 5 days at about 100 to about 2000 mg/m² per day in 5% dextrose and possibly 0.9% NaCl in water. Patients may also be administered narcotic analgesics and/or nonsteroidal anti-inflammatory agents if pain is a problem (Carr, B. I. et al. (1987), Cancer Research. 47:4199-4201 ; Creagan, E. T. et al. (1993), Am J. Clin. Oncol.. 16(3):243-244). It is also understood that the dosage and frequency may be adjusted according to patient tolerance of the drug as well as disease response.

AH publications, patents and patent documents are incorporated by reference herein, as though individually incorporated by reference. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions, methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope and concept of the invention as defined by the appended claims.

Claims

What Is Claimed Is:

1. A method of increasing expression of the androgen receptor in a prostate cancer cell comprising contacting said cell with 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof, under conditions effective to increase said expression.

2. A method of increasing E-cadherin expression in a prostate cancer cell comprising contacting said cell with 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof.

3. A method of inducing apoptosis in a prostate cell comprising contacting said cell with 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, 2 or 3, wherein said cell is a human neoplastic prostate cell.

5. The method of claim 1, 2 or 3, wherein said cell is contained in a subject.

6. The method of claim 1 wherein said effective conditions include an in vitro concentration of from a detectable level to about 10 μM of 5,6-dihydro-5- azacytidine, or said salt.

7. A method of treating a prostate tumor in a subject comprising administering 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof, to said subject in an amount effective to convert a hormone refractory tumor cell to a hormone sensitive tumor cell.

8. A method of treating a prostate tumor in a subject comprising administering 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof, to said subject in an amount effective to increase E-cadherin expression in a prostate tumor cell.

9. A method of treating a prostate tumor in a subject comprising administering 5,6-dihydro-5-azacytidine, or a pharmaceutically acceptable salt thereof, to said subject in an amount effective to increase apoptosis in a prostate tumor cell.

10. The method of claim 7, 8 or 9 wherein an effective amount is about 100 to about 2000 mg/m² per day.

1 1. The method of claim 10 wherein said administering is for a period of from about 5 to about 7 days.

12. The method of claim 7, further comprising administering hormone therapy to said subject.

13. A method for treating prostate cancer in a mammal, comprising administering to said mammal, an effective amount of 5,6-dihydro-5- azacytidine, or a pharmaceutically acceptable salt thereof.

14. A method for treating prostate cancer in a mammal, comprising administering to said mammal, an effective amount of 5,6-dihydro-5- azacytidine, or a pharmaceutically acceptable salt thereof, in combination with hormone therapy.

15. The method of claim 12 or 14 wherein the hormone therapy comprises an orchiectomy.

16. The method of claim 12 or 14 wherein the hormone therapy comprises administering an agonist of lutenizing hormone-releasing hormone.

17. A method for treating prostate cancer in a mammal, comprising administering to said mammal, 5,6-dihydro-5-azacytidine or a pharmaceutically acceptable salt thereof, in combination with an agent that inhibits the action of androgens.

18. The method of claim 15 or 16 further comprising administering an agent that inhibits the action of androgens.

19. The method of claim 16 wherein the agonist of lutenizing hormone- releasing hormone is leuprolide acetate or goserelin acetate.

20. The method of claim 17 or 18 wherein the agent that inhibits the action of androgens is bicalutamide or fiutamide.