WO2010120686A2 - Procédés destinés au diagnostic et au traitement de l'hyperplasie bénigne de la prostate - Google Patents

Procédés destinés au diagnostic et au traitement de l'hyperplasie bénigne de la prostate Download PDF

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WO2010120686A2
WO2010120686A2 PCT/US2010/030736 US2010030736W WO2010120686A2 WO 2010120686 A2 WO2010120686 A2 WO 2010120686A2 US 2010030736 W US2010030736 W US 2010030736W WO 2010120686 A2 WO2010120686 A2 WO 2010120686A2
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mcp
mab279
monoclonal antibody
bph
activity
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PCT/US2010/030736
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WO2010120686A3 (fr
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J. Kellogg Parsons
Christian P. Pavlovich
Kazutoshi Fujita
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Parsons J Kellogg
Pavlovich Christian P
Kazutoshi Fujita
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Publication of WO2010120686A2 publication Critical patent/WO2010120686A2/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1or LDCF-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention is directed to methods and devices for the diagnosis of benign prostatic hyperplasia, especially methods and devices for distinguishing benign prostatic hyperplasia from other diseases and conditions affecting the prostate, such as prostatitis and prostatic carcinoma.
  • BPH is characterized by hyperplasia of prostatic stromal and epithelial cells, resulting in the formation of large, fairly discrete nodules in the periurethral region of the prostate. When sufficiently large, the nodules compress the urethral canal to cause partial, or sometimes virtually complete, obstruction of the urethra which interferes with the normal flow of urine.
  • BPH The occurrence of BPH is definitely a function of age. Adenomatous prostate growth is believed to begin at approximately 30 years of age in most men. An estimated 50% of men have histological evidence of BPH by age 50 years and 75% by age 80 years. In 40-50% of these patients, BPH becomes clinically significant.
  • BPH can be a progressive disease, especially if left untreated. Incomplete voiding results in stasis of bacteria in the bladder residue, which can lead to an increased risk of urinary tract infections.
  • Urinary bladder stones can be formed from the crystallization of salts in the residual urine; the formation of such stones can lead to severe pain and other symptoms.
  • Urinary retention termed acute or chronic, is another form of progression of BPH. Acute urinary retention is the inability to void, while in chronic urinary retention the residual urinary volume gradually increases and the bladder distends. Some patients who suffer from chronic urinary retention may eventually progress to renal failure, a condition termed obstructive uropathy.
  • DHT Dihydrotestosterone
  • 5 ⁇ -reductase This enzyme is localized principally in the stromal cells; hence, these cells are the main site for the synthesis of DHT.
  • DHT can act in an autocrine fashion on the stromal cells or in paracrine fashion by diffusing into nearby epithelial cells. In both of these cell types, DHT binds to nuclear androgen and signals the transcription of growth factors that are mitogenic to the epithelial and stromal cells. DHT is 10 times more potent than testosterone because it dissociates from the androgen receptor more slowly. The importance of DHT in causing nodular hyperplasia is supported by clinical observations in which an inhibitor of 5 ⁇ -reductase is given to men with this condition. Therapy with 5 ⁇ -reductase inhibitor markedly reduces the DHT content of the prostate and in turn reduces prostate volume and, in many cases, BPH symptoms.
  • treatment is typically fairly conservative, such as lifestyle changes such as avoidance of caffeine and alcohol, and the use of medications such as alpha-blockers ( ⁇ i -adrenergic receptor antagonists) such as doxazosin, terazosin, alfuzosin, and tamsulosin, as well as 5 ⁇ -reductase inhibitors such as finasteride or dutasteride.
  • alpha-blockers ⁇ i -adrenergic receptor antagonists
  • doxazosin doxazosin
  • terazosin terazosin
  • alfuzosin alfuzosin
  • tamsulosin as well as 5 ⁇ -reductase inhibitors
  • 5 ⁇ -reductase inhibitors such as finasteride or dutasteride.
  • Saw palmetto extracts (Serenoa repens) and other herbal extracts may also be beneficial.
  • TUMT transurethral microwave thermotherapy
  • TUNA transurethral needle ablation
  • TRTP surgical transurethral resection of the prostate
  • This is a surgical procedure involving removal of part of the prostate through the urethra.
  • a procedure can cause complications such as bleeding, thrombosis, hyponatremia due to bladder irrigation, and urinary tract complications such as impotence.
  • prostatic carcinoma prostatic adenocarcinoma
  • Prostatic carcinomas while typically presenting with no significant symptoms or symptoms comparable to BPH, is a much more serious disease.
  • Prostatic carcinomas while often slow- growing, can grow more rapidly and metastasize to other organs, such as the bones, lymph nodes, rectum, and bladder.
  • Treatment of prostatic carcinoma is typically far more invasive and aggressive than for BPH, involving surgery, radiation, chemotherapy, and other treatment modalities.
  • Prostatitis is an inflammation of the prostate, frequently associated with acute or chronic bacterial infection, although it can result from other causes. Prostatitis can cause symptoms similar to those of BPH, although it can also be associated with other symptoms, such as pelvic pain in men.
  • such a diagnostic method would be precise and would avoid false positives (diagnosis of BPH when the patient does not have this condition) and false negatives (diagnosis of no BPH when, in fact, the patient has the condition).
  • such a diagnostic method would avoid subjective determinations based on imprecise impressions such as the size of the prostate as felt in rectal examination, would be noninvasive, and would be rapid and simple to perform.
  • MCP-1 Monocyte Chemoattractant Protein-1
  • One aspect of the invention is a method of diagnosing BPH comprising the steps of:
  • the assay for MCP-1 is an immunoassay, such as a sandwich immunoassay.
  • the sandwich immunoassay can be an ELISA assay.
  • Other immunoassay techniques such as the enzyme multiplied immunoassay technique (EMIT), a cloned enzyme donor immunoassay (CEDIA), an immuno- PCR assay, a phosphor immunoassay, a quantum dot immunoassay, a solid phase, light-scattering immunoassay, or a surface effect immunoassay, can be used. If the immunoassay is a sandwich immunoassay, it can be performed in a lateral flow device.
  • the reference value for the concentration of MCP-1 in prostatic fluid is about 5000 intensity units as measured in densitometry in a conventional MCP-1 immunoassay.
  • the method comprises determining a ratio of the level of MCP-1 in prostatic fluid to the total weight of the prostate gland, such that a ratio of greater than about 6.0 intensity units/g prostate gland weight is diagnostic of BPH.
  • the method comprises determining a ratio of the level of MCP-1 in prostatic fluid to the total protein in the prostatic fluid, such that a ratio of greater than about 400 intensity units/ ⁇ g total protein is diagnostic of the existence of BPH.
  • the sample can be urine. If the sample is urine, typically the urine sample is collected after prostatic massage. In one alternative, any detectable MCP-1 concentration is diagnostic of BPH. In another alternative, a MCP-1 concentration of at least 10 pg/ml is diagnostic of BPH.
  • Another diagnostic method within the scope of the invention is a method for diagnosing BPH comprising the steps of:
  • Another aspect of the present invention is a method for treating or preventing BPH comprising the step of administering a therapeutically effective quantity of an agent that inhibits the expression or activity of MCP-1 to a patient who has or is at risk of developing BPH in order to inhibit the expression or activity of MCP-1 to a sufficient degree that BPH is treated or its occurrence prevented.
  • the agent that inhibits the expression or activity of MCP-1 inhibits the expression of MCP-1
  • the agent is an inhibitory nucleotide selected from the group consisting of short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, and complementary DNA (cDNA).
  • the agent that inhibits the expression of MCP-1 is an angiotensin receptor blocker (ARB).
  • the agent that inhibits the expression or activity of MCP-1 inhibits the activity of MCP-1
  • the agent is an antibody that specifically binds MCP-1.
  • the agent is an antibody that specifically binds CCR2.
  • the agent is an antagonist of CCR2, such as, but not limited to, 1'-[2-[4-(trifluoromethyl)phenyl]ethyl]-spiro[4H- 3,1-benzoxazine-4,4'-piperidin]-2(1H)-one hydrochloride.
  • Another aspect of the present invention is a pharmaceutical composition for the treatment or prevention of BPH comprising:
  • the agent that inhibits the expression or activity of MCP-1 is as described above.
  • Another aspect of the present invention is a method of screening for a compound inhibiting the expression and/or activity of MCP-1 comprising the steps of:
  • the cells that demonstrate a detectable response to MCP-1 are cells that endogenously express and secrete MCP-1.
  • the cells that demonstrate a detectable response to MCP-1 are cells that express a receptor for MCP-1.
  • the response to MCP-1 that is measured can be cell proliferation.
  • the compound potentially inhibiting the expression and/or activity of MCP-1 can be selected from the group consisting of a protein, a peptide, a peptidomimetic, a nucleic acid, a steroid, an alkaloid, a prostaglandin or analogue thereof, a prostacyclin or analogue thereof, a receptor agonist, a receptor antagonist, a monosaccharide, a disaccharide, a carbohydrate larger than a disaccharide, or a small molecule that is other than a steroid, an alkaloid, a monosaccharide, a disaccharide, a prostaglandin or analogue thereof, and a prostacyclin or analogue thereof.
  • Figure 1 is a graph showing that conditioned medium from PrSC stimulated the proliferation of PrEC, and that this proliferation was completely inhibited by a specific MCP-1 inhibitor, RS-102895.
  • Figure 2 is a scatter plot of 147 cytokines with the average OD of each cytokine in an immunoassay specific for that cytokine from the large prostate group plotted on the y axis and the average OD of each cytokine from the small prostate group plotted on the x axis. The position of MCP-1 on the scatter plot is indicated.
  • Figure 3 is a plot of the detailed data for a number of cytokines, with the OD of the cytokine in an immunoassay specific for that cytokine on the y axis and the weight of the prostate gland being measured on the x axis.
  • A MCP-1 ;
  • B Activin A;
  • C FGF-7;
  • D FGF-4;
  • E Endoglin;
  • F IL-6;
  • G IL-7; and
  • H IL-1 ⁇ .
  • Figure 4 shows the results of analysis for MCP-1 and IL-1 ⁇ for prostatic fluid from patients in the category Gleason 6 with minimum disease and no hormonal treatment.
  • Panel (A) plots the results for concentration of MCP-1 as represented by the ratio of OD in an MCP-1 -specific immunoassay to total protein on the y axis and the weight of the prostate gland being measured on the x axis.
  • Panel (B) plots the results for concentration of MCP-1 as represented by the ratio of OD in an MCP-1 -specific immunoassay to total protein on the y axis in two groups, separated on the x axis: a first group with prostate gland weights ⁇ 50 g and a second group with prostate gland weights >80 g.
  • Panel (C) plots the concentration of MCP-1 as represented by the OD in a MCP-1 specific immunoassay on the x axis versus the I-PSS score on the y axis.
  • Panel (D) plots the results for concentration of IL-1 ⁇ as represented by the ratio of OD in an IL-1 ⁇ -specific immunoassay to total protein on the y axis versus gland weight on the x axis.
  • Panel (E) plots the concentration of IL- 1 ⁇ as represented by the OD in an IL-1 ⁇ -specif ⁇ c immunoassay on the x axis versus the I-PSS score on the y axis.
  • FIG. 5 shows the effect of MCP-1 on the proliferation of the human cell lines PrEC (primary prostate basal epithelial cell) and PrSC (primary prostate stromal cell).
  • Figure 6 shows Western blots for CCR2 in PrEC and PrSC.
  • FIG. 7 shows the results of immunoassays for the detection of MCP-1 in PrEC and PrSC cells.
  • MCP-1 concentration (determined by immunoassay) is shown in pg/ml.
  • MCP-1 concentration (determined by immunoassay) is shown in pg/ml for PrEC, the LNCap cell line (derived from androgen-sensitive human prostate adenocarcinoma cells), the C42B cell line (also derived from prostate adenocarcinoma cells), the PC3 cell line (also derived from prostate adenocarcinoma cells), and the hFOB cell line (a human fetal osteoblast progenitor cell line).
  • LNCap cell line derived from androgen-sensitive human prostate adenocarcinoma cells
  • C42B cell line also derived from prostate adenocarcinoma cells
  • PC3 cell line also derived from prostate adenocarcinoma cells
  • hFOB cell line a human fetal osteoblast pro
  • FIG. 8 shows that the specific antagonist of CCR2 WS 102895 inhibits the effects of MCP-1 on PrEC cells.
  • proliferation of PrEC cells is represented as OD 4 5 0 -OD 6 io in the proliferation assay (the WST- 1 assay described above). Starting from the leftmost bar, the bars represent: (1) no MCP-1 and no WS 102895; (2) 10 nM MCP-1 and no WS102895; (3) 10 nM MCP-1 and 2.5 ⁇ M WS102985; (4) 10 nM MCP-1 and 5.0 ⁇ M WS102985; and (5) 10 nM MCP-1 and 10 ⁇ M WS102985. These results clearly show that WS102895 in inhibits the effects of MCP-1 on PrEC cells, blocking their proliferation.
  • FIG. 9 shows that the specific antagonist of CCR2 WS102895 also blocks the growth effect of conditioned media from PrSC to PrEC.
  • proliferation of PrEC cells is represented as OD4 5 o-OD 6 i 0 in the proliferation assay. Starting from the leftmost bar, the bars represent: (1) no conditioned media and no WS 102895; (2) conditioned media and no WS 102895; (3) conditioned media and 2.5 ⁇ M WS102985; (4) conditioned media and 5.0 ⁇ M WS102985; and (5) conditioned media and 10 ⁇ M WS102985.
  • Figure 10 shows that a specific monoclonal antibody binding MCP- 1 blocks the effect of PrSC-conditioned media in stimulating proliferation of PrEC.
  • conditioned media was used as in Figure 9 and proliferation of PrEC cells is represented as OD450-OD610 in the proliferation assay. Starting from the leftmost bar, the bars represent: (1) no conditioned media and no anti-MCP-1 mAb; (2) conditioned media and no anti-MCP-1 mAb; (3) conditioned media and anti-MCP-1 mAb at 1.0 ⁇ g/ml; and (4) conditioned media and anti-MCP-1 mAb at 5.0 ⁇ g/ml.
  • the anti-MCP-1 mAb has a very minimal effect on the proliferation of PrEC cells, as shown in the bottom panel of Figure 10.
  • the bars represent, starting from the left: (1) no anti-MCP-1 antibody; (2) anti-MCP-1 antibody at 0.25 ⁇ g/ml; (3) anti-MCP-1 antibody at 1.0 ⁇ g/ml; and (4) anti-MCP-1 antibody at 5.0 ⁇ g/ml.
  • Figure 11 shows that MCP-1 and IL-1 ⁇ levels correlate with CD68, a macrophage-specific marker, in prostatic juice.
  • Panel (A) the ratio of CD68/ ⁇ -actin is plotted on the y axis and MCP-1 concentration in pg/ml is plotted on the x axis.
  • Panel (B) the ratio of CD68/ ⁇ -actin is plotted on the y axis and IL-1 ⁇ concentration in pg/ml is plotted on the x axis.
  • Panel (C) the concentration of IL-1 ⁇ in pg/ml is plotted on the x axis and the concentration of MCP-1 in pg/ml is plotted on the y axis.
  • Panel (D) the results are divided into two groups, those with low CD68 concentration and those with high CD68 concentration, and the I-PSS score for each sample within each group is plotted separately, showing a correlation between high CD68 concentration and higher I- PSS score.
  • Figure 12 shows that conditioned medium from monocytes/macrophages stimulates the proliferation of PrEC.
  • the left panel shows results with PrEC and the right panel shows results with PrSC.
  • the bars represent: (1) no conditioned medium from monocytes/macrophages and no MCP-1; (2) no conditioned medium from monocytes/macrophages and MCP-1; (3) conditioned medium from monocytes/macrophages and no MCP-1; and (4) conditioned medium from monocytes/macrophages and MCP-1.
  • the results show a substantially greater effect with PrEC than with PrSC.
  • Figure 13 shows that inflammatory cytokines also enhance the secretion of MCP-1 from PrEC and PrSC.
  • the results in the left panel of Figure 13 are for PrEC; in the right panel, for PrSC.
  • the bars represent: (1) control; (2) IL-1 ⁇ ; (3) IFN- ⁇ ; and (4) IL-2.
  • MCP-1 in pg/ml is shown on the y axis.
  • IFN- ⁇ has a substantial effect in stimulating MCP-1 secretion in PrEC cells
  • all of IL-1 ⁇ , IFN- ⁇ and IL-2 have a substantial effect on the secretion of MCP-1 in PrSC cells.
  • Figure 14 is a schematic showing the role of MCP-1 in BPH, showing the interactions with stromal cells, basal cells, luminal cells, macrophages, and T cells, including interactions mediated by IL-1 ⁇ and IFN- ⁇ .
  • Figure 15 shows the effect of the synthetic androgen methyltrienolone (R 1881) on the secretion of MCP-1 by PrSC and the expression of the MCP-1 gene.
  • R1881 was used as follows: (1) no R1881 ; (2) 0.1 nM R1881 ; (3) 1.0 nM R1881 ; and (4) 1.0 nM R1881.
  • results for real-time PCR are shown on the y axis, representing mRNA specific for MCP-1 ; again, starting with the leftmost bar, R1881 was used as follows: (1) no R1881 ; (2) 0.1 nM R1881; (3) 1.0 nM R1881 ; and (4) 1.0 nM R1881.
  • Figure 16 shows the level of MCP-1 protein in patients after massage.
  • Panel (A) of Figure 16 the data is grouped into two parts: on the left, I-PSS scores are plotted for those patients for which MCP-1 was undetectable in urine after massage, and, on the right, I-PSS scores are plotted for those patients for which MCP-1 was detectable in urine after massage.
  • Panel (B) of Figure 16 the data is again grouped into two parts: on the left, estimated prostate gland size is plotted for those patients for which MCP-1 was undetectable in urine after massage, and, on the right, estimated prostate gland size is plotted for those patients for which MCP-1 was detectable in urine after massage.
  • Panel (C) of Figure 16 the data is again grouped into two parts: on the left, the age of the patient is plotted for those patients for which MCP-1 was undetectable in urine after massage, and, on the right, the age of the patient is plotted for those patients for which MCP-1 was detectable in urine after massage.
  • Figure 17 presents additional analysis of the data from the patients of Figure 16.
  • MCP-1 on the x axis is plotted versus I- PSS score on the y axis.
  • MCP-1 on the x axis is plotted versus gland weight (in g) on the y axis.
  • MCP- 1/total protein on the x axis is plotted versus I-PSS score on the y axis.
  • MCP-1 /total protein on the x axis is plotted versus gland weight (in g) on the y axis.
  • Figure 18 presents an analysis of the MCP-1 concentration in serum for these patients.
  • MCP-1 concentration in pg/ml is shown for serum (x2 diluted).
  • the data is grouped into three groups: the leftmost group is patients considered "normal"; the center group is patients for which there has been treatment failure; the rightmost group is patients with an AUA score of >20.
  • Figure 19 shows results of a study of expression of the gene for MCP-1 as measured in 25 paired normal-tumor RRP (radical retropubic prostatectomy) tissues and 9 BPH samples. Expression ratios of test sample/BPH reference are color coded, with red indicating higher expression in test sample relative to BPH and green indicating lower expression in the test sample relative to BPH. These results indicate that expression of MCP-1 is characteristic of BPH and not of prostatic carcinoma.
  • the present invention is based on the discovery that the cytokine Monocyte Chemoattractant Protein-1 (MCP-1) is strongly associated with the development of BPH and can serve as a marker for its diagnosis, as well as a target for its treatment.
  • MCP-1 Monocyte Chemoattractant Protein-1
  • Human MCP-1 is a 76-amino-acid protein with a pi of 9.7.
  • the protein contains two disulfide bonds.
  • the protein has the amino acid sequence: QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPK QKWVQDSMDHLDKQTQTPKT (SEQ ID NO: 1).
  • Human MCP-1 is cleaved from a 99-amino acid precursor protein with the amino acid sequence: MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLASYRRITS SKCPKEAVIFKTIVAKEICADPKQKVWQDSMDHLDKQTQTPKT (SEQ ID NO: 2).
  • human MCP-1 protein has the SwissProt accession number P13500. Structurally, human MCP-1 has the tertiary structure of a dimmer consisting of two three-stranded Greek keys upon which lie two antiparallel ⁇ -helices. The structure of the human MCP-1 gene has three exons separated by introns; the gene is on chromosome 17q11.2-q21. MCP-1 is also known as CCL2.
  • MCP-1 is a monocyte/macrophage chemotactic factor and induces monocytes to produce proinflammatory cytokines such as IL-1 and IL-6. MCP-1 also induces the migration of smooth muscle cells in vitro and enhances in vitro collagen expression by fibroblasts, partly by enhancing TGF- ⁇ . MCP-1 also chemoattracts basophils and CD4-positive and CD8-positive T lymphocytes. MCP-1 also plays central roles in the pathogenesis of lung fibrosis and diabetic nephropathy.
  • BPH benign prostatic hyperplasia
  • BPH nodules frequently occur concurrently together with chronic inflammatory infiltrates, T cells, and macrophages.
  • BPH is characterized by increased expression of the cytokines IL- 2, IL-4, IL-8, IL-15 and IFN ⁇ .
  • BPH is also characterized by downregulation of macrophage inhibitory cytokine- 1 /prostate derived factor.
  • This invention therefore, is based on the hypothesis that MCP-1 is the key cytokine involved in BPH development. Accordingly, MCP-1 may induce epithelial and stromal proliferation and may also induce extracellular matrix deposition.
  • MCP-1 can be a specific marker for BPH and can be used to discriminate BPH from prostatic carcinoma.
  • the use of MCP-1 as a specific marker for BPH is described below.
  • MCP-1 can be a novel target for BPH therapy, either alone or in conjunction with other therapies. The use of MCP-1 as a target for BPH therapy is described below.
  • one aspect of the present invention is a method of diagnosing BPH comprising the steps of:
  • the reference value for the concentration of MCP-1 in prostatic fluid is about 5000 intensity units as measured in densitometry in a conventional MCP-1 immunoassay.
  • the assay for MCP-1 is an immunoassay.
  • many immunoassay formats are suitable for use in immunoassays in this method of the present invention. Because MCP-1 is a sufficiently large protein antigen, it is capable of binding multiple antibody molecules. This enables the use of a sandwich immunoassay format, in which the MCP-1 protein molecule is bound to both an unlabeled antibody, typically bound to a solid support, and a labeled antibody. The MCP-1 protein molecule is then detected by the label of the labeled antibody.
  • One particularly useful sandwich immunoassay format is the enzyme-linked immunosorbent (ELISA) assay.
  • ELISA enzyme-linked immunosorbent
  • a non- labeled antibody is attached to the surface of a solid phase, such as that of a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates the separation of bound labeled reactants from free labeled reactants; since only the bound labeled reactants (labeled antibody) bound to the antigen (MCP-1) in the sandwich need to be detected or determined.
  • MCP-1 antigen
  • enzyme-labeled antibody is added to form the sandwich complex as described above, i.e., solid phase Ab-Ag-Ab (enzyme-labeled). Excess unbound antibody is then washed away, and a suitable enzyme substrate is added, along with appropriate buffers and coenzymes if required. The enzyme then catalytically converts the substrate to a detectable product, the quantity of which is proportional to the quantity of antigen in the sample.
  • enzymes include, but are not limited to, alkaline phosphatase, horseradish peroxidase, glucose 6-phosphate dehydrogenase, and ⁇ -galactosidase.
  • Other enzyme labels are also known in the art.
  • Enzyme- labeled antibodies can be prepared by covalent coupling procedures involving reagents such as, but not limited to, glutaraldehyde, N-succinimidyl 3-[pyridyl] propionate, carbodiimides, carbonyldiimidazole, and other cross-linking reagents known in the art, such as those described in GT.
  • recombinant antibody conjugates can be prepared by genetic engineering techniques known in the art, the conjugates being produced by transcription and translation of gene fusions.
  • the enzyme in the enzyme-labeled antibody in ELISA produces a product that is monitored and/or quantitated photometrically, such as by spectroscopy.
  • the enzyme produces a product that is monitored and/or quantitated by other means, such as detection of fluorescence, bioluminescence, or chemiluminescence.
  • immunoassays that use horseradish peroxidase as the enzyme label can be assayed by the detection of chemiluminescence using a mixture of luminol, peroxide, and an enhancer such as p-iodophenol or by using an acridan derivative.
  • Umbelliferone phosphate is a nonfluorescent substrate that is converted to the highly fluorescent umbelliferone by the catalytic activity of the enzyme alkaline phosphatase.
  • Another sensitive assay using an alkaline phosphatase label uses a chemiluminescent adamantyl 1,2-dioxetane aryl phosphate substrate, which is dephosphorylated by the catalytic activity of alkaline phosphate and decomposes with a concomitant long-lived glow of light, such that the detection limit for alkaline phosphatase using this assay can be as low as 1 zeptomole (10 "21 moles).
  • an enzyme cascade is used.
  • the advantage of the use of an enzyme cascade is that it combines the amplification properties of two enzymes — the alkaline phosphatase present in the antibody label and the second enzyme, alcohol dehydrogenase, in the assay reagent, thereby producing an extremely sensitive assay.
  • the alkaline phosphatase can dephosphorylate NADP to NAD, which then takes part in a reaction catalyzed by alcohol dehydrogenase and the enzyme diaphorase in which ethanol is oxidized to acetaldehyde and p-iodonitrotetrazolium violet is reduced to the detectable formazan by the catalytic activity of diaphorase.
  • EMIT enzyme multiplied immunoassay technique
  • MCP-1 analyte
  • MCP-1 analyte
  • binding of the antibody and the MCP-1 occurs.
  • An aliquot of a conjugate of an enzyme that can catalyze a reaction involving the substrate and the MCP-1 is added to the sample.
  • the enzyme-MCP-1 conjugate then binds with any excess antibody to MCP-1, forming an antibody-enzyme-MCP-1 conjugate complex.
  • the enzyme In the absence of free MCP-1 in the original sample, the enzyme is inactivated, such as by the binding of the enzyme in the enzyme- MCP-1 conjugate to the antibody which physically blocks access of the substrate to the active site of the enzyme or by changing the conformation of the enzyme and thus altering the activity.
  • this assay which is a homogeneous assay not requiring separation of bound and free label, the greater the concentration of analyte (MCP-1), the greater is the quantity of enzyme activity.
  • CEDIA enzyme donor immunoassay
  • inactive fragments the enzyme donor and acceptor
  • the enzyme donor and acceptor are prepared by manipulation of the Z gene of the lac operon of the bacterium Escherichia coli. These two fragments spontaneously reassemble to form active enzyme, even if the enzyme donor is attached to an antigen, such as MCP-1.
  • binding of antibody to the enzyme donor inhibits reassembly, thus blocking the formation of active enzyme and preventing enzyme activity.
  • immunoassays can be performed using radiolabeled antibodies, antibodies labeled with fluorescent, chemiluminescent, electrochemiluminescent, or bioluminescent labels, antibodies labeled with colloidal metals such as gold or silver, as well as other labels known in the art, such as, but not limited to, Surface-Enhanced Resonant Raman Spectroscopy (SERRS) labels, or signaling aptamers.
  • SERRS Surface-Enhanced Resonant Raman Spectroscopy
  • signaling aptamers for example, for bioluminescent labels, native or recombinant apoaequorin from the bioluminescent jellyfish Aequorea can be used as the label; the label is activated by reactions with coelenterazine, and light emission at 469 nm is triggered by reaction with calcium ions, such as calcium chloride.
  • a fluorophore (donor)-labeled antigen competes with an antigen in the sample for binding sites on an antibody labeled with a fluorescent dye (acceptor). The fluorescence of the donor is quenched when it is bound to the acceptor-labeled antibody.
  • Another alternative assay format is immuno-PCR. Immuno-PCR is a heterogeneous immunoassay in which a piece of single- or double-stranded DNA is used as a label for an antibody in a sandwich assay. Bound DNA label is amplified using PCR.
  • LOCI luminescent oxygen channeling immunoassay
  • Phosphor immunoassay is a heterogeneous immunoassay in which an upconverting phosphor nanoparticle is used as a label.
  • the nanoparticle (200- to 400-nm diameter) is a crystalline lanthanide oxysulfide. It absorbs two or more photons of infrared light (980 nm) and produces light emission at a shorter wavelength (anti-Stokes shift). The phosphorescence is not influences by reaction conditions such as temperature or buffer composition and there is no upconverted signal from biological components in the sample, resulting in a low background.
  • Still another alternative assay format is quantum dot immunoassay.
  • Quantum dot immunoassay is a heterogeneous immunoassay in which a nanometer-sized (less than 10 nm) semiconductor quantum dot is used as a label.
  • a quantum dot is a highly fluorescent nanocrystal composed of CdSe, ZnSe, InP or InAs or a layer of ZnS or CdS on, for example, an CdSe core.
  • Yet another alternative assay format is solid phase, light-scattering immunoassay, in which indium spheres are coated on glass to measure an antibody binding to an antigen (MCP-1 in this example). Binding of antibody to antigen increases dielectric layer thickness, which produces a greater degree of scatter than in areas where only an antigen is bound; quantitation is achieved by densitometry.
  • Still another alternative assay format is surface effect immunoassay, in which an antibody is immobilized on the surface of a waveguide (a quartz, glass, or plastic slide, or a gold- or silver-coated prism), and binding of an antigen is measured directly by total internal reflection fluorescence, surface plasmon resonance, or attenuated total reflection.
  • a waveguide a quartz, glass, or plastic slide, or a gold- or silver-coated prism
  • Other assay formats are known in the art.
  • a sandwich assay can be performed in a lateral flow assay device as generally known in the art.
  • the assay platform is typically constructed of a solid support that provides lateral flow of a sample through the assay platform when a sample is applied to a sampling platform that is in operable contact with the assay platform.
  • the sampling platform and the assay platform are typically constructed of a material such as nitrocellulose, glass fiber, paper, nylon, or a synthetic nanoporous polymer. Suitable materials are well known in the art and are described, for example, in U.S. Patent No.
  • the term “antibody” encompasses both polyclonal and monoclonal antibodies, as well as genetically engineered antibodies such as chimeric or humanized antibodies of the appropriate binding specificity. As used herein, unless further defined, the term “antibody” also encompasses antibody fragments such as sFv, Fv, Fab, Fab' and F(ab)'2 fragments.
  • the antigen MCP-1 protein
  • binds to multiple antibody molecules at least one unlabeled antibody molecule and at least one labeled antibody molecule
  • suitable antibody fragments that can bind only one antigen molecule can be used in assays according to the present invention.
  • MCP-1 is a specific marker for the presence of BPH.
  • the level of MCP-1 can be compared against a number of measurements of prostatic gland size or function, as described below.
  • MCP-1 can be measured in either prostatic fluid or urine.
  • the level of MCP-1 in prostatic fluid in intensity units as measured in densitometry in a conventional MCP-1 immunoassay per ml of prostatic fluid is determined.
  • a ratio of greater than about 5,000 intensity units/ml prostatic fluid is diagnostic of the existence of BPH.
  • the level of MCP-1 in prostatic fluid in intensity units as measured in densitometry in a conventional MCP-1 immunoassay is compared to the total weight of the prostate gland, in grams, to generate a ratio of MCP-1 to prostate gland weight.
  • a ratio of greater than about 6.0 intensity units/g prostate gland weight is diagnostic of the existence of BPH.
  • the level of MCP-1 in prostatic fluid in intensity units as measured in densitometry in a conventional MCP-1 immunoassay is compared to total protein in prostatic fluid.
  • a ratio of 400 intensity units/ ⁇ g total protein is diagnostic of the existence of BPH.
  • concentration of MCP-1 in urine after prostatic massage is measured; concentration of MCP-1 is expressed in intensity units as measured in densitometry in a conventional MCP-1 immunoassay.
  • any detectable MCP-1 concentration is diagnostic of BPH.
  • a MCP-1 concentration of at least 10 pg/ml is diagnostic of BPH.
  • the quantity of MCP-1 protein expression can be correlated with the quantity of mRNA that encodes the MCP-1 protein and can be translated into MCP-1 protein
  • another aspect of the present invention is a method of determining the quantity of mRNA encoding the MCP-1 protein.
  • the upregulation of MCP-1 expression is strongly characteristic of the diagnosis of BPH.
  • this method comprises:
  • the concentration of MCP-1 can be correlated with the existence and severity of LUTS. This enables one skilled in the art to predict the course of LUTS and to prescribe appropriate treatment for a patient who has LUTS or may experience LUTS.
  • Another aspect of the present invention is a method for treating or preventing BPH.
  • this method comprises the step of administering a therapeutically effective quantity of an agent that inhibits the expression or activity of MCP-1 to a patient who has or is at risk of developing BPH in order to inhibit the expression or activity of MCP-1 to a sufficient degree that BPH is treated or its occurrence prevented.
  • the agent that inhibits the expression or activity of MCP-1 is an agent that inhibits the expression of MCP-1. Inhibition of the expression of MCP-1 can be carried out at either the level of transcription of the gene for MCP-1 into mRNA or the translation of mRNA for MCP-1 into the corresponding protein.
  • inhibitory nucleotides are used to suppress MCP-1 expression. These include short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, or complementary DNA (cDNA).
  • siRNA short interfering RNA
  • miRNA microRNA
  • shRNA synthetic hairpin RNA
  • cDNA complementary DNA
  • siRNA targeting MCP-1 expression is used. Interference with the function and expression of endogenous genes by double-stranded RNA such as siRNA has been shown in various organisms. See, e.g., A. Fire et al., "Potent and Specific Genetic Interference by Double-Stranded RNA in Caenorhabditis elegans," Nature 391 :806-811 (1998); J.R.
  • siRNAs can include hairpin loops comprising self-complementary sequences or double stranded sequences.
  • siRNAs typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches.
  • double-stranded RNA can be synthesized by in vitro transcription of single- stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands.
  • Double-stranded RNA targeting MCP-1 can also be synthesized from a cDNA vector construct in which a MCP-1 gene (e.g., human MCP-1 gene) is cloned in opposing orientations separated by an inverted repeat.
  • RNA interference mediated by siRNA, miRNA, or shRNA is mediated at the level of translation; in other words, these interfering RNA molecules prevent translation of the corresponding mRNA molecules and lead to their degradation. It is also possible that RNA interference may also operate at the level of transcription, blocking transcription of the regions of the genome corresponding to these interfering RNA molecules.
  • the structure and function of these interfering RNA molecules are well known in the art and are described, for example, in R.F. Gesteland et al., eds, "The RNA World” (3 rd ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2006), pp. 535-565, incorporated herein by this reference.
  • nucleic acid agents targeting MCP-1 can also be employed in the methods of the present invention, e.g., antisense nucleic acids.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific target mRNA molecule. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the translation of mRNA into protein, and, thus, with the expression of a gene that is transcribed into that mRNA.
  • Antisense methods have been used to inhibit the expression of many genes in vitro. See, e.g., CJ. Marcus- Sekura/Techniques for Using Antisense Oligodeoxyribonucleotides to Study Gene Expression," Anal. Biochem. 172:289-295 (1988); J. E. Hambor et al., "Use of an Epstein-Barr Virus Episomal Replicon for Anti-Sense RNA-Mediated Gene Inhibition in a Human Cytotoxic T-CeII Clone," Proc. Natl. Acad. Sci. U.S.A.
  • MCP-1 nucleotide sequences such as those derived from mRNA, are known in the art, including the human MCP-1 gene sequence reported as NM_002982 in GenBank, and described further in G. Pacheco-Rodriguez et al., "Chemokine-Enhanced Chemotaxis of Lymphangioleiomyomatosis Cells with Mutations in the Tumor Suppressor TSC2 Gene," J. Immunol. 182: 1270-1277 (2009), incorporated herein by this reference. Based on the known sequences, inhibitory nucleotides (e.g., siRNA, miRNA, or shRNA) targeting PAR1 can be readily synthesized using methods well known in the art.
  • inhibitory nucleotides e.g., siRNA, miRNA, or shRNA
  • Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integral number of base pairs between these numbers.
  • Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, WA) and Ambion, Inc. (Austin, TX).
  • the agent that inhibits the expression or activity of MCP-1 is an agent that inhibits the activity of MCP-1.
  • an agent can be an antibody that specifically binds either MCP-1 or its receptor, CCR2.
  • the preparation of such antibodies is well known in the art and need not be described further herein.
  • antibodies according to the present invention can be of any class, such as IgG, IgA, IgD, IgE, IgM, or IgY, although IgG antibodies are typically preferred.
  • Antibodies can be of any mammalian or avian origin, including human, murine (mouse or rat), donkey, sheep, goat, rabbit, camel, horse, or chicken. In some alternatives, the antibodies can be bispecific.
  • the antibodies can be modified by the covalent attachment of any type of molecule to the antibody.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylate, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or other modifications known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., "Antibodies: A Laboratory Manual", (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-CeII Hybridomas 563-681 (Elsevier, N.Y., 1981), or by other standard methods known in the art.
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term "monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • suitable antibodies can be produced by phage display or other techniques.
  • human antibodies can be made by a variety of techniques, including phage display methods using antibody libraries derived from human immunoglobulin sequences and by the use of transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the antibodies can also be produced by expression of polynucleotides encoding these antibodies. Additionally, antibodies according to the present invention can be fused to marker sequences, such as a peptide tag to facilitate purification; a suitable tag is a hexahistidine tag.
  • the antibodies can also be conjugated to a diagnostic or therapeutic agent by methods known in the art. Techniques for preparing such conjugates are well known in the art.
  • MCP-1 Monocyte Chemotactic Protein-1
  • CCL2 Mouse monoclonal anti-MCP-1 antibody designated MAB279, commercially available from R&D Systems, Minneapolis. MN.
  • MAB279 mouse monoclonal anti-MCP-1 antibody designated MAB279, commercially available from R&D Systems, Minneapolis. MN.
  • This antibody was produced from a hybridoma resulting from the fusion of a mouse myeloma with B cells obtained from a mouse immunized with Escherichia co//-derived, recombinant human CCL2 (rhCCL2).
  • the IgG fraction from ascites fluid was purified by Protein A affinity chromatography.
  • Suitable antibodies with appropriate binding specificity can be used to inhibit the activity of MCP-1. These antibodies include, but are not limited to:
  • a monoclonal antibody having complementarity-determining regions of its heavy and light chains that are identical with the complementarity- determining regions of a monoclonal antibody specifically binding the same epitope of MCP-1 as is bound by MAB279 and that competes at least about 80% as effectively on a molar basis for the binding of MCP-1 as the monoclonal antibody specifically binding the same epitope of MCP-1 as is bound by MAB279;
  • a monoclonal antibody having complementarity- determining regions of its heavy and light chains that are identical with the complementarity-determining regions of a monoclonal antibody specifically binding the same epitope of MCP-1 as is bound by MAB279 and that competes at least about 80% as effectively on a molar basis for the binding of MCP-1 as the monoclonal antibody specifically binding the same epitope of MCP-1 as is bound by MAB279;
  • the agent that inhibits the activity of MCP-1 is an antagonist of the MCP-1 receptor CCR2, also known as CD192.
  • MCP-1 receptor CCR2 also known as CD192.
  • One suitable antagonist is RS102895, which is a CCR2-selective chemokine receptor antagonist whose systemic chemical name is 1'-[2-[4-
  • RS102895 has a molecular weight of 426.86 g/mol; it is CAS: 300815-41-2. Its formula is
  • ARBs include, but are not limited to, candesartan (cilexetil), irbesartan, losartan, olmesartan (medoxomil), telmisartan, valsartan, eprosartan, and the salts, solvates, analogues, congeners, bioisosteres, and prodrugs thereof.
  • ARBs have been shown to inhibit the expression of MCP-1.
  • the agent that inhibits the activity of MCP-1 is an antibody that specifically binds CCR2. Typically, this antibody is a monoclonal antibody; it can alternatively be a chimeric or humanized antibody.
  • treatment includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., BPH), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder.
  • a disease e.g., BPH
  • Subjects in need of treatment include patients already suffering from the disease or disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.
  • the terms "treating,” or similar terminology do not imply a cure for BPH or any other disease or condition; rather, this terminology is used to refer to any clinically detectable improvement in the disease or condition being treated or alleviated, including, but not limited to, reduction in LUTS symptoms, reduction of urinary frequency, reduction of pain, reduction of urethral obstruction, reduction of urinary retention, improvement in subjective well-being experienced by the patient, or any other clinically detectable improvement.
  • therapeutically effective amount refers to the amount of a therapy (e.g., a prophylactic or therapeutic agent) which is sufficient to treat BPH as described above.
  • compositions which comprise the agent that inhibits the expression or activity of MCP-1 and/or other active agents in a therapeutically effective dose along with a pharmaceutically acceptable carrier, diluent or excipient in unit dosage form.
  • Pharmaceutically acceptable carriers are agents which are not biologically or otherwise undesirable, i.e., the agents can be administered to a subject along with the agent that inhibits the expression or activity of MCP-1 without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the pharmaceutical composition in which it is contained.
  • the compositions can additionally contain other therapeutic agents that are suitable for treating or preventing BPH as described above.
  • Pharmaceutically acceptable carriers enhance or stabilize the composition, or can facilitate preparation of the composition.
  • Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the pharmaceutically acceptable carrier should be suitable for various routes of administration described herein.
  • compositions according to the present invention for the treatment or prevention of BPH in a subject comprise:
  • the pharmaceutical composition can further comprise an additional therapeutic agent for the treatment or prevention of BPH.
  • a pharmaceutical composition containing an agent that inhibits the expression or activity of MCP-1 and/or other therapeutic agents can be administered by a variety of methods known in the art.
  • the routes and/or modes of administration vary depending upon the desired results.
  • the active therapeutic agent may be coated in a material to protect the compound from the action of acids and other compounds that may inactivate the agent.
  • Conventional pharmaceutical practice can be employed to provide suitable formulations or compositions for the administration of such antagonists to subjects.
  • Any appropriate route of administration can be employed, for example, but not limited to, intravenous, parenteral, intraperitoneal, intravenous, transcutaneous, subcutaneous, intramuscular, intraurethral, oral administration.
  • either systemic or localized delivery of the therapeutic agent can be used in the course of treatment.
  • local administration of the agent that inhibits the expression or activity of MCP-1 is desired in order to achieve the intended therapeutic effect.
  • Many methods of localized delivery of therapeutic agents can be used in the practice of the invention.
  • such agents can be administered directly to the lower urinary tract by suppository, by direct injection or infusion, or by other means known in the art.
  • compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20 th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes.
  • Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, e.g., polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or can be oily solutions for administration or gels.
  • the agent that inhibits the expression or activity of MCP-1 for use in the methods of the invention is typically administered to a subject in an amount that is sufficient to achieve the desired therapeutic effect (e.g., eliminating or ameliorating symptoms associated with BPH) in a subject in need thereof.
  • a therapeutically effective dose or efficacious dose of the agent that inhibits the expression or activity of MCP-1 is employed in the pharmaceutical compositions of the invention.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular agent that inhibits the expression or activity of MCP-1 , the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the severity of the condition, other health considerations affecting the subject, and the status of liver and kidney function of the subject. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular agent that inhibits the expression or activity of MCP-1 employed, as well as the age, weight, condition, general health and prior medical history of the subject being treated, and like factors.
  • a pharmaceutically effective dosage would be between about 0.001 and 100 mg/kg body weight of the subject to be treated.
  • the agent that inhibits the expression or activity of MCP-1 and, if desired, other therapeutic regimens described above are usually administered to the subjects on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the agent that inhibits the expression or activity of MCP-1 and the other therapeutic agents, if any, used in the subject. In some methods, dosage is adjusted to achieve a plasma compound concentration of 1- 1000 ⁇ g/ml and in some methods 25-300 ⁇ g/ml. Alternatively, the agent that inhibits the expression or activity of MCP-1 can be administered as a sustained release formulation, in which case less frequent administration is required.
  • Dosage and frequency vary depending on the half-life in the subject of the agent that inhibits the expression or activity of MCP-1 and the other drugs included in a pharmaceutical composition.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime.
  • treatment can be monitored by observing one or more of the improving symptoms associated with BPH or LUTS, such as decreased urinary frequency, decreased urinary retention, decreased pain, improved urinary flow, or any other symptom associated with improvement in BPH or LUTS.
  • improving symptoms associated with BPH or LUTS such as decreased urinary frequency, decreased urinary retention, decreased pain, improved urinary flow, or any other symptom associated with improvement in BPH or LUTS.
  • Preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • the pharmaceutical compositions contemplated by the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the agent that inhibits the expression or activity of MCP-1 in water-soluble form. Additionally, suspensions of the agent that inhibits the expression or activity of MCP-1 can be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or modulators which increase the solubility of the agent that inhibits the expression or activity of MCP-1 to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active modulators with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating modulators may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different doses of agent that inhibits the expression or activity of MCP-1.
  • Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the agent that inhibits the expression or activity of MCP-1 may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • antioxidants such as sodium citrate, ascorbyl palmitate, propyl gallate, reducing agents, ascorbic acid, vitamin E, sodium bisulfite, butylated hydroxytoluene, BHA, acetylcysteine, monothioglycerol, phenyl- ⁇ -naphthylamine, or lecithin
  • chelators such as EDTA can be used.
  • Other ingredients that are conventional in the area of pharmaceutical compositions and formulations, such as lubricants in tablets or pills, coloring agents, or flavoring agents, can be used.
  • conventional pharmaceutical excipients or carriers can be used.
  • the pharmaceutical excipients can include, but are not necessarily limited to, calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Other pharmaceutical excipients are well known in the art.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, any and/or all of solvents, including aqueous and non-aqueous solvents, dispersion media, coatings, antibacterial and/or antifungal agents, isotonic and/or absorption delaying agents, and/or the like. The use of such media and/or agents for pharmaceutically active substances is well known in the art.
  • compositions should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Office of Biologies Standards or by other regulatory organizations regulating drugs.
  • sustained-release formulations or controlled-release formulations are well-known in the art.
  • the sustained-release or controlled- release formulation can be (1) an oral matrix sustained-release or controlled- release formulation; (2) an oral multilayered sustained-release or controlled- release tablet formulation; (3) an oral multiparticulate sustained-release or controlled-release formulation; (4) an oral osmotic sustained-release or controlled-release formulation; (5) an oral chewable sustained-release or controlled-release formulation; or (6) a dermal sustained-release or controlled- release patch formulation.
  • formulations for controlled release or sustained release comprising an agent that inhibits the expression or activity of MCP-1 by modifying the formulations described above, such as according to principles disclosed in V. H. K. Li et al, "Influence of Drug Properties and Routes of Drug Administration on the Design of Sustained and Controlled Release Systems” in Controlled Drug Delivery: Fundamentals and Applications (J. R. Robinson & V.H.L. Lee, eds, 2d ed., Marcel Dekker, New York, 1987), ch. 1, pp. 3-94, incorporated herein by this reference.
  • This process of preparation typically takes into account physicochemical properties of the agent that inhibits the expression or activity of MCP-1 , such as aqueous solubility, partition coefficient, molecular size, stability of the agent that inhibits the expression or activity of MCP-1 , and binding of the agent that inhibits the expression or activity of MCP-1 to proteins and other biological macromolecules.
  • This process of preparation also takes into account biological factors, such as absorption, distribution, metabolism, duration of action, the possible existence of side effects, and margin of safety, for the agent that inhibits the expression or activity of MCP-1. Accordingly, one of ordinary skill in the art could modify the formulations in order to incorporate an agent that inhibits the expression or activity of MCP-1 into a formulation having the desirable properties described above for a particular application.
  • Another aspect of the present invention is a method of screening for a compound inhibiting the expression and/or activity of MCP-1 to identify additional agents suitable for treating BPH.
  • such a screening method comprises:
  • the cells that demonstrate a detectable response to MCP-1 can be cells that endogenously express and secrete MCP-1 , such as PrSC.
  • the cells that demonstrate a detectable response to MCP-1 can be cells that express a receptor for MCP-1 , i.e., the receptor CCR2; the cells need not endogenously express and secrete MCP-1.
  • the response to MCP-1 can be cell proliferation, which can be determined using the WST-1 assay as described below.
  • the compound potentially inhibiting the expression and/or activity of MCP-1 can be a protein, a peptide, a peptidomimetic, a nucleic acid, a steroid, an alkaloid, a prostaglandin or analogue thereof, a prostacyclin or analogue thereof, a receptor agonist, a receptor antagonist, a monosaccharide, a disaccharide, a carbohydrate larger than a disaccharide, or a small molecule that is other than a steroid, an alkaloid, a monosaccharide, a disaccharide, a prostaglandin or analogue thereof, or a prostacyclin or analogue thereof.
  • MCP-1 Monocyte Chemotactic Protein-1
  • PrEC primary prostate basal epithelial cell
  • PrSC primary prostate stromal cell
  • MCP-1 Monocyte Chemotactic Protein-1
  • CCR2 Monocyte Chemotactic Protein-1
  • Conditioned medium from PrSC stimulated the proliferation of PrEC as well, an effect that was completely inhibited by the MCP- 1 antagonist ( Figure 1), and also by a neutralizing monoclonal antibody against MCP-1 (ruling out direct toxic effect of the inhibitor).
  • Figure 1 proliferation of PrEC cells was stimulated by conditioned medium from PrSC, but this effect was inhibited by the MCP-1 antagonist RS-102895, as shown in the results of Figure 1.
  • RS-102895 is a CCR2-selective chemokine receptor antagonist whose systemic chemical name is 1 '-[2-[4-(Trifluoromethyl)phenyl]ethyl]- spiro[4H-3,1-benzoxazine-4,4'-piperidin]-2(1 H)-one hydrochloride. Its formula is
  • the cytokine MCP- 1 may play an important role in prostatic enlargement and BPH, and is both a growth factor for epithelial and stromal cells, and a known macrophage chemoattractant. MCP- 1 merits further study as a potential BPH marker and therapeutic target.
  • MCP-D Prostate Monocyte Chemotactic Protein
  • MCP-1 monocyte chemotactic protein-1
  • BPH benign prostatic hyperplasia
  • I- PSS International Prostate Symptom Score
  • MCP-1 may be a novel biomarker for LUTS associated with BPH independent of prostate volume, age, and PSA.
  • prostate-generated inflammatory mediators may substantially contribute to LUTS pathogenesis in men with BPH.
  • Table 1 shows the upregulation of cytokines in large prostates, with the ratio of the cytokine in large (>60 g) versus small (40 g) prostates indicated for each cytokine.
  • Figure 2 is a scatter plot of 147 cytokines with the average OD of each cytokine in an immunoassay specific for that cytokine from the large prostate group plotted on the y axis and the average OD of each cytokine from the small prostate group plotted on the x axis. The position of MCP-1 on the scatter plot is indicated.
  • Figure 3 is a plot of the detailed data for a number of cytokines, with the OD of the cytokine in an immunoassay specific for that cytokine on the y axis and the weight of the prostate gland being measured on the x axis.
  • A MCP-1;
  • B Activin A;
  • C FGF-7;
  • D FGF-4;
  • E Endoglin;
  • F IL-6;
  • G IL-7; and
  • H IL-Ip.
  • Panel (A) plots the results for concentration of MCP-1 as represented by the ratio of OD in an MCP- 1 -specific immunoassay to total protein on the y axis and the weight of the prostate gland being measured on the x axis.
  • Panel (B) plots the results for concentration of MCP-1 as represented by the ratio of OD in an MCP-1 -specific immunoassay to total protein on the y axis in two groups, separated on the x axis: a first group with prostate gland weights ⁇ 50 g and a second group with prostate gland weights >80 g.
  • Panel (C) plots the concentration of MCP-1 as represented by the OD in a MCP-1 specific immunoassay on the x axis versus the I-PSS score on the y axis.
  • Panel (D) plots the results for concentration of IL- 1 ⁇ as represented by the ratio of OD in an IL-1 ⁇ -specific immunoassay to total protein on the y axis versus gland weight on the x axis.
  • Panel (E) plots the concentration of IL-1 ⁇ as represented by the OD in an IL-1 ⁇ -specific immunoassay on the x axis versus the I-PSS score on the y axis.
  • MCP-1 concentration is not associated with prostatic size. This was apparent in multivariate regression, adjusting for PSA and age.
  • I-PSS score was not associated with prostate size (previously known from other work).
  • Increased MCP-1 concentration is associated with increased I-PSS score, independent of age, PSA, and prostate size.
  • the results of immunoassays for the detection of MCP-1 in PrEC and PrSC cells are shown in Figure 7 (left panel).
  • MCP-1 concentration (determined by immunoassay) is shown in pg/ml.
  • MCP-1 concentration (determined by immunoassay) is shown in pg/ml for PrEC, the LNCap cell line (derived from androgen-sensitive human prostate adenocarcinoma cells), the C42B cell line (also derived from prostate adenocarcinoma cells), the PC3 cell line (also derived from prostate adenocarcinoma cells), and the hFOB cell line (a human fetal osteoblast progenitor cell line).
  • LNCap cell line derived from androgen-sensitive human prostate adenocarcinoma cells
  • the C42B cell line also derived from prostate adenocarcinoma cells
  • the PC3 cell line also derived from prostate adenocarcinoma cells
  • the hFOB cell line a human fetal osteoblast progenitor cell line
  • conditioned media was used as in Figure 9 and proliferation of PrEC cells is represented as OD 450 -OD6io in the proliferation assay (the WST-1 assay described above). Starting from the leftmost bar, the bars represent: (1) no conditioned media and no anti-MCP-1 mAb; (2) conditioned media and no anti-MCP-1 mAb; (3) conditioned media and anti-MCP-1 mAb at 1.0 ⁇ g/ml; and (4) conditioned media and anti-MCP-1 mAb at 5.0 ⁇ g/ml.
  • the anti-MCP-1 mAb has a very minimal effect on the proliferation of PrEC cells, as shown in the bottom panel of Figure 10.
  • the bars represent, starting from the left: (1) no anti- MCP-1 antibody; (2) anti-MCP-1 antibody at 0.25 ⁇ g/ml; (3) anti-MCP-1 antibody at 1.0 ⁇ g/ml; and (4) anti-MCP-1 antibody at 5.0 ⁇ g/ml.
  • MCP-1 and IL-1 ⁇ levels correlate with CD68, a macrophage-specific marker, in prostatic juice.
  • the results are shown in Figure 11.
  • Panel (A) the ratio of CD68/ ⁇ -actin is plotted on the y axis and MCP-1 concentration in pg/ml is plotted on the x axis.
  • Panel (B) the ratio of CD68/ ⁇ - actin is plotted on the y axis and IL-1 ⁇ concentration in pg/ml is plotted on the x axis.
  • Panel (C) the concentration of IL-1 ⁇ in pg/ml is plotted on the x axis and the concentration of MCP-1 in pg/ml is plotted on the y axis.
  • Panel (D) the results are divided into two groups, those with low CD68 concentration and those with high CD68 concentration, and the I-PSS score for each sample within each group is plotted separately, showing a correlation between high CD68 concentration and higher I-PSS score.
  • a higher I-PSS score means a greater severity of LUTS symptoms.
  • Conditioned medium from monocytes/macrophages stimulates the proliferation of PrEC.
  • the results are shown in Figure 12.
  • the left panel shows results with PrEC and the right panel shows results with PrSC.
  • the bars represent: (1) no conditioned medium from monocytes/macrophages and no MCP-1; (2) no conditioned medium from monocytes/macrophages and MCP-1; (3) conditioned medium from monocytes/macrophages and no MCP-1 ; and (4) conditioned medium from monocytes/macrophages and MCP-1.
  • the results show a substantially greater effect with PrEC than with PrSC.
  • IL-1 ⁇ and interferons are known to synergistically induce the synthesis of MCP-1 (and MCP-2) in fibroblasts and epithelial cells. Also, it is known that IL-1 ⁇ is secreted from monocytes/macrophages.
  • Figure 14 is a schematic showing the role of MCP-1 in BPH, showing the interactions with stromal cells, basal cells, luminal cells, macrophages, and T cells, including interactions mediated by IL-1 ⁇ and IFN- ⁇ .
  • MCP- 1 is known to be chemotactic for cells playing a substantial role in inflammation.
  • Androgens may also have an effect on MCP-1 secretion from PrSC.
  • the results are shown in Figure 15 with the synthetic androgen methyltrienolone (R 1881).
  • R 1881 synthetic androgen methyltrienolone
  • results for ELISA for MCP-1 in the supernatant of PrSC are shown; MCP-1 in pg/ml is shown on the y axis, and starting with the leftmost bar, R1881 was used as follows: (1) no R1881; (2) 0.1 nM R1881; (3) 1.0 nM R1881; and (4) 1.0 nM R1881.
  • results for real-time PCR are shown on the y axis, representing mRNA specific for MCP-1; again, starting with the leftmost bar, R1881 was used as follows: (1) no R1881; (2) 0.1 nM R1881; (3) 1.0 nM R1881; and (4) 1.0 nM R1881.
  • the results shown in Figure 15 are for incubation for 4 days; the ELISA may have been skewed by the numbers of PrSC cells, because R1881 stimulates the growth of PrSC cells. With 2 days of incubation, there was no difference in mRNA of MCP-1 and in MCP-1 secretion from PrSC.
  • MCP-1 levels were measured in urine of patients after massage. A total of 28 patients were studied. All patients were confirmed as negative for prostatic carcinoma by biopsy. MCP-1 levels in urine were measured by ELISA as described above. I-PSS scores were also evaluated for these patients. Protein levels in the urine were measured by the Bradford method. The volumes of the prostate glands in these patients were also estimated from transurethral ultrasound (TRUS) or digital rectal examination (DRE).
  • TRUS transurethral ultrasound
  • DRE digital rectal examination
  • Panel (C) of Figure 16 the data is again grouped into two parts: on the left, the age of the patient is plotted for those patients for which MCP-1 was undetectable in urine after massage, and, on the right, the age of the patient is plotted for those patients for which MCP-1 was detectable in urine after massage.
  • Figure 17 presents additional analysis of data from these patients.
  • MCP-1 on the x axis is plotted versus I-PSS score on the y axis.
  • MCP-1 on the x axis is plotted versus gland weight (in g) on the y axis.
  • MCP-1 /total protein on the x axis is plotted versus I-PSS score on the y axis.
  • MCP-1/total protein on the x axis is plotted versus gland weight (in g) on the y axis.
  • Figure 18 presents an analysis of the MCP-1 concentration in serum for these patients.
  • MCP-1 concentration in pg/ml is shown for serum (x2 diluted).
  • the data is grouped into three groups: the leftmost group is patients considered "normal"; the center group is patients for which there has been treatment failure; the rightmost group is patients with an AUA score of >20.
  • First strand cDNA synthesis was performed with real-time PCR using the SuperArray inflammatory cytokine gene array. The array contained 84 disease-focused genes and several controls. Data was analyzed according to the ⁇ Ct method (Ct threshold cycle). Expression in PrEC cells was compared with expression in BPH- 1 cells and human BPH samples.
  • Results The BPH-1 cells demonstrated 380-fold increased expression of monocyte chemotactic protein (MCP-1), followed by IL-8-RB (95- fold) and IL-10-RA (100-fold), when compared to PrEC cells. Sufficient RNA was obtained from 4 of the human samples, which also revealed an average of 9000- fold increased expression of MCP-1 in 2 cases, when compared to PrEC cells.
  • MCP-1 monocyte chemotactic protein
  • IL-8-RB IL-8-RB
  • IL-10-RA 100-fold
  • Methods of diagnosis of BPH according to the present invention based on the concentration of MCP-1 or mRNA encoding MCP-1 , offer improved diagnosis of this condition while clearly distinguishing it from prostatic carcinoma and prostatitis.
  • the methods of diagnosis according to the present invention therefore enable individuals suspected of having BPH to get appropriate treatment more rapidly without the necessity of their being subjected to unneeded, inappropriate, and invasive therapies, particularly those intended to treat prostatic carcinoma.
  • treatment methods according to the present invention directed to the activity of MCP-1 are suitable for the treatment of diagnosed BPH and are specific for that condition, without serious side effects.
  • Diagnostic methods according to the present invention possess industrial applicability, such as for screening compounds for their effect on the progression of BPH, and can be performed in vitro.
  • Treatment methods according to the present invention also possess industrial applicability for the preparation of a medicament for the treatment of BPH.
  • the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise.
  • the invention encompasses any other stated intervening values and ranges including either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.

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Abstract

La chémokine MCP-1 est étroitement associée au développement de l'hyperplasie bénigne de la prostate (HBP) et la détermination de la concentration de MCP-1 dans les sécrétions prostatiques ou dans les urines après un massage de la prostate ou la détermination de l'expression du gène MCP-1 par détermination de la concentration de l'ARNm spécifique à la MCP-1 peut être utilisée pour diagnostiquer spécifiquement l'HBP et de la différencier d'autres maladies ou affections de la prostate, comme la prostatite et le cancer de la prostate. De plus, la MCP-1 peut servir de cible pour le traitement de l'HBP et des agents affectant l'expression ou l'activité de la MCP-1 peuvent servir d'agents thérapeutiques contre l'HBP.
PCT/US2010/030736 2009-04-13 2010-04-12 Procédés destinés au diagnostic et au traitement de l'hyperplasie bénigne de la prostate WO2010120686A2 (fr)

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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030087271A1 (en) * 1996-03-01 2003-05-08 Ebersole Richard C. Method for the amplification and detection of a nucleic acid fragment of interest
US20030092125A1 (en) * 1998-04-15 2003-05-15 Abgenix, Inc. Epitope-driven human antibody production and gene expression profiling
US20030207328A1 (en) * 1996-04-25 2003-11-06 Genicon Sciences Corporation Analyte assay using particulate labels
US20030211105A1 (en) * 2001-05-18 2003-11-13 Murphy William J Methods for reducing tumor growth and metastasis by inhibiting mcp-1 activity
US20040162329A1 (en) * 2002-07-29 2004-08-19 Lockwood Samuel Fournier Structural carotenoid analogs for the inhibition and amelioration of disease
US20060128034A1 (en) * 2004-12-10 2006-06-15 Petruno Patrick T Diagnostic test using gated measurement of fluorescence from quantum dots
US20060171947A1 (en) * 2001-09-06 2006-08-03 Burnham Institute Serine/threonine hydrolase proteins and screening assays
WO2007084839A2 (fr) * 2006-01-13 2007-07-26 Elixirin Corporation Methodes et compositions alimentaires pouvant ameliorer le metabolisme et reduire une espece d'oxygene reactive
US20080118437A1 (en) * 2006-03-01 2008-05-22 Regents Of The University Of Michigan Diagnosis and treatment O prostate cancer
US20080200385A1 (en) * 2005-05-21 2008-08-21 Proteosys Ag, A Corporation Of Germany, Annexin for Cancer Risk Assessment

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030087271A1 (en) * 1996-03-01 2003-05-08 Ebersole Richard C. Method for the amplification and detection of a nucleic acid fragment of interest
US20030207328A1 (en) * 1996-04-25 2003-11-06 Genicon Sciences Corporation Analyte assay using particulate labels
US20030092125A1 (en) * 1998-04-15 2003-05-15 Abgenix, Inc. Epitope-driven human antibody production and gene expression profiling
US20030211105A1 (en) * 2001-05-18 2003-11-13 Murphy William J Methods for reducing tumor growth and metastasis by inhibiting mcp-1 activity
US20060171947A1 (en) * 2001-09-06 2006-08-03 Burnham Institute Serine/threonine hydrolase proteins and screening assays
US20040162329A1 (en) * 2002-07-29 2004-08-19 Lockwood Samuel Fournier Structural carotenoid analogs for the inhibition and amelioration of disease
US20060128034A1 (en) * 2004-12-10 2006-06-15 Petruno Patrick T Diagnostic test using gated measurement of fluorescence from quantum dots
US20080200385A1 (en) * 2005-05-21 2008-08-21 Proteosys Ag, A Corporation Of Germany, Annexin for Cancer Risk Assessment
WO2007084839A2 (fr) * 2006-01-13 2007-07-26 Elixirin Corporation Methodes et compositions alimentaires pouvant ameliorer le metabolisme et reduire une espece d'oxygene reactive
US20080118437A1 (en) * 2006-03-01 2008-05-22 Regents Of The University Of Michigan Diagnosis and treatment O prostate cancer

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