AU2004203427B2 - Method of Treating Bladder and Lower Urinary Tract Urinary Syndromes - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name of Applicant: Address for Service: Duke University CULLEN CO.

Level 26 239 George Street Brisbane Qld 4000 Invention Title: Method of Treating Bladder and Lower Urinary Tract Urinary Syndromes Details of Original Application: 38830/99 The following statement is a full description of this invention, including the best method of performing it, known to us: METHOD OF TREATING BLADDER

AND

LOWER URINARY TRACT

SYNDROMES

TECHNICAL FIELD The present invention relates to bladder and lower urinary tract syndromes, particularly, irritative symptoms. and to a method of treating same using a,,-adrenergic receptor (adARI antagonists. The invention further relates to a method of screening compounds for their ability to serve as adAR antagonists.

BACKGROUND

Lower urinary tract symptoms (LUTS) resulting from bladder outlet 1C obstruction (BOO) remains one of the most commonly encountered disorders in urology, and can be secondary to fixed anatomical and/or functional causes (Steers et al, Voiding dysfunction: diagnosis. classification, and management in Adult and Pediatric Urology; Third Edition. J.Y. Gillenwater. et al.. Editors. 1996.

Mosby-Year Book, Inc.: St. Louis. p. 1220-1325.). Causes of BOO include prostatic enlargement (benign or malignant), bladder neck contracture, urethral 1 t stricture, and meatal stricture (Steers et al. Voiding dysfunction: diagnosis, classification, and management in Adult and Pediatric Urology: Third Edition, J.Y. Gilienwater, et al., Editors. 1996. Mosby-Year Book, Inc.: St. Louis. p. 1220- 1325.). Symptoms associated with BOO typically fall into obstructive or irritative categories; obstructive symptoms include hesitancy, poor stream, prolonged urination. and feelings of incomplete emptying, while irritative symptoms consist 2 of frequency, urgency, nocturia, and unstable bladder contractions. The bladder is functionally and anatomically divided into the detrusor (body and ventral base) and trigone (dorsal portion of base extending between the ureteral orifices and the bladder neck) (Zderic et al, Voiding function: relevant anatomy, physiology, pharmacology, and molecular aspects, in Adult and Pediatric Urology; Third Edition, J.Y. Gillenwater, et al., Editors. 1996. Mosby-Year Book, Inc.: St. Louis.

p. 1159-1219), with distinct histology, histochemistry. and pharmacology. In contrast, the prostate and trigone have similar vascular supply, innervation, and receptor expression (Gosling et al. Detrusor morphology in relation to bladder outflow obstruction and instability, in Benign Prostatic Hypertrophy. F. Hinman, Editor. 1983, Springer-Verlag: Berlin. p. 666-71).

The physiology of LUTS secondary to benign prostatic hypertrophy (BPH) has two components: a static component related to the increase in prostatic cellular mass and a dynamic component related to variations in prostatic smooth muscle tone (Caine et al. Brit. J. Urol. 47:193-202 (1975)).

Histologically BPH is characterized by glandular (epithelial) and stromal (fibromuscular) hyperplasia, with the latter being the dominant factor in the pathogenesis of clinically significant BPH (Shapiro et al, J. Urol. 147:1293-1297 2 0 (1992)). Therefore much attention has focused on the role of the sympathetic nervous system and a,-adrenergic receptors (a,ARs) in the dynamic component of BOO, leading to clinical studies of aAR antagonists as agents to relieve outlet obstruction. These studies have found that aAR antagonists relax prostatic smooth muscle, relieving obstructive symptoms (Chapple, Brit. J. Urol. 1:47-55 (1995), Caine, Urol. Clin. N. Am. 17:641-649 (1990). Kawabe and Niijima, Urol.

Int. 42:280-284 (1987), Lepor et al, J. Urol. 148:1467-1474 (1992), Reuther and Aagaard. Urol. Int. 39:312-313 (1984), Matyus and Horvath. Med. Res. Rev.

17:523-535 (1997)). In addition, a,AR antagonists have been found to relieve the irritative bladder symptoms in men (most often associated with BPH) and women (Matyus and Horvath. Med. Res. Rev. 17:523-535 (1997), Serels and Stein, Neurourol. Urodyn. 17:31-36 (1998)). While it is logical to assume that elimination of BOO would relieve irritative symptoms, a number of recent studies suggest that the relationship between bladder irritability and outlet obstruction is not straightforward (Caine, Urol. Clin. N. Am. 17:641-649 (1990), Chapple and Smith, Brit. J. Urol. 73:117-123 (1994), Steers and De, J. Urol. 140:864-71 (1988), Steers et al. Am. J. Physiol. 266:R20 (1994)).

a,ARs are members of the larger family of G protein-coupled adrenergic receptors which mediate actions of the endogenous catecholamines norepinephrine (NE) and epinephrine, resulting in smooth muscle contraction.

cDNAs encoding three distinct aAR subtypes a, b Id) have been cloned, expressed in cells, and resultant protein characterized pharmacologically (Schwinn et al, J. Pharmacol. Exper. Ther. 272:134-142 (1995), Hieble et al, Pharmacol. Rev. 47:267-70 (1995)). a,,ARs predorhinate in prostate and bladder trigone (Price et al, J. Urol. 150:546-551 (1993)). and have been shown to be functionally important in mediating prostate smooth muscle contraction (Forray et al, Mol. Pharmacol. 45:703-708 (1994). Lepor et al., J. Pharmacol. Exper. Ther.

270:722-727 (1994)). In addition to the three cloned aAR subtypes which have high affinity for the antagonist prazosin, a fourth type of a,AR with low affinity for prazosin (aL) has been postulated (Muramatsu et al, Brit. J. Urol. 74:572-578 (1994)). In spite of initial evidence suggesting a role for the Ca,AR in human prostate smooth muscle contraction (Ford et al, Mol. Pharmacol. 49:209-215 (1996)), more recent data suggests RS17053 (the compound used in these studies) detects a low affinity state of the ct,,AR in tissues rather than a distinct atAR (Ford et al, Br. J. Pharmacol. 121:1127-1135 (1997)). Since non-selective aAR antagonists currently used to treat BPH have undesirable side-effects including light headedness, dizziness, and asthenia (Carruthers, Drug Safety 11:12-20 (1994)), many investigators have suggested that a,AR subtype selective antagonists might be beneficial in improving BPH-related symptoms via relieving BOO (Matyus and Horvath, Med. Res. Rev. 17:523-535 (1997), Hieble and Ruffolo, Jr., Exp. Opin. Invest. Drugs 6:367-387 (1997)). However, this approach does not take into account that irritative symptoms may persist in spite of relief of outlet obstruction (Hieble and Ruffolo, Jr.. Exp. Opin. Invest. Drugs 6:367-387 (1997)).

Very little information exists regarding the role of acARs in human detrusor. One of the few studies addressing this issue suggests human bladder (dome) contains only a,,ARs (Walden et al, J. Urol. 157:1032-1038 (1997)).

However, since irritative bladder symptoms persist in some patients despite relief of BOO, nonselective (aAR antagonists may relieve the irritative effects of BPH through direct effects on bladder detrusor or other sites involved in micturation.

The present invention results from the realization that human detrusor expresses two (aAR subtypes This realization makes possible the identification of ctAR subtype selective antagonists that can be used to treat irritative symptoms.

SUMMARY OF THE INVENTION The present invention relates generally to bladder and lower urinary tract syndromes and, more particularly, to a method of identifying adAR antagonists that can be used to treat irritative symptoms. The invention also relates to a method of treating irritative symptoms using such agents.

02/01 2009 12:30 FAX +61 7 3229 3384 CULLEN CO. 1005/013 4a Definitions of the specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided a method of treating irritative symptoms of bladder or lower urinary tract disease comprising administering to a patient in need of such treatment an amount of an aid-adrenergic receptor (ajdAR) antagonist sufficient to effect said treatment.

According to a second embodiment of the invention, there is provided a method of screening for a candidate agent suitable for use in relieving irritative symptoms of bladder or lower urinary tract disease in a patient comprising: i) incubating a test compound with aitd-adrenergic receptor (aidAR), or portion thereof comprising the transmembrane domain of C dAR, ii) determining the amount of said test compound bound to said a idAR, or said portion thereof, and iii) identifying a test compound that binds said a IdAR, or said portion thereof, as said candidate agent.

[Text continues on page COMS ID No: ARCS-218700 Received by IP Australia: Time 13:35 Date 2009-01-02 Objects and advantages of the invention will be apparent from the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURES 1A-1C. Schematic of the location of crAR subtype probes.

Highlighted in bold are regions of a 1 (Fig. 1A), (Fig. 1B), and ad (Fig. IC) ARs encoded by probes used in RNase protection assays.

FIGURE 2. Representative saturation binding isotherm generated using increasing concentrations of the a,AR radiolabeled antagonist 2 1IJHEAT in human detrusor membranes. Kd is 130- 1.09 pM similar to that reported for cells stably expressing each cloned human a,AR subtype (Schwinn et al. J PharmacoL Exper. Ther. 272:134-142 (1995) al dAR of the reference refers to the a,,AR subtype described herein since the a,AR nomenclature used here is the IUPHAR nomenclature (Hieble et al, Phar. Rev. 97:267 (1995)).

FIGURE 3. RNase protection assays examining a,AR subtype expression in detrusor were performed in all patients 13). A representative RNase protection assay showing results from five patients is shown. In this experiment, radiolabeled probe for each a,AR subtype is shown at the far left along with (from left to right) protected fragments resulting from total RNA extracted from rat-1 fibroblast cells stably expressing each cloned human aAR subtype (20 mg; positive probe control), yeast tRNA (20 mg; negative control); and total RNA isolated from human detrusor (20 mg) from five patients (lanes 1- Gel exposure times are 24 hrs for probe and positive control lanes and 72 hrs for tRNA and human detrusor samples. Although the a,,AR subtype mRNA band is stronger than the a,,AR protected fragment, the a,,

A

R probe contains 73% more radiolabeled aUTP compared with the aId; hence, after normalization for radioactive label incorporation, two-fold predominance of the aldAR subtype in human detrusor is apparent.

FIGURES 4A and 4B. Results from RT-PCR experiments on human detrusor (Fig. 4A) and rat whole bladder (Fig. 4B) RNA. aAR subtype specific cDNA in plasmid vectors served as positive controls.

FIGURE 5. a,AR subtype expression in human detrusor was determined using competition analysis with the a,,AR-subtype selective ligand BMY7378.

Results from a representative curve are shown demonstrating a two-site fit with high affinity Ki corresponding to the aldAR.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the recognition of a,JAR as the aAR subtype responsible for irritative symptoms associated with bladder and lower urinary tract diseases. The invention provides, in one embodiment, a method of selecting a,,AR antagonists and, in a further embodiment, a method of treating irritative symptoms using a iAR antagonists. (The nomenclature used herein is the new nomenclature provided in Hieble et al, Phar. Rev. 97:267 (1995)).

The method of treatment to which the invention relates comprises administering to a patient suffering irritative symptoms an amount of an a,,AR antagonist sufficient to relieve such symptoms. In accordance with the invention, irritative symptoms include excessive frequency of urination, urgency of urination, nocturia and unstable bladder contractions. Patients amenable to treatment include men and women, children and adults. In males, a preferred antagonist is both an a,aAR and an a2dAR antagonist. In females, preferred antagonists are acAR specific antagonists. The amount of the antagonist to be administered and the treatment regimen will vary with the antagonist, the patient and the effect sought. Optimum doses and regimens, however, can be readily determined by one skilled in the relevant art.

The present invention also relates to a method of screening compounds for their ability to bind primarily to calAR and thereby to function, potentially, as i,,AR antagonists Preferred adAR selective antagonists show at least a two fold selectivity for acAR relative to a,.AR or albAR. Binding assays of this embodiment invention include cell-free assays in which a0dAR. or portion thereof relevant transmembrane portion see, generally. Hwa et al, J. Biol. Chem. 271:7956 (1996)).

is incubated with a test compound (proteinaceous or non-proteinaceous) which.

advantageously, bears a detectable label a radioactive or fluorescent label).

Preparations of membranes that bear a,,AR can be used in this assay. including commercially available preparations the NEN multireceptor kit (NET 1034)).

Following incubation, the adAR, or portion thereof, free or bound to test compound, can be separated from unbound test compound using any of a variety of techniques (for example, the oaIAR (or portion thereof) associated with a membrane) can be bound to a solid support a plate or a column) and washed free of unbound test compound). The amount of test compound bound to aIdAR, or portion thereof. is then determined using a technique appropriate for detecting the label used liquid scintillation counting in the case of a radiolabelled test compound). (See Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995).) Binding assays of this embodiment can also take the form of cell-free competition binding assays. Such assays can be conducted as described in the Examples that follow (see particularly Example 2 (the test compound being substituted for BMY 7378 see also Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995)). Alternatively, a,,AR, or portion thereof, can be incubated with a compound known to interact, specifically, with ctdAR BMY7378), which compound, advantageously, bears a detectable label a radioactive or fluorescent label). A test compound (proteinaceous or non-proteinaceous) is added to the reaction and assayed for its ability to compete with the known (labeled) compound for binding to aIdAR, or portion thereof. Free known (labeled) compound can be separated from bound known compound, and the amount of bound known compound determined to assess the ability of the test compound to compete. This assay can be formatted so as to facilitate screening of large numbers of test compounds by linking the a, 4 AR, or portion thereof or, to a solid support so that it can be readily washed free of unbound reactants.

cGIdAR, or portion thereof, suitable for use in the cell-free assays described above can be isolated from natural sources as membrane preparations derived from bladder, human bladder) or prepared recombinantly or chemically. The aidAR. or portion thereof, or can be prepared as a fusion protein using, for example.

known recombinant techniques. Preferred fusion proteins include a HIS tag, a FLAG tag, a GFP tag or other tag (moiety) suitable for use in colorimetric assays. Typically, the non-aldAR moiety is present in the fusion protein N-terminal to the acdAR, or 2 0 portion thereof domain, but it can also be C-terminal.

As indicated above, the cadAR, or portion thereof, can be present linked to a solid support, including a plastic or glass plate or bead, a chromatographic resin, a filter or a membrane. Methods of attachment of proteins, or membranes containing same, to such supports are well known in the art.

9 The binding assays of the invention also include cell-based assays in which XtAR, or portion thereof, is associated with the cell membrane of an intact cell. Cells suitable for use in such assays include cells that naturally express a,,AR and cells that have been engineered to express, advantageously, over express, cca,AR (or portion S thereof). Advantageously, cells expressing human ac,,AR are used. Suitable cells are preferably eucaryotic, including mammalian (human and nonhuman) cells, insect cells and yeast cells.

Cells can be engineered to express acdAR (advantageously. human cd,,AR, or portion thereof) by introducing into a selected host a eucaryotic host) an expression construct comprising a sequence encoding a,,AR. or portion thereof.

operably linked to a promoter. A variety of vectors and promoters can be used. (See Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995).) Introduction of the construct into the host can be effected using any of a variety of standard transfection/transformation protocols (see Molecular Biology, A Laboratory Manual, second edition. J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Press, 1989). Cells thus produced can be cultured using established culture techniques suitable for the involved host. Culture conditions can be optimized to ensure expression of the a,,AR (or portion thereof) encoding sequence.

While for the cell-based binding assays it is appropriate that the .cdAR (or portion thereof) be associated with the cell membrane, for other purposes the expression product can be secreted into the culture medium or present in the cell cytoplasm.

The cell-based binding assays of the invention can be carried out essentially as described above with respect to the cell free assays. Advantageously, the cell used expresses predominantly the acdAR subtype. By way of example, the cell-based binding assay can be carried out by adding test compound (advantageously, bearing a detectable radioactive or fluorescent) label), to medium in which the acdAR (or portion thereof) expressing cells are cultured, incubating the test compound with the cells under conditions favorable to binding and then removing unbound test compound and determining the amount of test compound associated with the cells.

As in the case of the cell-free assays, the cell-based assays can also take the form of competitive assays, as described above. For example, a compound known to bind a,,AR (and preferably labelled with a detectable label) can be incubated with cd,,AR (or portion thereof) expressing cells in the presence and absence of test compound. The affinity of a test compound for aldAR can be assessed by determining the amount of known compound associated with the cells incubated in the presence of the test compound, as compared to the amount associated with the cells in the absence of the test compound.

A test compound identified in one or more of the above-described assays as being capable of binding to a,dAR can, potentially. serve as an a,dAR antagonist and therefore be suitable for use in the irritative symptom treatment method of the invention. To determine the specific effect of any particular test compound selected on the basis of its ability to bind a various assays can be used including IP assays (see Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995)) and bladder (e.g.

human bladder) smooth muscle contraction assays (Ford et al, Mol. Pharm. 49:209 (1996)). Compounds suitable for use in treating irritative symptoms will be associated with antagonistic (inhibitory) effects in the IP assay and contraction inhibitory effects in the contraction assay.

In another embodiment, the invention relates to compounds identified using the above-described assays as being c,0dAR antagonist. The compounds identified in accordance with the above assays can be formulated as pharmaceutical compositions.

Such compositions comprise the compound and a pharmaceutically acceptable diluent or carrier. The compound can be present in dosage unit form as a tablet or capsule) or as a solution, preferably sterile, particularly when administration by injection is anticipated. The dose and dosage regimen will vary, for example, with the patient, the compound and the effect sought. Optimum doses and regimens can be determined readily by one skilled in the art.

In another embodiment, the invention relates to antibodies specific for ccadAR.

and antigen binding fragments thereof, including or F(ab) fragments. The antibodies can be monoclonal or polyclonal and can be prepared using standard techniques. The antibodies can be used in adAR purification protocols or the antibodies can be formulated as pharmaceutical compositions and used therapeutically as a,,AR antagonists.

In yet another embodiment, the present invention relates to a gene therapy approach to treating irritative symptoms. In this embodiment, oligonucleotides (constructs) are used that, upon administration, result in the production of a molecule that down regulates production of cadAR. In a related embodiment, the present invention relates to ad1AR antisense constructs and to a method of using same to treat irritative symptoms. Such constructs can be designed to target any of a variety of regions of the addAR gene, including the encoding sequence regions encoding the intracellular portion that interacts with G protein and participates in the signal transduction pathway) and the 5'-untranslated region.

Delivery of the above-described constructs can be effected using any of a variety of approaches, including installation into the bladder via the uretha) and introduction into the cerebrospinal fluid. The constructs can also be administered systemically, in which case targeting can be effected using, for example, smooth muscle bladder smooth muscle) specific promoters.

Effective vectors for use in the above-described gene therapy/antisense embodiments include viral vectors, such as retroviral vectors, adenoviral vectors and adenoassociated viral vectors. The constructs can also be present in association with a lipid, e.g. a liposome. (For details of antisense constructs and delivery systems. etc.

see, for example. Wagner Nature 372:333 (1994).) The amount of construct to be administered will vary, for example, with the construct, the patient and the effect sought. One skilled in the relevant art can readily optimize the dose and treatment regimen.

In yet another embodiment, the invention relates to kits, for example. kits suitable for conducting assays described herein. Such kits can include cadAR, or portion thereof, for example, bound to a solid support. The kit can include an c,,ARencoding sequence, a,dAR antisense construct or a,,AR-specific antibody. The kit can include any of the above components disposed within one or more container means. The kit can. further include ancillary reagents buffers) for use in the assays.

Certain aspects of the present invention are described in greater detail in the non-limiting Examples that follow.

EXAMPLES

The following experimental details are relevant to the specific Examples that follow.

Tissue preparation. Full-thickness human bladder detrusor was obtained as discarded "normal" tissue adjacent to tumor specimens (n=l radical cystectomy, n=12 radical cystoprostatectomy for transitional cell carcinoma of the bladder) with appropriate institutional approval. Each sample was inspected by a pathologist, and normal tissue confirmed. Detrusor smooth muscle was grossly teased from urothelial and serosal layers, snap frozen in liquid nitrogen within minutes of excision, and stored at -70°C for later use. Whole rat bladder was obtained from euthanized male Sprague-Dawley rats (Charles River Laboratories; Wilmington, MA) with institutional animal care committee approval. Rat tissue was harvested within two minutes of death, snap frozen in liquid nitrogen, and stored at -70 0 C for later use.

Human detrusor and rat bladder membrane preparation. Human detrusor and rat whole bladder was minced over dry ice, and suspended in cold lysis buffer (5mM Tris HCI and 5mM EDTA, pH7.4) with protease inhibitors benzamidine (I mg/ml), leupeptin (5mg/ml), and soybean trypsin inhibitor (Sigma Chemical Company; St. Louis, MO). A lysate was prepared with a Polytron PT 3000 (Brinkmann; Westbury, NY) at 10,000 rpm for seconds. After pelleting at 40,000 x g for 15 minutes (Sorvall SM24 rotor), membranes were suspended in cold resuspension buffer (150mM NaCI, Tris HCI, 5mM EDTA, pH 7 4 with protease inhibitors, and kept on ice for immediate use (or stored at -70 0 C for later use). Protein content was determined using the bicinchoninic assay (BCA) with bovine serum albumin (BSA) standards (Pierce; Rockford, IL).

Radioligand binding. Ail mRNA and protein studies described were performed using detrusor from each patient described above In order to conserve sample, and yet fully characterize aARs in human detrusor, additional full saturation binding isotherms were generated in human detrusor samples from a subset of patients using a buffer consisting of 150mM NaCI, Tris HCI and 5mM EDTA, pH7.4. with protease inhibitors. Each reaction was performed in triplicate, in a total volume of 0.25 ml. including diluted human detrusor membranes (50 to 100 mg protein) and the a,AR antagonist

['SI]HEAT

(NEN Research Products-DuPont; Boston, MA) ranging in concentration from 2- 900pM; nonspecific binding was measured in the presence of ImM prazosin (Sigma). The reaction proceeded at 25°C for 45 minutes, and was terminated with five-fold dilution of ice-cold 50mM Tris HC1, pH7.4 buffer, followed by rapid filtration over GF/C filters using a Brandel harvester. Dried filters were then counted in a gamma counter. Specific binding was calculated by subtracting nonspecific binding from total binding. Saturation curves were fit with noniterative regression analysis using InPlot software (GraphPad; San Diego, CA). Total a,AR density was then determined in each detrusor sample as described above, using a saturating concentration of 2 I]HEAT (300pM).

Results are reported as mean±SEM to two significant figures.

To determine Ki values in human detrusor for aAR subtype 2 5 discriminating ligands, competition binding was performed in triplicate in a total volume of 0.25ml using binding buffer (see saturation binding above). Human detrusor membranes (50 to 100 jg protein) were incubated with a K, concentration (120 pM) of the aAR antagonist 1 5 I]HEAT, and increasing concentrations (10 to 10'M) of the non-radiolabeled dAR-selective ligand BMY7378 (Research Biochemicals International; Natick, MA). Reaction conditions were as described above. Curves were fit with noniterative regression analysis using InPlot software (GraphPad).

Preparation of RNA. Total RNA was extracted from human detrusor or rat whole bladder samples using the RNazol method (Tel-Test, Inc.: Friendswood.

TX). RNA was quantitated using a spectrophotometer at 260/280 nm, and aliquoted into 20 mg samples for immediate use.

Human aAR cDNA constructs. The human ai.AR probe consists of a 0.326kb (PvulI/HindIII) fragment in pGEM-4Z (Promega Corporation; Madison, WI), corresponding to nucleotides 958-1283 of the cloned human aAR cDNA (GenBank #L31774). The human abAR probe consists of a 0.673kb (XhoI/BamHI) fragment in pGEM-4Z (Promega), corresponding to nucleotides 94-766 of the cloned human caAR cDNA (GenBank #L31773). The human &IdAR probe consists of a 0.377kb (EcoRI/Pstl) fragment, corresponding to nucleotides 520-896 of the cloned human cadAR cDNA (GenBank #L31772).

Figure 1 shows the location of each aAR subtype probe within a schematic of the encoded protein. The human cyclophilin probe consists of a 0.103kb (KpnI/EcoRI) fragment in pTRI (Ambion, Inc.; Austin, TX), corresponding to nucleotides 38-140 of the cloned human cyclophilin gene (GenBank #X52856).

Labeling of RNA probes. Antisense single-stranded radiolabeled RNA probes were generated from linearized a,AR cDNA constructs using RNA polymerase T7 cyclophilin) and SP6 (alb, aid) as described in the Promega Protocols and Applications Guide (Promega Corporation; Madison, WI). a,,AR and adAR cDNA constructs were linearized with EcoRI, and the ab AR cDNA construct was linearized with HindIII. "P-aUTP (NEN Research Products- DuPont) was incorporated into RNA probes at the time of probe synthesis. All probes were purified on a 5% polyacrylaminde gel (300V for 1.5hr); after exposure to film for 3 min, radiolabeled RNA probes were excised from the gel and passively eluted overnight into 400pl of RPA II kit (Ambion) elution buffer at 37°C.

RNase protection assays. RNase protection assays were conducted as previously described (Zinn et al, Cell 34:865-879 (1983)) with a few modifications. In brief, total RNA samples (20mg) were dissolved in 20ml of hybridization buffer containing >20-fold excess of radiolabeled probe (2 x 10 cpm/reaction for ac,, alb, ad, and 1 x 10 s cpm/reaction for cyclophilin), and incubated overnight at 55 0 C and 65 0 C (a d, cyclophilin). To ensure specificity of the synthesized radiolabeled antisense human aAR subtype selective probes, RNase protection assays were performed in tandom with total RNA extracted from rat-1 fibroblast cells stably expressing each cloned human a,AR subtype. As a negative control, RNase protection assays for each a,AR subtype selective probe were performed in tandom with yeast tRNA samples and other non-hybridizing aAR subtypes. Antisense radiolabeled probe to the highly conserved region of the constitutively expressed human cyclophilin gene was also utilized as a control to ensure identical amounts of total RNA in each assay. The final gel was exposed to X-Omat AR film (Eastman Kodak Company; Rochester, NY) for 24-72 hours.

aoAR mRNA quantitation in human detrusor smooth muscle from RNase protection assays. In order to quantitate relative aAR subtype mRNA, each RNase protection assay final gel was exposed to PhosphorImager plates (Molecular Dynamics; Sunnyvale, CA) for 24 hours. Volume integration of specific protected radiolabeled bands for each mRNA resulting from hybridization products was corrected for background, normalized for cyclophilin signal, and expressed as arbitrary density units, using ImageQuant gel image-analysis software (Molecular Dynamics). a,AR probes contained the following number of UTP sites for 32 P-aUTP incorporation: 88. 117, ld, 51. Arbitrary density units were normalized to the lowest 'P-aUTP incorporating probe (adAR) and then expressed as a fraction (±SEM) of total a,AR mRNA signal strength.

Polymerase Chain Reaction (PCR). RT-PCR was used to confirm expression of human detrusor a,AR subtypes (to ensure low concentrations of a subtype were not missed in human bladder) and to compare aAR subtype mRNA expression in rat whole bladder with previously published rat data (Walden et al, J. Urol. 157:1032-1038 (1997), Scofield et al, J. Pharmacol. Exper. Ther. 275:1035-1042 (1995)). Human and rat aAR subtype primers were synthesized at Duke University Medical Center. Reverse transcription of Img of DNasetreated human detrusor or rat bladder RNA was performed in triplicate in a reaction mixture containing 5mM MgCI,, ImM each of dATP, dCTP, dGTP, and dTTP, I0mM Tris-HC, 50mM KCI, 2ml DEPC treated water, 2.5mM random hexamers, 1 unit of RNase inhibitor, and 2.5 units of MuLV Reverse Transcriptase (Perkin Elmer: Foster City, CA); simultaneous control samples not treated with reverse transcriptase were used to rule out amplification of genomic DNA. Reverse transcriptase reactions were run for 60 min at 42 0 C, 5 min at and 10 min at 4°C. Each aAR mRNA subtype was amplified by PCR in triplicate in a 100ml reaction containing 50mM KC1, 10mM Tris'HC1, pH8.3, 2mM MgCL, 200mM each of dATP, dCTP, dGTP and dTTP, 15pM of sense and antisense primer, 5% DMSO, and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer). PCR reactions were performed in a DeltaCycler IITM temperature cycler (ERICOMP; San Diego, CA). The following conditions were established for all three rat primer sets: one denaturation cycle for 3 minutes at 95 0 C, cycles of 1 min at 95CC, 1 min annealing at 58°C, and a 1 min extension at 72°C.

The following conditions were established for all three human primer sets: one denaturation cycle for 3 minutes at 95°C, 35 cycles of 1 min at 95'C. 1 min annealing at 60 0 C for cta and and 68°C for aj, and a 1 min extension at 72°C.

A final extension cycle was performed for 10 min at 72°C. Reaction mixtures were then cooled at 4 0 C. 10ml of each PCR product was separated by gel electrophoresis in 0.8 agarose. Since PCR experiments were only confirmatory in nature by design, exact quantitation (requiring competitive PCR) was not performed. However, to ensure that any statement regarding relative mRNA levels is appropriate, it is important to note that conditions described above (e.g.

different annealing temperatures) were chosen after extensive preliminary analysis with each primer set to ensure optimal amplification conditions with similar primer product efficiency. Equality of reverse transcription efficiency for products was checked using equal concentrations of starting control cDNA; these 2 5 reactions also served as a positive control for use of correct primer sets. Thirtyfive cycles of amplification was chosen since it is at the upper end of the linear amplification range for all six primer sets (a 1 AR mRNAs are rare at baseline in many human tissues and in our hands 40 cycles of amplification is where the curve becomes non-linear).

EXAMPLE 1 Human Patient Population Human detrusor smooth muscle was obtained from male (n=12) and female patients. Patient age ranged from 56 to 76 years old (mean 59.6).

Significant past medical history included tobacco abuse, coronary artery disease, hypertension controlled with a,AR or PAR antagonists and a history of BOO necessitating previous transurethral resection of prostate. Comparison of results from patients with hypertension and/or BOO suggests medical history did not affect the results. A larger study would be required to make any definitive statement in this regard.

EXAMPLE 2 aAR Ligand Saturation Binding Pharmacological characteristics of a,ARs in human detrusor include a Kd for the radiolabeled a,AR antagonist [1SI]HEAT of 130±1.9 pM, similar to that 2 0 reported for cells stably expressing the cloned human a,,AR subtype ((Schwinn et al, J Pharmacol. Exper. Ther. 272:134-142 (1995)). A representative saturation binding isotherm is shown in Figure 2. Total aAR density as measured by saturation binding in human detrusor membrane preparations with the aAR antagonist 25 I]HEAT is 6.3±1.0 fmol/mg protein (mean±SEM. range 2.7-9.0, n=13). Although low (with corresponding high non-specific binding of 70-80% as expected), caAR expression is reproducible and consistent within and between patients.

EXAMPLE 3 Identification and Quantification of the acAR mRNA Subtypes in Human Bladder Detrusor In order to determine which aAR subtypes are present in human detrusor.

molecular approaches were chosen due to their sensitivity and specificity. To ensure specificity of the synthesized radiolabeled antisense human a, AR subtype selective probes, RNase protection assays were performed simultaneously with total RNA extracted from rat-1 fibroblast cells stably expressing each cloned human aAR subtype. Each a,AR subtype specific probe protects a single predominant fragment of predicted size without cross-hybridization (Figure 3.

positive control cells); a lack of cross-hybridization between subtypes with each probe (Price et al, Mol. Pharmacol. 46:221-226 (1994)). As was previously demonstrated a further negative control, RNase protection assays for each a,AR subtype selective probe were performed in tandom with yeast tRNA samples, where no hybridization is demonstrated (Figure 3, tRNA lane). Human detrusor contains CadAR>a,,AR mRNA, but no aLbAR mRNA in every patient studied (n=13; Figure 3 shows representative results from patients number 1 through This data, when corrected for background, normalized for cyclophilin content, and corrected for probe "P-aUTP incorporation, reveals that a,dAR mRNA constitutes 66±4.8% and ,,AR mRNA 34-4.8% of the total aAR mRNNA in human 2 5 detrusor.

EXAMPLE 4 Confirmation of aAR Subtype mRNA in Human Detrusor and Comparison with Rat Whole Bladder using RT-PCR In order to confirm results from RNase protection assays and to compare with another frequently used animal model (rat), aAR subtype expression was examined using RT-PCR in each patient. Primer nucleotide sequences, melting temperatures and primer positions relative to the cDNA sequence are shown in Table 1 and Table 2; these primers do not span an intron.

TABLE 1. Oligonucleoride primers used for rat a,AR subtype RT-PCR.

Rat a 1 AR primers jnucleotide sequences

T.

JS' 3' aA es GTAGCCAAGAGAAAGCCG 620C a 1 1 An ancisense LAACLLJACCACGATGCCCAG 66"C Primer position relative to cDNA 628-647 839-820 629-649 928-908 a, 6 AR sense

GCTCCTTCTACATCCCGCTCG

i cCtbAR antisense aldAR sense CdAR antisenseI

AGGGGAGCAACATAAGATGT-

CGTGTGCTCCTrCTACCT.ACC

GCACAGCACGAAGACACCCAC

62 0

C

67 0

C

68Q [1062-1042 TABLE 2. Oligonucleotide primers used for human a 1 ,AR subtype RT-PCR.

Human ;:AR primers cz,,,AR sense a 13 AR antisense a 1 tAR sense aIbAR antisense a 1 4 dAR sense a,, 4 AR antisense nucleotide sequens Primer position 5' 3! relative to cDNA ATCATCTCCATCGACCGCTACA 66, 0 C 355-376 TCACTTGCTCCGAGTCCGACnT 68 0 C 697-676 GCTCCTTCTACATCCCTCTGiG 68 0 C 629-649 AGGCTAGCCAGCACAAAGATGA 6 7 0 C 928-908 ACCACGCGCAGCCTCGAGGCAGGC 84 0 C 85-873 GAGCGAGCTGCGGAAGGTGTGCCC 82 0 C f 999-916

I

Although RNase protection assays are considered the "gold standard" for quantitating mRNA present in a given tissue, this approach is not as sensitive as PCR, therefore very small amounts of mRNA can be missed in a RNase protection assay but demonstrated by PCR. As shown in Figure 4, RT-PCR performed on human detrusor total RNA demonstrates the presence of c,,AR and a,,AR mRNA. and lack of ac,AR mRNA, consistent with data from the RNase protection assays. Of note, aidAR mRNA accounts for approximately 60-70% of total a,AR mRNA in human detrusor with alAR mRNA accounting for 30-40%, again confirming the RNase protection assay results. Species heterogeneity (human versus rat) of a;AR subtype mRNA expression has been previously reported for many tissues (Price et al. Mol. Pharmacol. 46:221-226 (1994), Price et al, Mol. Pharmacol. 45:171-175 (1994)). Indeed, as seen in Figure 4. RT-PCR performed on pooled rat bladder total RNA demonstrates the presence of all three az,AR mRNAs in roughly equal concentrations in rat.

EXAMPLE Determination of a,AR Subtype Expression at a Protein Level Competition analysis was used to determine a,AR subtype expression at a protein level in human detrusor. Since molecular studies demonstrate a predominance of aldAR mRNA, the adAR-selective compound BMY7378 was used in these studies. As graphically represented in Figure 5, a two-site fit was evident in every patient studied with high affinity binding predominating (high pK, 8.6±0.2 [66±3.1% total] vs. low pK, 4.9±0.2 [35+3.1% total]) (Table 3).

02/01 2009 12:30 FAX +61 7 3229 3384 CULLEN CO. Z006/013 24 TABLE 3. Results from competition binding experiments utilizing membranes from rat-i fibroblasts stably transfected with each caAR subtype (controls) and human detrusor Since no crAR was found in human detrusor by RNase protection assays and RT-PCR, one versus two site fit of the data was utilized.

BMY7378 (pK a,AR obAR aI,,AR High Lowv Human den-usor 4.9±0.2 8.6-0.2 66±3.1 35±3.1 Control a, 4.8±0.1 Control 0I, Control ad 8.5+0.1 All documents cited above are hereby incorporated in their entirety by reference.

One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, unless in the context or usage an exclusive interpretation of the term is required.

Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

COMS ID No: ARCS-218700 Received by IP Australia: Time 13:35 Date 2009-01-02

Claims (19)

1. A method of treating irritative symptoms of bladder or lower urinary tract disease comprising administering to a patient in need of such treatment an amount of an cala-adrenergic receptor (aodAR) antagonist sufficient to effect said treatment.

2. The method according to claim I wherein said patient suffers from a disease of the bladder.

3. The method according to claim 1 wherein said patient suffers from a disease of the lower urinary tract.

4. The method according to any one of claims 1 to 3 wherein the patient is a female.

The method according to claim 4 wherein said cidAR antagonist is specific for aclAR.

6. The method according to any one of claims 1 to 3 wherein the patient is male.

7. The method according to claim 6 wherein said CIdAR antagonist is an a1dAR antagonist and an otl.AR antagonist.

8. The method according to any one of claims 1 to 7 wherein said symptoms are selected from the group consisting of frequent urination, urgent urination. nocturia and unstable bladder contractions.

9. A method of screening for a candidate agent suitable for use in relieving irritative symptoms of bladder or lower urinary tract disease in a patient comprising: i) incubating a test compound with lad-adrenergic receptor (aodAR), or portion thereof comprising the transmembrane domain of l dAR, ii) determining the amount of said test compound bound to said c IdAR, or said portion thereof, and iii) identifying a test compound that binds said cldAR, or said portion thereof, as said candidate agent.

COMS ID No: ARCS-218700 Received by IP Australia: Time 13:35 Date 2009-01-02 02/01 2009 12:31 FAX +61 7 3229 3384 CULLEN CO. o008/013 26 The method according to claim 9 wherein said test compound bears a detectable label.

11. The method according to either claim 9 or 10 wherein said aldAR, or said portion thereof, is present in a cell membrane.

12. The method according to claim 11 wherein said membrane is the membrane of an intact cell.

13. The method according to claim 12 wherein said cell is a eucaryotic cell.

14. The method according to either claim 12 or 13 wherein said cell is a cell that has been engineered to express or over-express said aIdAR or said portion thereof.

The method according to any one of claims 9 to 14 wherein said test compound is incubated with said tdAR, or said portion thereof, in the presence of a known aldAR binding agent (orlaAR-BA), and the amount of said test compound that binds to said a 1 idAR, or said portion thereof, is determined indirectly by determining the amount of said known CidAR-BA that binds to said aldAR, or said portion thereof.

16. The method according to claim 15 wherein said known aidAR-BA bears a detectable label.

17. The method according to claim 15 wherein said known aldAR-BA is BMY7378 or tamsulosin.

18. A method of treating irritative symptoms of bladder or lower urinary tract disease as defined in claim 1, wherein said antagonist is subtantially as described in one or more of the accompanying examples.

19. A method of screening for a candidate agent suitable for use in relieving irritative symptoms of bladder or lower urinary tract disease as defined in claim 9, wherein said agent is substantially as described in one or more of the accompanying examples. Dated: 2 January 2009 COMS ID No: ARCS-218700 Received by IP Australia: Time 13:35 Date 2009-01-02