WO2007113243A2

WO2007113243A2 - Use of pde 5 inhibitors for the treatment of overactive bladder

Info

Publication number: WO2007113243A2
Application number: PCT/EP2007/053090
Authority: WO
Inventors: Iñigo SÁENZ DE TEJADA GORMAN; Javier Angulo Frutos
Original assignee: Investigación Y Clínica Andrológicas S.L.
Priority date: 2006-03-31
Filing date: 2007-03-30
Publication date: 2007-10-11
Also published as: WO2007113243A3

Abstract

The present invention relates to the use of an inhibitor of cyclic guanosine 3', 5'-monophosphate-specific phosphodiesterase type 5 (PDE 5) activity or of an inhibitor of PDE 5 expression for the manufacture of a medicament for the treatment and/or prophylaxis of overactive bladder disease. In addition, it further refers to pharmaceutical compositions containing said inhibitors of PDE 5 activity and expression.

Description

USE OF PDE 5 INHIBITORS FOR THE TREATMENT OF OVERACTIVE

BLADDER

FIELD OF THE INVENTION The present invention relates to the use of an inhibitor of cyclic guanosine 3', 5'- monophosphate-specific phosphodiesterase type 5 (PDE 5) activity or of an inhibitor of PDE 5 expression for the manufacture of a medicament for the treatment and/or prophylaxis of overactive bladder disease.

BACKGROUND OF THE INVENTION Overactive bladder (OAB) is a medical condition referring to the symptoms of urinary urgency and frequency, with or without urge urinary incontinence (Urology, 55 (supp. 5A): 1-2 (2000)). It can occur idiopathically, in the absence of local pathological, neurological or metabolic factors that would account for these symptoms. It can also occur as the result of neurological disease (for example, spinal cord injury, cerebrovascular disease, Parkinsonism or multiple sclerosis) or bladder outlet obstruction. The latter is most common in men with benign prostatic hyperplasia, and is sometimes referred to as lower urinary tract symptoms (LUTS). The following three symptomatic groups are included in overactive bladder: (1) increased urinary frequency and urinary urgency alone, (2) increased urinary frequency, urinary urgency and urinary incontinence, and (3) mixed-type urinary incontinence.

The normal bladder fills at a physiological rate dictated by the function of the kidneys. The bladder can accommodate large volumes of urine due to the physical properties of the bladder as well as a neural inhibitory system. The inhibitory mechanism is believed to involve inhibition of parasympathetic activity and, possibly, an increase in sympathetic tone to produce detrusor relaxation and allow filling to occur. During filling, the bladder neck and urethra are contracted, preventing leakage. Voiding, or micturition, is characterized by a relaxation of the outlet neck and the urethra followed by contraction of the detrusor muscle. When micturition is complete, the detrusor muscle relaxes and the bladder neck and urethra contract to seal off the bladder and allow bladder filling. Between 4 and 8% of the total population are estimated to suffer from urge urinary incontinence (UUI) at any point in time, although in most countries, only about 15% of such sufferers are diagnosed. Of those diagnosed, only about 70% receive medical treatment. UUI is more prevalent in the elderly, and 80% of the cases are female. Pads and other physical devices are regularly used by a large proportion of incontinent patients not receiving medical treatment. The US market for incontinence pads was estimated at $1.5 billion in 1997.

Muscarinic-cholinergic receptor antagonists (e.g. oxybutin hydrochloride, tolterodine tartrate, trospium chloride, darifenacin succinate, solifenacin ) are commonly prescribed for treatment for OAB in western countries. Propiverine and Flavoxate are sometimes prescribed in Japan. Estrogen and progesterone therapy has been studied and is believed to partially alleviate incontinence in some women. Other studies suggest alpha-adrenergic agonists, beta-adrenergic-receptor activating agents, cholinergic receptor-blocking compounds and cholinergic receptor-stimulating drugs may be beneficial. However, existing therapies are associated with side effects including constipation, visual-accommodation abnormalities, xerophthalmia (dry eyes) and xerostomia (dry mouth), which are poorly tolerated by some users. Therefore, despite the availability of existing treatments, there is a major unmet and growing need for an effective and acceptable medical treatment for UI and OAB.

SUMMARY OF THE INVENTION

It is an object of the present invention the use of an inhibitor of cyclic guanosine 3',5'-monophosphate-specific phosphodiesterase type 5 (PDE 5) activity or of an inhibitor of PDE 5 expression for the manufacture of a medicament for the treatment and/or prophylaxis of overactive bladder disease (OBD). More specifically, the inhibitors used in the invention allow treating OBD by inhibiting upstream targets in one or more biochemical pathways that modulate PDE 5 activity. The expression of PDE 5 is inhibited at one or more transcriptional, translational, or post-translational levels.

The PDE 5 inhibitor used in the present invention is selective with respect to the inhibition of other PDE's (e.g., PDE 1, PDE 2, PDE 3, PDE 4, PDE 6, PDE 7, PDE 8,

PDE 9, PDE 10, and PDE 11) in the target tissue(s) or organs(s). More particularly, PDE 5 inhibitor is at least 2 to 5 times more effective in inhibiting PDE 5 activity than inhibiting any other PDE activity in those target tissues or organs.

In a particular embodiment, the inhibitor of PDE 5 activity is a compound of formula (I):

(I) wherein R⁰ is selected from the group consisting of hydro, halo and C_1-6 alkyl; R¹ is selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci-6 alkyl, C_3-S cycloalkyl, C_3-S cycloalkyl Ci_-3 alkylene, and aryl-Ci_-3 alkylene, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, C_1-6 alkoxy, methylenedioxy, and heteroaryl Ci_-3 alkylene, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, and C_1-6 alkoxy; R² is an optionally substituted monocyclic aromatic ring selected from the group consisting of phenyl, thiophenyl, furyl, and pyridinyl, or an optionally substituted bicyclic ring:

attached to the rest of the molecule via one of the phenyl ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from oxygen, sulfur, and nitrogen; and

R³ represents hydro or Ci_-3 alkyl, or R¹ and R³ together represent a 3- or 4-membered alkyl or alkenyl chain component of a 5- or 6-membered ring; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof. In another particular embodiment, the inhibitor of PDE 5 activity is a compound of formula (II):

(H) wherein R⁴, R⁵, and R⁶ are independently selected from the group consisting of hydro, Ci-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-S cycloalkyl, and C_3-S cycloalkyl Ci_-3 alkylene; and

R⁷ and R⁸ are independently selected from the group consisting of hydro, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-s cycloalkyl, and C_3-s cycloalkyl Ci_-3 alkylene or together represent a 5 or 6 membered cycloalkyl or heterocycloalkyl.

In another particular embodiment, the inhibitor of PDE 5 activity is a compound of formula (III):

R 13

(in) wherein R⁹, R¹⁰, and R¹¹ are independently selected from the group consisting of hydro, Ci-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-s cycloalkyl, and C_3-s cycloalkyl Ci_-3 alkylene; and R¹² and R¹³ are independently selected from the group consisting of hydro, C_1-6 alkyl, C_2-O alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-S cycloalkyl, and C_3-S cycloalkyl Ci_-3 alkylene or together represent a 5 or 6 membered cycloalkyl or heterocycloalkyl.

In another particular embodiment, the inhibitor of PDE 5 activity is a compound of formula (IV):

R 18

(IV) wherein R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are independently selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-s cycloalkyl, C_3-s cycloalkyl Ci_-3 alkylene, C_3-s cycloheteroalkyl, and C_3-s cyclo he tero alkyl Ci_-3 alkylene.

In another particular embodiment, the inhibitor of PDE 5 activity is a compound of formula (V):

wherein

R⁰ is selected from the group consisting of hydro, halo, and C_1-6 alkyl; R¹ is selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci-6 alkyl, C_3-s cycloalkyl, C_3-s cycloalkyl Ci_-3 alkylene, and aryl Ci_-3 alkylene, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, C_1-6 alkoxy, methylenedioxy, and heteroaryl Ci_-3 alkylene, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, and C_1-6 alkoxy; R² is an optionally substituted monocyclic aromatic ring selected from the group consisting of phenyl, thiophenyl, furyl, and pyridinyl, or an optionally substituted bicyclic ring

attached to the rest of the molecule via one of the phenyl ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from oxygen, sulfur, and nitrogen; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof. In another particular embodiment, the inhibitor of PDE 5 activity is a compound of formula (VI):

(VI) wherein R⁰ is selected from the group consisting of hydro, halo, and C_1-6 alkyl;

R¹⁹ hydrogen, halogen, cyano, Het, NO₂, trifiuoromethyl, trifluoromethoxy, C_1-6 alkyl, CO₂H, or CO₂Ci_-6 alkyl; Ci_-6 alkyleneOR^a, C(=O)R^a, OC(=O)R^a, C(=O)OR^a, Ci_-4 alkyleneHet, Ci_-4 alkyleneC(=O)OR^a, OCi_-4 alkylene C(=O)OR^a, Ci_-4 alkylene OCi_-4 alkylene C(=O)OR^a, C(=O)NR^aSO₂R^a, C(=O)C_M alkyleneHet, Ci_-4 alkylene NR^aR^b, C_2-6 alkenylene NR^aR^b, C(=0)NR^aR^b, C(=O)NR^aCi_-4 alkylene 0R^b, C(=O)NR^aCi_-4 alkyleneHet, 0R^a, OC_2-4 alkyleneNR^aR^b, OCi_-4 alkylene CH(OR^a)CH₂NR^aR^b, OCi_-4 alkyleneHet, OC_2-6 alkyleneOR^a, OC_2-4 alkyleneNR^aC(=O)OR^b, NR^aR^b, NR^aCi_-4 alkyleneNR^aR^b, NR^aC(=0)R^b, NR^aC(=0)NR^aR^b, N(SO₂Ci_-4 alkyl)₂, NR^a(SO₂Ci_-4 alkyl), SO₂NR^aR^b, and OSO₂trifluoromethyl; R²⁰ is hydrogen, or R¹⁹ and R²⁰ are taken together to form a 3- or 4- membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;

R^a is selected from the group consisting of hydrogen, C_1-6 alkyl, C_3-6 cycloalkyl, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, heteroaryl, heteroaryl Ci_-3 alkyl, and Ci_-3 alkyleneheteroaryl;

R^b is selected from the group consisting of hydrogen, C_1-6 alkyl, C_3-s cycloalkyl,

Ci_-3 alkyleneN(R^a)2, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, and heteroaryl;

B is aryl or heteroaryl and is selected from the group consisting of optionally substituted 5- or 6-membered aromatic rings and optionally substituted fused bicyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur;

Het represents a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Ci_-4 alkyl or

C(=O)OR^a; m is 1, 2, 3 or 4; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

In another particular embodiment the inhibitor of PDE 5 activity is a compound of formula (VII):

(VII) wherein

R⁰ is selected from the group consisting of hydro, halo, and C_1-6alkyl; R¹⁹ hydrogen, halogen, cyano, Het, NO₂, trifiuoromethyl, trifluoromethoxy, C_1-6 alkyl, CO₂H, or CO₂Ci_-6 alkyl; Ci_-6 alkyleneOR^a, C(=O)R^a, OC(=O)R^a, C(=O)OR^a, Ci_-4 alkyleneHet, Ci_-4 alkylene C(=O)OR^a, OCi_-4 alkylene C(=O)OR^a, Ci_-4 alkyleneOCi_-4 alkyleneC(=O)OR^a, C(=O)NR^aSO₂R^a, C(O)CM alkyleneHet, Ci_-4 alkyleneNR^aR^b, C_2-6 alkenyleneNR^aR^b, C(=O)NR^aR^b, C(=O)NR^aCi_-4 alkyleneOR^b, C(=O)NR^aCi_-4 alkyleneHet, OR^a, OC_2-4 alkyleneNR^aR^b, OCi_-4 alkyleneCH(OR^a)CH₂NR^aR^b, OCi_-4 alkyleneHet, OC_2-6 alkyleneOR^a, OC_2-4 alkyleneNR^aC(=0)0R^b, NR^aR^b, NR^aCi_-4 alkyleneNR^aR^b, NR^aC(=0)R^b, NR^aC(=0)NR^aR^b, N(SO₂Ci_-4 alkyl)₂, NR^a(SO₂Ci_-4 alkyl), S0₂NR^aR^b, and

0 S O₂trifluoromethy 1;

R²⁰ is hydrogen, or R¹⁹ and R²⁰ are taken together to form a 3- or 4- membered alkyl ene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom; X is selected from the group consisting of C(=0), (CH₂)_nC(=O), C(=O)C≡C,

C(=O)C(R^a)=C(R^a), C(=S), SO, SO₂, SO₂C(R^a)=CR^a, CR^aR^b, CR^a=CR^a, C(=0)NR^a, and C(=N-0R^a); Y is selected from the group consisting of R^a, R^d, (CH₂)_nC(=0)R^c, N(R^b)(CH₂)_nR^c,

0(CH₂)_nR^c, N(R^b)C(=0)R^c, C(=0)N(R^a)(R^c), N(R^a)C(=0)R^c,

and N(R^a)S0₂R^c;

R^a is selected from the group consisting of hydrogen, Ci_-6 alkyl, C_3-6 cycloalkyl, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, heteroaryl, heteroaryl Ci_-3 alkyl, and Ci_-3 alkyleneheteroaryl;

R^b is selected from the group consisting of hydrogen, C_1-6alkyl, C_3-scycloalkyl, Ci- ₃ alkyleneN(R^a)₂, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, and heteroaryl;

R^c is selected from the group consisting of hydrogen, Ci_-6 alkyl, aryl, heteroaryl, aryl Ci_-3 alkyl, heteroaryl Ci_-3 alkyl, Ci_-3 alkyleneN(R^a)₂, Ci_-6 alkylenearyl, Ci_-6 alkyleneHet, halo Ci_-6 alkyl, C_3-s cycloalkyl, C_3-s heterocycloalkyl, Het, Ci_-3 alkyleneheteroaryl, Ci_-6 alkylene C(=O)OR^a, and Ci_-3 alkylene C_3-s heterocycloalkyl; or R^a and R^c are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;

R^d is a 5- or 6-membered ring or a bicyclic fused ring system, saturated or partially or fully unsaturated, comprising carbon atoms and optionally one to three heteroatoms selected from oxygen, sulfur, and nitrogen, and optionally substituted with one or more R¹⁹; Het represents a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Ci_-4 alkyl or C(=O)OR^a; n is 0 or 1, q is 0, 1, 2, 3, or 4, t is 1, 2, 3, or 4, m is 1, 2, 3 or 4; and pharmaceutically acceptable salt, solvate, hydrate, isomer and prodrugs thereof.

In another particular embodiment, the inhibitor of PDE 5 expression is an antisense oligonucleotide which negatively regulates PDE 5 expression.

In another particular embodiment, the inhibitor of PDE 5 expression is a ribozyme inhibitor.

In another particular embodiment, the inhibitor of PDE 5 expression is a doble- stranded RNA wherein one strand is complementary to a target region in PDE 5- encoding polynucleotide.

In another particular embodiment, the inhibitor of PDE 5 expression is a circular lasso RNA which inhibits the PDE 5-encoding polynucleotide.

Another object of the present invention refers to a pharmaceutical composition comprising at least of the PDE 5 activity inhibitors defined above, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

Finally, another object of the invention relates to a method of preventing and/or treating overactive bladder disease comprising administering to a patient in need thereof a therapeutically effective amount of a PDE inhibitor as the ones defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 outlines a generic experimental protocol for the assessment of tadalafil (a PDE 5 inhibitor) in treating experimental OAB. Figure 2 shows the effects of tadalafil (0.5 mg/kg; intravenously) on micturition frequency during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 mL/hr for 20 min in male rats. Data are expressed as the mean ± SEM of the number of micturition reflexes observed within the period of infusion, and n indicates the number of animals used. Figure 3 shows the effects of tadalafil (0.5 mg/kg; i.v.) on micturition volume during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 mL/hr for 20 min in male rats. Data are expressed as the mean ± SEM, and n indicates the number of animals used. Figure 4 shows the effects of tadalafil (0.5 mg/kg; i.v.) on intravesical pressure increases during micturition reflexes induced by infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 mL/hr rate for 20 min in male rats. Data are expressed as the mean ± SEM, and n indicates the number of animals used.

Figure 5 compares the effects of tadalafil (0.5 mg/kg; i.v.) and vehicle on micturition frequency during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 mL/hr for 20 min in male rats. Data are expressed as the mean ± SEM of the percentage of the number of micturition reflexes observed in the previous exposure to

CH₃COOH, before vehicle or tadalafil administration. Number of animals used is in parentheses. The difference between tadalafil and vehicle (control) was significant (p < 0.01, by unpaired Student's t-test).

Figure 6 compares the effects of tadalafil (0.5 mg/kg; i.v.) and vehicle on micturition volume during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 mL/hr for 20 min in male rats. Data are expressed as the mean ± SEM of the percentage of micturition volume observed in the previous exposure to CH₃COOH, before vehicle or tadalafil administration. Number of animals used is in parentheses. The difference between tadalafil and vehicle (control) was significant (p < 0.01 by unpaired Student's t-test).

Figure 7 compares the effects of tadalafil (0.5 mg/kg; i.v.) and vehicle on intravesical pressure (IVP) increases during micturition reflexes induced by infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate pf 5 mL/hr for 20 min in male rats. Data are expressed as the mean ± SEM of the percentage of IVP increases observed in the previous exposure to CH₃COOH, before vehicle or tadalafil administration. Number of animals used is in parentheses. The difference between tadalafil and vehicle (control) was not significant. Figure 8 shows the lack of effect of vehicle on micturition frequency, mean volume for micturition, and IVP increase during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 ml/hr for 20 min in male rats.

Figure 9 shows the effect of the anticholinergic oxybutynin under the same experimental conditions as used for obtaining data in Figure 8.

Figure 10 shows the effect of the PDE 5 inhibitor tadalafil under the same experimental conditions as used for obtaining data in Figure 8.

DETAILED DESCRIPTION OF THE INVENTION

An object of the present invention refers to the use of an inhibitor of PDE 5 activity or of an inhibitor of PDE 5 expression for the manufacture of a medicament for the treatment and/or prophylaxis of overactive bladder disease (OBD). Inhibiting of PDE 5 activity can be either direct or indirect and selective or non-selective.

"Direct inhibition" of PDE 5 activity refers to inhibiting PDE 5 activity through an interaction between PDE 5 and an inhibitor. "Indirect inhibition" of PDE 5 activity refers to inhibiting PDE 5 activity through inhibiting PDE 5 expression at one or more transcriptional, translational, or post-translational levels.

"Selectively inhibiting" PDE 5 activity refers to inhibiting the activity of PDE 5, either directly or indirectly, more effectively than inhibiting at least one other member of the PDE family.

"Non-selectively inhibiting" PDE 5 refers to inhibiting the activity of PDE 5, either directly or indirectly, while simultaneously inhibiting the activity of one or more other members of the PDE family at a comparable level.

"Ci-6 alkyl" as used herein refers to a straight or branched hydrocarbon chain radical consisting of carbon and hydrogen atoms, containing no insaturation, having one to six carbon atoms, and which is attached to the rest of the molecule by a single bond, e. g., methyl, ethyl, n-propyl, /-propyl, n-butyl, t-butyl, n-pentyl, etc. An alkyl group can be unsubstituted or substituted with one or two suitable substituents.

"C_2-O alkenyl" as used herein refers to a straight or branched chain alkenyl moiety consisting of carbon and hydrogen atoms, having two to six carbon atoms and at least one double bond of either E or Z stereochemistry where applicable, e.g., vinyl, allyl, 1- and 2-butenyl, and 2-methyl-2-propenyl. An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.

"C_2-O alkynyl" as used herein refers to a straight or branched chain alkynyl moiety consisting of carbon and hydrogen atoms, having two to six carbon atoms and at least one triple bond. The triple bond of an alkynyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkynyl groups include, but are not limited to alkynyl groups such as ethynyl, propynyl, butynyl and pentynyl An alkynyl group can be unsubstituted or substituted with one or two suitable substituents. "C3-8 cycloalkyl" as used herein refers to an alicyclic group consisting of carbon and hydrogen atoms, having three to eight carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. A cycloalkyl group can be unsubstituted or substituted with one or two suitable substituents

The term "heterocycloalkyl" as used herein refers to a stable 3-to 15 membered ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. For the purposes of this invention, the heterocycle may be a monocyclic, bicyclic or tricyclic ring system, which may include fused ring systems. Examples of such heterocycles include, but are not limited to, azepines, benzimidazole, benzothiazole, furan, isothiazole, imidazole, indole, piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran.

The term "aryl" as used herein refers to a phenyl or phenyl substituted with one to three substituents independently selected from halo, C_1-6 alkyl, C_1-6 alkoxy and methylenedioxy.

The term "heteroaryl" as used herein refers to thienyl, furyl or pyridyl, each optionally substituted with one to three substituents independently selected form halo, C i-6 alkyl, and C_1-6 alkoxy.

The term "C₁-C₆ alkoxy" as used herein refers to a radical of the formula -ORa, wherein R_a is an alkyl radical as defined above, e. g., methoxy, ethoxy, propoxy, etc.

The term "C_3-S cycloalkyl Ci_-3 alkylene" as used herein refers to a cycloalkyl group as defined above linked to a Ci_-3 alkyl group. Preferred examples include methylcyclopentyl and methylcyclohexyl.

The term "aryl Ci_-3 alkylene" as used herein refers to an aryl group linked to a Ci_-3 alkyl group. Preferred example is benzyl. The term "heteroaryl Ci_-3 alkylene" as used herein refers to a heteroaryl group as defined above linked to a Ci_-3 alkyl group.

The term "halo" as used herein refers to fluorine, chlorine, bromine or iodine.

Inhibitors of PDE 5 activity

The relative efficacies of inhibiting enzyme activity (or other biological activity) can be established by determining a decrease in the activity or expression of one PDE, e.g., PDE 5, and comparing the results to inhibition of another, e.g., non-PDE 5, PDE. Determination of the selectivity can be via the comparison of the enzyme activity of PDE 5 in a biochemical assay, level of mRNA transcription or level of protein expression. Such determinations are performed using conventional means, such as through measurement of IC50. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of an inhibitor under study. The experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used. The concentration at which 50% enzyme activity is inhibited (as compared to the activity in the absence of any effector or inhibitor) is taken as the IC50 value. Analogously, other inhibitory concentrations can be defined through appropriate determinations of activity. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., IC₉0, etc. It will be appreciated that determining an IC50 value is generally associated with direct inhibition of an enzyme, but that inhibition of an enzyme through indirect means also provides the same measurable endpoint, i.e., a decrease in enzyme activity. Thus, IC comparisons are contemplated for determining a degree of selective inhibition whether by direct or indirect mechanisms. In a particular embodiment, PDE 5 activity is selectively inhibited at least about

5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold more than any one or more members of the PDE family (e.g., PDE 1, PDE 2, PDE 3, PDE 4, PDE 6, PDE 7, PDE 8, PDE 9, PDE 10, and PDE 11). In another particular embodiment, PDE 5 activity is selectively inhibited at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 200-fold, at least about 250- fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold, more than any one or more members of the PDE family selectively inhibits PDE 5.

In a particular embodiment, the inhibitor of PDE 5 activity used in the present invention is a compound of formula (I):

(I) wherein R⁰ is selected from the group consisting of hydro, halo and C_1-6 alkyl; R¹ is selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci-6 alkyl, C_3-S cycloalkyl, C_3-S cycloalkyl Ci_-3 alkylene, and aryl-Ci_-3 alkylene, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, C_1-6 alkoxy, methylenedioxy, and heteroaryl Ci_-3 alkylene, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, and C_1-6 alkoxy;

R² is an optionally substituted monocyclic aromatic ring selected from the group consisting of phenyl, thiophenyl, furyl, and pyridinyl, or an optionally substituted bicyclic ring:

R³ represents hydro or Ci_-3 alkyl, or R¹ and R³ together represent a 3- or 4-membered alkyl or alkenyl chain component of a 5- or 6-membered ring; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

These beta-carboline compounds and derivatives thereof are described in U.S. Patents 5,859,006; 5,981,527; 6,825,197; 6,838,456; 6,858,620; 6,872,721; 6,878,711; 6,903,099; 6,911,542; 6,960,587; 6,962,918; 6,984,641; 6,001,847; 6,143,757; 6,043,252; 6,117,881; 6,306,870; 6,462,047; 6,992,192; and 7,022,856 as well as in U.S. Patent Applications Nos. 2003/0207867; 2004/0162291; 2004/0152705; 2004/0116458; and 2004/0147542, each of which is incorporated by reference herein in its entirety.

In a preferred embodiment, the compound of formula (I) is a compound of formula (Ia):

(Ia) which is interchangeably referred to throughout this disclosure as tadalafil, or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof. In another particular embodiment, the inhibitor of PDE 5 activity used in the present invention is a compound of formula (II):

R⁷ and R⁸ are independently selected from the group consisting of hydro, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-s cycloalkyl, and C_3-s cycloalkyl Ci_-3 alkylene or together represent a 5 or 6 membered cycloalkyl or heterocycloalkyl; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

Compounds of formula (II) are generally disclosed in US patents 5,250,534, 6,469,012 and 6,436,944; US patent applications 2004/087599 and 2001/044441 and PCT Publications WO05/049616, WO02/079203 and WO02/072586, each of which is incorporated herein in its entirety.

In a preferred embodiment, the compound of formula (II) is a compound of formula (Ha):

(Ha) which is interchangeably referred to throughout this disclosure as sildenafil, or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

In another particular embodiment, the inhibitor of PDE 5 activity used in the present invention is a compound of formula (III):

R¹³ (III) wherein R⁹, R¹⁰, and R¹¹ are independently selected from the group consisting of hydro, Ci-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-S cycloalkyl, and C_3-S cycloalkyl Ci_-3 alkylene; and

R¹² and R¹³ are independently selected from the group consisting of hydro, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-s cycloalkyl, and C_3-s cycloalkyl Ci_-3 alkylene or together represent a 5 or 6 membered cycloalkyl or heterocycloalkyl; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof. Compounds of formula (III) are generally disclosed in US patents 6,362,178 and

6,740,306 and PCT publications WO05/110420, WO06/015715 and WO01/19357, each of which is incorporated by reference herein in its entirety.

In a preferred embodiment, the compound of formula (III) is a compound of formula (Ilia):

(Ilia) which is interchangeably referred to throughout this disclosure as vardenafil, or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof. In another particular embodiment, the inhibitor of PDE 5 activity used in the present invention is a compound of formula (IV):

R 18

(IV) wherein R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are independently selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-S cycloalkyl, C_3-S cycloalkyl Ci_-3 alkylene, C_3-s cycloheteroalkyl, and C_3-s cyclo he tero alkyl Ci_-3 alkylene; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

Compounds of formula (IV) are generally disclosed in U.S. Patent Nos. 5,171,851 and 6,844,436, each of which is incorporated by reference herein in its entirety.

In a preferred embodiment, the compound of formula (IV) is a compound of formula (IVa):

which is interchangeably referred to throughout this disclosure as udenafil, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In another particular embodiment, the inhibitor of PDE 5 activity used in the present invention is a compound of formula (V):

(V) wherein

R⁰ is selected from the group consisting of hydro, halo, and C_1-6 alkyl; R¹ is selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci-6 alkyl, C_3-S cycloalkyl, C_3-S cycloalkyl Ci_-3 alkylene, and aryl Ci_-3 alkylene, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, C_1-6 alkoxy, methylenedioxy, and heteroaryl Ci_-3 alkylene, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, and C_1-6 alkoxy;

R² is an optionally substituted monocyclic aromatic ring selected from the group consisting of phenyl, thiophenyl, furyl, and pyridinyl, or an optionally substituted bicyclic ring

attached to the rest of the molecule via one of the phenyl ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from oxygen, sulfur, and nitrogen; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof. In another particular embodiment, the inhibitor of PDE 5 activity used in the present invention is a compound of formula (VI):

(VI) wherein

R⁰ is selected from the group consisting of hydro, halo, and C_1-6 alkyl; R¹⁹ hydrogen, halogen, cyano, Het, NO₂, trifiuoromethyl, trifluoromethoxy, C_1-6 alkyl, CO₂H, or CO₂Ci_-6 alkyl; Ci_-6 alkyleneOR^a, C(=O)R^a, OC(=O)R^a, C(=O)OR^a, Ci_-4 alkyleneHet, Ci_-4 alkyleneC(=O)OR^a, OCi_-4 alkylene C(=O)OR^a, Ci_-4 alkylene OCi_-4 alkylene C(=O)OR^a, C(=O)NR^aSO₂R^a, C(O)CM alkyleneHet, Ci_-4 alkylene NR^aR^b, C_2-6 alkenylene NR^aR^b, C(=0)NR^aR^b, C(=O)NR^aCi_-4 alkylene 0R^b, C(=O)NR^aCi_-4 alkyleneHet, 0R^a, OC_2-4 alkyleneNR^aR^b, OCi_-4 alkylene CH(OR^a)CH₂NR^aR^b, OCi_-4 alkyleneHet, OC_2-6 alkyleneOR^a, OC_2-4 alkyleneNR^aC(=O)OR^b, NR^aR^b, NR^aCi_-4 alkyleneNR^aR^b, NR^aC(=0)R^b, NR^aC(=0)NR^aR^b, N(SO₂Ci_-4 alkyl)₂, NR^a(SO₂Ci_-4 alkyl), S0₂NR^aR^b, and OSO₂trifluoromethyl; R²⁰ is hydrogen, or R¹⁹ and R²⁰ are taken together to form a 3- or 4- membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;

R^b is selected from the group consisting of hydrogen, Ci_-6 alkyl, C_3-s cycloalkyl, Ci_-3 alkyleneN(R^a)₂, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, and heteroaryl; B is aryl or heteroaryl and is selected from the group consisting of optionally substituted 5- or 6-membered aromatic rings and optionally substituted fused bicyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur; Het represents a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Ci_-4 alkyl or C(=O)OR^a; m is 1, 2, 3 or 4; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

In another particular embodiment the inhibitor of PDE 5 activity used in the present invention is a compound of formula (VII):

(VII) wherein

R⁰ is selected from the group consisting of hydro, halo, and Ci_6alkyl;

R¹⁹ hydrogen, halogen, cyano, Het, NO₂, trifluoromethyl, trifluoromethoxy, C_1-6 alkyl, CO₂H, or CO₂Ci_-6 alkyl; Ci_-6 alkyleneOR^a, C(=O)R^a, OC(=O)R^a, C(=O)OR^a, Ci_-4 alkyleneHet, Ci_-4 alkylene C(=O)OR^a, OCi_-4 alkylene C(=O)OR^a,

Ci_-4 alkyleneOCi_-4 alkyleneC(=O)OR^a, C(=O)NR^aSO₂R^a, C(O)C_M alkyleneHet, Ci_-4 alkyleneNR^aR^b, C_2-6 alkenyleneNR^aR^b, C(=0)NR^aR^b, C(=O)NR^aCi_-4 alkyleneOR^b, C(=O)NR^aCi_-4 alkyleneHet, 0R^a, OC_2-4 alkyleneNR^aR^b, OCi_-4 alkyleneCH(OR^a)CH₂NR^aR^b, OCi_-4 alkyleneHet, OC_2-6 alkyleneOR^a, OC_2-4 alkyleneNR^aC(=O)OR^b, NR^aR^b, NR^aCi_-4 alkyleneNR^aR^b, NR^aC(=0)R^b,

NR^aC(=0)NR^aR^b, N(SO₂Ci_-4 alkyl)₂, NR^a(SO₂Ci_-4 alkyl), SO₂NR^aR^b, and O S O₂trifluoromethy 1;

R²⁰ is hydrogen, or R¹⁹ and R²⁰ are taken together to form a 3- or 4- membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom;

X is selected from the group consisting of C(O), (CH₂)_nC(=O), C(=O)C≡C, C(=O)C(R^a)=C(R^a), C(=S), SO, SO₂, SO₂C(R^a)=CR^a, CR^aR^b, CR^a=CR^a, C(=0)NR^a, and C(=N-0R^a); Y is selected from the group consisting of R^a, R^d, (CH₂)_nC(=O)R^c, N(R^b)(CH₂)_nR^c,

O(CH₂)_nR^c, N(R^b)C(=O)R^c, C(=O)N(R^a)(R^c), N(R^a)C(=O)R^c,

and

N(R^a)SO₂R^c;

R^b is selected from the group consisting of hydrogen, C_1-6alkyl, C_3-scycloalkyl, Ci- ₃alkyleneN(R^a)₂, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, and heteroaryl;

R^c is selected from the group consisting of hydrogen, C_1-6 alkyl, aryl, heteroaryl, aryl Ci_-3 alkyl, heteroaryl Ci_-3 alkyl, Ci_-3 alkyleneN(R^a)₂, Ci_-6 alkylenearyl, Ci_-6 alkyleneHet, halo Ci_-6 alkyl, C_3-s cycloalkyl, C_3-s heterocycloalkyl, Het, Ci_-3 alkyleneheteroaryl, Ci_-6 alkylene C(=O)OR^a, and Ci_-3 alkylene C_3-s heterocycloalkyl; or R^a and R^c are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;

R^d is a 5- or 6-membered ring or a bicyclic fused ring system, saturated or partially or fully unsaturated, comprising carbon atoms and optionally one to three heteroatoms selected from oxygen, sulfur, and nitrogen, and optionally substituted with one or more R¹⁹; Het represents a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Ci_-4 alkyl or

C(=O)OR^a; n is 0 or 1, q is 0, 1, 2, 3, or 4, t is 1, 2, 3, or 4, m is 1, 2, 3 or 4; and pharmaceutically acceptable salt, solvate, isomer and prodrugs thereof.

Compounds of formula (V), (VI) and (VII) are also disclosed in U.S. Patents

5,859,006; 5,981,527; 6,825,197; 6,838,456; 6,858,620; 6,872,721; 6,878,711;

6,903,099; 6,911,542; 6,960,587; 6,962,918; 6,984,641; 6,001,847; 6,143,757;

6,043,252; 6,117,881; 6,306,870; 6,462,047; 6,992,192; and 7,022,856 as well as in U.S. Patent Applications Nos. 2003/0207867; 2004/0162291; 2004/0152705; 2004/0116458; and 2004/0147542, each of which is incorporated by reference herein in its entirety.

Inhibitors used in the invention embrace the compounds disclosed herein, compounds having similar inhibitory profiles, and compounds that compete with a disclosed inhibitor compound for binding to PDE 5, and in each case, conjugates and derivatives thereof.

Unless otherwise stated, the compounds used in the invention are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon or ¹⁵N-enriched nitrogen are within the scope of this invention.

The term "pharmaceutically acceptable salts" refers to any pharmaceutically acceptable salt which, upon administration to the recipient is capable of providing (directly or indirectly) a compound as described herein. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts.

For instance, pharmaceutically acceptable salts of compounds used in the invention are synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminium and lithium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine, glucamine and basic aminoacids salts. The preparation of salts and derivatives can be carried out by methods known in the art.

The term "solvate" as used herein means a compound or a pharmaceutically acceptable salt of a compound, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate".

The term "prodrug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art, and include, depending on the functional groups present in the molecule and without limitation, the following derivatives of the present compounds: esters, amino acid esters, phosphate esters, metal salts sulfonate esters, carbamates, and amides. Prodrug design is discussed generally in Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion is provided in Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium Series, and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987).

To illustrate, prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkage, thereby introducing or exposing a functional group on the resultant product. The prodrugs can be designed to react with an endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life. Alternatively, prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, amino acids, or acetate. The resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound. High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound.

Particularly favoured derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. The compounds used in the present invention may be in crystalline form either as free compounds or as solvates (e.g. hydrates).

The compounds of formula (I) to (VII) or their salts or solvates used in the invention are preferably in pharmaceutically acceptable or substantially pure form. By pharmaceutically acceptable form is meant, "inter alia", having a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and including no material considered toxic at normal dosage levels. Purity levels for the drug substance are preferably above 50%, more preferably above 70%, most preferably above 90%. In a preferred embodiment it is above 95% of the compounds of formula (I) to (VII), or of its salts, solvates or prodrugs.

Inhibitors of PDE 5 expression

In a particular embodiment, compounds that selectively negatively regulate PDE 5 mRNA expression more effectively than they do other isozymes of the PDE family, and that possess acceptable pharmacological properties are also contemplated for their use in the present invention. Representative polynucleotides encoding human PDE 5 are disclosed, for example, in Genbank Accession Nos. P16499, Pl 1541, NP 001074, NP_237223, NP_246273, and NP_236914, in each case the entire disclosures of which are incorporated herein by reference.

In another embodiment, antisense oligonucleotides which negatively regulate PDE 5 expression via hybridization to messenger RNA (mRNA) encoding PDE 5 can also be used in the present invention. Modifications of PDE 5 expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA,

PNA, or modified oligonucleotides) to the coding or regulatory regions of the gene encoding PDE 5. Such technology is well known in the art, and antisense oligonucleotides or larger polynucleotides can be designed from various locations along the coding or control regions of sequences encoding PDE 5. (See, e.g., Agrawal, S., ed.

(1996) Antisense Therapeutics, Humana Press Inc., Totawa, N.J.). In one embodiment, antisense oligonucleotides at least 5 to about 50 nucleotides in length, including all lengths (measured in number of nucleotides) in between, which specifically hybridize to mRNA encoding PDE 5 and inhibit mRNA expression, and as a result PDE 5 protein expression, are contemplated for their use in the invention. Antisense oligonucleotides include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo. It is understood in the art that, while antisense oligonucleotides that are perfectly complementary to a region in the target polynucleotide possess the highest degree of specific inhibition, antisense oligonucleotides that are not perfectly complementary, i.e., those which include a limited number of mismatches with respect to a region in the target polynucleotide, also retain high degrees of hybridization specificity and therefore also can inhibit expression of the target mRNA. Accordingly, antisense oligonucleotides that are perfectly complementary to a target region in a polynucleotide encoding PDE 5, as well as antisense oligonucleotides that are not perfectly complementary (i.e., include mismatches) to a target region in the target polynucleotide to the extent that the mismatches do not preclude specific hybridization to the target region in the target polynucleotide, are also used in the present invention. Preparation and use of antisense compounds is described, for example, in U.S. Patent No. 6,277,981, the entire disclosure of which is incorporated herein by reference (see also, Gibson (ed.), Antisense and Ribozyme Methodology, (1997), the entire disclosure of which is incorporated herein by reference).

In another particular embodiment, uses are also provided utilizing ribozyme inhibitors which, as is known in the art, include a nucleotide region which specifically hybridizes to a target polynucleotide and an enzymatic moiety that digests the target polynucleotide. Specificity of ribozyme inhibition is related to the length of the antisense region and the degree of complementarity of the antisense region to the target region in the target polynucleotide. Therefore, in one embodiment, methods are provided for ribozyme inhibitors comprising antisense regions from 5 to about 50 nucleotides in length, including all nucleotide lengths in between, that are perfectly complementary, as well as antisense regions that include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target PDE 5-encoding polynucleotide. Ribozymes used in the present invention include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo, to the extent that the modifications do not alter the ability of the ribozyme to specifically hybridize to the target region or diminish enzymatic activity of the molecule. Because ribozymes are enzymatic, a single molecule is able to direct digestion of multiple target molecules thereby offering the advantage of being effective at lower concentrations than non-enzymatic antisense oligonucleotides. Preparation and use of ribozyme technology is described in U.S. Patent Nos. 6,696,250; 6,410,224; and 5,225,347, the entire disclosures of which are incorporated herein by reference.

In another embodiment, uses are provided in which RNAi technology is utilized for inhibiting PDE 5 expression. In one embodiment, double-stranded RNA (dsRNA), wherein one strand is complementary to a target region in a target PDE 5-encoding polynucleotide, is employed. In general, dsRNA molecules of this type are less than 30 nucleotides in length and referred to in the art as short interfering RNA (siRNA). In another embodiment, however, use of dsRNA molecules longer than 30 nucleotides in length, and in certain embodiments, these longer dsRNA molecules can be about 30 nucleotides in length up to 200 nucleotides in length and longer, and including all length dsRNA molecules in between. As with other RNA inhibitors, complementarity of one strand in the dsRNA molecule can be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target PDE 5-encoding polynucleotide. As with other RNA inhibition technologies, dsRNA molecules include those comprising modified internucleotide linkages and/or those comprising modified nucleotides which are known in the art to improve stability of the oligonucleotide, i.e., make the oligonucleotide more resistant to nuclease degradation, particularly in vivo. Preparation and use of RNAi compounds is described in U.S. Patent Publication No. 2004/0023390, the entire disclosure of which is incorporated herein by reference. In another embodiment, inhibition of PDE 5 expression is effected using RNA lasso technology. Circular RNA lasso inhibitors are highly structured molecules that are inherently more resistant to degradation and therefore do not, in general, include or require modified internucleotide linkage or modified nucleotides. The circular lasso structure includes a region that is capable of hybridizing to a target region in a target polynucleotide, the hybridizing region in the lasso being of a length typical for other RNA inhibiting technologies. As with other RNA inhibiting technologies, the hybridizing region in the lasso may be a perfect match with the target region in the target polynucleotide, or may include mismatches to the extent that the mismatches do not preclude specific hybridization to the target region in the target PDE 5-encoding polynucleotide. Because RNA lassos are circular and form tight topological linkage with the target region, inhibitors of this type are generally not displaced by helicase action unlike typical antisense oligonucleotides, and therefore can be utilized as dosages lower than typical antisense oligonucleotides. Preparation and use of RNA lassos is described in U.S. Patent 6,369,038, the entire disclosure of which is incorporated herein by reference.

The inhibitors of PDE 5 activity and expression used in the present invention for the prevention and/or treatment of overactive bladder disease, are formulated in a suitable pharmaceutical composition, in a therapeutically effective quantity.

Such pharmaceutical compositions may be for administration for injection, or for oral, nasal, transdermal or other forms of administration, including, e.g., by intravenous, intradermal, intramuscular, intravesical, intraurethral, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drugs) or subcutaneous injection (including depot administration for long term release); by sublingual, anal, vaginal, or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea. Preferably, the pharmaceutical composition is administered transdermally and intravesically. The pharmaceutical composition may be administered in a single dose or a plurality of doses over a period of time. In general, pharmaceutical compositions comprise effective amounts of the active compound together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation. The pharmaceutical compositions optionally may include still other pharmaceutically acceptable liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media, including but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, starches, sucrose, dextrose, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the inhibitors. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference. The compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.

Contemplated for use herein are oral solid dosage forms, which are described generally in Chapter 89 of Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Alternatively, proteinoid encapsulation may be used (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673), or liposomal encapsulation may be used, the liposomes optionally derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of solid dosage forms for therapeutics in general is given in Chapter 10 of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, incorporated herein by reference. In general, the formulation will include a preparation of the invention and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.

If necessary, the compounds used in the invention may be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline (Abuchowski and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, (1981), pp 367-383; Newmark, et al, J Appl. Biochem. 4:185-189 (1982)). Other polymers that could be used are poly-l,3-dioxolane and poly-l,3,6-tioxocane.

For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compound. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See US Patent No. 5,792,451.

Compositions can be included in formulation as fine multiparticulates in the form of granules or pellets of particle size about, for example, one mm. The formulation of the material for capsule administration can also be as a powder, lightly compressed plugs or even as tablets. Compositions are optionally prepared by compression.

Colorants and flavoring agents may be included. For example, the preparation may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

The compositions can be diluted or increased in the volume with an inert material. Exemplary diluents include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA- Rx 1500, Emcompress and Avicell.

Compositions including disintegrants are further contemplated in solid dosage form compositions. Materials used as disintegrants include, but are not limited to, starch

(including the commercial disintegrant based on starch, Explotab), sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite. Another form of disintegrant is an insoluble cationic exchange resin. Powdered gums may also be used as disintegrants and as binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Pharmaceutical compositions including binders are further contemplated to hold the therapeutic agent together to form a hard tablet and exemplary binders include materials from natural products such as acacia, tragacanth, starch, and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent in a pharmaceutical composition is further contemplated to prevent sticking during the formulation process. Lubricants include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of a pharmaceutical composition during formulation and to aid rearrangement during compression are also provided. Exemplary glidants include starch, talc, pyrogenic silica and hydrated silicoaluminate. To aid dissolution of a composition into the aqueous environment, incorporation of a surfactant as a wetting agent is contemplated. Exemplary surfactants include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfo succinate, and dioctyl sodium sulfonate. Cationic detergents are contemplated, including for example and without limitation, benzalkonium chloride or benzethonium chloride. Compositions using as surfactants lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose are also contemplated. Compositions comprising these surfactants, either alone or as a mixture in different ratios, are therefore further provided. Optionally, additives are included in a pharmaceutical composition to enhance uptake of the compound, such additives including, for example and without limitation, fatty acids oleic acid, linoleic acid and linolenic acid.

In a particular embodiment, controlled release formulations are also provided. A preparation is incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices, e.g., alginates, polysaccharides, may also be incorporated into the formulation. Another form of a controlled release is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.

Other coatings may be used in compositions disclosed herein, including for example, a variety of sugars which could be applied in a coating pan. The compositions also include a film coated tablet and the materials used in this instance are divided into two groups. The first includes the nonenteric materials, such as and without limitation methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy- ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy- methyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials is also contemplated to provide the optimum film coating.

Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.

Also contemplated herein is pulmonary delivery of a preparation of the invention. The compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Pulmonary delivery is described in Adjei et al, Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44; Braquet et al. (1989), J Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146; Hubbard et al. (1989), Annals Int. Med. 3: 206-12; Smith et al. (1989), J Clin. Invest. 84: 1145-6; Oswein et al. (March 1990), "Aerosolization of Proteins", Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.; Debs et al. (1988), J Immunol. 140: 3482-8 and Platz et al, U.S. Pat. No. 5,284,656, the disclosures of which are incorporated herein by reference.

Also contemplated for practice of the invention is a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by

Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest

Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N. C; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for the dispensing a preparation of the invention. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.

For effective delivery to distal lung, the composition is prepared in particulate form with an average particle size of less than 10 μm (or microns), preferably form 0.5 to 5 μm.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the compounds used in the invention dissolved in water at a concentration of about 0.1 to 25 mg per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofiuorocarbon, a hydro fluorocarbon, or a hydrocarbon, including trichlorofiuorome thane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafiuoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the compound used in the invention and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.

Nasal delivery of preparations of the invention is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery via transport across other mucous membranes is also contemplated.

Buccal delivery of the compound is also contemplated. Buccal delivery formulations are known in the art for use with peptides.

Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from its use in derivatizing a compound). Dextrans, such as cyclodextran, may be used. Cyclodextrins may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.

The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is also contemplated. The compounds used in the invention are administered in a therapeutically effective amount. A therapeutically effective amount, as used herein, is an amount of a PDE 5 inhibitor sufficient to treat or prevent the symptoms, progression, or onset of OAB. The therapeutically effective amount will vary depending on the inhibitor, the state of the subject's OAB or its severity, and the age, weight, etc., of the subject to be treated. Dosage ranges contemplated are about 0.001 to about 20 mg/kg. Specific ranges include about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.005 to about 2 mg/kg, and about 0.01 to about 1 mg/kg. In one embodiment, a selective inhibitor is administered in an amount to selectively inhibit PDE 5.

A therapeutically effective amount can vary, depending on any of a number of factors, including, e.g., the specific inhibitor, the route of administration, the condition of the subject, as well as other factors understood by those in the art.

Toxicity and therapeutic efficacy of inhibitors of PDE 5 activity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Additionally, this information can be determined in cell cultures or experimental animals additionally treated with other therapies including but not limited to radiation, chemotherapeutic agents, photodynamic therapies, radio frequency ablation, anti-angiogenic agents, and combinations thereof.

Accordingly, dosage ranges are contemplated. In a particular ebodiment, where the PDE 5 inhibitor is tadalafil, the dosage range contemplated is about 0.02 to about

0.8 mg/kg. This dosage may be administered in commercially available tablets of 5 mg,

10 mg, or 20 mg tadalafil. Other tablet dosages contemplated include about 1, about 2, about 3, about 4, about 6, about 7, about 8, about 9, about 11, about 12, about 13, about

14, about 15, about 16, about 17, about 18, about 19, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 mg tadalafil.

In another particular embodiment, where the PDE 5 inhibitor is sildenafil, or its citrate salt, the dosage range contemplated is about 0.2 to about 1 mg/kg. This dosage is, in various embodiments, administered using commercially available tablets of 25 mg, 50 mg, or 100 mg sildenafil citrate. Other tablet dosages contemplated include about 5, about 10, about 15, about 20, about 30, about 35, about 40, about 45, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, and about 150 mg sildenafil citrate.

Still in another particular embodiment, where the PDE 5 inhibitor is vardenafil, or its hydrochloride salt, the dosage range contemplated is about 0.2 to about 0.4 mg/kg.

This dosage is, in various embodiments, administered using a commercially available tablets of 5 mg, 10 mg, or 20 mg vardenafil. Other tablet dosages contemplated include about 1, about 2, about 3, about 4, about 6, about 7, about 8, about 9, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 mg vardenafil.

In another particular embodiment, where the PDE 5 inhibitor is udenafil, the dosage range contemplated is about 1 to about 2 mg/kg. This dosage is administered, in certain embodiments, using available tablets of 100 mg or 200 mg udenafil. Other tablet dosages contemplated include about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 205, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, and about 300 mg udenafil. The compounds used in the present invention may also be administered with other therapeutic agents to provide a combination therapy. The other therapeutic agents may form part of the same composition or be provided as a separate composition. The other therapeutic agent can be administered simultaneously with the compound that inhibits PDE 5 or can be administered prior of after administration of the compound that inhibits PDE 5.

Contemplated therapeutic agents include, but are not limited to, estrogenic agents, progestational substances, alpha-adrenergic antagonists, beta-adrenergic receptor blocking agents, cholinergic-receptor blocking compounds, vasopressin analogs, 5-HT reuptake inhibitors, norepinephrine reuptake inhibitors, acetylcholine uptake inhibitors, botulinum toxins, calcitonin gene-related peptide receptor ligands, calcium channel blockers, cyclooxygenase-2 inhibitors, IL-I beta inhibitors, lipoxygenase inhibitors, and cholinergic-receptor-stimulating drugs. More preferably, the therapeutic agents are alpha adrenergic receptor agonists and/or beta-adrenergic receptor agonists. Examples of therapeutic agents contemplated include, but are not limited to, amsulosin hydrochloride, tamsulosin hydrochloride, oxybutynin chloride, desmopressin acetate, duloxetine hydrochloride, tolterodine tartrate, darifenacin hydrobromide, botulinum toxin type A, cizolirtine citrate, temiverine hydrochloride hydrate, ajulemic acid, solifenacin succinate, imidafenacin, trospium chloride, solabegron hydrochloride, capeserod hydrochloride, fesoterodine, casopitant hydrochloride, (+)-(S,S)-reboxetine, and ATD-oxybutynin. There are a number of diseases or conditions that have been correlated to OAB occurrence. Therefore, one aspect provides the use of an inhibitor of PDE 5 in the manufacture of a medicament for treating one or more correlated diseases or conditions in which OAB may arise. Such diseases or conditions include drug side effects, heavy metal poisoning, nerve damage, neurological disease, fibromyalgia, diabetes, irritable bowel syndrome, stroke, bladder hyperfiexia, post-surgical denervation of the bladder, disorders associated with serotonin 5-hydroxytryptamine (5-HT) metabolism, and nocturnal enuresis. Specific neurological diseases correlated with OAB include multiple sclerosis, Parkinson's disease, brain injury, and spinal cord injury. Anxiety disorders such as depression, anxiety, and attention deficit disorders have all been associated with the onset of O AB.

Finally, in another embodiment, the invention relates to a method of preventing and/or treating overactive bladder disease comprising administering to a patient in need thereof a therapeutically effective amount of a PDE inhibitor as the ones defined above.

The method disclosed herein may be used in conjunction with non- pharmaceutically based OAB therapies, including, but not limited to, bladder retraining and nerve stimulation therapy.

EXAMPLES

The following examples are provided to illustrate the invention, but are not intended to limit the scope thereof. Male Sprague-Dawley (300-350 g) rats were used. Rats were anaesthetized with urethane (1.2 g/kg Lp.). The left carotid artery was cannulated to continuously register systemic blood pressure by means of a pressure transducer (Abbott, Sligo, Ireland) connected to a MacLab data acquisition system (ADInstruments, Castle Hill, Australia). Heart rate was obtained from blood pressure signal. The urinary bladder was exposed by a midline abdominal incision. A polyethylene catheter was placed into the bladder lumen through a small incision in the bladder wall and fixed with a suture. The catheter was connected to a pressure transducer and to the data acquisition system to register intravesical pressure. The intravesical catheter is also connected to an infusion pump (Harvard Apparatus, Harvard, MA, USA). After a stabilization period, a continuous infusion of the bladder with physiological salt solution (0.9% NaCl; 5 mL/hr) is started and maintained for 20 minutes. The micturition frequency, the infused volume required for each micturition (micturition volume) and the intravesical pressure increase produced in each micturition reflex were determined. Figure 1 shows a pictorial representation of this experimental setup. Once basal urodynamic parameters were obtained, bladder overactivity was induced by intravesically infusing 0.9% NaCl containing 0.3% acetic acid (Kakizaki and de Groat. J Urol, 155(l):355-60 (1996); Woods et al. J Urol, 166(3): 1142-7 (2001)). After a stabilization period, urodynamic parameters under bladder hyperactivity conditions were evaluated during 20 min of infusion. Tadalafil was then intravenously administered and infusion of 0.9% NaCl containing 0.3% acetic acid was again started after 45 min. Urodynamic parameters were again evaluated during a 20 min infusion period. The number of micturition reflexes, the micturition volume and the IVP increase during micturition were measured for each animal during all three treatment periods (control, acetic acid induced OAB, and acetic acid induced OAB plus tadalafil treatment) were obtained (see Figures 2, 3, and 4).

The effects of vehicle (0.9% NaCl; i.v.) (0.9% NaCl; i.v.) on micturition frequency, micturition volume, and intravesical pressure (IVP) increase during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at rate of 5 mL/hr for 20 min in rats are shown in Figure 8. Data are expressed as the mean ± SEM, and n indicates the number of animals used. Acetic acid caused a significant increase in micturition frequency and a decrease in micturition volume (** and *** indicate p < 0.01 and p < 0.001, respectively, versus control, by one-way ANOVA followed by Student-Newman- Keuls test). Vehicle administration did not modify acetic acid-induced OAB.

The effects of oxybutynin chloride (1 mg/kg; i.v.) on micturition frequency, micturition volume and intravesical pressure (IVP) increase during infusion of 0.9%

NaCl containing 0.3% acetic acid (CH₃COOH) at rate of 5 mL/hr rate for 20 min in rats are shown in Figure 9. Data are expressed as the mean ± SEM, and n indicates the number of animals used. Again, acetic acid caused a significant increase in micturition frequency and a decrease in micturition volume. In this case, there was also a slight greater IVP increase after acetic acid treatment (* indicates p<0.05 versus control, by one-way ANOVA followed by Student-Newman- Keuls test). Oxybutynin did not significantly change micturition frequency or micturition volume. However, it did reduce IVP (f indicates p < 0.05 versus CH₃COOH without oxybutynin, by one-way ANOVA followed by Student-Newman-Keuls test).

The effects of tadalafil citrate (0.5 mg/kg; i.v.) on micturition frequency, micturition volume and intravesical pressure (IVP) increase during infusion of 0.9% NaCl containing 0.3% acetic acid (CH₃COOH) at a rate of 5 mL/hr for 20 min in rats are shown in Figure 10. Data are expressed as the mean ± SEM, and n indicates the number of animals used. Tadalafil significantly reduced micturition frequency and increased micturition volume (* indicates p < 0.05 versus control, and the f indicates p < 0.05 versus CH₃COOH by one-way ANOVA followed by Student-Newman-Keuls test).

The data obtained for oxybutynin (a known anti-cholinergic agent) and tadalafil (a PDE 5 inhibitor), differ in that tadalafil produces higher micturition volumes and lower micturition frequency (closer to control values) than oxybutynin. These results indicate a greater suppression of overactive bladder by tadalafil than by oxybutynin. The foregoing describes and exemplifies the invention but is not intended to limit the invention defined by the claims which follow. All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the materials and methods of this invention have been described in terms of specific embodiments, it will be apparent to those of skill in the art that variations may be applied to the materials and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those of ordinary skill in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. Use of an inhibitor of PDE 5 activity or of an inhibitor of PDE 5 expression for the manufacture of a medicament for the treatment and/or prophylaxis of overactive bladder disease.

2. Use according to claim 1 where the inhibitor of PDE 5 is selective with respect to the inhibition of other PDE's in the target tissue (s) or organ (s), in particular where the PDE 5 inhibitor is at least 2 to 5 times more effective in inhibiting PDE5 activity than inhibiting any other PDE activity in those target tissues or organs.

3. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (I):

(I) wherein

R⁰ is selected from the group consisting of hydro, halo, and C_1-6 alkyl; R¹ is selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-S cycloalkyl, C_3-S cycloalkyl Ci_-3 alkylene, and aryl Ci_-3 alkylene, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl,

Ci-6 alkoxy, methylenedioxy, and heteroarylCi_-3alkylene, wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, and C_1-6 alkoxy; R² is an optionally substituted monocyclic aromatic ring selected from the group consisting of phenyl, thiophenyl, furyl, and pyridinyl, or an optionally substituted bicyclic ring

R³ represents hydro or Ci_-3 alkyl, or R¹ and R³ together represent a 3- or 4- membered alkyl or alkenyl chain component of a 5- or 6-membered ring; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

4. Use according to claim 3, wherein the compound of formula (I) is a compound of formula (Ia):

(Ia)

or is a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

5. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (II):

(H) wherein:

R⁴, R⁵, and R⁶ are independently selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-S cycloalkyl, and C_3-S cycloalkyl Ci_-3 alkylene; and R⁷ and R⁸ are independently selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-s cycloalkyl, and C_3-s cycloalkyl Ci_-3 alkylene or together represent a 5 or 6 membered cycloalkyl or heterocycloalkyl; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

6. Use according to claim 4, wherein the compound of formula (II) is a compound of formula (Ha):

(Ha) or is a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

7. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (III):

R¹³

(III) wherein: R⁹, R¹⁰, and R¹¹ are independently selected from the group consisting of hydrogen, Ci-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-S cycloalkyl, and C_3-S cycloalkyl Ci_-3 alkylene; and

R¹² and R¹³ are independently selected from the group consisting of hydro, Ci_-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo Ci_-6 alkyl, C_3-s cycloalkyl, and C_3-s cycloalkyl

Ci_-3 alkylene or together represent a 5 or 6 membered cycloalkyl or heterocycloalkyl; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

8. Use according to claim 7, wherein the compound of formula (III) is a compound of formula (Ilia):

(Ilia) or is a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

9. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (IV):

R¹⁸

(IV) wherein R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ are independently selected from the group consisting of hydrogen, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-S cycloalkyl, C_3-S cycloalkyl Ci_-3 alkylene, C_3-s cycloheteroalkyl, and C_3-s cyclo he tero alkyl Ci_-3 alkylene; i or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

10. Use according to claim 8, wherein the compound of formula (IV) is a compound of formula (IVa):

(IVa) or is a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

11. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (V):

(V) wherein

R⁰ is selected from the group consisting of hydro, halo, and C_1-6 alkyl; R¹ is selected from the group consisting of hydro, C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl, halo C_1-6 alkyl, C_3-s cycloalkyl, C_3-s cycloalkyl Ci_-3 alkylene, and aryl Ci_-3 alkylene, wherein aryl is phenyl or phenyl substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, Ci-6 alkoxy, methylenedioxy, and

wherein heteroaryl is thienyl, furyl, or pyridyl, each optionally substituted with one to three substituents independently selected from the group consisting of halo, C_1-6 alkyl, and C_1-6 alkoxy; R² is an optionally substituted monocyclic aromatic ring selected from the group consisting of phenyl, thiophenyl, furyl, and pyridinyl, or an optionally substituted bicyclic ring

attached to the rest of the molecule via one of the phenyl ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring, saturated or partially or fully unsaturated, and comprises carbon atoms and optionally one or two heteroatoms selected from oxygen, sulfur, and nitrogen; or a pharmaceutically acceptable salt, solvate, hydrate, isomer or prodrug thereof.

12. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (VI):

(VI) wherein R⁰ is selected from the group consisting of hydro, halo, and C_1-6alkyl;

R¹⁹ hydrogen, halogen, cyano, Het, NO₂, trifluoromethyl, trifluoromethoxy, C_1-6 alkyl, CO₂H, or CO₂Ci_-6 alkyl; Ci_-6 alkylene OR^a, C(=O)R^a, OC(=O)R^a, C(=O)OR^a, Ci_-4 alkyleneHet, Ci_-4 alkyleneC(=O)OR^a, OCi_-4 alkyleneC(=O)OR^a, Ci_-4 alkylene OCi_-4 alkylene C(=O)OR^a, C(=O)NR^aSO₂R^a, C(O)CM alkyleneHet, Ci_-4 alkyleneNR^aR^b, C_2-6 alkenylene NR^aR^b, C(=0)NR^aR^b, C(=0)NR^aCi_-4 alkylene OR^b, C(=O)NR^aCi_-4 alkyleneHet, OR^a, OC_2-4 alkyleneNR^aR^b, OCi_-4 alkyleneCH(OR^a)CH₂NR^aR^b, OCi_-4 alkyleneHet, OC_2-6 alkyleneOR^a, OC_2-4 alkyleneNR^aC(=0)0R^b, NR^aR^b, NR^aCi_-4 alkyleneNR^aR^b, NR^aC(=0)R^b, NR^aC(=0)NR^aR^b, N(SO₂Ci_-4alkyl)₂, NR^a(SO₂Ci_-4 alkyl), S0₂NR^aR^b, and OSO₂trifluoromethyl;

R²⁰ is hydrogen, or R¹⁹ and R²⁰ are taken together to form a 3- or 4- membered alkyl ene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom; R^a is selected from the group consisting of hydrogen, Ci_-6 alkyl, C_3-6 cycloalkyl, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, heteroaryl, heteroaryl Ci_-3 alkyl, and Ci_-3 alkyleneheteroaryl;

R^b is selected from the group consisting of hydrogen, Ci_-6 alkyl, C_3-s cycloalkyl, Ci_-3 alkyleneN(R^a)₂, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, and heteroaryl; B is aryl or heteroaryl and is selected from the group consisting of optionally substituted 5- or 6-membered aromatic rings and optionally substituted fused bicyclic aromatic ring systems, either carbocyclic or containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur; Het represents a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with Ci_-4 alkyl or

13. Use according to claims 1 or 2, wherein the inhibitor of PDE 5 activity is a compound of formula (VII):

(VII) wherein R⁰ is selected from the group consisting of hydro, halo, and C_1-6alkyl; R¹⁹ hydrogen, halogen, cyano, Het, NO₂, trifiuoromethyl, trifluoromethoxy, C_1-6 alkyl, CO₂H, or CO₂Ci_-6 alkyl; Ci_-6 alkylene 0R^a, C(=O)R^a, OC(=O)R^a, C(=O)OR^a, Ci_-4 alkyleneHet, Ci_-4 alkyleneC(=O)OR^a, OCi_-4 alkyleneC(=O)OR^a, Ci_-4 alkyleneOCi.4 alkyleneC(=O)OR^a, C(=O)NR^aSO₂R^a, C(=O)C_M alkyleneHet,

Ci_-4 alkyleneNR^aR^b, C_2-6 alkenyleneNR^aR^b, C(=0)NR^aR^b, C(=0)NR^aCi_-4 alkyleneOR^b, C(=0)NR^aCi_-4 alkyleneHet, 0R^a, OC_2-4 alkylene NR^aR^b, OCi_-4 alkyleneCH(OR^a)CH₂NR^aR^b, OCi_-4 alkyleneHet, OC_2-6 alkylene 0R^a, OC_2-4 alkylene NR^aC(=0)0R^b, NR^aR^b, NR^aCi_-4 alkyleneNR^aR^b, NR^aC(=0)R^b, NR^aC(=0)NR^aR^b, N(SO₂Ci_-4 alkyl)₂, NR^a(SO₂Ci_-4 alkyl), S0₂NR^aR^b, and

OSO₂trifluoromethyl;

R²⁰ is hydrogen, or R¹⁹ and R²⁰ are taken together to form a 3- or 4- membered alkylene or alkenylene chain component of a 5- or 6-membered ring, optionally containing at least one heteroatom; X is selected from the group consisting of C(=0), (CH₂)_nC(=O), C(=O)C≡C,

0(CH₂)_nR^c, N(R^b)C(=0)R^c, C(=0)N(R^a)(R^c), N(R^a)C(=0)R^c,

and N(R^a)S0₂R^c;

R^a is selected from the group consisting of hydrogen, Ci_-6alkyl, C_3-6cycloalkyl, aryl, arylCi-₃ alkyl, Ci_-3 alkylenearyl, heteroaryl, heteroaryl Ci_-3 alkyl, and Ci_-3 alkyleneheteroaryl;

R^b is selected from the group consisting of hydrogen, Ci_-6 alkyl, C_3-s cycloalkyl, Ci_-3 alkyleneN(R^a)₂, aryl, aryl Ci_-3 alkyl, Ci_-3 alkylenearyl, and heteroaryl;

R^c is selected from the group consisting of hydrogen, Ci_-6 alkyl, aryl, heteroaryl, arylCi_-3 alkyl, heteroarylCi_-3 alkyl, Ci_-3 alkyleneN(R^a)₂, Ci_-6 alkylenearyl, Ci_-6 alkyleneHet, haloCi_-6 alkyl, C_3-s cycloalkyl, C_3-s heterocycloalkyl, Het, Ci_-3 alkyleneheteroaryl, Ci_-6 alkyleneC(=O)OR^a, and Ci_-3 alkyleneC_3-s heterocycloalkyl; or R^a and R^c are taken together to form a 5- or 6-membered ring, optionally containing at least one heteroatom;

R^d is a 5- or 6-membered ring or a bicyclic fused ring system, saturated or partially or fully unsaturated, comprising carbon atoms and optionally one to three heteroatoms selected from oxygen, sulfur, and nitrogen, and optionally substituted with one or more R¹⁹;

Het represents a 5- or 6-membered heterocyclic ring, saturated or partially or fully unsaturated, containing at least one heteroatom selected from the group consisting of oxygen, nitrogen, and sulfur, and optionally substituted with

or C(=O)OR^a; n is 0 or 1, q is 0, 1, 2, 3, or 4, t is 1, 2, 3, or 4, m is 1, 2, 3 or 4; and pharmaceutically acceptable salt, solvate, hydrate, isomer and prodrugs thereof.

14. Use according to any of the previous claims wherein the inhibitor of PDE 5 activity is selected from the group consisting of tadalafil, sildenafil, vardenafil, udenafil, zaprinast, mixtures thereof, and a salt, solvate, prodrug, and hydrate thereof.

15. Use according to claim 14 wherein the inhibitor of PDE 5 activity is tadalafil, in particular, tadalafil citrate.

16. Use according to claim 1 wherein the inhibitor of PDE 5 expression is an antisense oligonucleotide which negatively regulate PDE 5 expression.

17. Use according to claim 1 wherein the inhibitor of PDE 5 expression is a ribozyme inhibitor.

18. Use according to claim 1 wherein the inhibitor of PDE 5 expression is a doble- stranded RNA wherein one strand is complementary to a target region in PDE 5- encoding polynucleotide.

19. Use according to claim 1 wherein the inhibitor of PDE 5 expression is a circular lasso RNA which inhibits the PDE5-encoding polynucleotide.

20. A pharmaceutical composition comprising at least one of the inhibitors of PDE 5 activity defined in claims 3 to 15 or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

21. A pharmaceutical composition according to claim 20 which additionally to the inhibitor of PDE 5 activity contains one or more other therapeutic agents.

22. The pharmaceutical composition according to claim 21 wherein the other therapeutic agent is selected from the group consisting of anticholinergic agents as oxybutynin, tolterodine, trospium, solifenacin and darifenacin; antidiuretic hormones as desmopressin; serotonin reuptake inhibitors like duloxetine; serotonin receptor agonists; vanilloid receptor agonists like capsaicin and resiniferatoxin; botulinum toxin; neurokinin receptor antagonists; αi -adrenergic receptor antagonists as tamsulosin, alfuzosin and doxazosin; and phosphodiesterase type 4 inhibitors.

23. A pharmaceutical composition comprising at least one of the inhibitors of PDE 5 expression defined in claims 16 to 19.

24. The pharmaceutical composition according to any one of claims 20 to 23 which is administered transdermally and intravesically.

25. Method of preventing and/or treating overactive bladder disease comprising administering to a patient in need thereof a therapeutically effective amount of an inhibitor of PDE activity as defined in any of claims 3 to 15 or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

26. Method of preventing and/or treating overactive bladder disease comprising administering to a patient in need thereof a therapeutically effective amount of an inhibitor of PDE expression as defined in any of claims 16 to 19.