Field of the Invention

Treatment of dysfunction of urinary bladder activities. More particularly, treatment of neurogenic detrusor overactivity.

Background of the Invention

Neurogenic detrusor overactivity is a common sequel of suprasacral spinal cord injury. Subpopulations of C-afferent fibers, which are characteristically sensitive to capsaicin, have been implicated in the pathogenesis of this neurogenic detrusor overactivity, de Groat, W.C., et ah: Mechanisms underlying the recovery of urinary bladder function following spinal cord injury, J. Autonom. Nerv. Syst., 30 (Suppl): S71, 1990. Tachykinins are synthesized by C-type afferent neurons. Local release of tachykinins (substance P, neurokinin A, neurokinin B) and other peptides from sensory nerves in the bladder wall has been shown to produce diverse biological effects, such as smooth muscle contraction, facilitation of transmitter release from nerves, vasodilatation, and increased plasma permeability, Maggi, CA. : Tachykinins and calcitonin gene-related peptide (CGKP) as co- transmitters released from peripheral endings of sensory nerves, Progr. Neurobiol., 45: 1, 1995. The actions of the tachykinins are mediated by activation of three distinct receptor types termed NK-I , NK-2 and NK-3, Maggi, C.A.: The mammalian tachykinin receptors, Gen. Pharmacol., 26: 911, 1995. Specific tachykinin receptors are present in peripheral sensory nerves and smooth muscle of the urinary bladder of several species, including humans. Rat and guinea pig detrusors contain both NK-I and NK-2 receptors, however in humans the NK-2 receptor seems to be the only mediator of contractile response to tachykinins in bladder smooth muscle, Giuliani, S., et ah: Characterization of the tachykinin neurokini-2 receptor in the human urinary bladder by mean of selective receptor antagonists andpeptide inhibitors, J. Pharmacol. Exp. Ther., 267: 590, 1993; Zeng, X.P., et ah,: Characterization of tachykinin NK2 receptors in human urinary bladder, J Urol, 153: 1688, 1995, and Palea, S., et ah.: Pharmacological characterization of tachykinin NK2 receptors of isolated human urinary bladder, prostatic urethra and prostate, J. Pharmacol. Exp. Ther.,

277: 700, 1996. hi rats, it has been suggested that tachykinins modulate vesical tone by action on NK-2 receptors, Lecci, A., et ah: Bladder distension and activation of the efferent function of sensory fibers: similarities with the effect of capsaicin. Br. J. Pharmacol., 124: 259, 1998.

Experimental studies suggested that the voiding reflex in animals with spinal cord injury is mediated by unmyelinated C-fibers and that this pathway can be blocked by capsaicin.

Description of the Invention Bladder overactivity and hypersensitivity disorders cause by tachykinins released from sensory nerves are antagonized with drugs selective for NK-2 receptors.

We tested the efficacy of a neurokinin-2 receptor antagonist ("NK2RA"), ((S)-N-[2- (3 ₎ 4-dichlorophenyl)-4-[4-(2-oxoperhydro-pyrimidin-l-yl) piperidino]butyl]-N- methylbenzamide dihydrochloride), on neurogenic detrusor overactivity after spinal cord injury in rats and discovered that three weeks after spinal cord injury, neurogenic detrusor overactivity developed in all control paraplegic animals with a mean bladder capacity plus or minus standard deviation of 0.7 ± 0.2 ml. and a mean voiding pressure of 59 ± 14.2 cm.H ₂ 0. Neurogenic detrusor overactivity resolved in 50% and 83% of the animals that received the NK2RA for one week at doses of 0.3 mg./kg./day and 0.6 mg./kg./day, respectively. Mean cystometric bladder capacity was 1.2 ± 0.5 versus 1.3 ± 0.4 ml. and mean voiding pressure was 46.1 ± 10.8 versus 40 ± 9.9 cm. H ₂ O in animals that received 0.3 versus 0.6 mg./kg./day, respectively. The NK2RA compound produced better urodynamic results when given for 2 weeks rather than 1 week.

Accordingly, we have shown that a NK2RA is effective in the treatment of neurogenic detrusor overactivity after spinal cord injury in the rat and may thus provide an alternative clinical treatment option for neurogenic detrusor overactivity and urgency/frequency syndrome. This NK-2 selective antagonist has both time- and dose- response effects, which further suggests the potential for clinical application.

Brief Description of the Drawings

Figure 1: Cystometrography results obtained with a normal rat (A) and with a paraplegic rat

(B).

Figure 2: Mean basline (Basal) vesical pressure in animal groups. Asterisk indicates statistical significance (p < 0.05 vs paraplegics). Figure 3: Mean cystometric bladder capacity in animal groups. Asterisk indicates statistical significance (p < 0.05 vs paraplegics).

Figure 4: Cystometrography in paraplegic rat that received 0.3 mg/kg daily of a NK2RA for two weeks.

Figure 5: Mean frequency of detrusor hyperreflexia in animal groups. Asterisk indicates statistical significance (p < 0.05 vs paraplegics).

Figure 6: Mean amplitude of detrusor hyperreflexia (DH) in animal groups. Asterisk indicates statistical significance (p < 0.05 vs paraplegics). Figure 7: Mean micturition pressure in animal groups. Asterisk indicates statistical significance (p < 0.05 vs paraplegics).

Detailed Description of the Invention Materials and Methods: Animals.

In this study we used 48 female Sprague-Dawley rats (Charles River, Montreal, Quebec, Canada) weighing 200-250 gm. The animals were maintained and treated according to the guidelines of the Canadian Council on Animal Care. The experimental protocol was approved by the local Animal Care Committee. The animals were housed in a 12 x 12 light- dark cycle room with free access to food and water. Spinal Cord Injury,

Six rats served as sham operated control and received 1 ml of saline subcutaneously daily. The 42 remaining animals in this study underwent spinal cord transection at the level of the 10 ^th thoracic vertebra. The animals were anesthetized by intramuscular injection of ketamine (50 mg./kg. body weight) and xylazine (5 mg./kg. body weight). Body temperature was maintained between 37 ⁰ C and 38 ⁰ C during the surgery and for the first postoperative 48 hours using a controlled heating pad connected to an electronic rectal thermometer. After sufficient depth of anesthesia, the last rib was identified and the level of the corresponding vertebra was localized. A midline dorsal incision was made about 2-cm over the subcutaneous trunci to expose the vertebral spine and the paraspinous muscle. These muscles were split from their attachment to the spinal processes and retracted laterally. Laminectomy of TlO vertebra was done using a curved scissor. Using a sharp iris scissor, the spinal cord was completely transected under direct visual control aided by microscopic magnification. To ensure complete transection of the spinal cord, a 5 mm gap between the proximal and distal ends was created and a piece of Gel-foam (Upjohn Company of Canada, Don Mills, Ontario, Canada) was placed between the severed ends of the spinal cord. The overlying muscles were then approximated and the skin closed using 4-zero catgut sutures.

Six animals served as normal controls, while 32 underwent spinal cord transection at the 10 ^th thoracic vertebra. Two weeks after spinal cord injury, 6 animals underwent filling cystometrography to confirm neurogenic detrusor overactivity, while another 12 served as control paraplegics. The remaining 24 paraplegic animals were used to test the drug and were divided into 2 equal groups of 12. Group 1 received the drug in a dose of 0.3 mg./kg. daily, while group 2 received a dose of 0.6 mg./kg. daily. Each control paraplegic and treatment group was further subdivided into 2 subgroups of 6 rats. In subgroup 1, filling cystometrography was done 3 weeks after spinal cord injury, while in subgroup 2 it was done 4 weeks after spinal cord injury. Animal grouping and drug administration.

Two weeks after spinalization, 6 randomized animals underwent filling cystometrography to ensure development of neurogenic detrusor overactivity before drug administration. The 36 remaining paraplegic animals were randomly divided into 3 groups. The first group included 12 spinalized rats that served as control paraplegics. Other 24 paraplegic animals were used to test the drug and were divided into 2 equal groups of 12.

Group 1 received the drug at a dose of 0.3 mg./kg. daily, while group 2 received a dose of 0.6 mg./kg. daily. Each control paraplegic and treatment group was further subdivided into 2 subgroups of 6 rats. In subgroup 1, filling cystometrography was done 3 weeks after spinal cord injury, while in subgroup 2 it was done 4 weeks after spinal cord injury. ((S)-N-[2-(3,4-Dichlorophenyl)-4-[4-(2-oxoperhydro-pyrimidin -l-yl) piperidino]butyl]-N- methylbenzamide dihydrochloride) was dissolved in 5% polyethylene glycol 400 in saline and delivered via a Model 2004 Alzet osmotic minipump (Alza Corp., Palo Alto, California) inserted subcutaneously into a pouch created on the back of the animals. Compound. In the present studies, we evaluated the efficacy of a selective NK-2 antagonist on neurogenic detrusor overactivity in chronic spinalized rats. We chose this model of detrusor overactivity as an extreme but reliable and reproducible model. Results have shown that the effects of this NK-2 selective antagonist are dose and time dependent.

((S)-N-[2-(3,4-Dichlorophenyl)-4-[4-(2-oxoperhydro-pyrimi din-l-yl) piperidino]butyl]-N-methylbenzamide dihydrochloride) is a novel, orally potent and selective non-peptide neurokinin-2 (NK-2) receptor antagonist, Rumsey, W., et ah, Pharmacological Characterization ofZM274773: A Novel, Potent and Orally-Active Non-Peptide Neurokinin- A (NK-2) Receptor Antagonist, The FASEB J. 13(4): A365, 1999. M274773 binds with high-

affinity to cloned human NK-2 receptors (Ki = 2.4 nM) and has more than 50-fold reduced affinity for cloned human NK-I receptor. In vitro, the compound competitively antagonized the contractile responses evoked by neurokinin A (NKA) on human bronchus as well as responses evoked by [β-Ala ^s ]-NKA(4-10) on guinea-pig trachea (GPT) or rabbit pulmonary artery (RPA), with similar potency (-logKβ-9.5 and 9.0, respectively). This compound demonstrated lower potency to competitively antagonize contractile responses to Ac-[Arg ⁶ , Sar ⁹ , Met(O ₂ ) ⁿ ]-Substance P ₆ -π (NK-I ligand) in vitro (-LogK _B =4.9 and 6.0 in GPT and RPA, respectively) and it was essentially inactive against histaminergic or muscarinic receptors in GPT. M274773 was inactive in binding studies against 110 additional ion channels, enzymes, transporters and receptors including adrenergic, serotonergic, nicotinic, muscarinic, dopaminergic, gabaergic and histaminergic varities. Animal care.

Each rat was housed in a separate cage containing special bedding materials to prevent decubitus ulcer formation. The position of the animals was changed at least twice daily for the first week. The animals were given trimethoprim (2.2 mg./kg. body weight) subcutaneously twice daily for the first three days after spinal cord injury. The bladder was emptied manually every 8 hours after surgery until reflex voiding was recovered (first two weeks), after which the bladder was emptied twice daily. AU animals were weighed twice weekly. Animals in which ulcers, self- mutilation or significant weight loss developed were sacrificed.

Cystometrography.

Cystometrography was done in awake animals to study the detrusor response to filling after supraspinal transection and to evaluate the effect of different doses and intervals of M274773 on urinary bladder function after spinal cord injury. With the animal under general anesthesia (ketamine 50 mg./kg. body weight and xylazine 5 mg./kg. body weight), a PE-90 polyethylene catheter (Clay- Adam, Parsippany, New Jersey) was implanted into the bladder through the dome as previously described, Yaksh, TX. , et al.,: Micturition in rats: a chronic model for study of bladder function and effect of anesthetics, Am. J. Physiol., 251: Rl 177, 1986. The catheter was tunneled subcutaneously and exteriorized at the nape of the neck. Cystometrography was done 48 hours after tube implantation into the bladder. The bladder catheter was connected via a t-tube to a pressure transducer (Stetham Instruments, Inc., Oxnard, California) and a Flo-Gard 6200 volumetric infusion pump (Travenol Laboratories, Inc., Deerfield, Illinois). The conscious rat was placed without any restraint in a metabolic

cage. After the bladder had been emptied, room temperature saline was infused into the bladder at a rate of 100 μl. per minute. Intravesical pressure was recorded continuously using a Mac Lab/2e computer (AD Instruments, Castle Hill, Australia). At least three reproducible voiding cycles were recorded per animal. The variables recorded during cystometrography were resting bladder pressure, uninhibited contractions, frequency of uninhibited detrusor contractions per minute, amplitude of the uninhibited detrusor contractions in cm. H ₂ O, bladder capacity and spontaneous voiding pressure. A contraction of 15 cm. H ₂ O and more was considered unstable. The presence of unstable bladder contractions in this model of neurogenic bladder was considered neurogenic detrusor overactivity. Data analysis was done using 1-way analysis of variance. AU data were shown as the mean plus or minus standard deviation, with p < 0.05 considered statistically significant. Results Sham operated controls.

Mean baseline vesical pressure in sham operated control animals was 5.8 ± 1.6 cm.H ₂ 0, while mean cystometric capacity was 0.33 ± 0.01 ml. This bladder capacity was associated with a micturition pressure of 35.8 ± 8.9 cm.H ₂ 0. None of these animals showed uninhibited detrusor contractions (Fig. 1-A). Control paraplegics.

Mean baseline vesical pressure in control paraplegic animals 3 weeks after spinalization was 9 ± 1.7 cm.H ₂ 0, mean cystometric capacity was 0.7 ± 0.2 ml. and mean micturition pressure was 59 ± 14.2 cm.H ₂ 0. These control paraplegic animals showed neurogenic detrusor overactivity with a mean frequency of 3.6 ± 0.7 contractions per minute and a mean amplitude of 29 ± 4 cm. H ₂ O. Four weeks after spinalization, mean baseline vesical pressure in control paraplegic animals was 12 ± 2.1 cm. H ₂ O with a mean cystometric capacity of 0.56 ± 0.1 ml. and a mean micturition pressure of 64.6 ± 12 cm. H ₂ O. These animals showed neurogenic detrusor overactivity with a mean frequency of 5.2 ± 0.4 contractions per minute and mean amplitude of 31 ± 3 cm. H ₂ O (Fig.1 -B). Treatment with ((S)-N- [2-(3, 4-Dichlorophenyl)-4-[4-(2-oxoperhydro-pyrimidin~l-yl) piperidino] butyl] -N-methylbenzamide dϊhy dro chloride) . Mean basal vesical pressure in animals that received a dose of 0.3 mg./kg. M274773 daily was significantly reduced to 4.8 ±1.5 and 4.5 ± 1.2 cm. H ₂ O when the compound was given for 1 and 2 weeks, respectively (Fig.2). This decrease in the baseline pressure was

associated with a significant increase in the mean cystometric bladder capacity to 1.3 ± 0.5 and 1.6 ± 0.8 ml. after 1 and 2 weeks of treatment, respectively (Fig.3).

Neurogenic detrusor overactivity also disappeared in 50 % and 66 % of the animals that received a dose of 0.3 mg./kg. M274773 daily for 1 and 2 weeks, respectively (Fig. 4). Although neurogenic detrusor overactivity did not disappear in 50 % and 33% of the animals, there was a significant decrease in the mean frequency of these hyperreflexic contractions to 2,1 ± 0.7 and 1.1 ± 0.1 contractions per minute when M274773 was given at 0.3 mg./kg. daily for 1 and 2 weeks, respectively (Fig. 5). The significant decrease in the frequency of the hyperreflexic contractions was associated with a decrease in the mean amplitude of these contractions to 27 ± 3 and 20 ± 2.8 cm. H ₂ O after 1 and 2 weeks of treatment, respectively (fig. 6). However, the decrease in the mean amplitude of hyperreflexic contractions was only statistically different when the compound was given for 2 weeks. These changes in urodynamic parameters were associated with a reduction in mean micturition pressure to 46.1 ± 10.8 and 40 ± 7.9 cm. H ₂ O after 1 and 2 weeks of treatment, respectively (Fig. 7), values that approximate mean micturition pressures in sham-operated normal control rats.

Again, the decrease in the mean micturition pressure was only statistically different when the drug was given for 2 weeks.

Mean baseline vesical pressure was significantly reduced to 4.3 ± 1.5 and 3.6 ± 1.3 cm. H ₂ O when M274773 was given at 0.6 mg./kg. daily for 1 and 2 weeks, respectively (Fig. 2). This decrease in the baseline pressure was associated with a significant increase in the mean cystometric bladder capacity tol .3 ± 0.4 and 1.8 ± 0.4 ml. after 1 and 2 weeks of treatment, respectively (Fig. 3).

Only 1 animal (17%) showed neurogenic detrusor overactivity after 1 and 2 weeks of treatment (Fig. 4). The frequency of these hyperreflexic contractions was significantly reduced to 1.8 ± 0.2 and 1 ± 0.1 contractions per minute after 1 and 2 weeks of treatment, respectively (Fig. 5). Also, contraction amplitude was significantly reduced to 17 ± 2 and 15 ± 2 cm.H ₂ 0 after 1 and 2 weeks of treatment, respectively (Fig. 6). These changes in urodynamic parameters were associated with a significant reduction in mean micturition pressure to 40 ± 9.9 and 32.6 ± 6.3 cm. water after 1 and 2 weeks of treatment, respectively (Fig. 7).

Thus, it is contemplated that detrusor overactivity in a subject may be treated by administration of a therapeutically-effective amount of a neurokinin-2 receptor antagonist or a pharmaceutically-acceptable salt thereof to a subject.

More particularly, it is contemplated that detrusor overactivity in a subject may be treated by administration of a therapeutically-effective amount of (5)-iV-[2-(3 ₎ 4- dicb.lorophenyl)-4-[4-(2-oxo-piperidin-l-yl)piperidino]butyl ]-iV-metb.yl-2-fluorobenzamide or pharmaceutically-acceptable salt thereof to said subject. It is further contemplated that such treatment may be effective wherein said subject is a human.

It is further contemplated that such treatment may be effective wherein said pharmaceutically-acceptable salt is selected from a chloride, a sulfate, a tosylate, a mesylate, a napsylate, a besylate, a phosphate, a salicylate, a tartrate, a lactate, a citrate, a benzoate, a succinate, an acetate or a maleate.

It is further contemplated that such treatment may be effective when additionally comprising co-administering one or more other medically-compatible therapeutic agents.

It is further contemplated that such treatment may be effective wherein said other therapeutic agents are selected from, an estrogenic agent, a progestational substance, an alpha-adrenergic agonist, a beta-adrenergic receptor blocking agent, a cholinergic-receptor blocking compound or a cholinergic-receptor-stimulating drug.

It is further contemplated that such treatment may be effective wherein said compound or pharmaceutically-acceptable salt thereof is administered in a physiologically- acceptable manner, selected from topical application, ingestion, inhalation, insufflation or injection.

It is further contemplated that such treatment may be effective wherein said compound or pharmaceutically-acceptable salt thereof is administered topically.

It is further contemplated that such treatment may be achieved by administering by capsule, tablet, aqueous solution, aqueous suspension, non-aqueous suspension, suppository, aerosol or powder a daily dose range of 5 to 100 mg of said compound or said pharmaceutically-acceptable salt thereof, in a single dose or divided into two, three or four daily doses.

It is further contemplated that such treatment may be achieved with a pharmaceutical composition for treating or preventing detrusor overactivity comprising (5)-iV-[2-(3,4- dichlorophenyl)-4-[4-(2-oxo-piperidin-l-yl)piperidino]butyl] -iV-methyl-2-fluorobenzamide or pharmaceutically-acceptable salt thereof and at least one pharmaceutically-acceptable excipient or diluent.

It is further contemplated that (5)-iV-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydroρyrimidin-l-yl)piperidino]butyl]-iV-methylbenzam ide or a pharmaceutically- acceptable salt thereof may be used in the preparation of a medicament for treating or preventing detrusor overactivity. It is further contemplated that a pharmaceutically-acceptable salt may be selected from a chloride, a sulfate, a tosylate, a mesylate, a napsylate, a besylate, a phosphate, a salicylate, a tartrate, a lactate, a citrate, a benzoate, a succinate, an acetate or a maleate.

It is further contemplated that (,y)-iV-[2-(3,4-dichloroρhenyl)-4-[4-(2- oxoρerhydropyrimidin-l-yl)piperidino]butyl]-iV-methylbenzam ide or a pharmaceutically- acceptable salt thereof may be used in the preparation of a medicament suitable for coadministration with one or more other medically-compatible therapeutic agents.

It is further contemplated that said other medically-compatible therapeutic agents may be selected from, an estrogenic agent, a progestational substance, an alpha-adrenergic agonist, a beta-adrenergic receptor blocking agent, a cholinergic-receptor blocking compound or a cholinergic-receptor-stimulating drug.

It is further contemplated that (,S)-iV-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidin-l-yl)piperidino]butyl]-iV-methylbenzami de or a pharmaceutically- acceptable salt thereof may be used in a medicament suitable for administration orally, parenterally, rectally, by inhalation or by insufflation. It is further contemplated that such a medicament may comprise a tablet or capsule containing about 0.1 mg up to about 250 mg of said compound or pharmaceutically- acceptable salt thereof. More particularly it is contemplated that a medicament may comprise about 0.1 mg to about 250 mg of said compound suitable for administration by inhalation as a single dose or divided into two, three or four daily doses. It is further contemplated that a medicament comprising about 5 mg to about 100 mg of said compound may be suitable for administration by inhalation as a single dose or divided into two, three or four daily doses.

It is further contemplated that a medicament comprising about 5 mg to about 100 mg may be suitable for administration as a capsule, a tablet, an aqueous solution, an aqueous suspension, a non-aqueous suspension, a suppository, an aerosol or a powder.

It is further contemplated that (5)-7V-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidin-l-yl)piperidino]butyl]-N-methylbenzamid e or a pharmaceutically- acceptable salt thereof may be used for treating or preventing detrusor overactivity.

It is further contemplated that (5)-iV-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidm-l-yl)piperidino]butyl]-Λ ^r -methylbenzamide may be used for treating or preventing detrusor overactivity in the form of a pharmaceutically-acceptable salt selected from a chloride, a sulfate, a tosylate, a mesylate, a napsylate, a besylate, a phosphate, a salicylate, a tartrate, a lactate, a citrate, a benzoate, a succinate, an acetate or a maleate.

It is further contemplated that (.S)-iV-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidin-l-yl)piperidino]butyl]-iV-methylbenzami de or a pharmaceutically- acceptable salt thereof for treating or preventing detrusor overactivity may be used by coadministration with one or more other medically-compatible therapeutic agents. It is further contemplated that (ιS)-_V-[2-(3 ,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidin-l-yl)piperidino]butyl]-iV-methylbenzami de or a pharmaceutically- acceptable salt thereof for treating or preventing detrusor overactivity may be used with other medically-compatible therapeutic agents selected from, an estrogenic agent, a progestational substance, an alpha-adrenergic agonist, a beta-adrenergic receptor blocking agent, a cholinergic-receptor blocking compound or a cholinergic-receptor-stimulating drug.

It is further contemplated that (,S)-iV-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidin-l-yl)piperidino]butyl]-7V-methylbenzami de or a pharmaceutically- acceptable salt thereof for treating or preventing detrusor overactivity may be used by administrating orally, parenterally, rectally, by inhalation or by insufflation. It is further contemplated that (5)-iV-[2-(3,4-dichlorophenyl)-4-[4-(2- oxoperhydropyrimidin-l-yl)piperidino]butyl]-iV-methylbenzami de or a pharmaceutically- acceptable salt thereof for treating or preventing detrusor overactivity may be used by administering as a capsule, a tablet, an aqueous solution, an aqueous suspension, a nonaqueous suspension, a suppository, an aerosol or a powder.