WO2015021988A1

WO2015021988A1 - Pyrrolo-isoquinoline compounds for treatment of pain associated with interstitial cystitis/bladder pain syndrome

Info

Publication number: WO2015021988A1
Application number: PCT/DK2014/050239
Authority: WO
Inventors: Henrik BLOU; Gert BOLWIG
Original assignee: Aros Pharma Aps
Priority date: 2013-08-12
Filing date: 2014-08-11
Publication date: 2015-02-19

Abstract

The present invention relates to a pyrolo[3,2h]isoquinoline compound of Formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof, for use in the treatment of a disease, such as pain, associated with Interstitial Cystitis/Bladder Pain Syndrome in a mammal.

Description

PYRROLO-ISOQUINOLINE COMPOUNDS FOR TREATMENT OF PAIN ASSOCIATED WITH INTERSTITIAL CYSTITIS/BLADDER PAIN SYNDROME

Field of invention

The present invention relates to the use of compounds of Formula (I) for the treatment of a disease, such as pain, associated with Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). In particular the present invention relates to the use of compounds of Formula (I) for the treatment of pain associated with IC/BPS. Background of invention

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a chronic sterile disease of the bladder characterized by bladder pain and pelvic pain as well as increased urinary frequency, urinary urgency and nocturia (night-time urination). IC/BPS has a profound impact on quality of life of the sufferers and affects the ability to participate in the workplace as well as other activities.

IC/BPS is predominantly a disease that affects women with varying prevalence estimates that range up to 70 per 100,000. The ethology of IC/BPS is unknown but involves inflammation and damage to the urothelium, the inner lining of the bladder.

Management of IC/BPS includes physical therapy, lifestyle interventions and medications. Oral medications include antidepressants, such as amitryptiline, histamine H2-receptor antagonists, such as cimetidine, however these agents have limited efficacy. Bladder instillations are used and include preparations of dimethyl sulfoxide, and compositions containing pentosan polysulfate or heparin, sodium hyaluronate, and lidocaine. Other bladder coating therapies include bladder instillations with sodium hyaluronate and chondroitin.

Despite the available treatments many sufferers continue to experience pelvic and bladder pain. The compounds of the present invention have shown superior effect when used in the management of this disease.

Summary of invention

The present application addresses the problem of providing an efficient treatment of a disease, such as pain, associated with IC/BPS. Particularly the invention relates to the novel use of the compounds of the present invention in the treatment of a disease, such as pain, associated with IC/BPS. More particular, the invention relates to the novel use of the compounds of the present invention in the treatment of pain associated with IC/BPS.

Further, the invention relates to the novel use of compounds of the present invention in the treatment of urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia.

Furthermore, the present invention provides pharmaceutical compositions for use in the treatment of a disease, such as pain, associated with IC/BPS. Especially, the present invention provides pharmaceutical compositions for use in the treatment of pain associated with IC/BPS.

Additionally, the present invention provides pharmaceutical compositions for use in the treatment of urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia. Accordingly, the present invention relates to a compound of Formula (I), or a pharmaceutically acceptable salt, solvate or prodrug thereof. The compound is preferably 5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl- 1 H-pyrrolo[3,2h]isoquinoline-2(3H)-one. Detailed description of the invention

The present invention relates to treatment of a disease, such as pain, associated with IC/BPS. In particular the present invention relates to the novel use of the compounds of the present invention in the treatment of pain associated with IC/BPS. Further, the invention relates to the novel use of compounds of the present invention in the treatment of urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia.

Accordingly, the present invention relates to compounds of Formula (I)

or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein

F is selected from the group consisting of methyl and ethyl,

R₂ is selected from the group consisting of methyl and methoxy, and

R₃ is selected from the group consisting of fluoro, chloro and methyl,

for use in the treatment of a disease associated with IC/BPS by administering an effective therapeutic amount of said compound to a mammal subject. The compounds are preferably a compound selected from the group consisting of 5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one;

5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one;

5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one;

5-(2,5-dimethylphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one; and

5-(5-chloro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-ethyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one.

The most preferred compound of Formula (I) is 5-(5-fluoro-2-methoxyphenyl)-6,7,8,9- tetrahydro-3-(hydroxyimino)-8-methyl-1 H-pyrrolo[3,2h]isoquinoline-2(3H)-one. The compound of the invention may be provided in any form suitable for the intended administration. Suitable forms include pharmaceutically acceptable salts, solvates and prodrugs of the compound of Formula (I). All components must be "pharmaceutically acceptable" in the sense of being compatible with the other components of the formulation and not deleterious to the recipient thereof.

Pharmaceutically acceptable salts refer to salts of the compounds of the invention, which are considered to be acceptable for clinical and/or veterinary use. Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the invention with a mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition salts and base addition salts, respectively. It will be recognized that the particular counterion forming a part of any salt of this invention is not of a critical nature, so long as the salt as a whole is pharmaceutically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole. These salts may be prepared by methods known to the skilled person.

Examples of pharmaceutically acceptable addition salts include acid addition salts formed with inorganic acids e.g. hydrochloric, hydrobromic, sulfuric, nitric, hydroiodic, metaphosphoric, or phosphoric acid; and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, trifluoroacetic, malic, lactic, formic, propionic, glycolic, gluconic, camphorsulfuric, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), ethanesulfonic, pantothenic, stearic, sulfinilic, alginic and

galacturonic acid; and arylsulfonic, for example benzenesulfonic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid; and base addition salts formed with alkali metals and alkaline earth metals and organic bases such as N,N- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine and procaine; and internally formed salts.

Examples of suitable metal cations include sodium cation (Na+), potassium cation (K+), magnesium cation (Mg2+), calcium cation (Ca2+), and the like, (see, for example, Berge S.M. et al., "Pharmaceutical Salts," J. of Pharma. Sci., 1977;66: 1). As used herein, the term "solvate" means a compound of the invention or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and/or acceptable for administration to humans in trace amounts. The solvated forms, including hydrated forms, are equivalent to unsolvated forms and are encompassed within the scope of the present invention. As used herein, the term "prodrug" means a compound that is transformed to yield a compound of the present invention. The transformation may occur at any point after administration until the compound reaches the ultimate target site of desired action. The transformation may occur by various mechanisms, such as through hydrolysis.

The compounds may be prepared by any known synthesis, such as for example the syntheses described in International Patent Application WO 2007/059608. The invention further relates to a pharmaceutical composition comprising one or more compounds as defined herein. In one embodiment, the pharmaceutical composition only comprises one compound of Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof. In a particular embodiment the pharmaceutical composition only comprises one compound of the present invention, said compound being 5-(5- fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one. In other embodiments, the pharmaceutical composition comprises a mixture of two compounds of Formula (I) or pharmaceutically acceptable salts, solvates or prodrugs thereof. In yet other embodiments the pharmaceutical composition may comprise another active compound in addition to the one or more compounds of Formula (I) or pharmaceutically acceptable salts, solvates or prodrugs thereof.

The compounds and the pharmaceutical compositions according to the invention are useful in the treatment of a disease, such as pain, associated with IC/BPS. The term "a disease, such as pain, associated with IC/BPS" as used herein refers to any kind of pain, which is associated with the IC/BPS disease, especially bladder pain and/or pelvic pain, as well as urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia. It is noted that the disease "Bladder Pain Syndrome" is also known as "Painful Bladder Syndrome (PBS)".

In particular, the compounds and pharmaceutical compositions according to the present invention are useful in the treatment of pain associated with IC/BPS. The term "pain associated with IC/BPS" as used herein refers bladder and/or pelvic pain, i.e. to any kind of pain in the bladder and the surrounding pelvic region. In its broadest aspect, the term includes all cases of urinary pain that can be attributed to both IC/BPS as well as other causes such as for example infections and urinary stones. In some embodiments the compounds and pharmaceutical compositions are useful in the treatment of bladder pain. In other embodiments the compounds and pharmaceutical compositions are useful in the treatment of pelvic pain. In a preferred embodiment, the compounds and pharmaceutical compositions according to the present invention are useful for treating allodynia (decrease in nociceptive threshold) and/or hyperalgesia associated with IC/BPS.

The compounds and pharmaceutical compositions according to the present invention are further useful in the treatment urinary symptoms associated with IC/BPS, such as urinary frequency, urinary urgency and/or nocturia. In a more preferred embodiment of the present invention, the compounds and pharmaceutical compositions are capable of decreasing the urinary frequency and/or increasing the intercontraction interval (i.e. the time between two subsequent micturitions).

Thus, in a preferred embodiment where the compounds of the present invention are used for treatment of disease associated with IC/BPS, the treatment results in prevention, alleviation or treatment of one or more signs and symptoms associated with IC/BPS selected from the group consisting of pain (which may for example be bladder and/or pelvic pain, and which may include allodynia and/or hyperalgesia), urinary frequency, urinary urgency and/or nocturia. The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of reducing risk of, alleviating or relieving urinary symptoms, pain symptoms, discomfort, or unease in a patient suffering of IC/BPS.

The beneficial effect is achieved whenever the mammal is administered an effective pharmaceutical amount of the compound according to the present invention. By the term "effective pharmaceutical amount" is meant the amount required in order to induce a beneficial effect in terms of alleviating or relieving pain symptoms, discomfort, urinary symptoms or unease in a patient suffering of IC/BPS. The individual in need of treatment normally receives a dosage of one or more of the compounds according to the invention. In the context of the present invention, the term "dosage" is meant to describe the dosage required for an average human being having a weight of about 75 kg.

In one embodiment of the present invention the compound is given in a dosage lying in a range of from about 1 to about 1000 mg, such as for example about 1 to 500 mg, 5 to 500 mg, 5 to 250 mg or 10 to 200 mg. In a particular embodiment of the present invention the compound is given in a dosage lying in the range of from about 1 to about 10 mg, such as for example about 1 to 8 mg, 2 to 8 mg, 2 to 6 mg or 3 to 5 mg. In another embodiment the dosage is 3.75 mg.

A predetermined dosage regime does not exist. In contrary, the patient may be administered a dosage whenever the patient experiences pain. Hence, the dosage may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 times during a 24 hours period. The physician will in any event determine the actual dosage, which will be most suitable for any particular patient and it may vary with the severity of disease, age, weight and response of the particular patient. The above dosages are, of course only exemplary of the average case and there may be instances where higher or lower doses are merited and such are within the scope of the invention.

Another way of expressing the dosage level in accordance with the present invention is as mg/kg bodyweight. Accordingly, for administration to human patients the dosage levels of the compounds in accordance with the present invention, or pharmaceutically acceptable salts, solvates, or prodrugs thereof, will be in a range from about 0.01 to about 15 mg/kg bodyweight, such as for example from about 0.01 to about 10 mg/kg bodyweight, about 0.05 to about 10 mg/kg bodyweight, 0.05 to 5 mg/kg bodyweight or 0.1 to 2.5 mg/kg bodyweight.

In yet another way of expressing the dosage is as unit dosages. Such unit dosages may be administered one or more time during the day and is typically administered whenever the patient feels the need to be treated. In some embodiments the unit dosage of the compounds in accordance with the present invention will be in a range of from about 0.01 to about 0.2 mg/kg bodyweight, such as for example from about 0.01 to 0.10 mg/kg bodyweight, about 0.02 to 0.08 mg/kg bodyweight, about 0.03 to 0.07 mg/kg bodyweight or 0.04 to 0.06 mg/kg bodyweight. In another embodiment the dosage is 0.05 mg/kg bodyweight. The compounds for use in accordance with the present invention may be administered alone, or as part of a combination therapy. If a combination of active agents is administered, then it may be administered simultaneously, separately or sequentially. Depending on the disease and the state of the disease to be treated, it may be relevant to include one or more additional active compound in the medicament.

Examples of routes of administration which can be used for the compounds according to the present invention include parental administration. Parenteral administration is any administration route not being the oral/enteral route whereby the medicament avoids first-pass degradation in the liver. Accordingly, parenteral administration includes any injections and infusions, for example bolus injection or continuous infusion, such as intravenous administration, intramuscular administration,

subcutaneous administration. Furthermore, parenteral administration includes inhalations and topical administration. Accordingly, the compounds for use in accordance with the present invention may be administered topically to cross any mucosal membrane of an animal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, rectum, urethra or bladder, preferably the mucosa of the urethra or bladder, and accordingly, parenteral administration may also include buccal, sublingual, nasal, rectal, vaginal and intraperitoneal administration as well as pulmonary and bronchial administration by inhalation or installation. Also, the compounds for use in accordance with the present invention may be administered topically to cross the skin. Preferably, the compounds for use in accordance with the present invention may be used as a local treatment, i.e. be introduced directly to the site(s) of action as will be described below.

Accordingly, the compounds for use in accordance with the present invention may be applied to the skin or mucosa directly, or the compounds for use in accordance with the present invention may be injected into the site of action, for example into the diseased tissue or to an end artery leading directly to the diseased tissue. Thus the compounds according to the present invention may be applied directly to the urethral and/or bladder tissue or to an end artery leading directly to the urethral and/or bladder tissue. Such local application of the compounds for use according to the present invention may for example be performed by use of instillation and/or injection.

In a preferred embodiment, the compounds for use according to the present invention are administered by local application directly to the urethral and/or bladder tissue such as by injection and/or instillation, wherein bladder instillation is the most preferred.

The main route of administration is by bladder installation in order to introduce the agent to ultimately target the sites of desired action. Appropriate dosage forms for such administration may be prepared by conventional techniques.

The compounds of the present invention may be used to treat pain symptoms of IC/BPS sufferers in a bladder instillation. In such installation the compounds of the present invention may be suspended or dissolved in a liquid formulation with suitable excipients and administered by means of a urinary catheter. The instillation is held for varying periods of time, from a few seconds to 15 minutes or more (known as "dwell time"), before being drained or voided. Although most physicians use the "adult" size catheters, if patients have discomfort or difficulty with it, they can request the use of a smaller "pediatric" catheter.

The compounds of the present invention may also be used to treat pain symptoms of IC/BPS sufferers in a bladder instillation in a combination product containing other instillation agents such as dimethyl sulfoxide, pentosan polysulfate, heparin, sodium hyaluronate, or lidocaine as well as one (or more) compound(s) according to the present invention ranging from one to several hundred milligrams per dose. The compounds of the present invention may be suspended or dissolved in a liquid formulation with suitable excipients and administered by means of a urinary catheter. For use in the present invention the compounds may be administered alone, but will generally be administered in admixture with suitable pharmaceutical excipients, diluents or carriers selected with regard to the intended route of administration and standard pharmaceutical practice. When the compounds are formulated as a bladder installation liquid, the pharmaceutical excipients typically include substances such as for example hydoxypropyl^-cyclodextrin, dextrose and sodium phosphate buffer.

In addition to the above described formulations, medicaments containing a compound for use in accordance with the present invention may furthermore be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa.

Figures

Figure 1 : Effects of pretreatment with vehicle and Compound A ( g/ml) on basal cystometric parameters in CYP treated groups.

The white bar (left) denoted the group treated with saline/vehicle. Dark grey (middle) bar denotes the group treated with vehicle/CYP. The dark grey bar (right) denotes the group treated with Compound A/CYP. Double asterisks (**) symbols p<0.01 versus saline group, one way ANOVA followed by Dunnett's test. Hash (#) denotes p<0.05 versus vehicle /CYP (P2) group, unpaired Student t-test.

Examples

Example 1

The efficacy and utility of a preparation of compound A (5-(5-fluoro-2-methoxyphenyl)- 6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H-pyrrolo[3,2h]isoquinoline-2(3H)-one) for the treatment of pain associated with IC/BPS have been studied in a model of acute visceral hypersensitivity induced by CYP in female Sprague Dawley rats.

Cyclophosphamide (CYP)-induced bladder dysfunction is a commonly used experimental model for cystitis in rodents (Juszczak et al., 2007). It is known that in rats CYP treatment leads to accumulation of its toxic metabolite acrolein in the urinary bladder (Cox, 1979) mediating urinary bladder inflammation (Smaldone et al., 2009) and visceral pain (Boucher et al., 2000). It has been demonstrated that a single intraperitoneal (i.p.) injection of CYP induces visceral painful behaviors and urinary bladder inflammation that peak shortly after CYP injection and persist for up to 4 hours (Lluel et al., 2010; Auge et al., 201 1 and 2013). Thus, CYP-induced cystitis in rats is a relevant model to test therapeutic approaches for the treatment of urinary bladder inflammation and visceral pain.

The aim of the present study was to evaluate the effect of intravesical administration of Compound A on urinary bladder pain and urinary bladder weight modification in acute CYP-induced cystitis in female Sprague Dawley rats.

Test system

Animals

Adult female Sprague-Dawley rats (Janvier, Le Genest Saint Isle, France), weighing 205-285 g at the beginning of the experiments, were used. Animals were

acclimatized to the laboratory conditions for at least 3 days before the start of any experiments. They were housed in groups of 2 or 3 in polypropylene type III H cages (cage size: 806 cm², Charles River Laboratories) with free access to food (Rodent Maintenance Diet R04/10 from Safe, France) and water until tested. Beds of wood chips (29/12 Plus, Souralit, Girona, Spain) in rodent cages were changed once per week. The animal house was maintained under artificial lighting (12 hours) between 07h00 and 19h00 in a controlled ambient temperature of 21 ± 3°C, and relative humidity of 40-70%. All experimental protocols were carried out in accordance with the European Community Council Directive 2010/63/EU. They were performed in accordance with French legislation concerning the protection of laboratory animals and in accordance with a currently valid license for experiments on vertebrate animals, issued by the French Ministry for Agriculture and Fisheries to Dr. Philippe Lluel (UROsphere, N°31 -1 1-555-46 dated 17 May 2011 - validity = 5 years) and Dr. Nicolas Cenac (UMR1043 Inserm, Toulouse; N°31 -12-555-23 dated 03 August 2012 - validity = 5 years).

Laboratory equipment

Mechanical stimuli were performed using von Frey filaments of different forces (reference: NV-17775; BioSeb ID Tech, Vitrolles, France). Anesthesia was performed using a gas anesthesia station (TEM SEGA, Pessac, France).

Intravesical administrations were performed using Introcan Safety® catheters (reference: 4251628-01 ; B.Braun, Boulogne Billancourt, France).

Urinary bladder weights were measured using a microbalance (New classic MS105, METTLER TOLEDO, Viroflay, France). Substances

The test substance (Compound A) (25μg/mL) was prepared freshly each week of experimentation in the vehicle. The solution, protected from light, was stirred until complete dissolution of the substance (at least two hours). The pH was checked for each preparation and was found to be around 7.4.

Hydroxypropyl^-cyclodextrin (batch n° 01721 EJ-299), dextrose (batch n°

SLBC9345V), sodium dihydrogen phosphate monohydrate (batch n° BCBK0189V) and disodium hydrogen phosphate heptahydrate (batch n° 031 M0191V) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). CYP (batch n° A0302605) was purchased from Acros Organics via Fischer Scientific (lllkirch, France). Isoflurane (batch n° B45D1 2A) was supplied by Belamont (Northumberland, United Kingdom). Physiological saline (0.9% sodium chloride) (batch n° 3009851) was purchased from VWR (Fontenay- sous-Bois, France). Sodium pentobarbital (batch n° 145A1) was supplied by Ceva sante animale (La Ballastiere, France).

Vehicle was composed of 2.5% hydoxypropyl^-cyclodextrin, 3% dextrose and 25mM sodium phosphate buffer. Sodium phosphate buffer included sodium

dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate. CYP (prepared fresh daily) was dissolved in physiological saline at a final

concentration of 30 mg/mL (w/v).

Experimental groups

Three experimental groups of 10 animals were included in this study as described in Table 1 below. Table 1

Study design

Animals treatments

On the day of each experiment, rats were weighed and the weights recorded. Rats were then anesthetized with isoflurane (2-5%) and a catheter inserted into the bladders. Bladders were infused with 500μΙ_ of Compound A (25μg/mL) or its vehicle for 45 min.

Acute CYP-induced cystitis

To induce acute cystitis, a single i.p. injection of CYP at a dose of 150 mg/kg in a final volume of 5 mL/kg was performed.

Assessment of referred visceral pain using von Frey filaments

Visceral pain was measured by applying to the lower abdomen, close to the urinary bladder, 8 von Frey filaments of increasing forces of 1 , 2, 4, 6, 8, 10, 26 and 60 g with an interval of 5 seconds between each filament.

Prior to testing, the abdominal area for mechanical stimulation of each animal was shaved. Animals were then placed on a raised wire mesh floor under individual Plexiglas box and acclimatized for 30 minutes before starting the von Frey test. Filaments were then applied 1-2 seconds with enough strength to cause the filament to slightly bend. Each filament was tested 3 times with an interval of 5 seconds between each application.

Nociceptive behaviors were scored for each animal and each filament as follows in Table 2 below: Table 2

Protocol design

In order to decrease the level of stress due to the new environment, for 2 days prior to experiments (D-2 and D-1), rats were acclimatized to the individual Plexiglas box for a minimum of 30 min and to the von Frey filament application.

On the day of the experiment, DO, von Frey testing was first performed 60 min before CYP administration (DO, T=-60min), in order to obtain basal values.

Compound A or its vehicle was administrated 45 min prior to CYP administration (DO, T=-45min) and 2 hours after CYP administration (DO, T=+2h), von Frey testing was performed. Just after the last von Frey testing (+2h), rats were sacrificed and urinary bladder weights were recorded. The protocol design is summarized in the below table 3:

Table 3

Assessment of urinary bladder weight

At the end of the experiment, rats were sacrificed by a lethal pentobarbital i.p.

injection (54.7 mg/mL, 1 mL/rat) followed by cervical dislocation. Urinary bladders were rapidly collected and cleaned from lipoid tissue. Their weights were recorded using a microbalance. Analyzed parameters

Nociceptive parameters are expressed as follows in Table 4 below:

Table 4

AUC's were calculated using GraphPad Prism (Version 5; GraphPad Software Inc., La Jolla, CA, USA).

Urinary bladder weight is expressed both in mg and in % of body weight.

Analysis

Statistical analysis was performed using GraphPad Prism (Version 5; GraphPad Software Inc., La Jolla, CA, USA). A p value <0.05 was accepted for statistical significance.

Percentage of nociceptive scores:

Two-way ANOVA with repeated measures (RM) (Two-way RM ANOVA) was performed either to compare Vehicle/Saline and Vehicle/CYP injected groups (before or 2h after CYP/saline injection) or to compare Vehicle/CYP and Compound A/CYP treated groups (before or 2h after CYP injection). Nociceptive threshold, AUC, body weight and urinary bladder weight:

Before carrying out statistical tests, it was determined whether the data were normally distributed and their variance evaluated (See appendix 5 / Statistical analysis). The appropriate statistical test was applied in consequence:

- an Unpaired t-test was used as the parametric test to compare 2 groups before or 2h after CYP/saline injection.

a Mann Whitney test was used as non-parametric t-test to compare 2 groups before or 2h after CYP/saline injection.

Results

The effect of Compound A on urinary bladder pain induced by cyclophosphamide in rats is shown in Tables 5-8 below.

Table 5. Nociceptive threshold (g)

Group 1 Group 2 Group 3

Vehicle/Saline Vehicle/CYP Cm p. A/CYP

Rat no. Basal 2h Basal 2h Basal 2h

1 26 26 26 6 26 1

2 26 26 26 10 10 26

3 10 26 26 10 10 8

4 26 26 26 8 10 26

5 10 10 10 6 10 10

6 10 60 26 10 10 10

7 26 26 8 6 10 26

8 26 26 10 8 10 26

9 10 60 8 8 26 10

10 10 26 10 10 26 60 average 18 31 18 8 15 20 Table 6. Percentage of nociceptive score

Table 7A. Area under the curve (AUC) between 1 and 10g

Group 1 Group 2 Group 3

Vehicle/Saline Vehicle/CYP Cm p. A/CYP

Rat no. Basal 2h Basal 2h Basal 2h

1 0 0 0 77 0 60.5

2 0 0 0 11 11 0

3 11 0 0 11 11 55

4 0 0 0 44 11 0

5 11 11 11 99 11 11

6 11 0 0 11 11 11

7 0 0 44 66 0 0

8 0 0 22 44 22 0

9 11 0 33 66 33 11

10 11 0 11 22 11 0 average 5.5 1.1 12 45 12 15 Table 7B. Area under the curve (AUC) between 10 and 60g

Table 7C. Area under the curve (AUC) between 1 and 60g

Group 1 Group 2 Group 3

Vehicle/Saline Vehicle/CYP Cm p. A/CYP

Rat no. Basal 2h Basal 2h Basal 2h

1 1111 836 1111 2189 1386 1535

2 1111 1111 1111 1947 1210 1111

3 1485 649 1111 1947 1485 1705

4 1111 836 1386 1606 1485 1111

5 1485 1210 1210 1936 1210 1210

6 1485 374 1111 1210 1210 1397

7 836 836 1331 1628 1584 1111

8 1111 1111 1584 1606 1485 1573

9 1210 374 1507 1815 1111 1210

10 1485 836 1485 1859 1111 561 average 1243 817 1295 1774 1328 1252 Table 8. Effect on bladder weight

Effect of CYP on visceral pain

In order to confirm CYP-induced visceral pain, the nociceptive parameters between Saline- and CYP- injected groups (in presence of vehicle) were compared (Tables 5, 6, 7A, 7B and 7C, group 1 and 2). Before CYP or saline injection, no significant difference in the nociceptive parameters were observed between the Vehicle/Saline- and Vehicle/CYP- injected groups (p>0.05, Mann Whitney test or Two-way RM ANOVA).

Two hours after CYP or saline injection, a significant decrease in the nociceptive threshold was observed in CYP- versus Saline-injected rats (p<0.001 , Mann Whitney test, Table 5). In addition, significant increases in nociceptive scores (p<0.001 , Two-way RM ANOVA, Table 6) and corresponding AUC 1-60g (pO.001 , Unpaired t-test, Table 7C) were observed 2h after CYP injection compared to Saline- injected rats. The nociceptive response was characterized by significant increases in allodynia: AUCs 1-1 Og (p<0.001 , Mann Whitney test, Table 7A) and hyperalgesia: AUC 10-60g (pO.001 , Unpaired t-test, Table 7B).

Effect of CYP on urinary bladder weight

No significant differences were observed in rat body weight, recorded at the start of the experiments, between the Vehicle/Saline- and Vehicle/CYP-injected groups (p>0.05, unpaired t-test, Table 8, group 1 and 2). In CYP- versus Saline-injected rats, significant increases in urinary bladder weight in mg (p<0.01 , Mann Whitney test) and in % of body weight (p<0.01 , unpaired t-test) were observed. Effect of the Compound A treatment on CYP-induced visceral pain

In order to analyze the effect of Compound A on CYP-induced visceral pain, the nociceptive parameters were compared between Vehicle- and Compound A-treated groups (Table 5, 6, 7A, 7B and 7C, group 2 and 3). Before CYP injection, no significant differences in the nociceptive parameters were observed between

Vehicle/CYP- and Compound A/CYP- treated groups (p>0.05, Mann Whitney test or Two-way RM ANOVA). Two hours after CYP injection, Compound A treatment significantly increased nociceptive threshold compared to Vehicle-treated rats (p<0.05, Mann Whitney test, Table 5). In addition, a significant decrease in the nociceptive scores (p<0.01 , Two-way RM ANOVA, Table 6) and the corresponding AUCs 1-60g (pO.01 , Unpaired t-test, Table 7C), 1-1 Og (p<0.05, Mann Whitney test, Table 7A) and 10-60g (p<0.01 , Unpaired t-test, Table 7B) were observed. The Compound A effect was more significant for higher forces of von Frey filaments (p<0.01 for AUC 10-60g versus pO.05 for AUC 1-1 Og).

Effect of the Compound A treatment on CYP-induced urinary bladder weight increase No significant difference in rat body weight, recorded at the start of the

experiments, was observed between the Vehicle/CYP- and Compound A/CYP- treated groups (p>0.05, Unpaired t-test, Table 8, group 2 and 3). In Compound A- versus Vehicle-treated rats, significant decreases of urinary bladder weight in mg and in % of body weight were observed 2 hours after CYP injection (p<0.05, Unpaired t- test).

Conclusion

CYP injection (150 mg/kg, i.p.) induced visceral hypersensitivity in female rats, 2h after administration. The CYP effects were characterized by both allodynia (decrease in nociceptive threshold in response to innocuous von Frey forces) and hyperalgesia (increased nociceptive scores in response to noxious von Frey forces). It is noteworthy that the CYP-induced allodynia was lower in comparison to previous studies. This difference may be due to either the isoflurane anesthesia duration (45 min) or the intravesical route of administration.

As in previous studies, urinary bladder weight was significantly increased 2 hours after CYP injection. This increase probably reflects an inflammatory response including leukocytes infiltration and/or oedema formation (Auge et al., 2013). A single intravesical administration of Compound A reversed the referred visceral allodynia and hyperalgesia induced by the CYP injection. Furthermore, Compound A treatment slightly but significantly attenuated the CYP-induced increases in urinary bladder weight suggesting an anti-inflammatory effect.

In conclusion, these data clearly show that intravesical Compound A treatment reduces referred visceral pain in a model of acute cystitis. Example 2

The efficacy and utility of a preparation of compound A (5-(5-fluoro-2-methoxyphenyl)- 6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H-pyrrolo[3,2h]isoquinoline-2(3H)-one) for the treatment of urinary bladder function associated with IC/BPS have been studied in a model of acute CYP-induced cystitis in female Sprague Dawley rats.

Cyclophosphamide (CYP)-induced bladder dysfunction is a commonly used experimental model for cystitis in rodents (Juszczak et al., 2007). It is known that in rats CYP treatment leads to accumulation of its toxic metabolite acrolein in the urinary bladder (Cox, 1979) mediating urinary bladder inflammation (Smaldone et al., 2009) and visceral pain (Boucher et al., 2000).

The results demonstrate that when compound A (25 μg/mL) was administered before CYP, bladder hyperactivity was significantly reduced; increasing the in inter-contraction interval (ICI) and decreasing the micturition frequency (MF) compared to vehicle (measured 2 hours after CYP administration).

Test System

Animals

Female Sprague-Dawley rats weighing 208-290 g at delivery were used (Janvier Labs, Le Genest Saint Isle, France). Animals were delivered to the laboratory at least 3-5 days before the experiments during which time they were acclimatized to laboratory conditions. They were housed in groups of 3-4 in polypropylene type E cages (size: 1032 cm2, Charles River Laboratories) with free access to food (Teklad 2016 global rodents, Harlan, Gannat, France) and water until tested. Beds of wood chips (29/12 Plus, Souralit, Girona, Spain) in rodent cages were changed twice weekly. The animal house was maintained under artificial lighting (12 hours) between 7h30 and 19h30 in a controlled ambient temperature of 21 ±3 °C, and relative humidity of 40-70%. Information related to any clinical signs and mortality were archived with the study materials.

Laboratory equipment

For cystometry experiments, bladder catheters were connected via a T-tube to a strain gauge MX 860 Novatrans III Gold (Medex Medical SARL, Nantes-Carquefou, France) and a syringe pump (70-2208 Model 11 plus, Harvard Apparatus, Les Ullis, France). Intravesical pressure was recorded continuously using a Powerl_ab/8e interface (AD Instruments Pty Ltd, Castle-Hill, Australia) and Chart® software running on a personnel computer. Data were analyzed with Microsoft Excel ® software.

Preparation of test article

Compound A was diluted in 2.5% hydroxypropyl^-cyclodextrin, 3% dextrose and 25 mM sodium phosphate buffer at a final concentration of 25 μg/mL. After weighing the necessary quantity of Compound A, the final volume of vehicle was added and maintained under magnetic agitation until complete dissolution (for 2 hours minimum). The solution was prepared weekly and stored at 4°C and protected from light.

The vehicle (1) was 2.5% hydroxypropyl^-cyclodextrin, 3% dextrose and 25 mM sodium phosphate buffer at approximately pH 7.5. To prepare this vehicle, the following compounds were used :

2- hydroxypropyl^-cyclodextrin, Lot 01721 EJ-299

Dextrose, Lot SLBC9345V

Sodium phosphate monobasic monohydrate, Lot BCBK0189V

Sodium phosphate dibasic heptahydrate, Lot 031 M0191V

All of the above compounds were in the form of a white powder and purchased from Sigma Aldrich.

To prepare the vehicle, 80% of the final volume of sterile water (autoclaved water) was added in a calibrated flask. The necessary quantities of sodium phosphate monobasic monohydrate and sodium phosphate dibasic heptahydrate were added and stirred under magnetic agitation until complete dissolution. Then necessary quantities of dextrose and 2-hydroxypropyl^-cyclodextrin were added and stirred until complete dissolution. The final volume was completed with sterile water and the solution was stirred. The pH was controlled and was approximately 7.5.

Substance-induced cystitis: cyclophosphamide

Cyclophoshamide, lot A0302605 in the form of a white powder from Acros Organics was used for the study.

Preparation of solution

Solution was prepared freshly each day of experimentation. Cyclophosphamide monohydrate was dissolved in physiological saline at a concentration of 30 mg/mL and intraperitoneally administered at a constant volume of 5 rnUkg in order to obtain a final dose of 150 mg/kg.

Physiological saline (NaCI 0.9%) (batch n° 13056406 and 13085407) was purchased from B-Braun via Centravet (Lapalisse, France). Water for injection (batch n° 2A165) was purchased from Lavoisier (Paris, France). Isoflurane (Aerrane®, batch n° 13F28A36) was purchased from Baxter Laboratories (Maurepas, France). Sodium pentobarbital (batch n° 157A1) was purchased from CEVA via Centravet (Lapalisse, France).

Rats were anesthetized with isoflurane (1.5-3%). After a midline incision of the abdomen, the urinary bladder was implanted with a polyethylene catheter (0.58 and 0.96 mm of internal and outer diameter, respectively) through the bladder dome and exteriorized at the scapular level.

Study design

Experimental groups

Three experimental groups of 10 animals were included in this study as described in Table 9 below.

A single i.p. dose of CYP 150 mg/kg or its vehicle (physiological saline) was administered at 5 ml_/kg. Table 9

Cystometry started 24 h after CYP treatment. The bladder was continuously infused at 2 ml/hr with physiological saline for at least 45 min (basal values). Then physiological saline was switched with Compound A (group 3) or their common vehicle (group 2) and the bladder infused for 90 min.

The following cystometric parameters were analyzed:

- Amplitude of bladder micturition contractions (AM), i.e. pressure between Threshold pressure (ThP) and Maximal pressure (MP) (mmHg),

- Micturition Frequency (MF), i.e. number of micturition contractions per 15 minutes intervals (peaks/15 minutes),

- Intercontraction interval (ICI), i.e. time between two subsequent micturitions (sec), Effects of pretreatment with vehicle and Compound A (25 Mg/mL) on basal (saline infusion) cystometric parameters (AM, ICI and MF) in CYP (2 hours)-treated- groups

Compared to the saline/vehicle group (of a parallel control experiment), a significant decrease in inter-contraction interval (ICI) values was observed in the two groups treated with CYP (vehicle and Compound A), (p<0.01 , one way ANOVA followed by Dunnett's test). A significant increase in ICI was observed in the Compound A group compared to its vehicle (p<0.05, unpaired Student t test, Figure 1 panel A, Tables 10- 12 below).

Compared to the saline/vehicle group (of a parallel control experiment), a significant increase in Micturition Frequency (MF) values was observed in the vehicle group (p<0.01 one way ANOVA followed by a Dunnett's test). A significant decrease in MF was observed in the Compound A group compared to its vehicle (p<0.05, unpaired Student t test) whereas no difference was observed with the saline/vehicle group (of a parallel control experiment). (Figure 1 panel B, Tables 10-12).

No statistical difference was observed in amplitude of bladder micturition contractions (AM) parameters between the 2 groups treated with CYP and the saline/vehicle group of a parallel control experiment (one way ANOVA followed by a Dunnett test, Figure 1 panel C, Tables 10-12 below).

The results demonstrate that when compound A (25 μg/mL) was administered before CYP, bladder hyperactivity was significantly reduced since the in inter-contraction interval (ICI) was increased and the micturition frequency (MF) was reduced compared to vehicle (measured 2 hours after CYP administration).

Table 10: Effect of Saline on cystometric parameters

Saline Vehicle Infusion (2ml/hr)

Infusion

Parameters (2ml/hr)

Basal

0-30 min 30-60 min 60-90 min Values

mean 19.13 16.92 16.41 20.36

AM (mmHg)

sem 1.77 1.51 1.39 2.74 mean 562.06 519.81 602.48 627.74

ICI (sec)

sem 53.86 67 05 89 06 11E .92

Basal

VI I D lO/ U U/40 40/OU Wl/ 7c/nn

Values

MF (peaks/ mean 2.39 1.91 2.09 1.55 2.00 2.27 2.00 15 min) sem 0.42 0.56 0.34 0.49 0.27 0.36 0.45

Table 1 1 : Effect of CYP/Vehicle on cystometric parameters

Table 12: Effect of Compound A/Vehicle on cystometric parameters

References

Auge C. et al., 2013. Relevance of the cyclophosphamide-induced cystitis model for pharmacological studies targeting inflammation and pain of the bladder. Eur. J. Pharmacol. 707(1 -3):32-40. Auge C. et ai, 201 1. Pharmacological modulation of urinary bladder

inflammation in a rat model of cyclophosphamide-induced acute bladder pain.

Inflamm. Res. 60 (Suppl 1): S248. Boucher M., 2000. Cyclophosphamide-induced cystitis in freely-moving

conscious rats: behavioral approach to a new model of visceral pain. J. Urol.

164(1):203-8.

Cox PJ., 1979. Cyclophosphamide cystitis-identification of acrolein as the causative agent. Biochem. Pharmacol. 28(13):2045-9.

Juszczak K. et ai, 2007. Animal models of overactive bladder:

cyclophosphamide (CYP)-induced cystitis in rats. Folia Med. Cracov. 48(1 -4): 113-23. LIuel P. et ai, 2010. Cyclophosphamide-induced cystitis in conscious female rats: development and pharmacological validation of an experimental model of referred visceral pain. Annual meeting of the International Continence Society (ICS), Toronto, Canada. Smaldone MC. et ai, 2009. Multiplex analysis of urinary cytokine levels in rat model of cyclophosphamide-induced cystitis. Urology. 73(2):421 -6.

Claims

1. A compound of Formula (I)

Formula (I) or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein

F is selected from the group consisting of methyl and ethyl,

R₂ is selected from the group consisting of methyl and methoxy, and

R₃ is selected from the group consisting of fluoro, chloro and methyl,

for use in the treatment of a disease associated with Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) by administering an effective therapeutic amount of said compound to a mammal subject.

2. The compound according to claim 1 , wherein the compound is selected from the group consisting of:

5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one;

3. The compound according to any of claims 1 or 2, wherein the compound is 5-(5- fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8-methyl-1 H- pyrrolo[3,2h]isoquinoline-2(3H)-one.

4. The compound according to any of the preceding claims for use in the treatment of a disease associated with IC/BPS, wherein the disease is selected from the group consisting of bladder pain and/or pelvic pain as well as urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia.

5. The compound according to any of the preceding claims for use in the treatment of a disease associated with IC/BPS, wherein the disease is selected from the group consisting of bladder pain and/or pelvic pain.

6. The compound according to any of the preceding claims for use in the treatment of a urinary symptom associated with IC/BPS, such as urinary frequency, urinary urgency and/or nocturia.

7. The compound according to any of the preceding claims wherein said treatment is capable of decreasing the urinary frequency and/or increasing the intercontraction interval in a subject in need thereof.

8. The compound according to any of the preceding claims, wherein said administration is by local application directly to the urethra and/or bladder.

9. The compound according to any of the preceding claims, wherein said administration is by injection and/or instillation directly to the urethra and/or bladder.

10. The compound according to any of the preceding claims, wherein said

administration is by bladder instillation.

1 1. The compound according to any of the preceding claims, wherein said

administration is by parental administration.

12. The compound according to any of the preceding claims for use in the treatment of a disease associated with IC/BPS in a mammal subject, where the mammal subject is human being.

13. The compound according to any of the preceding claims, wherein the compound is given in a dosage lying in the range of from about 1 to about 1000 mg, such as for example from about 1 to 500 mg, 5 to 500 mg, 5 to 250 mg or 10 to 200 mg.

14. A pharmaceutical composition comprising a compound according to any of claims 1-3.

15. The pharmaceutical composition according to claim 14, wherein the composition further comprises one or more additional active compound(s).

16. The pharmaceutical composition according to any of claims 14-15, wherein the compound is 5-(5-fluoro-2-methoxyphenyl)-6,7,8,9-tetrahydro-3-(hydroxyimino)-8- methyl-1 H-pyrrolo[3,2h]isoquinoline-2(3H)-one or a pharmaceutically acceptable salt, solvate or prodrug thereof.

17. The pharmaceutical composition according to any of claims 14-16, wherein the pharmaceutical composition is formulated as a liquid for intravesical administration into the bladder by use of a bladder installation.

18. A method of treating a disease associated with IC/BPS in a mammal subject in need thereof, said method comprising administering an effective therapeutic amount of a compound according to any of claims 1 to 3 to the mammal subject.

19. The method according to claim 18, where the mammal subject is a human being.

20. The method according to any of claims 18 to 19, where the disease associated with IC/BPS is selected from the group consisting of bladder pain and/or pelvic pain as well as urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia.

21. The method according to any of claims 18 to 20, where the effective therapeutic amount equals a range of from about 1 to about 1000 mg, such as for example from about 1 to 500 mg, 5 to 500 mg, 5 to 250 mg or 10 to 200 mg.

22. The method according to any of claims 18-21 , where the compound according to any of claims 1-3 is administered by local application directly to the urethra and/or bladder.

23. The method according to any of claims 18 to 21 , where the compound according to any of claims 1 to 3 is administered by use of a bladder installation.

24. Use of a compound according to any of claims 1 to 3 for the manufacture of a medicament for treating a mammal subject suffering of a disease and/or urinary symptom associated with IC/BPS.

25. Use according to claim 24, where the disease associated with IC/BPS is selected from the group consisting of bladder pain and/or pelvic pain as well as urinary symptoms, such as urinary frequency, urinary urgency and/or nocturia.

26. Use according to any of claims 24 and 25, where the mammal subject is a human being.

27. The use according to any of claims 24 to 26, where the compounds are

administered by local application directly to the urethra and/or bladder.

28. Use according to any of claims 24 to 27, where the compounds are formulated as a liquid for intravesical administration into the bladder by use of a bladder installation.