TREATMENT AND DIAGNOSIS OF CHRONIC INFLAMMATORY CONDITIONS IN THE LOWER URINARY TRACT

Abstract

A method for treating chronic inflammatory conditions in the lower urinary tract, the method comprising administering to a patient in need thereof, an effective amount of a reagent selected from the group consisting of IL-1β inhibitors and MMP inhibitors, or proteins selected from ASC or NLRP-3 is provided. Diagnostic methods are also described and claimed. The present disclosure further relates to an IL-1 inhibitor for use in a method of treating, alleviating or reducing pain and pain related symptoms of chronic pelvic pain syndrome. The IL-1 inhibitor may be an IL-1 receptor antagonist. The IL-1 inhibitor may be anakinra. The chronic pelvic pain syndrome may be a urological pain syndrome, a gynecological pain syndrome of the external genitalia, an internal pelvic pain syndrome and a gastrointestinal pelvic pain syndrome.

Claims

1. An agent that modulates the IL-1 pathway, particularly an agent selected from an IL-1 inhibitor, an MMP inhibitor and an NK1 inhibitor, or a pharmaceutical composition comprising the one or more agents, for use in the treatment of a chronic inflammatory condition in the lower urinary tract and/or for the treatment of CPPS.

2. An agent according to claim 1, wherein the chronic inflammatory condition is chronic cystitis, chronic pelvic pain syndrome and/or bladder-associated pelvic pain.

3. An agent according to claim 1 or claim 2, wherein the agent is an interleukin-1 receptor antagonist.

4. An agent according to any preceding claim, wherein the interleukin-1 receptor antagonist is an IL-1β inhibitor.

5. An agent according to any preceding claim, wherein the interleukin-1 receptor antagonist is anakinra, or an active fragment or active variant thereof.

6. An agent according to claim 1 or claim 2, wherein the agent is an MMP inhibitor.

7. An agent according to claim 6, wherein the MMP inhibitor is an MMP7 inhibitor.

8. An agent according to claim 6 or claim 7, wherein the MMP inhibitor is batimastat.

9. An agent according to any preceding claim, wherein the agent is for use in a subject that has a variation in an IL-1 related gene.

10. A method for treating chronic inflammatory conditions in the lower urinary tract or for treating CPPS, the method comprising administering to a subject in need thereof an effective amount of an agent that modulates the IL-1 pathway, particularly an agent selected from an IL-1 inhibitor, an MMP inhibitor and an NK1 inhibitor.

11. A method according to claim 10 wherein the condition is chronic cystitis, chronic pelvic pain syndrome and/or bladder-associated pelvic pain.

12. A method according to claim 10 or 11 wherein the reagent is an interleukin-1 receptor antagonist.

13. A method according to any of claims 10 to 12 wherein the interleukin-1 receptor antagonist is an IL-1β inhibitor.

14. A method according to any of claims 10 to 13, wherein the interleukin-1 receptor antagonist is anakinra, or an active fragment or active variant thereof.

15. A method according to claim 10 or 11, wherein the reagent is an MMP inhibitor.

16. A method according to claim 15, wherein the MMP inhibitor is an MMP7 inhibitor.

17. A method according to claim 15 or 16, wherein the MMP inhibitor is Batimastat.

18. A method according to any of claims 10 to 17, wherein the subject has a variation in an IL-1 related gene

19. A method for diagnosing chronic inflammatory conditions in the lower urinary tract, said method comprising detecting elevated levels of Substance P in urine of a subject.

20. A method for diagnosing susceptibility to chronic inflammatory conditions in the lower urinary tract, said method comprising detecting a mutation in a gene encoding a protein selected from ASC or NLRP-3 which results in downregulation of said gene and/or in the expression of inactive protein.

21. A method for preventing or treating chronic inflammatory conditions in the lower urinary tract in a patient susceptible thereto as a result of a mutation which impacts on the expression of functional ASC or NLRP-3, which method comprises administering to said patient a protein selected from ASC or NLRP-3 or a functional fragment or variant thereof and/or administering an effective amount of a reagent selected from the group consisting of interleukin-1 receptor antagonists and MMP inhibitors.

22. A protein selected from ASC or NLRP-3 or a functional fragment or variant thereof, for use in the treatment of patients suffering from or susceptible to chronic inflammatory conditions in the lower urinary tract as a result of a mutation which impacts on the expression of functional ASC or NLRP-3 respectively.

23. An IL-1 inhibitor for use in a method of treating, alleviating or reducing pain and pain related symptoms of chronic pelvic pain syndrome.

24. The IL-1 inhibitor for use according to claim 23, wherein the IL-1 inhibitor is an IL-1 receptor antagonist.

25. The IL-1 inhibitor according to claim 23 or 24, wherein the IL-1 inhibitor is anakinra.

26. The IL-1 inhibitor according to any of claims 23 to 25, wherein the IL-1 inhibitor is administered at a dose of 1-8 mg/kg body weight, preferably 1-4 mg/kg body weight, more preferably 1-2 mg/kg body weight.

27. The IL-1 inhibitor for use according to any of claims 23 to 26, wherein the IL-1 inhibitor is administered at an interval of 24 hours to 6 months, preferably 48 hours to 2 months, more preferably, 72 hours to 1 month.

28. The IL-1 inhibitor for use according to any of claims 23 to 26, wherein the IL-1 inhibitor is administered on-demand.

29. The IL-1 inhibitor for use according to any of claims 23 to 28, wherein the IL-1 inhibitor is administered by subcutaneous injection, intravenous injection, intramuscular injection.

30. The IL-1 inhibitor for use according to any of claims 23 to 29, wherein said chronic pelvic pain syndrome is chosen from a urological pain syndrome, a gynecological pain syndrome of the external genitalia, an internal pelvic pain syndrome and a gastrointestinal pelvic pain syndrome.

31. The IL-1 inhibitor for use according to any of claims 23 to 30, wherein said urological pain syndrome is chosen from prostate pain syndrome, bladder pain syndrome, scrotal pain syndrome, testicular pain syndrome, epididymal pain syndrome, penile pain syndrome, urethral pain syndrome, post-vasectomy scrotal pain syndrome.

32. The IL-1 inhibitor for use according to any of claims 23 to 31, wherein said urological pain syndrome is chosen from prostate pain syndrome, bladder pain syndrome and urethral pain syndrome.

33. The IL-1 inhibitor for use according to claim 30 or 32, wherein said bladder pain syndrome is bladder pain syndrome type 3c.

34. The IL-1 inhibitor for use according to claim 30, wherein said gynecological pain syndrome of the external genitalia is chosen from vulvar pain syndrome, generalized vulvar pain syndrome, localized vulvar pain syndrome, vestibular pain syndrome and clitoral pain syndrome.

35. The IL-1 inhibitor for use according to claim 30, wherein said internal pelvic pain syndrome is chosen from endometriosis associated pain syndrome, chronic pelvic pain syndrome with cyclical exacerbations and dysmenorrhea.

36. The IL-1 inhibitor for use according to claim 30, wherein said gastrointestinal pelvic pain syndrome is chosen from irritable bowel syndrome, chronic anal pain syndrome and intermittent chronic anal pain syndrome.

37. The IL-1 inhibitor according to any preceding claim, wherein the inhibitor is for administration to a subject having a variation in an IL-1 related gene.

38. A method for diagnosing chronic inflammatory conditions in the lower urinary tract, or a chronic pelvic pain syndrome, or a predisposition thereto, comprising identifying a variation in one or more of IL1A, IL1B, IL1RN, IL1R1, NLRP3, PYCARD, MMP7, TAC1 and TACR1 in a sample obtained from a subject, when compared to the sequence found in the majority of subjects, wherein the presence of the variation is indicative of the presence of or predisposition to chronic inflammatory conditions in the lower urinary tract, or a chronic pelvic pain syndrome.

39. The agent according to claim 9, the method according to claim 18 or 38, or the IL-1 inhibitor according to claim 37, wherein the subject has more than one variation in one or more IL-1 related genes.

40. The agent according to claim 9 or 38, the method according to claim 18, 38 or 39, or the IL-1 inhibitor according to claim 37 or 39, wherein the subject has a variation in one or more of IL1A, IL1B, IL1RN, IL1R1, NLRP3, PYCARD, MMP7, TAC1 and TACR1.

41. The agent according to claim 9, 39 or 40, the method according to claim 18, 38, 39 or 40, or the IL-1 inhibitor according to claim 37, 39 or 40, wherein the subject has a variation in IL1A.

42. The agent according to claim 9, 37, 39, 40 or 41, the method according to claim 18, 38, 39, 40 or 41, or the IL-1 inhibitor according to claim 37, 39, 40 or 41, wherein the subject has a variation in IL1B.

43. The agent according to any of claims 9, 37 and 39 to 42, the method according to any of claims 18 and 38 to 42, or the IL-1 inhibitor according to any of claims 37 and 39 to 41, wherein the subject has a variation in IL1RN.

44. The agent according to any of claims 9, 37 and 39 to 43, the method according to any of claims 18 and 38 to 43, or the IL-1 inhibitor according to any of claims 37 and 39 to 43, wherein the subject has a variation in IL1R1.

45. The agent according to any of claims 9, 37 and 39 to 44, the method according to any of claims 18 and 38 to 44, or the IL-1 inhibitor according to any of claims 37 and 39 to 44, wherein the subject has a variation in NLRP3.

46. The agent according to any of claims 9, 37 and 39 to 45, the method according to any of claims 18 and 38 to 45, or the IL-1 inhibitor according to any of claims 37 and 39 to 45, wherein the subject has a variation in PYCARD.

47. The agent according to any of claims 9, 37 and 39 to 46, the method according to any of claims 18 and 38 to 46, or the IL-1 inhibitor according to any of claims 37 and 39 to 46, wherein the subject has a variation in MMP7.

48. The agent according to any of claims 9, 37 and 39 to 47, the method according to any of claims 18 and 38 to 47, or the IL-1 inhibitor according to any of claims 37 and 39 to 47, wherein the subject has a variation in TAC1.

49. The agent according to any of claims 9, 37 and 39 to 48, the method according to any of claims 18 and 38 to 48, or the IL-1 inhibitor according to any of claims 37 and 39 to 48, wherein the subject has a variation in TACR1.

50. The agent according to any of claims 9, 37 and 39 to 49, the method according to any of claims 18 and 38 to 49, or the IL-1 inhibitor according to any of claims 37 and 39 to 49, wherein the subject has a variation at one or more of rs113540343 (IL1A), rs4251972 (IL1RN) and rs10754558 (NLRP3), rs145268073 (NLRP3), and rs45507693 (IL1RN).

51. The agent according to any of claims 9, 37 and 39 to 50, the method according to any of claims 18 and 38 to 50, or the IL-1 inhibitor according to any of claims 37 and 39 to 50, wherein the subject has a variation at rs113540343 (IL1A).

52. The agent according to any of claims 9, 37 and 39 to 51, the method according to any of claims 18 and 38 to 51, or the IL-1 inhibitor according to any of claims 37 and 39 to 51, wherein the subject has a variation at rs4251972 (IL1RN).

53. The agent according to any of claims 9, 37 and 39 to 52, the method according to any of claims 18 and 38 to 52, or the IL-1 inhibitor according to any of claims 37 and 39 to 52, wherein the subject has a variation at rs10754558 (NLRP3).

54. The agent according to any of claims 9, 37 and 39 to 53, the method according to any of claims 18 and 38 to 53, or the IL-1 inhibitor according to any of claims 37 and 39 to 53, wherein the subject has a variation at rs145268073 (NLRP3).

55. The agent according to any of claims 9, 37 and 39 to 54, the method according to any of claims 18 and 38 to 54, or the IL-1 inhibitor according to any of claims 37 and 39 to 54, wherein the subject has a variation at rs45507693 (IL1RN).

Description

FIGURE LEGENDS

[0114] The invention will now be described by way of example only with reference to the following figures, in which:

[0115] FIG. 1 shows a treatment outline for patients with chronic pelvic pain;

[0116] FIG. 2 is a chart showing elevated urine Substance P (SP) levels in patients with chronic cystitis before treatment;

[0117] FIG. 3 is a series of charts showing urine SP levels is reduced in after Anakinra treatment in patients with chronic cystitis;

[0118] FIG. 4 is a chart showing no change in urine IL-1β in patients with chronic cystitis before Anakinra treatment

[0119] FIG. 5 is a series of charts showing urine IL-1β levels do not change after Anakinra treatment in patients with chronic cystitis;

[0120] FIG. 6 is a schematic showing the IL-1β/Substance P loop as described in “Neuroepithelial control of mucosal inflammation in acute cystitis” by Butler, D. S. C. et al. (2018), Scientific Reports.

[0121] FIG. 7 shows a treatment outline for patients with chronic pelvic pain syndrome (FIG. 7a), patient descriptions (FIG. 7b), pain scores (FIG. 7c), frequency scores (FIG. 7d), quality of life scores (FIG. 7e) and urine SP concentrations (FIG. 7f).

[0122] FIG. 8. shows the summary of study protocol and results in example 3.

[0123] FIG. 9. Therapeutic effects of IL-1RA treatment in patients with bladder pain syndrome.

[0124] a) Ten patients with a long history of BPS were enrolled after informed consent and instructed to fill in a questionnaire detailing frequency of urination, pain, and quality of life. Laboratory samples were obtained at diagnosis, after the initiation of Anakinra treatment, during the “treatment break” and during long term follow up. b) Study outcome variables demonstrating an increased quality of life, due to reduced pain and frequency (Red=pretreatment samples. Blue=post treatment samples). Urine SP levels were markedly decreased. The data is shown for individual patients and lines indicate group medians. Wilcoxon signed rank test, P-values. c) Table showing treatment effects, side effects and long term treatment regimes in each patient.

[0125] FIG. 10. IL-1RA treatment alters gene expression in patients with bladder pain syndrome.

[0126] a, Inhibition of gene expression, following Anakinra treatment. Heat map of significantly regulated pathways in individual patients. Orange=upregulated, Blue=downregulated (cut off FC 1.5, compared to individual pre-treatment samples). Inhibited pathways included neuro-inflammation, IL-1- and inflammasome signalling, pattern recognition and adaptive immunity. b, IL-1R1 dependent gene expression network of PI showing significant inhibition of downstream genes (red=activated genes, blue=inhibited genes). c, Histograms of SNPs in rs113540343, rs4251972, rs10754558 and rs10199359 comparing frequencies of minor and major alleles between patients and the control NOMAD population. d, Table of the 10 most different SNPs between patients and the 1000 genomes control population.

[0127] FIG. 11. Structural views of rs145268073 (NLRP3) and rs45507693 (IL1RN).

[0128] a) IL1RN (magenta) in complex with the ILR1 (cyan) (PDBID: 1IRA) is shown with the position of rs45507693 boxed. b) Local environment of rs45507693 with side chains reaching within 4 Å of the alanine 90 beta carbon (magenta) are shown. The mutation to threonine (yellow) will decrease the local hydrophobicity. c) NLRP3 shown in complex with the NEK7 kinase (salmon) (PDB ID: 6NPY) with the position of rs145268073 boxed. The mutation is positioned close to the ADP binding site and interface between the NB1-HD1 (red) and WHD-HD2-LRR (blue) modules. d) Local environment of rs145268073 illustrating its close proximity to the WHD-HD2-LRR (blue)/NB1-HD1 (red) interface that undergoes large structural changes upon inflammasome formation. Shown are also side chains reaching within 4 Å of the arginine 488 distal guanidinium group and the ADP molecule in the NB1-HD1 module.

DETAILED DESCRIPTION OF THE INVENTION

Example 1: IL1 Inhibitors for the Treatment of CPPS

[0129] The present teaching relates to the treatment of pain and pain related symptoms of chronic pelvic pain syndrome, wherein the method comprises administration of an IL1 inhibitor to a subject in need thereof.

[0130] The present teaching also relates to the alleviation of pain and pain related symptoms of chronic pelvic pain syndrome, wherein the method comprises administration of an IL1 inhibitor to a subject in need thereof.

[0131] The present teaching also relates to a method of reducing pain and pain related symptoms of chronic pelvic pain syndrome, wherein the method comprises administration of an IL1 inhibitor to a subject in need thereof.

[0132] According to one specific embodiment, an IL-1 inhibitor, such as an IL1-receptor antagonist, is used in a method of treating, alleviating or reducing pain and pain related symptoms of chronic pelvic pain syndrome. The chronic pelvic pain syndrome may be a urological pain syndrome, a gynecological pain syndrome of the external genitalia, an internal pelvic pain syndrome or a gastrointestinal pelvic pain syndrome. Specifically, the chronic pelvic pain syndrome may be prostate pain syndrome, bladder pain syndrome or urethral pain syndrome. The IL-1 receptor antagonist is preferably administered at a dose of 1-8 mg/kg body weight, preferably 1-4 mg/kg body weight, more preferably 1-2 mg/kg body weight. The IL-1 receptor antagonist is administered at an interval of 24 hours to 6 months, preferably 48 hours to 2 months, more preferably, 72 hours to 1 month. In certain cases, the IL-1 receptor antagonist is administered on-demand. The IL-1 receptor antagonist is administered by subcutaneous injection, intravenous injection, intramuscular injection, preferably as a subcutaneous injection. The IL-1 inhibitor may be chosen from anakinra, kanakinumab and diacerein.

[0133] According to another specific embodiment, the IL-1 inhibitor anakinra is used in a method of treating, alleviating or reducing pain and pain related symptoms of chronic pelvic pain syndrome. The chronic pelvic pain syndrome may be a urological pain syndrome, a gynecological pain syndrome of the external genitalia, an internal pelvic pain syndrome or a gastrointestinal pelvic pain syndrome. Specifically, the chronic pelvic pain syndrome may be prostate pain syndrome, bladder pain syndrome or urethral pain syndrome. Anakinra is administered at a dose of 1-8 mg/kg body weight, preferably 1-4 mg/kg body weight, more preferably 1-2 mg/kg body weight. Anakinra is administered at an interval of 24 hours to 6 months, preferably 48 hours to 2 months, more preferably, 72 hours to 1 month. In certain cases, anakinra is administered on-demand. Anakinra is administered by subcutaneous injection, intravenous injection, intramuscular injection, preferably as a subcutaneous injection. Generally, anakinra is administered as subcutaneous injection of 100 mg at an interval between 1 injection every two weeks to 2 injections per week. Preferably, the dose of anakinra is 100 mg, or less, as a subcutaneous injection, administered daily as a maximum, or intermittently, with shorter or longer intervals (days to months) according to the need of reducing symptoms and following the clinical response.

[0134] In other words, the IL-1 receptor inhibitor anakinra may be administered at a dose of 1-8 mg/kg body weight, preferably 1-4 mg/kg body weight, more preferably 1-2 mg/kg body weight. Thus, anakinra may be administered at a dose of 75-600 mg/dose, preferably 75-300 mg/dose, more preferably 75-150 mg/dose. Thus, anakinra may be administered at a dose of 100 mg/dose.

[0135] The dose of anakinra may be adjusted if the patient is over- or underweight. Thus, an overweight patient may receive a dose higher than 100 mg, and an underweight person may receive a dose lower than 100 mg.

[0136] Anakinra may be administered at an interval of 24 hours to 6 months, preferably 48 hours to 2 months, more preferably, 72 hours to 1 month. Importantly, the specific time interval between administration of anakinra is determined for each patient, depending on the specific condition, pain intensity and effect of anakinra. Thus, anakinra may be administered once a day. If administered once a day, anakinra is preferably administered at approximately the same time point each day. Thus, anakinra may be administered at an interval of 24 hours.

[0137] Anakinra may be administered 1-2 times a week or at an interval of 1-3 weeks or at an interval of 1-6 months, such as 2-5 months or 3-4 months.

[0138] Further, anakinra may be administered on-demand.

[0139] Anakinra may be administered by subcutaneous injection, intravenous injection, intramuscular injection. Preferably, anakinra is administered by subcutaneous injection.

[0140] Specifically, the present teaching discloses anakinra for use in a method of treating, alleviating or reducing pain and pain related symptoms associated with urological pain syndrome. The urological pain syndrome may prostate pain syndrome, bladder pain syndrome, scrotal pain syndrome, testicular pain syndrome, epididymal pain syndrome, penile pain syndrome, urethral pain syndrome or post-vasectomy scrotal pain syndrome, particularly prostate pain syndrome, bladder pain syndrome or urethral pain syndrome. Preferably, the dose of anakinra is 100 mg, or less, as a subcutaneous injection, administered daily as a maximum, or intermittently, with shorter or longer intervals (days to months) according to the need of reducing symptoms and following the clinical response.

[0141] Thus, the present teaching discloses anakinra for use in a method of treating, alleviating or reducing pain and pain related symptoms associated with bladder pain syndrome (BPS). The BPS may be bladder pain syndrome type 3c. Preferably, the dose of anakinra is 100 mg, or less, as a subcutaneous injection, administered daily as a maximum, or intermittently, with shorter or longer intervals (days to months) according to the need of reducing symptoms and following the clinical response.

[0142] The present teaching also discloses anakinra for use in a method of treating, alleviating or reducing pain and pain related symptoms associated with prostate pain syndrome (PPS). Preferably, the dose of anakinra is 100 mg, or less, as a subcutaneous injection, administered daily as a maximum, or intermittently, with shorter or longer intervals (days to months) according to the need of reducing symptoms and following the clinical response.

[0143] Furthermore, the present teaching discloses anakinra for use in a method of treating, alleviating or reducing pain and pain related symptoms associated with urethral pain syndrome (UPS). Preferably, the dose of anakinra is 100 mg, or less, as a subcutaneous injection, administered daily as a maximum, or intermittently, with shorter or longer intervals (days to months) according to the need of reducing symptoms and following the clinical response.

Example 2: Anakinra for Use in Chronic Inflammatory Conditions

[0144] Here we identify that IL-1RA treatment may be useful to alleviate bladder pain. The hypothesis was evaluated by enrolling ten patients with chronic pelvic pain syndrome, (CPPS) in an investigator driven open trial (see below and FIG. 7). The patients received daily anakinra treatment (100 mg) until a stable response was observed. Treatment was then transiently interrupted and when the symptoms returned, treatment was resumed. Patient 10 remained asymptomatic.

[0145] Dramatic clinical effects were observed in 9/10 patients, who experienced a reduction in symptom score (pain and frequency, P<0.01) and increase in quality of life (P<0.02). Urine neuropeptide levels were markedly reduced (P<0.001) and at the RNA level, anakinra treatment inhibited neuroinflammation and IL-1-dependent gene expression. One patient did not respond.

[0146] Current therapeutic options in CPPS patients include antibiotics, non-steroidal anti-inflammatory drugs, opioids such as morphine and antidepressants. Bladder ulcers may be surgically removed, but in most cases, surgery has no lasting effect. This study suggests that IL-1R inhibition may offer a new molecular approach to treating cystitis and bladder pain, with the potential to improve the quality of life in patients, who so far has been offered little to alleviate their suffering.

[0147] The applicants have performed an open Anakinra trial in patients with chronic inflammatory conditions in the lower urinary tract, specifically chronic cystitis and CPPS. This structured observational study to investigate the effect of treatment with Anakinra in patients suffering from chronic cystitis and CPPS.

Overview of Patient Visits

[0148] Inclusion visit: Patient is informed about the study and is included after signed informed consent is obtained. A structured interview and clinical investigation is performed. The disease severity is evaluated using a symptom score. Follow-up visits are booked.

[0149] Samples: Blood and urine samples for investigation of inflammatory processes as well as genetic and proteomic analyses.

Visit 1:

[0150] A structured interview and clinical investigation is performed. Symptom severity is recorded.

[0151] Treatment with Anakinra is started and continued for seven days (until next visit).

[0152] Samples: Blood and urine samples for investigation of inflammatory processes as well as genetic and proteomic analyses.

Visit 2:

[0153] Evaluation of effect after one week of treatment with Anakinra. A structured interview and clinical investigation are performed. Symptom severity is evaluated. Patients with no treatment effect are excluded from further treatment.

[0154] Patients exhibiting a positive effect of the treatment make a pause in the treatment for two weeks (or at least long enough for the clinical effect of the treatment to wane).

[0155] Samples: Blood and urine samples for investigation of inflammatory processes as well as genetic and proteomic analyses.

Visit 3:

[0156] Follow-up after treatment pause. A structured interview and clinical investigation is performed. Symptom severity is evaluated. If the patient has relapse, Anakinra treatment is resumed.

[0157] Samples: Blood and urine samples for investigation of inflammatory processes as well as genetic and proteomic analyses.

Visit 4:

[0158] Follow-up with evaluation of effect after three weeks of treatment with Anakinra. A structured interview and clinical investigation is performed. Symptom severity is evaluated.

[0159] Patients pause the treatment for two weeks (or at least long enough for the clinical effect of the treatment to wane).

[0160] Samples: Blood and urine samples for investigation of inflammatory processes as well as genetic and proteomic analyses.

Visit 5:

[0161] Final visit after pause of treatment with Anakinra. A structured interview and clinical investigation is performed. Symptom severity is evaluated.

[0162] Samples: Blood and urine samples for investigation of inflammatory processes as well as genetic and proteomic analyses.

[0163] The visit programme is set out diagramatically in FIG. 1.

Methods

Clinical Investigation of Symptom Severity:

[0164] During patient visits, structured interviews evaluating symptoms (including consumption of medications) and clinical investigations are performed. Throughout the study period, the patients make daily symptom scores regarding pain, urination, sense of urgency and quality of life following a structured formula.

Inflammatory Parameters:

[0165] For evaluation of the levels of systemic and local inflammatory processes, analyses of CRP, neutrophil count and Interleukins are performed in serum and urine.

Genomics and Proteomics

[0166] Blood samples for DNA- and RNA-analyses as well as urine samples for proteomics are obtained throughout the study.

Monitoring:

[0167] Results are registered continuously within the clinical chart of each patient. Patient visits in the study are, to a large extent, part of regular patient management visits.

Results

Clinical Investigation of Symptom Severity

[0168] The treatment was proven effective in 60% of the patients as determined by a decrease in symptom scoring. Interestingly, all nine responding patients had Hunner's lesions, indicating that treatment might be more effective in this patient group. The patients were subsequently taken off treatment to investigate if the treatment was curative or whether symptoms would reappear. The symptoms ultimately came back, and the patients were again put back on anakinra treatment.

TABLE-US-00002 TABLE 1 Trial Results Patients Symptom Score Decrease Symptom Score No Decrease Recruited at 1 week of treatment at 1 week of treatment 15 9 6

Inflammatory Parameters:

[0169] To be able to define a treatment group where anakinra might serve as an effective therapeutic option, we searched for urine biomarkers. In addition to patient urine samples, samples were collected from healthy controls to compare urine concentrations of IL-1β and Substance P (SP). Surprisingly there was no IL-1β present in the urine of patients during maximum symptoms, nor in the control urine (13.2 vs 11.6 pg/ml) (FIG. 4) and urine IL-1β levels do not change after Anakinra treatment in patients (FIG. 5). In contrast, SP concentrations was increased in patients during maximum symptoms (209 vs 75 pg/ml, P=0.003), indicating a biomarker for bladder inflammation (FIG. 2). SP concentrations was subsequently analysed during the time of maximum symptom with paired samples from the time of minimum symptom showing a marked decrease after treatment (243 vs 136 pg/ml, P=0.02) (FIG. 3).

Study Protocol and Results

[0170] Ten patients with a long history of CPPS were enrolled after informed consent (age, gender, disease profile) and instructed to fill in a questionnaire detailing frequency of urination, pain, frequency of pain medication and quality of life.

[0171] The time to symptom relief after the first dose varied (1-6 hours) as did the duration (0.5-8 days), resulting in individualised treatment regimens (FIG. 7a). Two patients reported local irritation at the injection site and one patient developed neutropenia (FIG. 7b). A shared effect pattern included an initial response, resurgence of pain when treatment was interrupted and a return of pain relief and ease of urination when therapy was resumed.

[0172] A significant reduction in pain, urgency and frequency of urination was recorded in 9/10 patients (FIGS. 7c, 7d and 7e). Laboratory samples were obtained at diagnosis, after the initiation of anakinra treatment (n=10), during the “treatment break” and after continuation of anakinra treatment (n=9). Urine neuropeptides involved in the pain response (Substance P) were inhibited, with a biphasic response following the symptom score (FIG. 7f).

[0173] Gene expression analysis of peripheral blood RNA revealed a reduction in neuro-inflammation, following anakinra treatment.

Clinical Results

[0174] The effect of anakinra in the treatment of CPPS was studied in a clinical study. Inclusion criteria were CPPS mainly with the Bladder Pain phenotype, but also the with the Urethral and Prostate Pain phenotype. Exclusion criteria were uncontrolled diabetes mellitus, treatment for malignant disease within the preceding 10 years, pregnancy and age <18 years and ongoing infectious diseases, including urinary tract infections, and neutropenia (<1.5×10.sup.9/L). Patients gave informed consent for the use of unidentified personal clinical data for research purposes. Included patients were examined using clinical standard methods to rule out any ongoing disease. Blood and serum for hemoglobulin, liver and kidney function tests and White Blood Cell (WBC) counts were analysed. Urine was obtained for dip stick test (leukocytesteras for WBC estimation, the nitur test for ruling out bacteriuria), standard urinary Interleukins (IL6, IL8 and IL1b) and for urine culture. Cystoscopy and CT urography were performed in all patients. Blood and serum analysis and cystoscopy were repeated approximately two weeks after the start of treatment, and after approximately three months. Nine patients were recruited (Table 2).

[0175] After inclusion, patients were treated with anakinra 100 mg by self-administrated subcutaneous injection, repeated once daily as a maximum, or (if relief of symptoms and pain) on demand when symptoms or pain recurred. Regularly symptom scoring on frequency, supra-pubic/bladder pain/prostate/urethral pain and Quality of Life was performed to evaluate treatment effect.

TABLE-US-00003 TABLE 2 Patient characteristics and diagnosis Age Cystoscopy (years) CPPS/ Symptom finding Concomitant Gender CIC/ main duration Type 3c diseases, No (M/F) Cath subtype (year/s) yes/no Histology comment 1  61 M CIC BPS 2 yes inflamm. partial Type 3c tetraparesis, neurogenic bladder, kidney stone 2  67 M — BPS >10 yes inflamm. benign Type 3c prostate hyperplasia 3 77 F Cath BPS 2 yes inflamm. small Type 3c contracted bladder, previous irradiation due to gynecological cancer, COPD 4 67 F — BPS >10 yes nd healthy Type 3c otherwise 5 39 F — BPS 1 no nd healthy otherwise 6 70 F — BPS >10 no nd Mb Dercum 7 20 F — UPS <1 no nd healthy otherwise 8  70 M — PPS <1 no nd healthy otherwise 9  65 M — BPS >10 yes cystitis unspecified cystica intermittent fever, arthralgia Notes to Table 2 M = Male, F = Female; CIC = Clean Intermittent Catheterization; Cath = Indwelling catheter, CPPS = Chronic Pelvic Pain Syndrome; BPS Type 3c = Bladder Pain syndrome Type 3c; BPS = Bladder pain Syndrome; UPS = Urethral Pain Syndrome; PPS = Prostate Pain syndrome; Cystoscopy finding Type 3c = finding of Hunner's ulcera; inflamm. = inflammation corresponding with Hunner's ulcera. In patient 5 a non-specific scattering of red lesions was found at cystoscopy. Histological specimen was not taken. Repeat cystoscopy after 2 weeks of treatment revealed a normal bladder mucosa. In patient 9 cystoscopy revealed a “cystitis cystica”, or cystitis cystica glandularis, a specific benign inflammation due to chronic irritation or by unknown causes. In all other patients cystoscopy was normal. All cystoscopy findings refer to findings at inclusion in the study, except Patient 3 who was diagnosed with Hunner's ulcera one year prior to inclusion in the study (se detailed patient description below), nd = not determined; COPD = chronic obstructive pulmonary disease

[0176] Notably, all patients responded with dramatically reduced symptoms as measured by symptom scoring (Table 2). The maximum effect on symptoms after one s.c. inj. of 100 mg anakinra was seen within 1 to 24 hours and the maximum effect generally lasted 2-3 days. One patient (No 5) experienced the maximum effect for 8 days. In one patent (No 3), the maximum effect lasted only up to 13 hours, but generally, the maximum effect remained for several days. Thus, for the majority of patients, anakinra is administered one to two times a week at a dose of 100 mg s.c.

[0177] Notably, the number of micturitions during daytime was reduced in all patients and also number of micturitions at night-time was generally reduced.

[0178] The pain experienced by the patients was decreased dramatically and five out of the nine patients were even completely free from pain.

[0179] In addition, the experience quality of life (QoL) increased in all patients.

TABLE-US-00004 TABLE 3 Effect and duration of anakinra treatment in patients with Chronic Pelvic Pain/Bladder//Urethral/Prostate Pain Syndrome Evaluation of anakinra treatment effect Frequency QoL Onset Duration (nycturia) Pain reduction Side No (hours) (d/h) pre post pre post pre post effect 1 2 2-3 d.sup.   8 5 5 0 5 0 not reported (1-2) (0) 2 4-6 1-2 d.sup.  12 5 5 2 5 2 local s.c. irritation (5) (1) 3 1-2 7-13 h .sup.   — — 5-6 1 5-6 1 not reported 4 1-2 1-3 d.sup.  11 4 6 0 6 0 not reported (12) (3) (1) 5 1 8 d 12 6 4 0 4 0 headache post inj. (3) (1) 6 2 3 d 12 6 4 0 4 0 local s.c. irritation (5) (1) 7 24  3 d 12 6 4 1 4 1 not reported (4) (1) 8 2 2 d  8 8 4 2 6 2 not reported (5) (2) 9 3 3 d  7 4 5 0 5 0 not reported (1) (1) Notes to Table 3 Treatment was given all patients with anakinra 100 mg as a subcutaneos (s.c.) injection (inj.) with a maximum frequency of once daily, or after recurrence of Symptoms Obs./Treatm. = Observation/Treatment; Onset = time for maximum effect on symptoms after one s.c. inj. of 100 mg anakinra; Duration = time of lasting maximum effect after one s.c. inj. of 100 mg anakinra; d/h = days/hours; Frequency (nycturia) = Number of micturations during daytime and nighttime (the latter number within paranthesis); QoL = Quality of Life; Pain and QoL reduction estimated by interview and scored by 0-6 with 0 meaning symptom-free/not reduced QoL and 6 being severe pain and severely reduced QoL Side effect = reported side effects were mild. Local s.c. irritation = approx. 4 cm big red and edematous skin reaction at the place of injection, symptomatically; treated with hydrocortison application. Headache post inj. = moderate headache treated with analgetics such as paracetamol.

[0180] Long-term follow-up (Table 3) revealed that all nine patients experienced a highly increased, or increased, quality of life. The treatment effect in all patients was consistent during long-term follow-up.

[0181] Estimated WBC and Interleukins in urine were not significantly increased prior, during or after treatment.

[0182] If cystoscopy revealed Hunner's ulcera prior to treatment, this finding was unchanged at follow up cystoscopy. In one patient (patient 5), a non-specific scattering of small red lesions disappeared after treatment.

[0183] Side effects were sparse and mild; two cases of skin reaction at the place of injection, treated by hydrocortisone application, and one case of headache post injection (patient 5), treated occasionally with paracetamol and NSAID.

TABLE-US-00005 TABLE 4 Long-term follow-up of patients Anakinra Obs (s.c. 100 mg) time long-term No (days) treatment regimen Comment 1 397 1 per week highly increased QoL; after 6 months: treatment paus (ureteric stone) 2 274 1 per 2-4 weeks increased QoL, after 3 months long lasting symptom free 3 203 1 per day highly increased QoL, planned cystectomy postponed 4 173 1 per week highly increased QoL 5 121 1 per 2-3 days highly increased QoL 6 111 1 per 2-3 days highly increased QoL 7 38 1 per week increased QoL 8 33 1 per 2-3 days increased QoL 9 24 1 per week highly increased QoL,

[0184] Below is a detailed description of each patient.

[0185] Patient 1: 61 year old male with a previous history of traumatic spinal cord injury resulting in a neurogenic bladder disorder (hyperreflexia in combination with low detrusor function) treated with clean intermittent catherization. Two years prior to inclusion the patient had onset of dysuria, urge and pain from the bladder, with urine cultures only intermittently showing uropathogenic growth. Antibiotic treatment did not relieve the symptoms. Cystoscopy demonstrated a Hunner's ulcera. Treatment with transurethral resection and coagulation of the lesion as well as intravesical therapy with intravesical glycosaminoglycan (GAG) layer replenishment therapy did not result in subjective improvement. The patient was treated with little benefit with maximum dosages of peroral drugs against urgency (Mirabegron and anticholinergics) and with non-morphine analgesics. After administration of anakinra 100 mg s.c. (subcutaneous) with a dosage of approximately one injection per week, the patient was subjectively significantly improved with a clear reduction in frequency and local pain from the bladder. He is at the present under intermittent on-demand self-treatment with anakinra since more than one year with a stable treatment effect and without side effects.

[0186] Patient 2: Male 67 years old. History of more than 20 years of Bladder Pain Syndrome and benign prostate hyperplasia with outflow obstruction. Five years prior to inclusion a transurethral resection of the prostate including a coagulation/resection of a Hunner's ulcera in the bladder gave long-term symptom relief. Since one year recurring of symptoms with bladder pain, urge, and frequent nycturia, which the patient managed with the use of non-morphine analgesics and non-steroid anti-inflammatory drugs (NSAID). Cystoscopy revealed an incipient Hunner's ulcera. Anakinra treatment was initiated, which was followed by significant symptom and pain relief. The patient is at the present under on demand self-treatment, with approximately 1 injection of 100 mg anakinra (100 mg) per 2-3 weeks.

[0187] Patient 3: Female 77 years old. More than 20 years prior to inclusion successfully treated for gynecological cancer with irradiation and hysterosalpingo-oophorectomy. Chronic obstructive pulmonary disease. One year prior to inclusion start of urge and frequency, including local pain. Cystoscopy revealed a Hunner's ulcera in the bladder. Treatment with transurethral resection and coagulation of the lesion as well as intravesical therapy with intravesical glycosaminoglycan (GAG) layer replenishment therapy did not result in subjective improvement. After a few months, the bladder contracted and the patient was forced to be treated by indwelling catheter. The bladder pain was worsened and treated with morphine and corticosteroids. At inclusion the patient was on a waiting list for cystectomy. Cystoscopy prior to treatment did not reveal any bladder inflammation, other than normally found when indwelling catheter is used. After start of anakinra treatment pain relief followed immediately, and morphine and corticosteroid, treatment was ceased. At present the patient is on regular once daily anakinra self-administrated treatment, and the cystectomy is postponed.

[0188] Patient 4: Female 67 years old. Otherwise healthy female with a history of Bladder Pain Syndrome since more than 10 years. After an initial resection and coagulation, the patient has been symptom free. One year prior to inclusion the symptoms with urge and local pain recurred but treatment with non-morphine analgesics and NSAID gave little effect. Cystoscopy revealed an incipient Hunner's ulcera and treatment with anakinra was started which resulted in a constant relief of symptoms. The patient is at the present on one weekly injection of anakinra 100 mg s.c., which gives complete symptom relief.

[0189] Patient 5: Female 39 years old. Otherwise healthy, but with occasionally migraine attacks. Six months prior to inclusion slowly worsening urge and frequency, including local pain from the bladder. Cystoscopy revealed small red lesions in the bladder but without inflammation. The patient was treated with little benefit with maximum dosages of per oral drugs against urgency (Mirabegron and anticholinergics) and with non-morphine analgesics. Anakinra treatment resulted in constant relief of symptoms. The patient is at present under self-administrated treatment with anakinra 100 mg s.c. once per 1-2 weeks. After each injection, the patient gets minor headache, treated with NSAID and paracetamol. Repeated cystoscopy has demonstrated a normal bladder mucosa.

[0190] Patient 6: Female 70 years old. More than 20 years prior to inclusion diagnosed with Mb Dercum (adiposa dolorosa) and during the same period bladder pain including urge and frequency resulting in micturition once per hour, also during night-time. On cystoscopy, a normal bladder mucosa was found. Treatment with non-morphine analgesics, corticosteroids, anticholinergic drugs and mirabegron was not effective. After anakinra treatment started the patient did not have symptoms, or nearly no symptoms, from her adiposa dolorosa, and the urge, frequency and bladder pain dramatically decreased. She is at present on anakinra treatment 100 mg s.c with two injections per week.

[0191] Patient 7: Female 20 years old. Otherwise healthy. Six months prior to inclusion she had urethral pain and increased urgency, resulting in micturition once per 2 hours day and night. Treatment with local corticosteroid instillation in the urethra, non-morphine analgesics, corticosteroids, anticholinergic drugs and mirabegron was not effective. Cystoscopy was normal. After anakinra treatment, her symptoms urgency was dramatically reduced. The urethral pain was significantly reduced but recurred transiently. She is at present on anakinra treatment 100 mg s.c one injection per week.

[0192] Patient 8: Male 70 years old. Under treatment for hypertonia and atrial fibrillation. Six months prior to inclusion significant symptoms with severe urge and frequency in combination with prostate pain. The patient was forced to micturition every hour day and night. Examinations revealed a normal but tender benign prostate, cystoscopy and MR of the small pelvis and the prostate were all normal. Treatment with antibiotics (ex juvantibus), anticholinergic drugs and mirabegron was not effective. After starting anakinra treatment 100 mg s.c. injection the urgency and frequency was dramatically reduced. The prostate pain was reduced significantly, but recurred transiently. At present, the patient is under on-demand treatment with anakinra 100 mg s.c. injection.

[0193] Patient 9: Male 65 years old. Since more than 10 years with intermittent fever and arthralgia, without known cause in spite of extensive investigations by the rheumatoid department. Ten years prior to inclusion diagnosis of cystitis cystica (benign granulomatous inflammation) in the bladder. Increased urgency and frequency, but without the need for treatment. After anakinra treatment with 100 mg s.c. injection, the patient reported relief of fever attacks and arthralgia, as well as reduced urgency and frequency. At present the patient is under anakinra treatment 100 mg. s.c. injection twice per week.

Example 3: Treatment of BPS

[0194] The bladder pain syndrome (BPS) destroys careers, social life and sexual health, as patients experience debilitating pain, extreme frequency and urgency of urination. Numerous therapeutic approaches have been tested but except for the use of antibiotics to treat super-infections or pain medications, therapeutic success has been limited and patients are often severely handicapped. The molecular basis of BPS has also remained an enigma, precluding the development of more specific therapeutic alternatives.

[0195] By analysing the inflammatory phenotype in acute cystitis, the recently identified IL-1 as a cause of acute bladder pathology, aggravated by a genetic predisposition affecting the inflammasome constituents Asc and Nlrp3. The IL-1 receptor antagonist, Kineret (anakinra), dramatically reduced bladder inflammation and increased bacterial clearance in a murine cystitis model (ref). IL-1R blockade also affected the pain receptor NK1R in the bladder mucosa, linking IL-1 dependent inflammation to the pain response.

[0196] The inventors have tested the hypothesis that IL-1RA treatment may be useful to alleviate bladder pain. Ten patients with BPS were enrolled in an investigator-driven open trial. The patients received daily Kineret treatment (100 mg) for seven days. Treatment was then transiently interrupted and if the symptoms returned, treatment was resumed.

[0197] Dramatic clinical improvement was observed in 9/10 patients. A shared effect pattern included an initial response, resurgence of pain when treatment was interrupted and a return of pain relief and ease of micturition when therapy was resumed. One patient responded only transiently.

[0198] The patients experienced a reduction in symptom score (pain and frequency, P<0.01) and a marked increase in quality of life (P<0.02). At enrolment, elevated levels of neuropeptides controlling the pain response were detected in urine. A rapid reduction occurred after the initial Kineret treatment period and again when treatment was resumed after the treatment break (P<0.001). Gene expression was also inhibited, affecting neuroinflammation, Toll-like receptor dependent pattern recognition and IL-1-signalling.

[0199] Current therapeutic options in BPS patients include non-steroidal anti-inflammatory drugs, opioids such as morphine and experimental intravesical treatments. Bladder ulcers may be surgically removed, but in most cases, surgery has no lasting effect. This study suggests that IL-1R inhibition may offer a new molecular approach to treating cystitis and bladder pain, with the potential to improve the quality of life in patients, who so far has been offered little to alleviate their suffering.

[0200] a) Schematic of the study protocol. Ten patients with a long history of BPS (ref EAU guidelines) were enrolled after informed consent and instructed to fill in a questionnaire detailing frequency of urination, pain, frequency of pain medication and quality of life. Laboratory samples were obtained at diagnosis, after the initiation of Kineret treatment (n=10), during the “treatment break” and after continuation of Kineret treatment (n=9). Neuropeptides involved in the pain response (Substance P) were quantified in urine. Peripheral blood RNA was subjected to genome wide transcriptomic analysis to define molecular effects of Kineret treatment.

[0201] b) Study outcome variables demonstrating an increased quality of life and a reduction in frequency, pain and urine SP levels (Red=pretreatment samples. Blue=post treatment samples). The time to symptom relief after the first dose varied (1-6 hours) as did the duration (0.5-8 days), resulting in individualised treatment regimens. Two patients reported local irritation at the injection site, which was treated with local steroids. The data is represented as individual patients and the median. The data was analysed using Wilcoxon signed rank test, P-values less than 0.05 was considered significant.

[0202] c) Patient characteristics, underlying disease and outcome. Inflammation of the bladder in six patients was defined by cystoscopy and confirmed by histology. The mucosal cell infiltrate included mast cells and lymphocytes ref).

[0203] d) Inhibition of gene expression, following Kineret treatment. Heat map of significantly regulated pathways in individual patients Orange=upregulated, Blue=downregulated (cut off FC 1.5, compared to each individual pre-treatment sample). Inhibited pathways included neuro-inflammation, IL-1- and inflammasome signalling, pattern recognition and adaptive immunity. Fold change of significantly regulated genes are shown in Table 2 (means of nine patients).

Example 4

[0204] The immune response to infection must be exquisitely controlled and balanced. Deficiencies affecting cellular immune functions create an inadequate defense and increase the susceptibility to infection. Conversely, over-active immune responses are classically associated with diseases like asthma, rheumatoid arthritis and diabetes and treatments aim to return the immune balance and hinder disease progression.

[0205] Innate immunity controls the immediate response to infection, especially at mucosal surfaces where the initial contact between microbes and their host usually takes place. Detailed genetic studies have clearly shown that single gene defects control both the susceptibility to acute infection and the risk for chronicity and tissue damage. Specific immune activation pathways and immune effector functions have been identified and include Toll-like receptors, transcription factors, inflammasome, inflammatory cells.

[0206] The damaging potential of innate immune hyper-activation is highly relevant for the urinary tract, which is exposed to bacteria in everyone but leads to disease only in some. UTIs are extremely common, as about 50% of all women experience at least one episode during their lifetime. Many develop socially debilitating recurrent infections and therapeutic options are becoming extremely limited, due to the increase in antibiotic resistance.

[0207] The molecular basis of acute cystitis has been unresolved until recently, when Interleukin 1 (IL-1) hyper-activation was shown to drive acute disease severity and tissue damage. Paradoxically, disruption of the NACHT, LRR, PYD domains-containing protein 3 (Nlrp3) inflammasome created severe acute cystitis in infected mice, accompanied by IL-1 hyper-activation and a pain-sensing loop involving neurokinin 1 receptor (NK1R) and substance P (SP). Asc.sup.−/− and NIrp3.sup.−/− mice developed rapid, fulminant bladder inflammation and tissue damage, accompanied by massive neutrophil infiltration, hyper-activation of IL-1 beta and IL-1 beta-dependent gene networks. The severe disease phenotype was explained by a non-canonical processing mechanism in the Nlrp3 inflammasome-deficient mice, where overexpression of Matrix Metallopeptidase 7 (MMP7), resulted in highly efficient pro-IL-1beta processing. Finally, Asc and Nlrp3 were identified as transcriptional mmp7 repressors explaining why MMP7 is overexpressed in Asc.sup.−/− and NIrp3.sup.−/− mice. In stark contrast, Il1.sup.−/− mice were protected from infection, and did not show any signs of bladder inflammation, further emphasizing the importance of IL-1 as a driver of symptoms and pathology.

[0208] The IL-1 receptor antagonist (IL1-RA) Anakinra (brand name Kineret) is a biological immunomodulant, with an excellent safety record when used for indications such as rheumatoid arthritis. The therapeutic potential of IL-1 inhibition was examined in inflammasome-deficient mice developing severe cystitis. Anakinra was shown to dramatically reduce bladder inflammation and accelerate bacterial clearance. IL-1R blockade also reduced the expression of the pain receptor NK1R and its ligand SP in the bladder mucosa, linking IL-1 dependent inflammation to the pain response in the bladder mucosa.sup.3. The dramatic disease phenotype in NLRP3 inflammasome deficient mice and the therapeutic effect of Anakinra suggested that these mechanisms might be relevant in patients with chronic pelvic pain, which is often attributed to recurrent episodes of cystitis.

[0209] In this study, we addressed if IL-1RA treatment might be efficient in patients with chronic bladder pain. We conducted an explorative, open label trial in patients with BPS (Table 5). Patients received daily subcutaneous injections of Anakinra (100 mg) for seven days and the therapeutic effects were recorded. To address potential placebo effects, treatment was transiently interrupted for a maximum of 14 days and when the symptoms returned, treatment was resumed and continued with individual treatment regimes. At enrolment, cystoscopy revealed signs of Hunner's lesions in six of the patients, confirmed by histopathology. In the remaining patients, bladder pathology was not detected. During the study, patients were instructed to keep a symptom journal recording the sensation of pain and the frequency of micturition.

[0210] The immediate subjective response of the patients was remarkable. Dramatic clinical improvement was observed in 9/10 patients. A shared initial effect pattern included rapid symptom relief at the onset of treatment (median=2 hours) with lasting therapeutic effects (3 days). Dramatic clinical improvement was observed in all patients after the first 7 days, who experienced a reduction in symptom score (pain and frequency, P<0.02), leading to an increased quality of life (P<0.002). One 47-year old patient, with very severe BPS/ICS since the age of 17, noted a relief of symptoms within 1-2 hours after the first injection, and reported a return to normal life. The patient with slowest onset reported improvement after one week of treatment. One patient reported a partial response. Nine of the patients reported a complete, or close to complete clinical response and these patients remain on Anakinra treatment and have been followed for a median of 428 days (range 80-706 days). One patient responded only transiently, and discontinued treatment after 80 days.

[0211] This clinical response was accompanied by a change in urine neuropeptide levels. The pain sensor SP is activated during acute cystitis.sup.3 and inhibitors of the SP receptor NK1R reduce inflammation and pain in susceptible mice. SP levels were reduced in 9/10 patients during the first treatment round (FIG. 9). As treatment was interrupted, the resurgence of pain, frequency was accompanied by an increase in SP levels and when therapy was resumed, a return of pain relief and ease of micturition was accompanied by a reduction in SP levels.

[0212] To further understand the molecular basis of these effects, gene expression was analyzed longitudinally in RNA samples obtained; at onset, when the patients were in pain; at the time of maximum treatment effect in the first round; when pain returned during the treatment break; after reintroduction of Anakinra and at follow up, after 1-2 years of treatment. Gene expression, including canonical pathways involved in neuro-inflammation, TLR dependent pattern recognition and IL-1 signaling, was inhibited by treatment. Due to the pronounced effect on IL-1 related genes, we further investigated a gene network downstream of IL-1R1. In 8 out of 10 patients a marked reduction in IL-1R1 dependent gene expression was detected, primarily genes including CXCL1, IL1RAP CXCL3 and the IL1R1 gene itself.

[0213] By whole-exome sequencing, single nucleotide polymorphism (SNPs) were detected in IL-1-related genes including IL1A, IL1B, IL1RN, IL1R1, NLRP3, PYCARD, MMP7, TAC1 and TACR1. Allele frequencies in the patient group were compared to the NOMAD European database as well as allele frequencies at 1000 Genomes. Several SNPs were found in introns of these genes which included strong differences between the patients and the control populations for rs113540343 (IL1A), rs4251972 (IL1RN) and rs10754558 (NLRP3).

[0214] Several SNPs were identified in the IL1RN gene, which encodes the human IL-1 receptor antagonist after which Anakinra is designed. This suggests that the receptor antagonist might be defective in these patients, and hence the need for a correctly expressed IL-1 receptor antagonist in the form of anakinra. PYCARD encodes the inflammasome constituent ASC, which was identified as a cystitis susceptibility gene in mice. NLRP3 encodes the inflammasome component NLRP3 protein. Finally, SNPs were detected in TAC1 and TACR1, which encode the precursor of SP and its receptor NK1R, which drive pain sensing from the urinary bladder to dorsal root ganglia. The results suggest a genetic link between patients with bladder pain and IL-1 related genes that needs to be further evaluated in larger patient cohorts.

[0215] To further analyze the functional consequences of the SNPs we evaluated the effects of specific SNPs on experimentally determined 3-dimensional structures. Of the identified SNPs, rs145268073 and rs45507693 predict a change in the amino acid sequence of NLRP3 and IL1RN, respectively. In ILR1N, aa 99 was changed from an Alanine to a Threonine and in NLRP3, Arginine 488 was replaced by to a lysine or threonine. The atomic structures of both proteins have been solved individually and in complex with biologically binding partners (FIG. 11). The structure of ILR1N in complex with ILR1 (FIG. 11A, PDB ID: 1IRA) shows that the mutated A99 is situated in a central hydrophobic pocket with several relatively short non-polar contacts flanking the alanine's beta carbon (FIG. 11B). It is not unthinkable that the addition of a polar hydroxyl moiety might interfere sufficiently with this environment to indirectly alter the conformation of the nearby loop interfacing to ILR1.

[0216] In the complex structure of NLRP3 and activator, the mitotic NEK7 kinase, Arg488 is positioned in close proximity to key functional features of NLRP3 (FIG. 11C) (Sharif, H., et al. Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome. Nature 570, 338-343 (2019). NLRP3 is found in an inactive conformation, but earlier studies of related NLRC4 shows that activation and inflammasome formation is likely accomplished through a 90 degree rigid body movement of the NBD-HB1 module relative to the remainder WHD-HD2-LRR module of NLRP3 (Zhang, L., et al. Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization. Science 350, 404-409 (2015)). The exact mechanisms that triggers this event are unknown, but it has been shown that ADP increases the stability of the NEK7/NLRP3 complex. R488, which reaches into the central pocket of WHD, has no apparent contacts to ADP and is seemingly not directly part of the interface between the WHD-HD2-LRR and NBD-HB1 modules but it is positioned at a junction central to NLRP3 inflammasome formation. It may be speculated that this residue may play a central role in the formation of active NLRP3, for which no structure yet is available.

TABLE-US-00006 TABLE 5 Patient characteristics Symptom Other Duration Inflammation Concomitant ongoing ID Age M/F Cath Year(s) Cytoscopy Histology disease treatments Bladder Pain Syndrome with mucosal inflammation (Hunner's Ulcer) PI 77 F Cath 2 Positive Positive Small Morphine contracted bladder, previous irradiation due to gynecological cancer, chronic obstructive pulmonary disease PII 67 M — >10 Positive Positive Gout Allopurinol PIII 61 M CIC 2 Positive Positive Partial — paraparesis, neurogenic bladder disorder, kidney stone PIV 67 F — >10 Positive Positive Arthrosis — PV 67 M — >2 Positive Positive Gout Allopurinol PVI 78 F — 1 Positive Positive Hydrothyroidosis Levothyroxine Bladder Pain Syndrome without mucosal inflammation PVII 47 F — >10 Negative ND Otherwise — healthy PVIII 70 M — <1 Negative ND Prostate pain, — Artrial fibrillation PIX 39 F — 1 Negative ND Otherwise — healthy PX 70 F — >10 Negative ND Mb Dercum —