A pharmaceutical composition for treating or relieving ic / bps and use thereof

Abstract

The application discloses a kind of pharmaceutical composition for treating or relieving IC / BPS and application thereof, first disclose the application of the combination of Aprepitant and NLRP3 inflammasome selective inhibitor MCC950, especially in the application in the preparation of treating interstitial cystitis / bladder pain syndrome (IC / BPS) drug.The combination of Aprepitant and MCC950 can produce significant synergistic effect, by simultaneously blocking neurokinin-1 receptor (NK1R) mediated neurogenic inflammation and NLRP3 inflammasome mediated immune inflammation cascade, effectively inhibit the release of macrophage TNF-alpha, to fundamentally relieve the core symptoms such as bladder pain, frequent urination of IC / BPS.Compared with single drug, the combination administration scheme provided by the application has significant synergistic effect, provides a new strategy and more optimal administration scheme from symptomatic treatment to etiological treatment for the treatment of IC / BPS.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a pharmaceutical composition for treating interstitial cystitis / bladder pain syndrome (IC / BPS) and its application, particularly to the combined use of aprepitant and the NLRP3 inhibitor MCC950. Background Technology

[0002] Interstitial cystitis / bladder pain syndrome (IC / BPS) is a chronic, refractory urinary tract disease with unknown etiology and complex mechanisms. Its clinical features include pain related to bladder filling, urinary frequency, and urgency, which seriously affect the patient's quality of life.

[0003] In current technologies, treatment strategies for intrabladder-acquired pneumonia (IC / BPS) are mainly symptomatic, with various methods only providing temporary relief and failing to effectively block the core pathological link in the development of IC / BPS (i.e., the neuro-immuno-inflammatory circuit). Numerous studies have shown that pain and urinary dysfunction in IC / BPS are closely related to local neurogenic inflammation of the bladder and the overactivation of immune inflammasomes (especially the NLRP3 inflammasome). Neuropeptides such as substance P (SP) released from sensory nerve endings can activate NK1R, inducing neurogenic inflammation; simultaneously, the activation of the NLRP3 inflammasome in immune cells such as macrophages in the bladder tissue releases large amounts of pro-inflammatory factors such as IL-1β and TNF-α, forming an inflammatory cascade that sensitizes sensory nerves, exacerbating pain and abnormal urination reflexes.

[0004] MCC950 (CP-456773) is currently the most specific and widely used selective inhibitor of the NLRP3 inflammasome. This compound was previously studied in Phase II clinical trials for diseases such as gout and cardiovascular inflammation. However, clinical development was terminated due to undesirable metabolic properties, safety concerns, and limitations in drug development. Currently, it is only used in preclinical basic and animal studies. In urinary tract and pain-related models, MCC950 significantly reduces chronic bladder inflammation, decreases nerve sensitization, and improves urinary abnormalities and pain behaviors. It is a classic tool for validating the role of the NLRP3 pathway in the intracranial pressure (IC / BPS) system and provides crucial evidence for drug development targeting the NLRP3 inflammasome.

[0005] Aprepitant is currently the only clinically approved and most widely used oral NK1R antagonist, primarily used to prevent chemotherapy-induced nausea and vomiting, and can also be used for postoperative antiemetics. It has good safety and tolerability. It can cross the blood-brain barrier, blocking central and peripheral SP-NK1R signaling, and inhibiting pain sensitization and inflammation amplification.

[0006] Although existing studies have suggested that NK1R antagonists (such as aprepitant) or NLRP3 inhibitors (such as MCC950) have potential therapeutic value in bladder inflammation or pain models, the prior art has never disclosed or implied the use of the two in combination for the treatment of IC / BPS, let alone revealed whether the combination of the two can produce a synergistic effect, the specific synergistic mechanism, and the preferred dosing regimen. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a pharmaceutical composition for treating or alleviating IC / BPS and its application. It targets the core pathological features of IC / BPS (inflammatory cascade response and sensory nerve sensitization), while simultaneously blocking sensory neuropeptide SP and inflammasomes, thereby intervening in IC / BPS at the disease mechanism level, rather than simply alleviating symptoms. It strives to achieve a paradigm shift in the treatment of IC / BPS from symptomatic to etiological.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a pharmaceutical composition for treating or alleviating IC / BPS, comprising aprepitant and the NLRP3 inflammasome inhibitor MCC950.

[0009] Aprepitant is currently the only widely approved oral NK1R antagonist in clinical practice. It has good safety and blood-brain barrier permeability, and can effectively block the central and peripheral SP-NK1R signaling pathways.

[0010] MCC950 is currently the most specific selective small molecule inhibitor of the NLRP3 inflammasome and a classic tool for validating the role of the NLRP3 pathway in various inflammatory diseases.

[0011] Furthermore, the molar ratio of aprepitant to the NLRP3 inflammasome inhibitor MCC950 is 1:1.

[0012] Furthermore, the concentrations of both aprepitant and the NLRP3 inflammasome inhibitor MCC950 are 10 μM.

[0013] This invention not only proposes the concept of combination therapy, but also provides, through extensive experimental exploration, preferred drug types (aprepitant and MCC950), preferred dosage ratio (1:1), preferred concentration (10μM), preferred dosage (30mg / kg), and preferred dosing regimen (once every 2 days, for 3 consecutive times), providing solid technical support for subsequent clinical translation and drug development.

[0014] A second aspect of the present invention provides a combination medicament for treating or alleviating IC / BPS, comprising aprepitant and the NLRP3 inflammasome inhibitor MCC950 administered separately, simultaneously, or sequentially.

[0015] Furthermore, the molar ratio of aprepitant to the NLRP3 inflammasome inhibitor MCC950 is 1:1.

[0016] Furthermore, the concentrations of both aprepitant and the NLRP3 inflammasome inhibitor MCC950 are 10 μM.

[0017] Specifically, the combined administration route of aprepitant and MCC950 is intraperitoneal injection. However, depending on clinical needs, it can also be administered orally, intravenously, subcutaneously, transdermally, or via intravesical instillation.

[0018] A third aspect of the present invention provides the use of the above-described pharmaceutical composition or the combination of the above-described pharmaceutical compositions in the preparation of a medicament for treating or alleviating IC / BPS.

[0019] This invention reveals for the first time the synergistic mechanism of aprepitant combined with MCC950: the combination synergistically inhibits the release of TNF-α from macrophages (especially Cd11b⁺ / F4 / 80⁺ macrophages in bladder tissue). TNF-α is a key pro-inflammatory cytokine linking neurogenic inflammation and immune inflammation. By synergistically inhibiting TNF-α, this invention effectively blocks the vicious cycle of neuro-immune inflammation, thereby alleviating pain and urinary dysfunction in patients with intracranial hypertension (IC / BPS) at its root.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention demonstrates for the first time through in vivo animal experiments and in vitro cell experiments that aprepitant and MCC950 can produce a synergistic effect when used together. In animal models, the combined drug group is significantly better than the groups that use aprepitant or MCC950 alone in terms of relieving pain (open field test, stereotyped behavior score), improving urinary frequency (urine spot test) and reducing bladder tissue pathological damage (HE staining).

[0021] (2) This invention precisely targets the two core pathological mechanisms of IC / BPS: SP-NK1R-mediated neurogenic inflammation and NLRP3 inflammasome-mediated immune inflammation. By synergistically inhibiting the release of the key pro-inflammatory factor TNF-α, it effectively blocks the neuro-immune inflammatory circuit, which is expected to fundamentally change the disease process and overcome the limitations of existing symptomatic treatments.

[0022] (3) This invention not only proposes the concept of combined drug use, but also provides preferred drug types (aprepitant and MCC950), preferred dosage ratio (1:1), preferred concentration (10μM), preferred dosage (30mg / kg) and preferred dosing regimen (once every 2 days, for 3 consecutive times) through a large number of experiments, providing solid technical support for subsequent clinical translation and drug development.

[0023] (4) Aprepitant is already a clinically approved drug with complete safety data; although the clinical development of MCC950 was terminated due to safety issues, its effectiveness as a tool drug has been verified. This invention, through combination therapy, may reduce the dosage of MCC950 while achieving the same therapeutic effect, thereby avoiding its toxicity, or pointing the way for the development of novel and safe combination regimens of NLRP3 inhibitors and NK1R antagonists. Attached Figure Description

[0024] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0025] Figure 1 A statistical comparison chart of the total movement distance of mice in the open field experiment for each group; Figure 2 A statistical comparison chart of stereotyped behavior scores for each group of mice; Figure 3 A statistical chart showing the number of urine spots in each group of mice after 24 hours of urine spot experiment; Figure 4 HE-stained pathological images of bladder tissue from each group of mice; Figure 5 The expression levels of TNF-α in the culture supernatant of THP-1 cells after treatment with different concentrations of aprepitant; Figure 6 The expression level of TNF-α in the culture supernatant of THP-1 cells after treatment with different concentrations of MCC950 is shown in the figure. Figure 7 This is a statistical comparison of TNF-α expression levels in the supernatant of THP-1 cells in the control group, aprepitant monotherapy group, MCC950 monotherapy group, and combination therapy group in in vitro cell experiments. Figure 8 This is a statistical comparison of the median fluorescence intensity of TNF-α in Cd11b⁺ / F4 / 80⁺ macrophages in bladder tissue detected by flow cytometry in in vivo animal experiments. Figure 9 A comparison of the effect fractions (Fa) of aprepitant group, MCC950 group and combination therapy group on TNF-α release from THP-1 cells; Figure 10The figure shows the effect of different aprepitant to MCC950 ratios (3:1, 2:1, 1:1, 1:2, 1:3) on the TNF-α release fraction in THP-1 cells at the same total drug concentration (20 μM). Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example 1

[0027] Establishment of IC / BPS animal model and validation of the efficacy of combined drug administration regimen: 1. Laboratory animals and grouping SPF-grade female C57BL / 6J mice (6-8 weeks old, weighing 18-22g) were selected and, after 1 week of acclimatization feeding, were randomly divided into the following four groups, with 10 mice in each group.

[0028] IC / BPS model group (IC): IC / BPS model was established by intraperitoneal injection of cyclophosphamide (CYP), followed by an equal volume of solvent control (0.9% physiological saline).

[0029] IC model + aprepitant monotherapy group: CYP modeling, while aprepitant intervention was given.

[0030] IC model + Aprepitant + MCC950 group (IC + Aprepitant + MCC950): CYP modeling, with combined intervention of aprepitant and MCC950.

[0031] Blank control group: No treatment was given, and an equal volume of physiological saline was administered.

[0032] 2. IC / BPS model establishment and dosing regimen Model establishment: In the model group, aprepitant monotherapy group, and combination therapy group, an intraperitoneal injection of cyclophosphamide (CYP, purchased from Sigma-Aldrich) was used to establish an IC / BPS model. Specifically, mice were injected intraperitoneally with 100 mg / kg of CYP on days 1, 3, 5, and 7, for a total of four times, to induce typical IC / BPS symptoms such as bladder inflammation, pain, and urinary frequency.

[0033] Dosage regimen: Aprepitant monotherapy group (IC+Aprepitant): Aprepitant (purchased from Selleck Chemicals) was administered concurrently with CYP injection. The administration route was intraperitoneal injection at a dose of 30 mg / kg body weight, given every two days for a total of three doses.

[0034] The combination therapy group (IC + Aprepitant + MCC950): Aprepitant and MCC950 were administered concurrently after CYP injection for combined intervention. Both drugs were administered intraperitoneally at a dose of 30 mg / kg body weight, every two days for a total of three administrations.

[0035] Model group (IC): Control group receiving an equal volume of physiological saline or solvent at the same time point.

[0036] 3. Behavioral assessment The day after the last administration, behavioral tests were performed on mice in each group to assess the drug’s effect on improving IC / BPS-related symptoms (pain, urinary frequency).

[0037] 3.1 Open field test (assessing pain and anxiety-like behaviors) An open field box (40 cm × 40 cm × 40 cm) was placed in a quiet, softly lit environment. Mice from each group were placed in the center of the open field box, and their movement was continuously recorded over 5 minutes using a video tracking system (such as EthoVision XT). Behavioral analysis software was used to quantify the mice's total movement distance, time spent in the central area, and other indicators to assess spontaneous activity, anxiety, and pain stress responses.

[0038] Test results are as follows Figure 1 As shown, compared with the blank control group, the total movement distance of the model group (IC) mice was significantly reduced, indicating that the model was successfully established and the mice exhibited a state of pain stress. Compared with the model group (IC), the total movement distance of the aprepitant monotherapy group (IC+Aprepitant) mice increased, indicating that monotherapy could partially relieve pain. The total movement distance of the combination therapy group (IC+Aprepitant+MCC950) mice increased further, showing a highly significant difference compared with the model group (IC), and its effect was significantly better than that of the aprepitant monotherapy group (IC+Aprepitant). This indicates that the combination of aprepitant and MCC950 can significantly synergistically alleviate pain-related behaviors in IC / BPS mice.

[0039] 3.2 Stereotyped behavior score (assessing persistent pain) Mice were placed individually in transparent observation boxes and observed via video for 10 minutes. Two observers, unaware of the grouping, rated the mice's stereotyped behaviors such as licking, scratching, and circling according to the following criteria, and the average score was taken. Scoring criteria: 0 points, no stereotyped behavior; 1 point, occasional licking or scratching, lasting <30 seconds; 2 points, frequent licking, scratching, and circling, lasting 30 seconds to 3 minutes; 3 points, stereotyped behavior lasting >3 minutes, affecting normal activity.

[0040] Test results are as follows Figure 2 As shown, the stereotyped behavior scores of the model group (IC) mice were significantly higher than those of the blank control group. The stereotyped behavior scores of the combination therapy group (IC+Aprepitant+MCC950) were significantly lower than those of the model group (IC) and the aprepitant monotherapy group (IC+Aprepitant), indicating that the combination therapy can more effectively relieve the persistent pain of IC / BPS.

[0041] 3.3 Urine Spot Test After cleaning and disinfecting the metabolic cages, mice in each group were fasted but allowed free access to water for 12 hours. They were then placed individually in the metabolic cages, and urine was collected continuously over 24 hours in a quiet environment. Urine frequency, total urine area, and other indicators were recorded and quantified using a urine stain image analysis system.

[0042] Test results are as follows Figure 3 As shown, the number of urinary spots in the model group (IC) mice was significantly increased compared to the blank control group, confirming that the IC / BPS model mice exhibit severe urinary frequency symptoms. Compared to the model group (IC), the number of urinary spots was significantly reduced in the aprepitant monotherapy group (IC+Aprepitant). The number of urinary spots in the combination therapy group (IC+Aprepitant+MCC950) was further significantly reduced, showing a highly significant difference compared to the model group (IC), and was significantly superior to the aprepitant monotherapy group (IC+Aprepitant). These results strongly demonstrate that the combination administration of aprepitant and MCC950 has a significant synergistic effect in improving urinary frequency symptoms in IC / BPS mice.

[0043] 3.4 Histopathological examination: After behavioral testing, mice were euthanized, and bladder tissue was completely removed. The bladder tissue was fixed in 4% paraformaldehyde, routinely dehydrated, embedded in paraffin, sectioned (4 μm thick), and stained with hematoxylin and eosin (HE). Pathological changes in the bladder tissue were observed under a light microscope, with a focus on assessing the integrity of the bladder mucosal epithelium and the degree of inflammatory cell infiltration.

[0044] The results are as follows Figure 4As shown: In the blank control group, the bladder mucosal epithelium was intact, and the submucosa and muscle layer structures were clear, with no obvious inflammatory cell infiltration. In the model group (IC), the bladder mucosal epithelium was significantly defective and thinned, with a large number of diffuse inflammatory cells infiltrating the submucosa and muscle layer, and the tissue structure was disordered. In the aprepitant monotherapy group (IC+Aprepitant), the bladder mucosal epithelium was partially repaired, but epithelial thinning and local inflammatory cell infiltration were still visible, and the pathological damage was reduced compared to the model group. In the combination therapy group (IC+Aprepitant+MCC950), the bladder tissue morphology and structure were the most intact, the mucosal epithelium was continuous and intact, and there was very little inflammatory cell infiltration in the submucosa and muscle layer, with histological morphology close to that of the blank control group. The above pathological results confirm at the tissue level that the combination therapy can most effectively reduce local bladder inflammatory damage in IC / BPS, which is consistent with the behavioral results. Example 2

[0045] To further explore the molecular mechanism by which aprepitant and MCC950 produce a synergistic effect, this invention conducted in vitro cell experiments, focusing on the effect of the combination on the release of the key inflammatory factor TNF-α.

[0046] 1. Cell Culture and Macrophage Differentiation Human acute monocytic leukemia cell line THP-1 (purchased from ATCC) was cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) at 37°C in a 5% CO2 incubator. When the cell density reached 8 × 10⁶ cells / year... 5 At a concentration of 10 cells / mL, macrophages were seeded into 6-well plates and stimulated with phorbol ester (PMA, Sigma-Aldrich) at a final concentration of 20 ng / mL for 48 hours to induce differentiation into M0 macrophages. The expression of F4 / 80 and CD68 was detected by flow cytometry, and subsequent experiments were only conducted after confirming a differentiation efficiency >90%.

[0047] 2. Drug treatment and experimental grouping Successfully differentiated macrophages were randomly divided into the following treatment groups (each group had 3 replicates): LPS stimulation control group: only lipopolysaccharide (LPS, 1 μg / mL) was administered for stimulation.

[0048] Aprepitant monotherapy group: Aprepitant was pretreated for 1 hour with different concentrations (0, 1, 2.5, 5, 10, 20 μM) for 1 hour, followed by stimulation with LPS (1 μg / mL).

[0049] MCC950 monotherapy group: different concentrations (0, 1, 2.5, 5, 10, 20 μM) of MCC950 were pretreated for 1 hour, followed by stimulation with LPS (1 μg / mL).

[0050] Combined drug treatment group: Fixed concentration ratio experiment: Aprepitant and MCC950 were both 10 μM (molar ratio 1:1), pretreated together for 1 hour, and then LPS (1 μg / mL) was added for stimulation.

[0051] Experiments with different ratios: Under the premise of a fixed total drug concentration of 20 μM, different molar ratios of aprepitant to MCC950 were set up, including: 3:1, 2:1, 1:1, 1:2, and 1:3. Each group was pretreated with the drug for 1 hour, and then LPS (1 μg / mL) was added for stimulation.

[0052] All cells were cultured for another 24 hours after LPS stimulation. The cell culture supernatant was collected, centrifuged at 1000g for 10 minutes at 4°C to remove cell debris, and the supernatant was used for subsequent detection.

[0053] 3. ELISA method for detecting TNF-α release levels A commercially available mouse / human TNF-α ELISA kit was used, and the concentration of TNF-α in the cell supernatant of each group was measured strictly according to the instructions. The absolute content of TNF-α in each group was calculated based on the standard curve. For the evaluation of synergistic effect, the effect score (Fa) was calculated. The higher the Fa value, the stronger the inhibitory effect on TNF-α release. When the measured Fa value of the combination drug group was significantly higher than the sum of the Fa values ​​of the two single drug groups, it was determined to have a synergistic effect.

[0054] 4. Results of in vitro experiments Results of single-drug concentration-dependent experiments are as follows Figure 5 and Figure 6 As shown, both aprepitant and MCC950 monotherapy can inhibit the release of TNF-α from THP-1 macrophages in a concentration-dependent manner. Within the concentration range of 0-10 μM, the inhibitory effect significantly increases with increasing concentration; however, when the concentration exceeds 10 μM, the inhibitory efficiency plateaus, with no significant increase. These results indicate that 10 μM is the saturation concentration at which aprepitant and MCC950 monotherapy achieve maximum inhibitory efficiency.

[0055] The experimental results of combination therapy are as follows: Figure 7 and Figure 8As shown, under LPS stimulation, both 10 μM aprepitant monotherapy and 10 μM MCC950 monotherapy significantly inhibited TNF-α release. However, when the two were used in a 1:1 molar ratio (10 μM each), the inhibitory effect on TNF-α release far exceeded expectations. The TNF-α concentration in the combination therapy group was significantly lower than that in either monotherapy group, as shown in the figure. Figure 9 As shown, its effect score (Fa) is much higher than the arithmetic sum of the Fa values ​​of the two single-drug groups, indicating that the combination of the two drugs produced a synergistic effect rather than a simple additive effect.

[0056] like Figure 10 As shown, when the total drug concentration was fixed at 20 μM, the synergistic effects of different ratios differed significantly. The 1:1 ratio of aprepitant to MCC950 resulted in the highest Fa value, significantly better than other ratios (3:1, 2:1, 1:2, 1:3). Specifically, when the ratio deviated from 1:1, whether the proportion of aprepitant was too high (3:1, 2:1) or the proportion of MCC950 was too high (1:2, 1:3), the Fa value decreased significantly, indicating that an imbalance in the proportion of single drugs weakens the synergistic effect. This suggests that a 1:1 equimolar ratio is a key technical parameter for achieving optimal synergistic effects.

[0057] The aforementioned in vitro cell experiments revealed at the molecular level that aprepitant and MCC950 exert their anti-inflammatory and analgesic effects by synergistically inhibiting the release of the key pro-inflammatory factor TNF-α from macrophages. This mechanism constitutes the core scientific basis for the combination therapy of IC / BPS in this invention.

[0058] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A pharmaceutical composition for treating or alleviating IC / BPS, characterized in that, It contains aprepitant and the NLRP3 inflammasome inhibitor MCC950.

2. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of aprepitant to the NLRP3 inflammasome inhibitor MCC950 is 1:

1.

3. The pharmaceutical composition according to claim 2, characterized in that, The concentrations of both aprepitant and the NLRP3 inflammasome inhibitor MCC950 were 10 μM.

4. A combination drug for treating or alleviating IC / BPS, characterized in that, It contains aprepitant and the NLRP3 inflammasome inhibitor MCC950, which can be administered separately, simultaneously, or sequentially.

5. The combination drug according to claim 4, characterized in that, The molar ratio of aprepitant to the NLRP3 inflammasome inhibitor MCC950 is 1:

1.

6. The combination drug according to claim 5, characterized in that, The concentrations of both aprepitant and the NLRP3 inflammasome inhibitor MCC950 were 10 μM.

7. Use of the pharmaceutical composition of any one of claims 1-3 or the combination of pharmaceuticals of any one of claims 4-6 in the preparation of a medicament for treating or alleviating IC / BPS.

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