Method for preventing or treating urological chronic pelvic pain syndrome

Abstract

To provide a method for preventing or treating urological chronic pelvic pain syndrome (UCPPS) in a subject.SOLUTION: Provided is a method for preventing or treating urological chronic pelvic pain syndrome (UCPPS) in a subject that includes administering an effective amount of cerium oxide nanoparticles (CeNPs) to the subject. Also provided is a method for preventing or treating an UCPPS in a subject that includes administering to the subject a pharmaceutical composition comprising an effective amount of the CeNPs and a pharmaceutically acceptable carrier thereof.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] This disclosure relates to methods for preventing or treating chronic pelvic pain syndrome, and in particular to methods for preventing or treating urological chronic pelvic pain syndrome. [Background technology]

[0002] Chronic urological pelvic pain syndrome (UCPPS) is a debilitating chronic visceral disorder characterized by chronic lower abdominal and pelvic pain, as well as frequent and emergency urination. The prevalence of UCPPS is approximately 10% in the general population (8–11.5% in the male population[1]). Several hypotheses have been proposed regarding the etiology of UCPPS. There is no consensus on the mode of onset of the disease, but chronic inflammation and continuous induction may play an essential role in its progression[2]. UCPPS has a significant socioeconomic impact of over $70 billion annually, and the cost of treating UCPPS accelerates at a compound annual growth rate of approximately 5%. In addition to the associated healthcare costs, UCPPS has adverse effects on psychosocial health, causing sleep disturbances, anxiety, and depression[3].

[0003] Visceral pain is caused by ischemia, inflammation, and excessive activity of the walls of hollow organs. This is characteristic of many conditions, including treatment-resistant pain such as bladder pain syndrome and chronic pancreatitis, and contributes to a significant decline in quality of life. To date, the mechanisms of visceral pain, including UCPPS, are not as well understood as those of somatic pain, and chronic visceral pain conditions are usually difficult to alleviate effectively.

[0004] Non-invasive or minimally invasive therapies, including physiotherapy, pharmacological management, and nerve blockade, are used to treat UCPPS. However, their effectiveness is limited to the duration of treatment. For example, pentosan polysulfate sodium (PPS), also known as Elmiron, is an FDA-approved oral medication for UCPPS, but because it is an anticoagulant, it may increase the risk of bruising and bleeding. Furthermore, non-steroidal anti-inflammatory drugs (NSAIDs) have only short-term effects in treating UCPPS and need to be administered frequently (e.g., more than three times a day), which can lead to side effects such as kidney damage and allergies. For this reason, this type of treatment is listed as a second-line treatment by the American Urological Association due to the need for long-term oral administration and its low effectiveness. Hyaluronic acid (HA) is used to treat UCPPS by intravesical instillation due to its excellent biocompatibility. However, urination accelerates HA loss, requiring patients to undergo monthly perfusions, increasing treatment costs and negatively impacting quality of life.

[0005] Therefore, the medical need for alternative treatments and preventive measures for UCPPS that can overcome the aforementioned shortcomings remains unmet. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Clemens, JQ, et al., Urologic chronic pelvic pain syndrome: insights from the MAPP Research Network. Nature reviews. Urology, 2019. 16(3): p. 187-200. [Non-Patent Document 2] Nunez-Badinez, P., et al., Preclinical models of endometriosis and interstitial cystitis / bladder pain syndrome: an Innovative Medicines Initiative-PainCare initiative to improve their value for translational research in pelvic pain. PAIN, 2021. 162(9): p. 2349-2365. [Non-Patent Document 3] Lai, H.H., et al., Characterization of Whole Body Pain in Urological Chronic Pelvic Pain Syndrome at Baseline: A MAPP Research Network Study. Journal of Urology, 2017. 198(3): p. 622-631. [Non-Patent Document 4] Zhang, F., Q. Jin, and S.-W. Chan, Ceria nanoparticles: Size, size distribution, and shape. Journal of Applied Physics, 2004. 95(8): p. 4319-4326. [Non-Patent Document 5] Walczak, M.S., et al., Determining the chemical composition of corrosion inhibitor / metal interfaces with XPS: minimizing post immersion oxidation. JoVE (Journal of Visualized Experiments), 2017(121): p. e55163. [Non-Patent Document 6] Teng, Y.-N., et al., Etoposide Triggers Cellular Senescence by Inducing Multiple Centrosomes and Primary Cilia in Adrenocortical Tumor Cells. Cells, 2021. 10(6): p. 1466.

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[0007] In view of the above-mentioned problems in the art, the Disclosure provides a method for preventing or treating urological chronic pelvic pain syndrome (UCPPS), comprising administering an effective amount of cerium oxide nanoparticles (CeNPs) to a subject in need. In at least one embodiment of the Disclosure, the subject is a mammal, e.g., human. In some embodiments, administration of CeNPs can alleviate pain and / or reduce urinary frequency in a subject suffering from UCPPS.

[0008] Cerium oxide (CeO2) nanoparticles (CeNPs) offer several promising advantages for the treatment of UCPPS, including long-term effects, low cost, few harmful side effects, and a unique underlying mechanism of action. Specifically, CeNPs utilize oxidized CE 3+ and CE 4+ The coexistence of these states, along with the reversible redox switch between them, gives it unique antioxidant and / or catalytic properties. More specifically, the bulk crystal of CeO2 is mainly composed of CE 4+ It is composed of, but when the size of CeO2 is reduced to nanoscale, CE 3+The relative amount increases significantly, resulting in a higher catalytic effect. Therefore, in at least one embodiment of this disclosure, CeNPs may exhibit exceptional antioxidant properties by decomposing reactive oxygen species (ROS) and / or reactive nitrogen species (RNS), removing free radicals, and maintaining activity in tissues over long periods through spontaneous redox reactions. In some embodiments, CeNPs can prevent or treat oxidative stress-related UCPPS due to their remarkable antioxidant properties. In some embodiments, a decrease in heme oxygenase 1 (HO-1) expression in the bladder of a subject indicates that UCPPS may reduce oxidative stress in the bladder of a subject.

[0009] In at least one embodiment of this disclosure, CeNPs are administered to a subject to reduce inflammation in the subject. In some embodiments, administration of CeNPs may reduce pro-inflammatory factors or inflammatory agents in the subject's bladder, including but not limited to IRF7, IRF9, serpin B2, CXCL10, IL-6, and / or TNFα. For example, at least one of IL-6, TNFα, serpin B2, CXCL10, and heme oxygenase 1 (HO-1). In some embodiments, CeNPs are administered to a subject to improve edema and bleeding in the subject's bladder.

[0010] In at least one embodiment of this disclosure, the particle size of the CeNPs is about 10 nm to about 35 nm. In some embodiments, the particle size of the CeNPs is about 10 nm to about 30 nm, for example, about 15 nm to about 25 nm, about 20 nm to about 25 nm, or about 22 nm to about 25 nm, but this disclosure is not limited to these.

[0011] In at least one embodiment of this disclosure, the average particle size of the CeNPs is about 22 nm to about 25 nm. In some embodiments, the average particle size of the CeNPs is about 22 nm to about 24 nm, for example, about 22.5 nm to about 23.5 nm and about 22.5 nm to about 23 nm. In one embodiment of this disclosure, the average particle size of the CeNPs is about 22.5 nm, about 22.51 nm, about 22.52 nm, about 22.53 nm, about 22.54 nm, about 22.55 nm, about 22.56 nm, about 22.6 nm, about 22.7 nm, about 22.8 nm, about 22.9 nm and about 23.0 nm, but this disclosure is not limited thereto.

[0012] In at least one embodiment of this disclosure, the effective dose of CeNPs is about 3 mg / kg to about 100 mg / kg. In some embodiments, the effective dose of CeNPs is about 10 mg / kg to about 100 mg / kg, for example, about 10 mg / kg to about 90 mg / kg, about 10 mg / kg to about 80 mg / kg, about 15 mg / kg to about 70 mg / kg, about 20 mg / kg to about 70 mg / kg, about 20 mg / kg to about 50 mg / kg, about 25 mg / kg to about 40 mg / kg, and about 25 mg / kg to about 35 mg / kg, but this disclosure is not limited to these.

[0013] In at least one embodiment of this disclosure, CeNPs are administered to a subject 1 to 4 times per week, 1 to 4 times per month, or 1 to 4 times per year. In some preferred embodiments, CeNPs are administered to a subject once every 2 to 4 weeks, for example, once every 3 weeks.

[0014] In at least one embodiment of this disclosure, CeNPs are administered to a subject orally, intraperitoneally, intravenously, intradermally, intramuscularly, subcutaneously, or percutaneously. In some preferred embodiments, CeNPs are administered to a subject intraperitoneally or intravenously.

[0015] The present disclosure also provides a method for preventing or treating UCPPS, which includes administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the above-mentioned CeNPs and a pharmaceutically acceptable carrier thereof. In some embodiments, the pharmaceutical composition is administered orally, intraperitoneally, intravenously, intradermally, intramuscularly, subcutaneously, or transdermally.

[0016] The present disclosure further provides the use of a pharmaceutical composition in the manufacture of a medicament for preventing or treating UCPPS, the pharmaceutical composition comprising an effective amount of the above-mentioned CeNPs and a pharmaceutically acceptable carrier thereof.

Brief Description of the Drawings

[0017] The patent or patent application file includes at least one color drawing. A copy of this patent or patent application including the color drawing will be provided by the Patent Office upon request and payment of the required fees. The present disclosure can be more fully understood by referring to the following description of embodiments in conjunction with the accompanying drawings. [Figure 1] Figures 1A - 1D show the characterization of CeNPs. Figure 1A is a scanning electron microscope (SEM) imaging of cerium oxide nanoparticles (CeNPs). The CeNPs were observed at a magnification of 200,000× (scale bar: 200 nm). Figure 1B shows the hydrodynamic size distribution of CeNPs. Figure 1C is a transmission electron microscope (TEM) imaging of CeNPs at a magnification of 800,000× (scale bar: 2 nm). Figure 1D shows that the XPS spectrum of CENP reveals the binding energy region of cerium. The second, third, and eighth peaks indicated by the arrows are Ce3+, and the first, fourth to seventh peaks indicated by the arrows are Ce4+. [Figure 2]Figures 2A–2D show CeNPs tests using human urothelial T24 cells in vitro. Figure 2a shows T24 cells treated with 4-HC at concentrations of 0, 12.5, 25, 37.5, 50, 75, and 100 μM for 4 hours. Cell viability was examined using the WST-1 assay (MK400, Takara, Mountain View, California, USA). At a concentration of 37.5 μM of 4-HC, the viability of T24 cells decreased to approximately 50% (n=3). Figure 2B shows that the loss of T24 viability due to 4-HC was mitigated by treatment with cerium oxide nanoparticles (CeNPs) (n=3). Figure 2C shows the dichlorodihydrofluorescein diacetate (DCFDA) assay of T24 cells. Fluorescence microscopy images of each group: (1) Control group; (2) 4-HC only group: 37.5 μM 4-HC was added for 4 hours to induce cellular oxidative stress; (3) CeNPs treatment group: 5 μg / mL CeNPs were applied for 24 hours before induction with 37.5 μM 4-HC for 4 hours; (4) CeNPs only group: 5 μg / mL CeNPs alone were applied for 24 hours. Figure 2D shows that IL-6 and TNFα were significantly improved by 4-HC. (1) Control group; (2) 4-HC group: 37.5 μM 4-HC was added for 4 hours to induce cellular oxidative stress; (3) CeNPs + 4-HC group: 5 μg / mL CeNPs were applied for 24 hours before induction with 37.5 μM 4-HC for 4 hours; (4) CeNPs group: 5 μg / mL CeNPs alone were applied for 24 hours. Values ​​are expressed as mean ± SD. Data were analyzed by Tukey's multiple comparison test and one-way ANOVA. *P<0.05, **P<0.01. ns: No statistically significant difference (n=3). [Figure 3]Figures 3A and 3C show the results of the mechanical sensitivity test and the urination spot assay, respectively. Figure 3A shows the pain thresholds for the control, CYP, and CeNPs pretreatment groups. Figure 3B shows the quantification of the number of individual urination spots in the control, CYP, and CeNPs pretreatment groups. Numerical values ​​are expressed as mean ± standard error. Data were compared using Tukey's post-hoc test and one-way ANOVA. *P<0.05, **P<0.01. P>0.05: no significant difference (ns), n=3. Figure 3C shows representative urination patterns in the control, CYP, and CeNPs pretreatment groups. Urination spots are identified by *. [Figure 4] Figures 4A–4C show histological changes in mouse bladder in CYP-induced cystitis and the protective effect of CeNPs pretreatment. Figure 4A shows gross and hematoxylin-eosin (H&E) staining. Arrows indicate the suburothelial region. Figure 4B is a chart of edema scores. Urinary bladder from animals with cyclophosphamide (CYP)-induced cystitis shows vascular hypertrophy and increased suburothelial thickness compared to the control group (CTL) and the cerium oxide nanoparticle (CeNPs) pretreatment group. Figure 4C shows high-power field and scanning electron microscopy (SEM) images. Only the cyptobladder shows incomplete mucosa and naked urothelial cells, indicated by white arrows. Exposed urothelial cells (arrows) were observed in the cyptobladder. Complete cell membrane structures (arrows) were identified in the control and CeNPs pretreatment groups. Numerical values ​​are expressed as mean ± SE. The data were analyzed using Tukey's multiple comparison tests and one-way ANOVA. *P<0.05, **P<0.01. P>0.05: No statistical difference (ns) (n=3). [Figure 5] Figure 5 shows the relative levels of serpine B2, CXCL10, and heme oxygenase 1 (HO-1) in the bladder of the control group (CTL), the cyclophosphamide group (CYP), and the cerium oxide nanoparticle (CeNPs) pretreatment group. Values ​​are expressed as mean ± SE. Data were analyzed using Tukey's multiple comparison test and one-way ANOVA. *P<0.05, **P<0.01. P>0.05: No statistically significant difference (ns) (n=3). [Figure 6]Figure 6 shows the relative levels of heme oxygenase 1 (HO-1) in the bladder of the control group (CTL), the cyclophosphamide group (CYP), the cerium oxide nanoparticle (CeNPs) pretreatment group, and the CeNPs posttreatment group. [Figure 7] Figure 7 shows the relative levels of serpine B2, CXCL10, IRF7, IRF9IL-6, and TNFα in the bladder of the control group (CTL), the cyclophosphamide group (CYP), and the cerium oxide nanoparticle (CeNPs) pretreatment group. Values ​​are expressed as mean ± SD. Data were analyzed by Tukey's multiple comparison test and one-way ANOVA. ***P<0.001, (n=3). [Modes for carrying out the invention]

[0018] The following embodiments are used to illustrate the present disclosure. Those skilled in the art will readily conceive of other advantages and effects of the present invention based on the inventions herein. The present invention may also be carried out or applied as described in different embodiments. The following embodiments for carrying out the present invention may be adjusted or modified to suit different aspects and applications without departing from the scope thereof.

[0019] As used herein, the singular forms “a,” “an,” and “the” include multiple referents unless explicitly and obviously limited to a single referent. The term “or” is used interchangeably with “and / or” unless the context explicitly indicates otherwise.

[0020] Where used herein, the phrase “at least one” should be understood to mean, with respect to a list of one or more elements, at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of each element listed in the list of elements, nor excluding combinations of elements in the list of elements. Furthermore, this definition allows for the presence of elements other than those identified in the list of elements to which the phrase “at least one” refers, regardless of whether they are related to the identified elements. Thus, as a non-restrictive example, “at least one of A and B” (or equivalently “at least one of A or B,” or equivalently “at least one of A and / or B”) means, in one embodiment, that it optionally includes at least one A and B is absent (and optionally includes elements other than B); in another embodiment, that it optionally includes at least one B and A is absent (and optionally includes elements other than A); and in yet another embodiment, that it optionally includes at least one A and at least one B (and optionally includes other elements).

[0021] As used herein, the terms “comprising” (and any form of “comprise,” such as “comprises,” “comprises,” etc.), “having” (and any form of “have,” such as “has,” etc.), “including” (and any form of “includes,” such as “includes,” etc.), and “containing” (and any form of “contains,” such as “contains,” etc.) are inclusive or open-ended and do not exclude additional unlisted elements or steps of methods. For example, when describing an object as “containing,” unless otherwise specified, it may include other elements, structures, areas, parts, devices, systems, steps, or connections, and should not exclude other limitations.

[0022] The terms “about” or “approximately” mean within the tolerance range for a particular value as determined by those skilled in the art, which depends in part on how the value is measured or determined, i.e., on the limitations of the measuring system. For example, “about” may, by convention in the art, mean within 1 or a standard deviation greater than 1. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, such as within 5 times or 2 times the value. If a particular value is stated in the application and claims, unless otherwise specified, the term “about” should be assumed to mean within the tolerance range of that particular value.

[0023] The numerical ranges used herein are inclusive and combinable, and any number within a numerical range herein can be considered the maximum or minimum value for deriving the next range. For example, the numerical range "10nm to 35nm" should be understood to include any next range between the minimum value of 10nm and the maximum value of 35nm, such as 10nm to 20nm, 25nm to 35nm, and 22nm to 23nm. Furthermore, multiple numbers used herein can be arbitrarily selected as the maximum and minimum values ​​to derive numerical ranges. For example, the numerical ranges 10nm to 25nm, 10nm to 35nm, and 25nm to 35nm can be derived from the numbers 10nm, 25nm, and 35nm.

[0024] As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as diluents, disintegrants, suspending agents, wetting agents, fragrances, thickeners, fillers, preservatives, binders, lubricants, flow enhancers, and surfactants, which do not impair the biological activity or properties of the active ingredient (e.g., CeNPs as used herein) and are relatively non-toxic. That is, the vehicle does not cause undesirable biological effects when administered to a subject, nor does it interact in a harmful manner with the components of the medical composition in which it is contained.

[0025] As used herein, the terms “treat,” “treating,” and “treatment” mean the attainment of a desired pharmacological and / or physiological effect, for example, reducing or eliminating one or more symptoms of a disorder, disease, or condition, or a disorder, disease, or condition, or reducing or eliminating the (or multiple) causes of the disorder, disease, or condition itself. The effect may be preventive in that it completely or partially prevents the disease or its symptoms, or therapeutic in that it completely or partially cures, reduces, alleviates, improves, or improves a disease or a side effect caused by the disease or its symptoms.

[0026] As used herein, the terms “prevent,” “preventing,” and “prevention” mean methods of delaying and / or eliminating the onset of a disability, disease, or condition and / or symptoms associated therewith, methods of preventing a subject from contracting a disability, disease, or condition, or methods of reducing the risk of a subject contracting a disability, disease, or condition.

[0027] As used herein, the term “subject” refers to mammals such as humans, but may also refer to other animals such as livestock (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.) or laboratory animals (e.g., monkeys, rodents, mice, rabbits, guinea pigs, etc.). The term “patient” refers to a “subject” who is suspected of having or suffering from a disease or condition. Here, “subject in need of it” or “patient in need of it” refers to a subject or patient who has been diagnosed with, is at risk of having, has, is predetermined to have, or is suspected of having, a disease or disorder.

[0028] As used herein, the term “effective dose” means the amount of the active agent required to impart a desired preventive or therapeutic effect (e.g., reduction of oxidative stress or inflammation in a subject suffering from UCPPS) to a subject in need. In at least one embodiment of this disclosure, the effective dose of CeNPs is about 3 mg / kg to about 100 mg / kg, for example, about 10 mg / kg to about 100 mg / kg, about 10 mg / kg to about 90 mg / kg, about 10 mg / kg to about 80 mg / kg, about 10 mg / kg to about 70 mg / kg, about 15 mg / kg to about 60 mg / kg, about 15 mg / kg to about 50 mg / kg, about 20 mg / kg to about 40 mg / kg, and about 25 mg / kg to about 35 mg / kg. In some embodiments, the effective dose of CeNPs has a lower limit selected from 3 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, and 30 mg / kg, and an upper limit selected from 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 35 mg / kg, 34 mg / kg, 33 mg / kg, 32 mg / kg, 31 mg / kg, and 30 mg / kg.

[0029] In at least one embodiment of this disclosure, the particle size of the CeNPs is approximately 10 nm to approximately 35 nm. In some embodiments of this disclosure, the particle size of the CeNPs is approximately 10 nm to approximately 30 nm, for example, approximately 15 nm to approximately 25 nm, approximately 20 nm to approximately 25 nm, and approximately 22 nm to approximately 25 nm. In another embodiment, the particle size of the CeNPs has a lower limit selected from 10 nm, 15 nm, 20 nm, 22 nm, 22.4 nm, 22.47 nm, 22.48 nm, 22.49 nm, 22.50 nm, 22.51 nm, 22.52 nm, and 22.53 nm, and an upper limit selected from 35 nm, 30 nm, 25 nm, 22.70 nm, 22.60 nm, 22.59 nm, 22.58 nm, 22.57 nm, 22.56 nm, 22.55 nm, 22.54 nm, and 22.53 nm.

[0030] In at least one embodiment of this disclosure, the average particle diameter of the CeNPs is approximately 22 nm to approximately 25 nm. In one embodiment of this disclosure, the average particle diameter of the CeNPs is approximately 22 nm to approximately 24 nm, for example, approximately 22.5 nm to approximately 23.5 nm, and approximately 22.5 nm to approximately 23 nm. In one embodiment of this disclosure, the average particle diameter of the CeNPs is approximately 22.5 nm, approximately 22.51 nm, approximately 22.52 nm, approximately 22.53 nm, approximately 22.54 nm, approximately 22.55 nm, approximately 22.56 nm, approximately 22.6 nm, approximately 22.7 nm, approximately 22.8 nm, approximately 22.9 nm, and approximately 23.0 nm.

[0031] In one embodiment of this disclosure, ICR mice were treated with CeNPs once or twice a week to simulate the duration of human treatment (the weekly dose in ICR mice may correspond to a 3-month dose in humans), and experimental data showed that CeNPs administration has a long-term therapeutic effect for the prevention or treatment of UCPPS. In some preferred embodiments, CeNPs are administered to subjects once every 2-4 weeks or once every 3 weeks.

[0032] The following examples are provided to those skilled in the art to provide a complete description of the invention and how to prepare and use the assays, screenings, and therapeutic methods of the present invention, and are not intended to limit the scope of the invention to what the inventors consider to be their own invention.

[0033] Examples

[0034] Synthesis and Characterization of CeNPs

[0035] CeNPs were synthesized as previously described under the following conditions [4]: ​​Equivolute solutions of 0.0375 mol / L Ce(NO3)3·6H2O (99.5%, Alfa Aesar, Ward Hill, Massachusetts, USA) and 0.5 mol / L hexamethylenetetramine (HMTA) (99.9%, Alfa Aesar) were mixed and stirred at room temperature (22±1℃) for 24 hours. The solution was then centrifuged at 9,000 rpm for 30 minutes to obtain the CeNPs precipitate. The precipitate was washed twice with deionized water and ethanol (95%, Alfa Aesar).

[0036] The morphology and particle size of CeNPs were measured using a scanning electron microscope (SEM) (Hitachi S-4800 field emission scanning electron microscope and energy-dispersive X-ray spectrometer QUANTAX Annular XFlash® QUAD FQ5060, Hitachi, Tokyo, Japan) at an operating voltage of 10kV. SEM images of CeNPs (n=10) were analyzed using Nano Measure 1.2.5 software (National Cheng Kung University, Tainan, Taiwan) to calculate the average particle size of CeNPs. For morphological analysis using SEM, the elemental composition of the sample was analyzed using the accompanying accessory EDX (QUANTAX annularXflash® QUAD FQ5060). SEM images of CeNPs were observed at 200,000× magnification (scale bar 200nm).

[0037] The particle size and interplanar spacing of CeNPs were analyzed using a transmission electron microscope (TEM). CeNPs were dispersed in 95% ethanol, dispersed under ultrasound for 10 minutes, and then 2–10 μL were dropped onto a copper grid and air-dried. The particle size distribution of the nanoparticles was studied using TEM (Jeol 2010F, Jeol, Tokyo, Japan). The interplanar spacing, particle size (n=10), and selected region electron diffraction patterns were measured and analyzed using DigitalMicrograph 3 software (Gatan Inc., Pleasanton, California, USA).

[0038] The hydrodynamic size of CeNPs was characterized using a Zetasizer Nanozs (Malvern Instruments, Worcestershire, UK) and dynamic light scattering (DLS). The intensity of the scattered light was detected at 90° with respect to the incident beam. To confirm the dispersibility of the synthesized CeNPs in different solvents, CeNPs were dispersed in double-distilled water, sonicated for 10 min, and measured at 25 °C (n = 3). The acquired data were analyzed using the manufacturer-provided free software (Malvern Instruments, Zetasizer software).

[0039] The surface of CeNPs was characterized by X-ray photoelectron spectroscopy (XPS) using an AlKα source (Theta Probe, Thermo Fisher Scientific, Massachusetts, USA) to identify the oxidation state of cerium and quantify the Ce 3+ / CE 4+ ratio. The acquired data were input into XPSPEAK41 software [5] to process peak fitting.

[0040] Characterization of CeNPs

[0041] Using Nano Measurer software, the average particle size was found to be 22.53 nm (n = 10) (Figure 1A). The hydrodynamic size distribution of CeNPs in double-distilled water was 93.2 nm, and the PdI was 0.25 (Figure 1B). The high-resolution TEM image of CeNPs showed lattice fringes and d-spacing related to the (111) and (200) crystal planes, which was equivalent to the morphology of CeNPs (Figure 1C). In XPS, chemical bonding peaks corresponding to Ce 3+ (the 2nd, 3rd, and 8th peaks indicated by the arrows in Figure 1D) and Ce 4+ (the 1st, 4th - 7th peaks indicated by the arrows in Figure 1D) were shown. The peak between 875 eV and 895 eV corresponded to Ce3d 5 / 2 and the peak between 895 eV and 910 eV corresponded to Ce3d 3 / 2This corresponds to the degenerate level. The deconvolution peaks located at 875.100, 900.650, and 909.000 eV are Ce 3+ This corresponds to the oxidation state of the cerium ion. The deconvolution peaks observed at 881.240, 890.900, 893.400, 897.300, and 915.550e correspond to the Ce 4+ This could be due to the condition.

[0042] Timeline for evaluating CeNPs treatment in mice with CYP-induced cystitis

[0043] Table 1 shows the timeline for testing the effects of CeNPs in vivo. ICR mice were divided into four groups: a control group, a CYP group, a CeNPs pre-treatment group, and a CeNPs post-treatment group. Each group consisted of eight mice. Except for the control group, ICR mice were intraperitoneally administered CYP at a dose of 80 mg / kg to induce cystitis on days 7, 9, 11, and 13. In the CeNPs pre-treatment group, mice were intraperitoneally injected with 30 mg / kg of CeNPs on days 0 and 4. Alternatively, mice in the control and CYP groups were injected with PBS on days 0 and 4. In the CeNPs post-treatment group, mice were administered 30 mg / kg of CeNPs by intraperitoneal injection on days 8 and 13. On day 14, before sacrificing all mice, mechanical sensitivity testing and urination spot assays were performed. After CO2 euthanasia, the mice's bladders were collected, frozen in liquid nitrogen, and subjected to further histological and biochemical analysis.

[0044] [Table 1]

[0045] Example 1: Effects of 4-HC on human urothelial T24 cell viability, CeNPs treatment, and intracellular ROS measurement.

[0046] Human urothelial T24 cells (#60062, BCRC, Taiwan) were cultured in 90% McCoy 5a medium (Sigma, St. Louis, Missouri, USA) with 1.5 mM L-glutamine, 10% (v / v) fetal bovine serum, and 1% (v / v) PS. The cells were maintained at 37°C in a humidified incubator containing 5% CO2 [6], and 4-HC was used to induce oxidative stress in the cells. T24 cells were cultured for 10 4 Cells were seeded in 96-well plates at a cell / well density and incubated for 24 hours until fully adhered. In the wells containing the treated T24 cells, various concentrations of CeNPs were added to the culture medium and incubated for another 24 hours. Next, various concentrations of 4-HC (0, 12.5, 25, 37.5, 50, 75, and 100 μM) were added to the culture medium and incubated for 4 hours. T24 cell viability was reduced by 4-hydroperoxycyclophosphamide (4-HC) treatment, inducing the generation of intracellular reactive oxygen species (ROS). At a concentration of 37.5 μM, 4-HC reduced cell viability to 50% (IC50) and was selected as the induction concentration for subsequent experiments (Figure 2A). Different concentrations of CeNPs were applied prior to 4-HC induction. After a series of experiments, a concentration of 5 μg / mL of CeNPs was able to effectively alter the cell viability reduced by 4-HC (Figure 2B).

[0047] To confirm the inhibitory effect of CeNPs on the 4-HC-induced cellular oxidative stress response, T24 cells were cultured under four conditions using the dichlorodidifluorescein diacetate (DCFDA) assay: (1) control group; (2) 4 hours of exposure to 37.5 μM 4-HC to induce cellular oxidative stress; (3) 24 hours of exposure to 5 μg / mL CeNPs before 4 hours of exposure to 37.5 μM 4-HC; and (4) 24 hours of exposure to 5 μg / mL CeNPs alone. The amount of green fluorescence (DCF) was positively correlated with intracellular ROS content. CeNPs had an inhibitory effect on the 4-HC-induced cellular oxidative stress response (Figure 2C).

[0048] Total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlbad, California, USA). GAPDH was used as an endogenous control. cDNA was synthesized using the SensiFAST cDNA synthesis kit (Bioline, London, UK) with random hexamers and poly(DT) primers. PCR was performed using a qPCR system (Stepone® software v2.2). Each sample was analyzed in triplicate for both the target gene and the endogenous control. The mean CT value of the triplicate was used for further analysis. The Ct values ​​of the target gene from each sample were normalized relative to its endogenous control. -ΔΔCt The relative gene expression levels were converted using a specific method. The qPCR primers (5'-3') are shown in Table 2. The qPCR results are shown in Figure 2D. IL-6 and TNFα were significantly upregulated by 4-HC, but they were downregulated in the CeNPs + 4-HC group. In the CeNPs group, cells did not show excessive expression of inflammation-related genes, and it was confirmed that no inflammation was induced in cells even after 24 hours of application of 5 μg / mL of CeNPs.

[0049] [Table 2]

[0050] Example 2: Mechanical susceptibility test

[0051] Mechanical sensitivity was assessed using Semmes-Weinstein monofilaments (Ugo Basile, Comelio, Italy). Mice were tested in individual cages with stainless steel wire grid floors. Stimulation was limited to the lower abdomen, a common area of ​​the bladder, and performed in separate areas within this area to prevent desensitization. Three types of behavior were observed as positive responses to monofilament stimulation: 1) jumping, 2) immediately licking or scratching the monofilament stimulation area, or 3) rapidly contracting the abdomen. Each monofilament was applied for 1–2 seconds with a 5-second interval between stimulations. The 50% threshold (T50) was determined using the "up and down" method. Based on the most reliable threshold calculation, six consecutive attempts with different filaments were required [7]. T50 was determined by applying the formula: T50 = Xf + kd (where Xf is the last monofilament used, k is the Dixon table coefficient, and d is the average filament interval). Animals with CYP-induced cystitis showed a reduced pain threshold (n=3 per group, *P<0.05), which was mitigated in the CeNPs pretreatment group (n=3 per group, *P<0.05) (Figure 3A).

[0052] Example 3: Void Spot Assay

[0053] On the day of sacrifice, mice were placed in individual wire-bottom cages on filter paper (Whatman No. 1, AW1001-00917, Sigma-Aldrich, St. Louis, Missouri, USA) and left undisturbed in a quiet room for 3 hours with restricted food and water.[8] The filter paper was collected and dried and imaged under 365 nm ultraviolet light using a FluorChem digital imaging system (Alpha Innotech Corporation, San Leardro, California, USA). Volume was calculated by comparing the area of ​​the spots to a calibration curve. The area and number of void spots were calculated by analyzing the images using Void Whizzard and ImageJ.[9] Animals with CYP-induced cystitis showed increased urination frequency (n=3 per group, *P<0.05; 3c), which was mitigated in the CeNPs pretreatment group (n=3 per group, *P<0.05) (Figures 3B and 3C).

[0054] Example 4: Histological changes in mouse bladder after CYP-induced cystitis and protective effect of CeNPs pretreatment

[0055] After CO2 euthanasia, the mice's bladders were removed, placed in labeled tubes frozen in liquid nitrogen, and stored at -80°C for further histochemical and biochemical analysis. To assess morphological changes in the bladders, bladder sections (16 μm thick) were stained using the standard hematoxylin-eosin (H&E) protocol. They were instilled in modified Harris hematoxylin solution for 4 minutes and then in eosin Y solution (alcohol) (Sigma-Aldrich) for 1 minute.

[0056] Each bladder was sectioned by excising the bladder dome and clearly observing the internal cavity. Eight slides (all for H&E) were collected, each containing eight sections. This section represents the thickest central portion of the bladder. Previous studies have shown that, at a macroscopic level, urinary bladders from animals treated with CYP have increased suburothelial thickness compared to the control group, based on edema scores

[10] . For SEM, samples were fixed in 2.5% phosphate-buffered glutaraldehyde (0.1 m pH 7.4) for 24 hours, fixed in 1% OsO4 for 1 hour, dehydrated in a stepwise alcohol series, placed in amyl acetate, pressurized and dried with liquid CO2 in a critical point dryer, and coated with gold particles.

[0057] At a macroscopic level, the urine bags of animals treated with CYP showed greater edema and hemorrhage compared to the control group. Furthermore, increased thickness of the suburothelial layer was observed in CYP-treated animals compared to the control group

[10] (Figures 4A and 4B). These samples were observed using SEM. H&E staining revealed that the urine bags of animals with CYP-induced cystitis showed loss of urothelial membrane integrity and almost complete detachment of the urothelium compared to the control group (CTL) and the CeNPs-pretreated group (Figure 4C). Only the bladder of the CYP group showed incomplete mucosa and naked urothelial cells, indicated by whitish arrows. Exposed urothelial cells (arrows) were observed in the CYP group. Complete cell membrane structures (arrows) were identified in the control group and the CeNPs-pretreated group.

[0058] Example 5: Analysis of the expression levels of serpine B2, CXCL10, and heme oxygenase 1 (HO-1) using Western blotting.

[0059] The expression of serpine B2, CXCL10, and HO-1 was analyzed using Western blotting. Primary antibodies, anti-serpine B2 (#ab269275, Abcam, Cambridge, Massachusetts, USA), anti-CXCL10 (#ab9938, Abcam), anti-HO-1 (#ab13248, Abcam), and anti-β-actin (#26276, GeneTex, Irvine, California, USA), were incubated overnight at 4°C at a 1:7,000 dilution. The membranes were then washed three times with washing buffer for 30 minutes each, and incubated with a secondary anti-rabbit (#213110-01, GeneTex) or anti-mouse (#213110-01, GeneTex) IgG antibody at a 1:6,000 dilution at 37°C for 1 hour. Finally, the membrane was washed with immobilon Western Chemiluminescence HRP substrate (WBKLS0500, Millipore, Billerica, Massachusetts, USA), and protein bands were analyzed using a luminescence imaging system (Model: M3-8068; Hansor, Taichung, Taiwan).

[0060] Western blotting showed increased levels of serpin b2, Cxcl10, and HO-1 in the urine bags of CYP-treated animals compared to animals observed in the control and CeNPs pretreatment groups (Figure 5). qPCR results showed increased mRNA levels of serpin B2, CXCL10, IRF7, IRF9, IL-6, and TNFα in the bladder of CYP-treated animals compared to animals observed in the control and CeNPs pretreatment groups (Figure 7). Similar results were observed in the CeNPs posttreatment group. HO-1 levels in the CeNPs posttreatment group were significantly lower than those in the CYP group (Figure 6).

[0061] In summary, this disclosure provides a method for preventing or treating UCPPS by administering CeNPs to subjects in need. The administration of CeNPs not only overcomes oxidative stress and inflammation in the subjects' bladder but also produces analgesic and urination-reducing effects. Furthermore, compared to existing treatments for UCPPS, the method of this disclosure has long-term efficacy, low cost, fewer adverse side effects, and minimal invasiveness.

Claims

1. A medicament for preventing or treating urological chronic pelvic pain syndrome (UCPPS) in a subject in need thereof, comprising an effective amount of cerium oxide nanoparticles (CeNPs).

2. The method of claim 1, wherein the urological chronic pelvic pain syndrome is selected from the group consisting of painful bladder syndrome, interstitial cystitis, chronic prostatitis, chronic pelvic pain syndrome, and any combination thereof.

3. The agent of claim 1, which is administered to a subject to reduce oxidative stress in the subject.

4. The cerium oxide nanoparticles are in an oxidation state of Ce to reduce oxidative stress through a redox reaction. ３＋ and Ce ４＋ The drug of claim 1 , wherein the drug is a medicament having the coexistence of:

5. The agent of claim 4, which is administered to a subject to reduce expression of heme oxygenase 1 (HO-1) in the subject's bladder.

6. The method of claim 1 , wherein the method is administered to a subject to reduce inflammation in the subject.

7. The drug of claim 5 , which is administered to a subject to improve bladder edema and bleeding in the subject.

8. The method of claim 1 , wherein the method is administered to a subject to relieve pain in the subject.

9. The method of claim 1, wherein the method is administered to a subject to reduce urination frequency in the subject.

10. The method of claim 1, wherein the cerium oxide nanoparticles have a particle size of about 10 nm to about 35 nm.

11. 2. The method of claim 1, wherein the cerium oxide nanoparticles have an average particle size of about 22 nm to about 25 nm.

12. 2. The method of claim 1, wherein the therapeutically effective amount of the cerium oxide nanoparticles is about 3 mg / kg to about 100 mg / kg.

13. 10. The method of claim 1, wherein the method is administered to the subject 1 to 4 times per week, 1 to 4 times per month, or 1 to 4 times per year.

14. The method of claim 1, wherein the method is administered to the subject once every 2 to 4 weeks.

15. The method of claim 1 , wherein the method is administered to the subject orally, intraperitoneally, intravenously, intradermally, intramuscularly, subcutaneously, or transdermally.

16. The method of claim 1, wherein the subject is a mammal.

17. The method of claim 1 , wherein the subject is a human.

18. A pharmaceutical composition for preventing or treating urological chronic pelvic pain syndrome (UCPPS) in a subject in need thereof, comprising an effective amount of cerium oxide nanoparticles (CeNPs) and a pharmaceutically acceptable carrier thereof.

19. Use of a pharmaceutical composition in the manufacture of a medicament for preventing or treating urological chronic pelvic pain syndrome (UCPPS), wherein the pharmaceutical composition comprises an effective amount of cerium oxide nanoparticles (CeNPs) and a pharmaceutically acceptable carrier thereof.

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