Application of berberine hydrochloride in preparation of medicine for treating or improving interstitial cystitis

Abstract

The invention belongs to the field of medicines, and particularly relates to application of berberine hydrochloride in preparation of medicines for treating or improving interstitial cystitis. The invention shows that berberine hydrochloride not only shows a better treatment effect on interstitial cystitis, but also obviously improves inflammation and pain response of interstitial cystitis of a patient, and berberine hydrochloride creates a microenvironment more tending to anti-inflammation and tissue repair locally, so that bladder pain and inflammatory injury are relieved, and the effect of treating the interstitial cystitis is achieved. And the berberine hydrochloride has no side effect, so that the berberine hydrochloride can be used for treating or improving interstitial cystitis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of berberine hydrochloride in the preparation of a drug for treating or improving interstitial cystitis. Background Art

[0002] Interstitial Cystitis (IC) is a clinically difficult-to-treat, easily relapsing urinary system disease with an incidence rate as high as 6.5%. The number of female patients is about five times that of male patients. The main clinical manifestations are progressively aggravated frequency of urination, urgency of urination, and pain or discomfort in the pelvic region, which seriously affect the quality of life of patients. Although there is still no complete unified understanding of the pathophysiological mechanism of IC, it is currently considered to be related to multiple factors, mainly including: damage to the glycosaminoglycan (GAG) layer, urothelial damage, occult infection, abnormal neuromodulation, and autoimmune reaction, etc. Once the GAG layer on the surface of the bladder mucosa is damaged, tissue stimulators such as potassium ions and amines in urine are easily invaded into the bladder wall, inducing an inflammatory reaction; at the same time, a variety of immune cells aggregate locally and further cause deep inflammation and pain reactions in the bladder wall by releasing a variety of cytokines and neurotransmitters (ATP, acetylcholine, prostaglandin, nitric oxide, etc.). The cascade reaction of inflammatory factors is the pathological basis for the intractability and recurrence of pain in interstitial cystitis.

[0003] The currently adopted comprehensive treatment mode includes behavioral / non-pharmacological measures, oral medications (such as amitriptyline, pentosan polysulfate, hydroxyzine, etc.), bladder perfusion, minimally invasive surgery, and major surgery, etc. Among them, intravesical perfusion such as lidocaine combined with sodium bicarbonate, hyaluronic acid combined with chondroitin sulfate, etc. is an important route of local drug delivery, but the clinical treatment effect is uncertain and cannot meet the clinical needs.

[0004] Berberine hydrochloride is an isoquinoline alkaloid, which was initially used to treat diarrhea, inflammatory bowel disease, diabetes, atherosclerosis, etc. Due to its multiple pharmacological characteristics such as anti-inflammatory, antibacterial, and promoting tissue repair, it has been gradually applied to inflammation-related diseases in recent years. For example, berberine has applications in the treatment of acute cystitis and cystitis. However, since acute cystitis and cystitis are mostly caused by bacterial infection, which is quite different from the pathological factors of interstitial cystitis, there is currently no report on the application of berberine hydrochloride in the treatment of interstitial cystitis.

[0005] Studies have confirmed that during the pathological process of IC, the number and functional status of macrophages have a decisive impact on the progression of the inflammatory response. Macrophages in the M1 type can release various pro-inflammatory cytokines such as tumor necrosis factor TNF-α, IL-1β, and IL-6, further recruiting immune cells and exacerbating the destruction of the mucosal barrier. Type II (M2 type) macrophages can secrete cytokines such as IL-10 and TGF-β that have immunosuppressive and tissue repair effects, playing a balanced regulatory role between maintaining tissue homeostasis and the body's defense ability. Therefore, promoting the transformation of macrophages from the M1 type to the M2 type, or simultaneously inhibiting the over-activation of the M1 type, may be a potential effective way to relieve chronic inflammation. Summary of the Invention

[0006] To solve the above problems, the present invention provides the use of berberine hydrochloride in the preparation of a drug for treating or improving interstitial cystitis.

[0007] The above object is achieved through the following technical solutions:

[0008] The present invention provides the use of berberine hydrochloride in the preparation of a drug for treating or improving interstitial cystitis.

[0009] As a further optimized scheme of the present invention, the drug contains berberine hydrochloride and a pharmaceutically acceptable carrier.

[0010] As a further optimized scheme of the present invention, the drug dosage form is one or more of tablets, capsules, lozenges, injections, suspensions, suppositories, ointments, bladder instillations, oral preparations, suppositories, or sustained-release preparations.

[0011] As a further optimized scheme of the present invention, the dosage of berberine hydrochloride is 5-10 g / 100 ml per single dose, and bladder instillation is performed once every 3 weeks.

[0012] As a further optimized scheme of the present invention, the carrier is one or more of liposomes, nanoparticles, hydrogels, or microspheres.

[0013] The present invention also provides the use of the combination of berberine hydrochloride and a temperature-sensitive hydrogel in the preparation of a drug for treating or improving interstitial cystitis.

[0014] As a further optimized scheme of the present invention, the temperature-sensitive hydrogel is a PLGA-PEG-PLGA triblock copolymer.

[0015] The present invention also provides a composition for treating or improving interstitial cystitis, characterized in that the composition includes berberine hydrochloride and a PLGA-PEG-PLGA triblock copolymer.

[0016] As a further optimized solution of the present invention, the preparation method of the composition is to mix berberine hydrochloride with an aqueous solution of PLGA-PEG-PLGA triblock copolymer to obtain a gel-state composition.

[0017] As a further optimized solution of the present invention, the preparation method of the PLGA-PEG-PLGA triblock copolymer is as follows: Using PEG as an initiator and SN(OCT)2 as a catalyst, under nitrogen protection, ring-opening polymerization reaction of LA and GA is carried out, and the reaction product is obtained after freeze-drying.

[0018] The beneficial effects of the present invention are as follows: Berberine hydrochloride, as the main active ingredient of the traditional Chinese medicine berberine, has definite anti-inflammatory, bactericidal and astringent effects. The present invention for the first time discovers that berberine hydrochloride shows good therapeutic effects on interstitial cystitis, and significantly improves the inflammation and pain response of patients with interstitial cystitis. Berberine hydrochloride promotes the transformation of macrophages from M1 type to M2 type during the treatment of IC. By mobilizing the repair potential of macrophages, berberine hydrochloride creates a microenvironment more conducive to anti-inflammation and tissue repair locally, thereby alleviating bladder pain and inflammatory damage, and no side effects are seen. Therefore, berberine hydrochloride can be used as a drug for treating or improving interstitial cystitis.

[0019] The combination of PLGA-PEG-PLGA triblock copolymer and berberine hydrochloride forms a hydrogel, which provides ideal characteristics for the application in the berberine hydrochloride delivery system, especially in cases where controlled release or stimulus response is required, further enhancing the therapeutic and pain-improving effects of berberine hydrochloride. Description of the Drawings

[0020] Figure 1 It is a sectional view of the construction of the rat interstitial cystitis model in Example 1;

[0021] Figure 2 It is a comparison chart of the effects of each treatment group on the pain threshold of interstitial cystitis in Example 1;

[0022] Figure 3 It is a display chart of the maximum isobaric bladder capacity of rats in each group of Example 1;

[0023] Figure 4 It is a display chart of the isovolumetric bladder pressure of rats in each group of Example 1;

[0024] Figure 5 It is a comparison chart of the effects of reducing rat inflammatory factors (20×, 40×) in the model group and the high-dose berberine hydrochloride group in Example 1;

[0025] Figure 6 It is a comparison chart of toluidine blue staining of each treatment group in Example 1;

[0026] Figure 7 To implement Example 1 for detecting the effect of berberine hydrochloride on the tissue microenvironment by spatial transcriptomics;

[0027] Figure 8 It is an analysis diagram of the spatial transcriptome sequencing results by Cellchat;

[0028] Figure 9 It is a diagram of the mechanism of action of berberine hydrochloride against interstitial cystitis;

[0029] Figure 10 It is the appearance change of PLGA and Berberine Hydrochloride-PLGA hydrogels at different temperatures in Example 2;

[0030] Figure 11 It is the rheological properties of PLGA and Berberine Hydrochloride-PLGA hydrogels in Example 2. Specific Embodiments

[0031] The following further describes the present application in conjunction with the accompanying drawings. It is necessary to point out here that the following specific embodiments are only used for further illustration of the present application and cannot be construed as limiting the protection scope of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0032] The preparation methods used in this embodiment are all conventional methods known to those skilled in the art unless otherwise specified. Materials or reagents are all commercially available products unless otherwise specified.

[0033] Example 1: Therapeutic effect of berberine hydrochloride on an animal model of interstitial cystitis

[0034] 1. Materials

[0035] SD rats, female, weighing 100-180 g, were purchased from [Guangdong Provincial Medical Experimental Animal Center]. Berberine hydrochloride (purity ≥ 98%) was purchased from [Berberine Hydrochloride 633-65-8 B802465-100g Brand: Macklin China - Guidechem.com].

[0036] By intraperitoneal injection of CYP 40 mg / kg + bladder perfusion of LPS 750 μg, protamine sulfate 10 mg, clamping the urinary catheter, retaining for 30 min, once every other day, for 2 weeks. An animal model of interstitial cystitis in rats (hereinafter referred to as the model group) was established. The model construction method is shown in Table 1.

[0037] Table 1 Rat interstitial cystitis model construction method

[0038]

[0039]

[0040] After construction, the bladder tissue was taken for HE staining, and the results are shown in Figure 1 , in group A, the texture of the bladder mucosa and the muscular layer of the bladder wall was normal; in group B, a large number of microvessels infiltrated the bladder mucosa and muscular layer; in group C, a large number of immune cells infiltrated the tissue microenvironment. Pathological examination of rat bladder tissue showed mucosal edema, hemorrhage, ulcer formation and inflammatory cell infiltration, and the number of degranulated mast cells in the bladder tissue of the model group increased significantly, which is one of the typical pathological features of IC.

[0041] 2. Methods

[0042] SD rats (female, 180 - 200 g) were randomly divided into: blank control group (without any treatment, n = 10), model group (only establishing the interstitial cystitis model, without drug treatment, n = 10), positive control group (sodium hyaluronate, 1 mg / kg, n = 10), low-dose berberine hydrochloride group (5 g / 100 ml, n = 10), high-dose berberine hydrochloride group (10 g / 100 ml, n = 10).

[0043] Rats in each treatment group were continuously administered by intravesical perfusion for 6 weeks at the corresponding doses, once every 3 weeks. Bladder perfusion fluid: Berberine hydrochloride was dissolved in an aqueous solution of polymer or physiological saline, and uniformly dispersed by stirring or ultrasonic treatment.

[0044] During the experiment, the general state of the rats was observed and recorded, and their diet, activity and mental state were all normal.

[0045] 3. Detection indexes

[0046] (1) Pain and functional detection

[0047] The pain threshold was measured by the Von Filament method. The plantar surface of the hind limb of rats in each treatment group was stimulated, and the paw withdrawal threshold (g) was recorded. The average value was taken after repeating 3 times.

[0048] Taking the pain thresholds of the model group and the blank control group as the standardized thresholds, the standardized threshold of the model group was 0, and the standardized threshold of the control group was 1. The results are as Figure 2 shown. The threshold of the berberine hydrochloride group was significantly higher than that of the sodium hyaluronate group (p = 0.015). It shows that berberine hydrochloride has a significant therapeutic effect on the control of pain in interstitial cystitis, and the effect is better than that of sodium hyaluronate.

[0049] (2) Urodynamic detection (LABORIE system)

[0050] The bladder of rats in each group was perfused at the same pressure of 25 cmH2O, and the bladder capacity under the same pressure was measured. The results are as Figure 3 shown.

[0051] With the same bladder irrigation volume (1 ml), the changes in the intravesical pressure of rats in each group were measured, and the results are as Figure 4 shown.

[0052] (3) Berberine hydrochloride reduces the secretion of inflammatory cytokines

[0053] After 2 weeks, the rats were sacrificed, and bladder tissues were taken for pathological section examination to detect the expression of macrophage-related inflammatory factors (IL-10, IL-1β, IL-4) and macrophage-related anti-inflammatory factors (TNF-α, IL-6, IL-12) in the tissue microenvironment of the model group and the high-dose berberine hydrochloride group. As Figure 5 shown, in the high-dose group (shown as berberine hydrochloride), the expression of pro-inflammatory and anti-inflammatory factors in the microenvironment of the bladder mucosa tissue was higher than that in the model group, and the expression of anti-inflammatory factors was lower. As Figure 6 shown, the number of degranulated mast cells was significant in the model group, and the number of mast cells in the low-dose group and the high-dose group was significantly lower than that in the positive control group.

[0054] 4. Conclusion

[0055] In the model group, the pelvic pain threshold decreased. After 6 bladder irrigations in the positive control group, the pain relief standard was reached, and the pain relief rate reached 67%. After 4 bladder irrigations in the berberine hydrochloride group, the pain threshold of rats increased significantly, and the pain relief rate reached 80%. Berberine hydrochloride has a better effect on relieving the pain of interstitial cystitis than the existing drugs.

[0056] Compared with the model group and the positive control group, the infiltration of inflammatory cells in the bladder tissues of rats in the berberine hydrochloride group was significantly reduced, and the degree of mucosal damage decreased. The expression levels of inflammatory factors IL-6 and TNF-α decreased significantly. The urine volume of rats in the berberine hydrochloride group increased, indicating that berberine hydrochloride can effectively improve the micturition function of rats with interstitial cystitis.

[0057] 5. The mechanism of action of berberine hydrochloride on interstitial cystitis

[0058] Omics analysis of the bladder tissue of IC model rats was performed using single-cell sequencing. The composition of macrophage subsets changed significantly between groups: in the IC model group, the proportion of M1 macrophages was relatively high, expressing high levels of pro-inflammatory genes (such as TNF-α, IL-1β, IL-6, etc.), secreting high levels of cytotoxic molecules such as nitric oxide, and further aggravating local inflammation; while the proportion and activity of M2 macrophages (CD163 and IL-10) were relatively low. On the contrary, in the berberine hydrochloride treatment group, the expression of M1 markers decreased, and the M2 markers were significantly up-regulated, indicating that the macrophage population shifted towards type II polarization. This shift contributed to tissue repair, secretion of anti-inflammatory factors, and restoration of local immune homeostasis. Thus, while inhibiting the over-activation of M1 type, berberine hydrochloride tended to maintain or promote the function of M2 macrophages, creating a microenvironment conducive to inflammation resolution and tissue repair.

[0059] As Figure 7 shown, the expressions of CD86 and TLR4 were relatively high in the model group, while the expressions of CD206 and CD163 were relatively high in the berberine hydrochloride group. Thus, it can be seen that in the microenvironment of the bladder mucosal tissue in the model group, macrophages mainly had a pro-inflammatory function, and conversely, in the berberine hydrochloride group, the function of macrophages was mainly anti-inflammatory.

[0060] In-depth analysis of spatial transcriptome sequencing and results of cell-to-cell communication clearly revealed that berberine hydrochloride promoted the transformation of macrophages from M1 type to type II during the treatment of IC. By mobilizing the repair potential of macrophages, berberine hydrochloride created a microenvironment more conducive to anti-inflammation and tissue repair locally, thus alleviating bladder pain and inflammatory damage.

[0061] To further explore the mechanism by which berberine hydrochloride promotes macrophage type II polarization, based on the analysis of cell-to-cell communication results from spatial transcriptome sequencing data, as Figure 8 shown, the epithelial-macrophage communication in the model group was significantly stronger than that in the control group, and the HMGB1-TLR4 signal was particularly prominent. In the berberine hydrochloride group, the intensity of this signal communication was significantly inhibited.

[0062] The expressions of both HMGB1 and TLR4 increased in the bladder tissue of rats in the model group. Moreover, the surface molecule CD68 of type I macrophages and the expressions of MAPK and NF-κB signaling pathways were significantly up-regulated. The opposite results were seen in the berberine hydrochloride group, where the signal pathway mediated by HMGB1-TLR4 was significantly inhibited. Therefore, the inflammatory cascade reaction amplified by macrophage type I polarization mediated by epithelial-derived HMGB1 is an important pathological process affecting the microenvironment of IC tissue. As Figure 9As shown in the figure, on the one hand, berberine hydrochloride can inhibit the HMGB1 exosomes secreted by epithelial cells, reduce their over-activation in the TLR4 / MyD88 signaling pathway of macrophages, thereby promoting M2 polarization and alleviating chronic inflammation and pain in IC. On the other hand, berberine hydrochloride promotes M2 polarization of macrophages in the tissue microenvironment by inhibiting the activation of the HMGB1-TLR4 signaling pathway derived from bladder mucosal epithelial cells, thereby alleviating the inflammatory response and clinical symptoms of IC.

[0063] In summary, berberine hydrochloride can be used as a drug for treating or improving interstitial cystitis. The drug contains berberine hydrochloride and a pharmaceutically acceptable carrier. The drug dosage form is any one of tablets, capsules, lozenges, injections, suspensions, suppositories, ointments, bladder irrigation agents, oral preparations, suppositories or sustained-release preparations. The carrier can be any one of liposomes, nanoparticles, hydrogels or microspheres.

[0064] Example 2: Temperature sensitivity of the combination of thermosensitive hydrogel and berberine hydrochloride under different temperature conditions

[0065] 1. Materials

[0066] Thermosensitive hydrogel: PLGA-PEG-PLGA triblock copolymer. Prepared by ring-opening polymerization (ROP) of lactic acid LA and glycolide GA, with polyethylene glycol PEG as the initiator and stannous octoate SN(OCT)2 as the catalyst under nitrogen protection. Specifically:

[0067] The three-necked flask was filled with nitrogen for degassing, 5.02 g of PEG1500 was added, and it was stirred at 150 °C for 2 hours. Then, GA (1.05 g, 9 mmol) and LA (9.0 g, 62.5 mmol) were added to the three-necked flask, and the mixture was kept at 150 °C under nitrogen for 0.5 h. Next, after adding 30 μl of SN(OCT)2, the above mixture and reaction were carried out at 150 °C under nitrogen protection for 10 hours. The resulting solution was immediately poured into deionized water at 4 °C and stirred overnight. Then, the obtained product was heated to 80 °C in a water bath 3 times to remove unreacted monomers and water-soluble low molecular weight by-products. Finally, the PLGA-PEG-PLGA copolymer was obtained after freeze-drying for 24 hours. Among them, the weight ratio of (LA + GA) / PEG is 2:1, and the molar ratio of LA / GA is 7:1.

[0068] 2. Methods

[0069] Take out the freeze-dried powder of berberine hydrochloride from the refrigerator, let it stand at room temperature for a while to return to room temperature, drop in a little deionized water to dissolve it, and achieve uniform dispersion by stirring (magnetic stirring at 1000 rpm, 4 °C). Filter through a filter to ensure the purity of the perfusion solution and avoid clogging the perfusion catheter.

[0070] Slowly mix berberine hydrochloride (10 g) with 100 ml of an aqueous solution of PLGA-PEG-PLGA triblock copolymer (MW 30 kDa, abbreviated as PLGA). Gently shake the bottle to ensure thorough mixing. The final product is a pale yellow gel state, as Figure 10 shown. The prepared perfusion solution should be used immediately and not stored for a long time to avoid affecting the drug activity.

[0071] As Figure 10 shown, the appearance changes of PLGA and Berberine Hydrochloride-PLGA hydrogels at different temperatures confirm the mutual conversion from liquefaction to gelation of these two substances when the temperature changes. Figure 10 A and Figure 10 B respectively show the states of these two solutions at 25 °C and 37 °C. In Figure 10 A, the PLGA solution appears transparent and homogeneous at 25 °C, while the Berberine Hydrochloride-PLGA hydrogel presents a yellow solution, confirming the presence of the Berberine Hydrochloride component. Interaction with PLGA at room temperature may affect its gel state transformation. As the temperature rises to 37 °C, in Figure 10 B, we observe that the PLGA solution gels and remains transparent, while the Berberine Hydrochloride-PLGA solution gels, confirming that the addition of Berberine Hydrochloride does not significantly change the structure of PLGA, and both can gel at around 30 °C - 37 °C.

[0072] As Figure 11 shown, the rheological properties of PLGA and Berberine Hydrochloride-PLGA hydrogels change at different temperatures. Figure 11 A and Figure 11Figure C shows the changes in storage modulus (G') and loss modulus (G") of PLGA and Berberine Hydrochloride-PLGA complexes as the temperature increases. Both PLGA and Berberine Hydrochloride-PLGA hydrogels show significant changes in the temperature range of approximately 30 °C to 40 °C, exhibiting a similar sol-gel transition phenomenon. The sharp increase in G' and G" indicates that the change in temperature causes a phase behavior transition of the material, which may be caused by the interaction and structural change of polymer chains. This transition of PLGA occurs at around 37 °C, while in the Berberine Hydrochloride-PLGA hydrogel, although the transition temperature is slightly shifted and decreased to approximately 28 °C, the change trend is similar, indicating that the interaction between Berberine Hydrochloride and PLGA affects the thermal response characteristics of the hydrogel and also confirms the successful loading of Berberine Hydrochloride onto PLGA. In addition, Figure 11 Band Figure 11 Figure D shows the changes in viscosity (η) of these two solutions. As the temperature increases, the viscosities of PLGA and Berberine Hydrochloride-PLGA change significantly near 37 °C and 34 °C, respectively. The change in viscosity in the Berberine Hydrochloride-PLGA hydrogel is more significant, indicating that the fluidity of this complex has a stronger response to temperature changes.

[0073] 3. Conclusions

[0074] This similar sol-gel transition behavior shows the temperature sensitivity of PLGA and Berberine Hydrochloride-PLGA under different temperature conditions, which may provide ideal characteristics for their application in drug delivery systems, especially in cases where controlled release or stimulus response is required. Combining with Example 1, Berberine Hydrochloride-PLGA can further enhance the therapeutic and pain-improving effects of berberine hydrochloride.

[0075] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, several improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

Claims

1. Use of berberine hydrochloride in the preparation of a drug for treating or improving interstitial cystitis.

2. The application according to claim 1, wherein The drug contains berberine hydrochloride and a pharmaceutically acceptable carrier.

3. The application according to claim 2, wherein The dosage form of the drug is one or more of tablets, capsules, lozenges, injections, suspensions, suppositories, ointments, bladder instillation agents, oral preparations, suppositories or sustained-release preparations.

4. The application according to claim 1, characterized in that, The dosage of berberine hydrochloride is 5 - 10 g / 100 ml per single dose, and bladder instillation is performed once every 3 weeks.

5. The application according to claim 2, wherein The carrier is one or more of liposomes, nanoparticles, hydrogels or microspheres.

6. Use of the combination of berberine hydrochloride and a temperature-sensitive hydrogel in the preparation of a drug for treating or improving interstitial cystitis.

7. The application according to claim 6, characterized in that, The temperature-sensitive hydrogel is a PLGA-PEG-PLGA triblock copolymer.

8. A composition for treating or improving interstitial cystitis, characterized in that, The composition includes berberine hydrochloride and a PLGA-PEG-PLGA triblock copolymer.

9. The composition according to claim 8, wherein The preparation method of the composition is to mix an aqueous solution of berberine hydrochloride with a PLGA-PEG-PLGA triblock copolymer to obtain a gel-state composition.

10. The composition according to claim 9, wherein The preparation method of the PLGA-PEG-PLGA triblock copolymer is as follows: Using PEG as an initiator and SN(OCT)2 as a catalyst, under nitrogen protection, ring-opening polymerization reaction of LA and GA is carried out, and the reaction product is obtained after freeze-drying.

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