Application of cyclic dipeptide in preparation of medicine for treating diseases related to damage of calcium-sensitive receptor

Abstract

The invention discloses an application of cyclic dipeptide in preparation of drugs for treating diseases related to damage of calcium-sensitive receptors, the dipeptide can directly activate CaSR activity of damaged organ cells in a targeted manner, and can enter the cells to promote expression of CaSR upstream genes, so that the related diseases caused by damage of CaSR are effectively reversed. Animal experiments prove that Cyclo (L-Pro-L-Trp) can effectively improve the symptoms of hypocalcemia, intestinal tract calcium absorption damage, parathyroid hormone excessive secretion, osteoporosis and the like caused by CaSR damage, and non-target organ toxicity is controllable (liver and kidney function indexes are not obviously changed). Cell experiments prove that Cyclo (L-Pro-L-Trp) can effectively activate CaSR of parathyroid gland main cells and intestinal epithelial cells and various calcium ion internal flow channels and promote proliferation of the parathyroid gland main cells and the intestinal epithelial cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of cyclic (proline-tryptophan) dipeptide in the preparation of drugs for treating diseases related to impaired calcium-sensitive receptor (CaSR). Background Technology

[0002] Calcium-sensitive receptors (CaSRs) are G protein-coupled receptors widely distributed in the parathyroid glands, intestines, kidneys, and bone tissue. They play a central role in maintaining systemic calcium homeostasis and are involved in processes such as cell proliferation, differentiation, and hormone secretion. CaSRs sense extracellular calcium levels... 2+ Changes in CaSR concentration regulate parathyroid hormone (PTH) secretion, intestinal calcium absorption, and renal calcium reabsorption, serving as a core regulatory hub for calcium metabolism homeostasis. Studies have shown that abnormal CaSR function is closely related to various diseases. For example, in patients with chronic kidney disease (CKD) and secondary hyperparathyroidism (SHPT), renal failure leads to hypocalcemia. Long-term hypocalcemia triggers compensatory hyperplasia of the parathyroid glands, while the downregulation of CaSR expression creates a vicious cycle, resulting in excessive secretion of parathyroid hormone, further exacerbating calcium and phosphorus metabolism disorders and bone mineral loss. Defects in the CaSR signaling pathway lead to increased osteoclast activity, with bone resorption exceeding bone formation, resulting in a significant decrease in bone density and increased risk of fractures due to osteoporosis (OP). In CKD, impaired CaSR function promotes the transformation of vascular smooth muscle cells into osteoblast-like cells, accelerating vascular calcification and ectopic calcium deposition, increasing cardiovascular mortality.

[0003] Existing clinical treatments, such as oral calcium supplements, vitamin D analogs, and calcimimetic agents (e.g., cinacalcet), can partially alleviate symptoms, but they have significant limitations. Long-term use of calcium and vitamin D can easily lead to side effects such as hypercalcemia and vascular calcification, and they cannot repair CaSR function. While CaSR agonists can directly activate receptors, patients are prone to developing drug resistance, and their effect on improving intestinal calcium absorption is limited. In summary, developing novel drug therapies that target and repair CaSR function is a key breakthrough in solving the clinical challenges associated with calcium metabolism disorders. Summary of the Invention

[0004] To address the current lack of effective treatments for diseases related to impaired calcium receptors, this invention provides the application of a cyclic dipeptide in the preparation of drugs for treating these diseases. This dipeptide can directly target and activate the CaSR activity of damaged multi-organ cells, and simultaneously enter the cell to promote the expression of upstream genes of CaSR, thereby effectively reversing the related diseases caused by CaSR damage.

[0005] To achieve the above objectives, the present invention provides, in one aspect, the use of a cyclic dipeptide, and its pharmaceutically acceptable salt or acid in any of the following:

[0006] (1) Prepare drugs or health foods that improve diseases related to impaired calcium-sensitive receptors;

[0007] (2) Prepare drugs or health foods for treating diseases related to impaired calcium-sensitive receptors;

[0008] The cyclic dipeptide is a cyclic (proline-tryptophan) dipeptide with the structural formula Cyclo(L-Pro-L-Trp) and the molecular formula C. 16 H 17 N3O2, CAS number: 67889-75-2.

[0009] Specifically, the pharmaceutically acceptable salt is a salt formed by the combination of a cyclic dipeptide and a pharmaceutically acceptable acid. The pharmaceutically acceptable acid includes at least one of organic acids, inorganic acids, and amino acids, including but not limited to hydrochloric acid, sulfuric acid, citric acid, succinic acid, aspartic acid, lysine, etc.

[0010] Preferably, the drug comprises pharmaceutically acceptable excipients, and the dosage form is selected from tablets, capsules, oral liquids, sustained-release microspheres, or targeted delivery microcapsules.

[0011] Furthermore, the drug is secreted by Lactobacillus and extracted from its culture supernatant, and the pharmaceutically acceptable excipients include Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus murineis and / or supernatant culture medium.

[0012] Specifically, the calcium-sensitive receptor-related diseases include chronic renal failure, secondary hyperparathyroidism, malabsorption syndrome, and osteoporosis.

[0013] Specifically, the disease is characterized by decreased activity and / or expression of calcium-sensitive receptors (decreased expression levels of CaSR mRNA and protein), hypocalcemia (<2.1 mmol / L), elevated parathyroid hormone concentration (>65 pg / mL), parathyroid tissue hyperplasia (multiple small nodules on ultrasound of parathyroid glands, and persistent radionuclide concentration on radionuclide imaging), decreased intestinal calcium absorption (decreased intracellular calcium concentration in calcium mobilization experiments), and osteoporosis (femoral bone mineral density T-score <-2.5).

[0014] Specifically, the drug or health food repairs calcium-sensitive receptor activity and / or promotes calcium-sensitive receptor expression by regulating epithelial cells including, but not limited to, parathyroid chief cells, distal convoluted tubules and collecting ducts of the kidneys, intestinal epithelial cells, osteoclasts, osteoblasts and mammary epithelial cells.

[0015] The cyclic (pro-chromo) dipeptide exerts its effects through the following mechanisms: upregulating the expression of CaSR in cells, activating the activity of CaSR in the cell membrane, stimulating various calcium ion channels, such as transient receptor potential vanillin receptor channel protein family (TRPV), voltage-dependent calcium channel protein family (TPCN), voltage-dependent calcium ion channel α1A subunit (CACNA1A), sodium-calcium exchange protein (SLC8A1) channel, etc., promoting calcium ion influx; at the same time, it can regulate intestinal flora homeostasis, increase the abundance of probiotics, especially the abundance of species that can secrete this cyclic dipeptide, such as Lactobacillus johnsonii, Lactobacillus reuteri, and Lactobacillus murineis.

[0016] Preferably, the drug targets the disease characteristics of clinical patients, including an abnormally reduced abundance of bacterial species that secrete the cyclic dipeptide in the gut, such as Lactobacillus johnsonii, Lactobacillus reuteri, and Lactobacillus murineis.

[0017] Preferably, the drug has controllable toxicity to non-target organs, as evidenced by no statistically significant changes in liver function indicators (ALT, AST, creatinine, and blood urea nitrogen), and no damage to pathological features observed in H&E staining and Sirius red staining.

[0018] Through the above technical solution, the present invention achieves the following beneficial effects:

[0019] 1. This invention utilizes a cyclic (proline-tryptophan) dipeptide to target and repair the expression of cellular calcium saturation receptors (CaSRs), upregulate their activity, and synergistically promote extracellular calcium ion influx in conjunction with other calcium ion channels. Simultaneously, it can effectively regulate gut microbiota homeostasis, promote an increase in the abundance of microbiota capable of secreting endogenous cyclic (proline-tryptophan) dipeptides, and thereby regulate related disease symptoms through the gut-microbe-organ axis.

[0020] 2. Animal experiments have demonstrated that Cyclo(L-Pro-L-Trp) can effectively improve symptoms such as hypocalcemia, impaired intestinal calcium absorption, excessive parathyroid hormone secretion, and osteoporosis caused by CaSR damage, with controllable non-target organ toxicity (no significant changes in liver and kidney function indicators). Cellular experiments have confirmed that Cyclo(L-Pro-L-Trp) can effectively activate CaSR and various calcium ion influx channels in parathyroid chief cells and intestinal epithelial cells, and promote their proliferation.

[0021] 3. The cyclic (proline-tryptophan) dipeptide secreted by Lactobacillus disclosed in this invention is suitable for the prevention and treatment of multi-organ CaSR damage and its related complications, and has significant clinical translational potential. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0023] Figure 1 This is a standard curve test diagram of the cyclic (proline-tryptophan) dipeptide.

[0024] Figure 2 This is a graph showing the correspondence between the indicators in Table 2;

[0025] Figure 3 This is a diagram showing the expression and inhibition of calcium-sensitive receptors in in vitro cells;

[0026] Figure 4 This is a graph showing the correspondence between the indicators in Table 3;

[0027] Figure 5 These are H&E staining images of liver, kidney, and other tissues in mice after gavage administration of cyclic dipeptides. Detailed Implementation

[0028] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0029] Example 1: Preparation and Identification of Cyclic (Proline-Tryptophan) Dipeptide

[0030] Cyclic (proline-tryptophan) dipeptides were prepared by microbial fermentation: Lactobacillus johnsonii (LJ) strain was anaerobically cultured in MRS medium at 37°C for 48 hours, the supernatant was collected by centrifugation, and the crude extract was obtained by macroporous resin adsorption, ethanol gradient elution, and freeze drying, which was further purified by silica gel column chromatography.

[0031] The test data of the standard curve of the cyclic (proline-tryptophan) dipeptide are shown in Table 1.

[0032] Table 1. Test data for the standard curve of the cyclic (proline-tryptophan) dipeptide.

[0033]

[0034] From Table 1 and Figure 1 The LJ sample prepared by microbial fermentation showed a high degree of agreement with the curve of the commercial cyclic (proline-tryptophan) dipeptide standard (R2 = 0.9981), confirming that we successfully prepared and isolated a high-purity cyclic (proline-tryptophan) dipeptide (m / z = 284.1394).

[0035] Example 2: Therapeutic effect of cyclic (proline-tryptophan) dipeptide on a mouse model of calcium-sensitive receptor impairment.

[0036] The experimental animals used in this invention were SPF-grade C57BL / 6 mice (6-7 weeks old, male mice weighing 27±2g and female mice weighing 22±1g), with half males and half females, totaling 48 mice. They were randomly divided into a control group (CON), a model group (Sham), and a drug-treated group (Pro-Trp), with 16 mice in each group. The calcium-sensitive receptor damage model mouse was induced in vivo by 5 / 6 nephrectomy. Four weeks after surgery, long-term renal function damage led to calcium and phosphorus metabolism disorders and toxin accumulation, gradually destroying the activity of calcium-sensitive receptors in cells. Drug administration was started by gavage four weeks after surgery and continued for eight weeks.

[0037] Control group (CON): No surgery was performed; only normal saline was administered via gavage.

[0038] Model group (Sham): 5 / 6 nephrectomy was performed for induction, and normal saline was administered by gavage only.

[0039] The treatment group (Pro-Trp) underwent 5 / 6 nephrectomy induction and was administered cyclic (proline-tryptophan) dipeptide (30 mg / kg) by gavage. The cyclic (proline-tryptophan) dipeptide was dissolved in a 0.5% (w / w) sodium carboxymethyl cellulose (CMC-Na) solution and ultrasonically dispersed to form a homogeneous suspension, at which point the concentration of the cyclic (proline-tryptophan) dipeptide was 1 mg / mL.

[0040] Table 2. Therapeutic effects of cyclic (proline-tryptophan) dipeptides

[0041]

[0042]

[0043] From Table 2 and Figure 2 It was found that after 5 / 6 nephrectomy induction treatment, the model group, compared with the control group, exhibited typical pathological features of calcium-sensitive receptor damage: significantly reduced fluorescence expression intensity of cellular calcium-sensitive receptors, severe renal function impairment, and an imbalance in the serum calcium-to-phosphorus ratio; significant hyperplasia of parathyroid tissue and excessively high parathyroid hormone concentration, exhibiting symptoms of secondary hyperparathyroidism; significant damage to the intestinal barrier, weakened intestinal calcium absorption, reduced abundance of Lactobacillus flora in the intestine, and dysbiosis; and severe osteoporosis with significant bone loss. The cyclic (proline-tryptophan) dipeptide administration group significantly reversed the various symptoms caused by the above-mentioned calcium-sensitive receptor damage, alleviated renal function impairment, significantly inhibited secondary hyperparathyroidism, restored intestinal barrier function, promoted intestinal calcium absorption, maintained intestinal flora homeostasis, increased bone trabecular density, and alleviated osteoporosis.

[0044] Example 3: Activation effect of cyclic (proline-tryptophan) dipeptide on CaSR in intestinal epithelial cells

[0045] Mouse intestinal epithelial cells (MODEK) were divided into a model group (Sham, normal culture medium with CaSR siRNA interference) and an experimental group (Pro-Trp, normal culture medium with 10 μM Cyclo(L-Pro-L-Trp)). After culturing for 1 hour, the calcium influx intensity of the cells was detected using the Fluo-4 AM fluorescent probe.

[0046] Yuwa Figure 3 The results showed that, compared with the model group, the mRNA expression levels of CaSR and various calcium ion-related channels TRPV4, TPCN1, CACNA1A, and SLC8A1 in the experimental group cells were significantly increased, and the calcium influx intensity was 2 times higher than that in the model group (P < 0.01).

[0047] Further addition of the TRPV4 inhibitor (HC-067047) blocked the calcium influx effect, indicating that Cyclo (L-Pro-L-Trp) can promote calcium influx by synergistically activating the CaSR / TRPV4 channel. 2+ internal flow.

[0048] Example 4: Design of cyclic (proline-tryptophan) dipeptide administration and efficacy verification

[0049] Tablet formulation: Cyclo (L-Pro-L-Trp) 100mg, microcrystalline cellulose 200mg, lactose 50mg, magnesium stearate 5mg. After mixing, directly compress into tablets, each weighing 355mg. Take one tablet once daily.

[0050] Targeted microcapsules and their formulation: Cyclo (L-Pro-L-Trp) 50mg is encapsulated in pH-sensitive chitosan-sodium alginate microspheres (particle size 200nm). After oral administration, it is targeted and released into the intestines, enhancing the local CaSR activation effect. Take one capsule once daily.

[0051] Probiotic Complex Capsules and their Formula: A mixture of freeze-dried Lactobacillus johnsonii (5×10⁸ CFU / capsule) and Cyclo (L-Pro-L-Trp) 50mg. Synergistically regulates intestinal flora and promotes the secretion of endogenous cyclic dipeptides. Take one capsule once daily.

[0052] Table 3. Therapeutic effects of different administration methods of cyclic (proline-tryptophan) dipeptides

[0053]

[0054]

[0055] From Table 3 and Figure 4 Observations showed that after treatment with cyclic (proline-tryptophan) dipeptide using different administration methods, the calcium-sensitive receptor impairment characteristics were significantly improved in all three groups: oral tablets, targeted microcapsules, and probiotic complex capsules. This indicates that cyclic (proline-tryptophan) dipeptide can exert similar effects under different administration methods. These results effectively confirm that cyclic (proline-tryptophan) dipeptide can be customized according to clinical patient needs, thereby improving patient compliance and treatment efficacy.

[0056] Example 5: Evaluation of non-target organ toxicity of cyclic (proline-tryptophan) dipeptide

[0057] Experimental methods: Forty SD mice (half male and half female) were randomly divided into a control group (CON, administered via gavage with physiological saline) and an experimental group (Pro-Trp, administered via gavage with 25 mg / kg Cyclo (L-Pro-L-Trp)). The mice were observed for 14 days, and body weight, organ indices (heart, liver, spleen, lung, and kidney), and pathological sections (H&E staining) were recorded.

[0058] Table 4. Detection factor expression levels in mesenteric fibroblasts of different groups

[0059]

[0060] As shown in Table 4, there were no deaths in either the experimental or control groups, with a survival rate of 100%. LD50 50 >6000mg / kg, classified as non-toxic. There was no significant difference in body weight change between the experimental and control groups (P>0.05). Meanwhile, Figure 5 H&E staining showed no inflammatory infiltration or necrosis in any organ, meaning that the liver, kidney, and heart indices were all normal.

[0061] Conclusion: Cyclo (L-Pro-L-Trp) can safely and effectively repair the function of CaSR in multiple organs and is suitable for the prevention and treatment of diseases such as chronic renal failure, secondary hyperparathyroidism, intestinal calcium absorption dysfunction, and osteoporosis.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. The use of a cyclic dipeptide, its pharmaceutically acceptable salt or acid, in any of the following: (1) Prepare drugs or health foods that improve diseases related to impaired calcium-sensitive receptors; (2) Prepare drugs or health foods for treating diseases related to impaired calcium-sensitive receptors; in, The cyclic dipeptide is a cyclic (proline-tryptophan) dipeptide with the structural formula Cyclo(L-Pro-L-Trp) and the molecular formula C. 16 H 17 N3O2, CAS number: 67889-75-2.

2. The application according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the combination of a cyclic dipeptide and a pharmaceutically acceptable acid, wherein the pharmaceutically acceptable acid includes at least one of organic acids, inorganic acids, and amino acids.

3. The application according to claim 2, characterized in that, Pharmaceutically acceptable acids include hydrochloric acid, sulfuric acid, citric acid, succinic acid, aspartic acid, and lysine.

4. The application according to claim 1, characterized in that, The drug contains pharmaceutically acceptable excipients and is available in a dosage form selected from tablets, capsules, oral liquids, sustained-release microspheres, or targeted delivery microcapsules.

5. The application according to claim 4, characterized in that, The drug is secreted by Lactobacillus and extracted from its culture supernatant, and the pharmaceutically acceptable excipients include Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus murineis and / or supernatant culture medium.

6. The application according to claim 1, characterized in that, The diseases associated with impaired calcium-sensitive receptors include chronic renal failure, secondary hyperparathyroidism, malabsorption syndrome, and osteoporosis.

7. The application according to claim 6, characterized in that, The disease is characterized by decreased activity and / or expression of calcium-sensitive receptors, hypocalcemia, elevated parathyroid hormone levels, parathyroid tissue hyperplasia, decreased intestinal calcium absorption, and osteoporosis.

8. The application according to claim 1, characterized in that, The drug or health food product repairs calcium-sensitive receptor activity and / or promotes calcium-sensitive receptor expression by regulating epithelial cells including parathyroid chief cells, distal convoluted tubules and collecting ducts of the kidneys, intestinal epithelial cells, osteoclasts, osteoblasts and mammary epithelial cells.

9. The application according to any one of claims 1 to 8, characterized in that, The cyclic dipeptide can upregulate the expression of CaSR in cells, activate the activity of CaSR in the cell membrane, stimulate multiple calcium ion channels, and promote calcium ion influx; at the same time, it can regulate the homeostasis of intestinal flora and increase the abundance of probiotics.

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