A method for modifying human calcitonin, modified human calcitonin and use thereof
By modifying human calcitonin with O-acetylglucosamine glycosylation on threonine, serine, or tyrosine residues, the problem of self-aggregation of human calcitonin was solved, and the structural stability and bioactivity were improved over a long period of time. This makes it suitable for preparing more effective therapeutic agents for osteoporosis and other bone resorption-related diseases.
Patent Information
- Application Number
- CN202511046229.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2026-05-15
- Estimated Expiration
- 2045-07-29
AI Technical Summary
In the prior art, human calcitonin is prone to self-aggregation in solution, forming fibrous aggregates with a β-sheet structure, which leads to reduced biological activity, shortened half-life in vivo, and may trigger immunogenic reactions, thus limiting its formulation development and clinical application.
Human calcitonin is formed by O-acetylglucosamine glycosylation modification of threonine, serine or tyrosine residues, preferably on the threonine residue at position 21.
The modified human calcitonin maintained its monomeric state for 72 hours and retained its active monomeric structure for up to 96 hours, significantly improving structural stability and biological activity, which is superior to existing nitration modification methods.
Smart Images
Figure CN120943928B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedicine, specifically relating to a method for modifying human calcitonin, the modified human calcitonin and its application. Background Technology
[0002] Human calcitonin (hCT) is a polypeptide hormone secreted by thyroid C cells. It lowers serum calcium levels by specifically binding to calcitonin receptors, inhibiting osteoclast activity and reducing bone resorption. It is an important drug for the clinical treatment of osteoporosis, hypercalcemia, and bone metastases, among other bone resorption-related diseases. However, natural hCT readily aggregates in solution, forming fibrous aggregates with a β-sheet structure. This reduces its biological activity, shortens its in vivo half-life, and may trigger immunogenic reactions, severely limiting its formulation development and clinical application.
[0003] To address the aggregation problem of hCT, various chemical modification or sequence alteration strategies have been explored in existing technologies. For example, Chinese patent CN110922469A discloses a method for inhibiting aggregation by nitrifying the tyrosine residue at position 12 of human calcitonin (i.e., hCT(3N)), which can maintain the monomeric state of hCT for approximately 24 hours. Although such modifications can delay aggregation to some extent, they still have significant limitations: the inhibitory effect is short-lived, and aggregated particles will still form after long-term incubation (e.g., more than 24 hours); the stability is insufficient, failing to meet the requirements for long-term transportation and administration, and clinical translation still faces significant obstacles.
[0004] Therefore, developing a modification strategy that can more efficiently and effectively inhibit hCT aggregation while maintaining structural stability and bioactivity is of great significance for improving the stability, therapeutic efficacy, and reducing side effects of hCT drugs, and remains a key technical problem that urgently needs to be solved in this field. Summary of the Invention
[0005] Based on this, the present invention provides a human calcitonin modified by O-acetylglucosamine glycosylation modification of threonine / serine / tyrosine residues of human calcitonin, which has a much better effect than the human calcitonin modified by nitration of the tyrosine residue at position 12 of human calcitonin as described in patent CN110922469A.
[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:
[0007] The present invention provides a method for modifying human calcitonin, the modification method comprising: performing O-acetylglucosamine glycosylation modification on the amino acid residues of human calcitonin, wherein the amino acid residues are threonine residues, serine residues or tyrosine residues.
[0008] Preferably, in the above modification method, O-acetylglucosamine glycosylation modification is performed on the threonine residue at position 21 of human calcitonin.
[0009] Another aspect of the present invention provides a modified human calcitonin, which is obtained by the above-described modification method.
[0010] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described modified human calcitonin.
[0011] In another aspect, the present invention provides a medicament comprising the above-described modified human calcitonin or the above-described pharmaceutical composition.
[0012] Preferably, the dosage form of the above-mentioned drug is an injection solution, an injection powder, or an injection suspension.
[0013] In another aspect, the present invention provides the use of the above-described modified human calcitonin or the above-described pharmaceutical composition in the preparation of a medicament having one or more of the following effects:
[0014] (i) The drug has therapeutic effects on osteoporosis;
[0015] (ii) The drug has therapeutic effects on Paget's disease;
[0016] (iii) The drug has the effect of treating hypercalcemia or musculoskeletal pain caused by malignant tumors.
[0017] Preferably, in the above applications, the dosage form of the drug is an injection solution, an injection powder, or an injection suspension.
[0018] In another aspect, the present invention provides a method for inhibiting the aggregation of human calcitonin, characterized in that the method includes: performing O-acetylglucosamine glycosylation modification on the amino acid residues of human calcitonin, wherein the amino acid residues are threonine residues, serine residues, or tyrosine residues.
[0019] Preferably, in the above method, O-acetylglucosamine glycosylation modification is performed on the threonine residue at position 21 of human calcitonin.
[0020] The beneficial effects of this invention include:
[0021] (1) The human calcitonin modified by the modification method provided by the present invention can maintain the monomer state for 72 hours, while the human calcitonin modified by nitration of the tyrosine residue at position 12 of the calcitonin described in patent CN110922469A can only maintain the monomer state for 24 hours. This shows that the human calcitonin modification method in the present invention is far superior to the modification method of nitration of the tyrosine residue at position 12 of the calcitonin described in patent CN110922469A.
[0022] (2) The modified human calcitonin obtained by the modification method provided by the present invention can be detected by circular dichroism spectroscopy. The modified human calcitonin can maintain the active monomer structure configuration of hCT. This ability to maintain the active monomer configuration can even reach 96h, showing quite good structural stability. Attached Figure Description
[0023] Figure 1a Fluorescence kinetics curves of human calcitonin (hCT) and its modified variants;
[0024] Figure 1b Representative fluorescence curves of human calcitonin (hCT) and its modified variants at 72 h;
[0025] Figure 2 To examine the aggregation morphology of human calcitonin (hCT) and its modified variants after 24 h and 72 h of transmission electron microscopy;
[0026] Figure 3 To detect the aggregation of human calcitonin (hCT) and its modified variants using Nu-PAGE gel electrophoresis;
[0027] Figure 4a Dynamics of human calcitonin (hCT) and its modified variants within 0 h circular dichroism;
[0028] Figure 4b Dynamics of human calcitonin (hCT) and its modified variants within 6 hours using circular dichroism spectroscopy.
[0029] Figure 4c Dynamics of human calcitonin (hCT) and its modified variants within 12 hours using circular dichroism spectroscopy.
[0030] Figure 4d Dynamics of human calcitonin (hCT) and its modified variants within 24 hours using circular dichroism spectroscopy.
[0031] Figure 4e Dynamics of human calcitonin (hCT) and its modified variants within 36 hours using circular dichroism spectroscopy.
[0032] Figure 4f Dynamics of human calcitonin (hCT) and its modified variants within 48 hours using circular dichroism spectroscopy.
[0033] Figure 4g Dynamics of human calcitonin (hCT) and its modified variants within 60 hours using circular dichroism spectroscopy.
[0034] Figure 4h Dynamics of human calcitonin (hCT) and its modified variants within 72 hours using circular dichroism spectroscopy.
[0035] Figure 4iDynamics of human calcitonin (hCT) and its modified variants within 96 hours using circular dichroism. Detailed Implementation
[0036] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.
[0037] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.
[0038] In a first aspect, embodiments of the present invention provide a method for modifying human calcitonin, the modification method comprising: performing O-acetylglucosamine glycosylation modification on amino acid residues of human calcitonin, wherein the amino acid residues are threonine residues, serine residues or tyrosine residues.
[0039] In some specific examples, O-acetylglucosamine glycosylation modification was performed on the threonine residue at position 21 of human calcitonin.
[0040] It should be noted that the human calcitonin modified by the method provided in this invention can maintain its monomeric state for 72 hours, while the human calcitonin modified by nitration of the tyrosine residue at position 12 described in patent CN110922469A can only maintain its monomeric state for 24 hours. This indicates that the human calcitonin modification method in this invention is far superior to the modification method of nitration of the tyrosine residue at position 12 described in patent CN110922469A.
[0041] Secondly, embodiments of the present invention provide a modified human calcitonin, obtained by the above-described modification method.
[0042] It should be noted that the modified human calcitonin obtained by the modification method provided by the present invention can be detected by circular dichroism spectroscopy kinetics. The modified human calcitonin can maintain the active monomer structure configuration of hCT. This ability to maintain the active monomer configuration can even reach 96 hours, showing quite good structural stability.
[0043] Thirdly, embodiments of the present invention provide a pharmaceutical composition comprising the above-described modified human calcitonin.
[0044] It should be noted that the modified human calcitonin in this invention can be combined with other components having the same effect to form a pharmaceutical composition. Other components having the same effect are known in the art, such as one or more of calcium carbonate, calcium acetate, calcium gluconate, sodium alendronate, zoledronic acid, risedronate, salmon calcitonin, or eccalcitonin.
[0045] Fourthly, embodiments of the present invention provide a drug comprising the above-described modified human calcitonin or the above-described pharmaceutical composition.
[0046] It should be noted that the modified human calcitonin or the above-mentioned pharmaceutical composition in this invention can be prepared into a drug by adding excipients, which is more conducive to its use in different scenarios. Among them, the excipients are those known in the art, such as lyophilization protectants, buffers, osmotic modifiers or stabilizers, etc.
[0047] In some specific examples, the dosage form of the above-mentioned drugs is an injection solution, an injection powder, or an injection suspension.
[0048] It should be noted that the dosage forms of the above-mentioned drugs are well known in the art, such as injection solutions, injection powders, or injection suspensions.
[0049] Fifthly, embodiments of the present invention provide the use of the above-described modified human calcitonin or the above-described pharmaceutical composition in the preparation of a medicament, the medicament having one or more of the following effects:
[0050] (i) The drug has therapeutic effects on osteoporosis;
[0051] (ii) The drug has therapeutic effects on Paget's disease;
[0052] (iii) The drug has the effect of treating hypercalcemia or musculoskeletal pain caused by malignant tumors.
[0053] In some specific examples, the dosage form of the drug in the above applications is an injection solution, injection powder, or injection suspension.
[0054] In a sixth aspect, embodiments of the present invention provide a method for inhibiting the aggregation of human calcitonin, characterized in that the method includes: performing O-acetylglucosamine glycosylation modification on the amino acid residues of human calcitonin, wherein the amino acid residues are threonine residues, serine residues, or tyrosine residues.
[0055] In some specific examples, the above method involves O-acetylglucosamine glycosylation modification of the threonine residue at position 21 of human calcitonin.
[0056] It should be noted that the human calcitonin modified by the method provided in this invention can maintain its monomeric state for 72 hours, while the human calcitonin modified by nitration of the tyrosine residue at position 12 described in patent CN110922469A can only maintain its monomeric state for 24 hours. This indicates that the human calcitonin modification method in this invention is far superior to the modification method of nitration of the tyrosine residue at position 12 described in patent CN110922469A.
[0057] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.
[0058] Preparation Examples
[0059] Example 1
[0060] This invention provides a human calcitonin (hCT21-GlcNAc) obtained by modifying the 21st threonine residue of human calcitonin with O-acetylglucosamine glycosylation (O-GlcNAc glycosylation), which was synthesized by Shanghai Ketai Biotechnology Co., Ltd.
[0061] Comparative Example 1
[0062] Human calcitonin (hCT) without any modification was used as Comparative Example 1.
[0063] Comparative Example 2
[0064] Human calcitonin with a nitrated tyrosine residue at position 12 (denoted as hCT(3N)) as described in patent CN110922469A was used as Comparative Example 2.
[0065] Related tests
[0066] The following methods were used to measure ThT fluorescence spectra: During the experiment, 20 μM incubation solutions were prepared using 50 mM PB for hCT, hCT(3N), and hCT21-GlcNAc peptides, and then incubated in a 37°C water bath. Afterwards, equal amounts of sample were taken at 0, 4, 8, 12, 24, 36, 48, 60, and 72 hours, diluted 10-fold with PB containing 10 μM ThT, and the corresponding fluorescence absorbance values were measured on an LS55 Luminescence Spectrometer (PerkinElmer, USA). The excitation wavelength for ThT was set to 450 nm. Each experiment was repeated three times, and the average value was used as the final measurement result for plotting.
[0067] The transmission electron microscopy (TEM) testing methods are as follows: Samples for TEM observation were prepared by incubating 20 μM hCT, hCT(3N), and hCT21-GlcNAc peptide solutions at 37°C for 24 h and 72 h, respectively. Then, 30 μL of each sample was placed on a carbon-coated copper mesh (200 mesh) for absorption for 5 minutes. After removing excess liquid, the copper mesh was immersed in 30 μL of 5% uranium acetate for 10 minutes. Excess liquid was then removed, and the copper mesh was air-dried overnight. Finally, the dried copper mesh was used for TEM observation.
[0068] The gel electrophoresis experiments in the following tests were performed as follows: Nu-PAGE gel electrophoresis was conducted on a Mini Gel Tank using 4%-12% Bis-Tris protein precast gels. Samples were prepared by incubating 20 μM hCT, hCT(3N), and hCT21-GlcNAc peptide solutions at 37°C for 12, 24, and 72 h, respectively. Then, 40 μL of each sample solution was added to 12.5 μL of 4×LDS Nu-PAGE loading buffer, mixed thoroughly, and loaded onto the gel, with 20 μL loaded into each well. Electrophoresis was then performed in MES buffer. After electrophoresis, the gel was removed and silver stained. The specific steps for silver staining are as follows: First, the PAGE gel was washed twice with deionized water. Then, the gel was fixed three times with fixative, 20 minutes each time. After fixation, the PAGE gel was washed three times with 50% EtOH, 20 minutes each time. Next, it was pretreated with 0.02% Na₂S₂O₃ for one minute, followed by rinsing three times with deionized water for 20 seconds each time. Then, the gel was stained with staining solution for 20 minutes, followed by rinsing three times with deionized water for 20 seconds each time. Immediately afterward, the developing solution was added for development. Once all bands appeared, development was stopped immediately, and the washing process was repeated. Finally, the reaction was terminated by incubation with stop solution for 10 minutes. The gel was then removed, cleaned with deionized water, and the image was recorded using a scanner.
[0069] The following tests used the circular dichroism spectroscopy kinetic detection method: The CD sample preparation method was the same as the experimental method described above. Secondary structure curves of 40 μM hCT, hCT(3N), and hCT21-GlcNAc peptides were collected at 10 mM PB for 0, 6, 12, 24, 36, 48, 60, 72, and 96 h on a Jasco J-1500CD spectrophotometer (Jasco Corp., Japan). The specific instrument parameters during testing were as follows: optical path length, 1 mm; scan range, 190-260 nm; scan speed, 500 nm / min; data acquisition interval, 0.2 nm; bandwidth, 1 nm; number of scans, 3. All tests were performed at room temperature, and the obtained spectral results were corrected using a 10 mM PB blank curve.
[0070] (I) ThT fluorescence spectroscopy test
[0071] The hCT21-GlcNAc prepared in Example 1, the hCT in Comparative Example 1, and the hCT(3N) prepared in Comparative Example 2 were subjected to ThT fluorescence spectroscopy detection, and the results are as follows: Figure 1a and Figure 1b As shown in the figure. The results showed that the hCT21-GlcNAc prepared in Example 1 did not exhibit any fluorescence absorption value within 72 h, indicating that the O-GlcNAc glycosylation modification completely inhibited the formation of the β-sheet structure of hCT.
[0072] (II) Transmission electron microscopy observation
[0073] The morphological differences of the aggregates formed by hCT21-GlcNAc prepared in Example 1, hCT in Comparative Example 1, and hCT(3N) prepared in Comparative Example 2 during solution incubation were observed using transmission electron microscopy. The results are as follows: Figure 2 As shown, the results indicate that hCT alone exhibited a large number of long, overlapping fibers after 24 hours of incubation. In contrast, hCT(3N) showed a better inhibition effect, with only a small number of aggregated particles observed. However, no aggregated fibers, protofibrils, or aggregated particles were observed in the hCT21-GlcNAc sample. This demonstrates that O-GlcNAc glycosylation modification can indeed effectively inhibit hCT aggregation. Furthermore, only a few particles were observed in the hCT21-GlcNAc sample after 72 hours of incubation, while both hCT and hCT(3N) transformed into mature fibers. This further illustrates the superior inhibitory effect of O-GlcNAc glycosylation modification on hCT aggregation.
[0074] (III) Gel Electrophoresis Experiment
[0075] Gel electrophoresis experiments were performed on hCT21-GlcNAc prepared in Example 1, hCT in Comparative Example 1, and hCT(3N) prepared in Comparative Example 2, respectively. The results are as follows: Figure 3 As shown in the figure, the results showed that O-GlcNAc glycosylation-modified human calcitonin consistently exhibited a distinct band around 3.4 kDa (the theoretical molecular weight of hCT is 3.417 kDa), while the bands at the monomer positions of hCT and hCT(3N) completely disappeared after incubation for 24 and 72 h, respectively. This indicates that O-GlcNAc glycosylation modification can not only significantly inhibit the aggregation of human calcitonin but also maintain it in monomeric form over a long period of time (up to 72 h).
[0076] (iv) Dynamic detection of circular dichroism spectroscopy
[0077] To investigate the effects of O-GlcNAc glycosylation modification on the secondary structure (which significantly influences the binding and activation of hCT to the calcitonin receptor) during hCT aggregation, circular dichroism spectroscopy was also performed. The results are as follows: Figures 4a to 4i As shown in the figure. The results show that O-GlcNAc glycosylation modification can enable hCT to maintain the active monomer structure configuration, and this ability to maintain the active monomer configuration can be as high as 96 hours, demonstrating quite good structural stability.
[0078] The above test results indicate that O-GlcNAc glycosylation modification is a better strategy for preparing human calcitonin analogues with superior homology, and can produce better human calcitonin therapeutic agents for osteoporosis and other bone resorption-related diseases.
[0079] 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 it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. A method for modifying human calcitonin, characterized in that, The modification methods include: performing O-acetylglucosamine glycosylation modification on the amino acid residues of human calcitonin, wherein the amino acid residues are threonine residues; and performing O-acetylglucosamine glycosylation modification on the threonine residue at position 21 of human calcitonin.
2. A modified human calcitonin, obtained by the modification method described in claim 1.
3. A pharmaceutical composition, characterized in that, Including the modified human calcitonin as described in claim 2.
4. A drug, characterized in that, Includes the modified human calcitonin of claim 2 or the pharmaceutical composition of claim 3.
5. The drug according to claim 4, characterized in that, The drug is available in the form of an injection solution, an injection powder, or an injection suspension.
6. The use of the modified human calcitonin of claim 2 or the pharmaceutical composition of claim 3 in the preparation of a medicament, wherein the medicament has one or more of the following effects: (i) the medicament has the effect of treating osteoporosis; (ii) The drug has therapeutic effects on Paget's disease; (iii) The drug has the effect of treating hypercalcemia or musculoskeletal pain caused by malignant tumors.
7. The application according to claim 6, characterized in that, The drug is available in the form of an injection solution, an injection powder, or an injection suspension.
8. A method for inhibiting the accumulation of human calcitonin, characterized in that, The methods include: O-acetylglucosamine glycosylation modification was performed on the amino acid residues of human calcitonin, wherein the amino acid residues were threonine residues; O-acetylglucosamine glycosylation modification was performed on the threonine residue at position 21 of human calcitonin.