Effect of recombinant human fibroblast growth factor-18 protein pharmaceutical composition on osteoporosis

Abstract

The present application provides a kind of pharmaceutical composition applicable in osteoporosis disease treatment, it includes bioactive substance recombinant human fibroblast growth factor-18 (rhFGF18) protein and type I collagen, can resist the apoptosis of osteoblast caused by oxidative damage, improve bone metabolism, and increase osteogenesis, especially for the significant effect of estrogen deficiency caused bone loss, with wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of protein / peptide drug biotechnology. More specifically, this invention relates to a pharmaceutical composition containing recombinant human fibroblast growth factor-18 protein and its application in the treatment of osteoporosis. Background Technology

[0002] With the increasing aging of the population, osteoporosis has become a common and prevalent disease among middle-aged and elderly people, and its incidence is rising year by year. According to incomplete statistics, the incidence of osteoporosis in people over 60 years old is 60.72% for men and as high as 90.84% ​​for women. The annual incidence of fractures due to osteoporosis is approximately 10%. Currently, there are 200 million osteoporosis patients worldwide, with about 90 million in my country suffering from varying degrees of osteoporosis, ranking first in the world. Elderly people suffering from osteoporosis experience significantly reduced bone density, leading to severe back pain and limited mobility in mild cases, and paralysis in severe cases, seriously affecting their health and quality of life, and imposing a heavy economic burden on families and society.

[0003] Osteoporosis is particularly prevalent in postmenopausal women and is termed postmenopausal osteoporosis (PMOP), which is the most common type of osteoporosis (Black DM et al. N Engl J Med, 2016, 374(3): 254-262.). Studies have shown that estrogen deficiency increases bone turnover, affecting the normal development of trabecular bone (loss of connectivity) and cortical bone (thinning of the cortex), characterized by decreased bone mineral density (BMD) and pathological fractures (Eastell R et al. Nat Rev Dis Primers, 2016, 2: 160-169; Weitzmann MN et al. Journal of Clinical Investigation. 2006, 116(5): 1186-1194.). Furthermore, oxidative stress caused by estrogen deficiency can promote the development of postmenopausal osteoporosis (Almeida M et al. J Biol Chem, 2007, 282(37): 85-97; Lean JM et al. Journal of Clinical Investigation, 2003, 112(6): 915-923; Manolagas SC. Endocr Rev, 2010, 31(3): 266-300; Shi C et al. Bone, 2015, 79: 94-104.). Increasing research evidence suggests that excessive ROS can enhance osteoclast differentiation on the one hand, and inhibit osteoblast proliferation and differentiation on the other, ultimately leading to increased bone resorption. Although progress has been made in understanding how estrogen deficiency and oxidative stress induce PMOP, its potential pathogenic mechanisms remain complex and multifaceted (Weitzmann MN et al. Journal of Clinical Investigation. 2006, 116(5): 1186-1194.).

[0004] Fibroblast growth factor (FGF) is a class of cytokines that play a crucial role in cell protection, cell mitosis, angiogenesis, and tissue repair. Studies have shown that most FGFs are also closely related to bone development and growth. For example, FGF-FGFR plays a key role in the development of limb buds, intrachondral and intramembranous mesenchymal coagulation, and regulates cartilage formation, osteogenic processes, and bone and mineral homeostasis (Degnin et al. Journal of Cellular Biochemistry, 2010, 110(5):1046-1057; Ornitz et al. Genes Dev, 2015, 29(14):1463-1486.). As a member of the FGF family, FGF18 plays an important role in bone growth and development. It regulates bone development, cartilage formation, and stimulates the proliferation of osteoblasts and osteoclasts by modulating FGFR, BMP (bone morphogenetic protein), and multiple signaling pathways. It has potential clinical application value for treating bone diseases such as cartilage disorders, chondrodysplasia, and bone repair, and is receiving increasing attention (Moore EE et al. Osteoarthritis Cartilage, 2005, 13: 623-631; Gigout A et al. Osteoarthritis Cartilage, 2017, 25(11): 1858-1867; Ditte Reker et al. J Transl Med, 2017, 15(1): 250; D Reker et al. Sci Rep, 2020, 10(1): 6011. Jia Li et al. Expert Opin Investig Drugs, 2021, 30(9): 923-930.). Current research on the biological effects of FGF18 focuses mainly on cartilage and osteoarthritis, while there are no relevant research reports on osteoporosis, which is an important cause of osteoblast dysfunction and death.

[0005] Building upon previous work on FGF18 gene recombinant expression, protein purification, and activity identification, the inventors developed stable lyophilized formulations and investigated their effects on osteoporosis and related mechanisms through in vitro and in vivo experiments. FGF18 is a heparin-binding protein, and its amino acid composition contains a large number of basic amino acids such as lysine and arginine, leading to unstable peptide bonds that are easily broken. During the screening of lyophilized formulations, it was discovered that the addition of a certain concentration of type I collagen plays a crucial role in the stability of the protein's biological activity. Long-term stability tests showed that adding 5 times the amount of type I collagen as a protective agent to the lyophilized composition effectively protected the long-term stability of FGF18's biological activity.

[0006] In vitro studies showed that administration of FGF18 protein to the MC3T3-E1 osteoblast model induced by H2O2 oxidative damage effectively protected osteoblast viability and significantly improved osteoblast apoptosis induced by oxidative damage by inhibiting caspase 3 protein expression in osteoblasts and reducing the Bax / Bcl-2 ratio. In vivo studies showed that administration of FGF18 protein to the PMOP model established by ovariectomy (OVX) in ICR mice significantly increased serum estradiol E2 and bone alkaline phosphatase (BALP) levels, and decreased tartrate-resistant acid phosphatase (TRACP) levels, indicating that rhFGF18 can improve bone metabolism in OVX mice. Further micro-CT and HE results showed that rhFGF18 treatment significantly altered the trabecular structure, bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and structural pattern index (SMI) of the femur in OVX mice. Its main mechanism of action is that FGF18 can effectively improve estrogen deficiency-induced oxidative stress and the level and activity of antioxidant enzymes, protect osteoblast apoptosis caused by oxidative damage, improve bone metabolism, and increase bone formation. Summary of the Invention

[0007] The purpose of this invention is to provide a pharmaceutical composition for treating osteoporosis, particularly suitable for postmenopausal osteoporosis (PMOP). Furthermore, this invention also provides pharmaceutical formulations comprising this pharmaceutical composition, applications, etc.

[0008] Specifically, in a first aspect, the present invention provides a pharmaceutical composition for treating osteoporosis, comprising recombinant human fibroblast growth factor-18 (rhFGF18) protein and type I collagen, L-arginine, and lysine hydrochloride that have a protective effect on protein stability.

[0009] Because FGF18 contains a large number of basic amino acids in its amino acid sequence, its peptide bonds are prone to instability, including the N-terminus and C-terminus, which are easily broken. Existing technologies focus on truncating or mutant FGF18. However, the inventors have discovered that the natural FGF18 sequence possesses better biological activity. Therefore, in the specific embodiments of this invention (except for Example 1), FGF18 is the complete natural human sequence, and its preparation method is described in the already granted patent ZL201510702841.1.

[0010] Preferably, in the pharmaceutical composition of the first aspect of the present invention, the biologically active components include rhFGF18 protein and type I collagen. Type I collagen can act as a stabilizer and protectant for rhFGF18, and also has a certain proliferative effect on osteoblasts.

[0011] In a second aspect, the present invention provides a lyophilized pharmaceutical formulation for treating osteoporosis, comprising the pharmaceutical composition of the first aspect of the present invention and excipients, stabilizers, protectants, buffer systems, or other pharmaceutical excipients required for the lyophilized formulation. Examples include mannitol as an excipient, trehalose, Tween 80, and disodium EDTA as protein stabilizers and protectants, and PBS (pH 7.0) as a buffer system.

[0012] Preferably, in the pharmaceutical formulation of the present invention, the excipients used are injection-grade pharmaceutical excipients. That is, the pharmaceutical formulation of the second aspect of the present invention is an injection formulation. During application, the lyophilized pharmaceutical composition of the present invention can be dissolved or diluted with physiological saline or sterile water for injection to prepare an injection.

[0013] In a third aspect, the present invention provides the use of FGF18 in the treatment of postmenopausal osteoporosis. The method comprises administering a lyophilized formulation of an FGF18 pharmaceutical composition, wherein the FGF18 pharmaceutical composition is administered subcutaneously or intraperitoneally three times a week, preferably once every other day.

[0014] Preferably, in the application of the third aspect of the invention, FGF18 and type I collagen are used as active pharmaceutical ingredients. Specifically, the third aspect of the invention preferably provides the use of a lyophilized pharmaceutical composition containing FGF18 and type I collagen as active pharmaceutical ingredients in osteoporosis. The pharmaceutical composition contains 1–10 mg of FGF18 protein and 10–30 mg of type I collagen, preferably 3–9 mg of FGF18 protein and 15–25 mg of type I collagen, and most preferably 4 mg of FGF18 protein and 20 mg of type I collagen.

[0015] The beneficial effects achieved by this invention are as follows: the lyophilized formulation of the recombinant human fibroblast growth factor-18 drug composition of this invention has good biological activity and can be used to treat osteoporosis, especially postmenopausal osteoporosis; the drug is administered subcutaneously or intraperitoneally, and by effectively reducing oxidative stress levels and regulating the levels and activities of antioxidant enzymes, it slows down the decline in estrogen levels, protects osteoblast apoptosis, improves bone metabolism, and increases bone formation, thereby improving osteoporosis, especially postmenopausal osteoporosis. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0017] Figure 1 Biological activities of recombinant human fibroblast growth factor-18 natural sequence protein (rhFGF18), N-terminal truncated protein (rhFGF18ΔN), and C-terminal truncated protein (rhFGF18ΔC) on MC3T3-E1 osteoblasts;

[0018] Figure 2 Effects of different concentrations of type I collagen on the biological activity stability and proliferative biological activity of osteoblasts in recombinant human fibroblast growth factor-18 lyophilized composition.

[0019] Figure 3 Effect of recombinant human fibroblast growth factor-18 protein on the viability of MC3T3-E1 osteoblasts in a model of oxidative damage induced by H2O2.

[0020] Figure 4 The effect of recombinant human fibroblast growth factor-18 protein on apoptosis of MC3T3-E1 osteoblasts in a model of oxidative damage induced by H2O2.

[0021] Figure 5 Effect of lyophilized recombinant human fibroblast growth factor-18 drug composition on body weight in OVX mice;

[0022] Figure 6 Effects of lyophilized recombinant human fibroblast growth factor-18 pharmaceutical composition on bone metabolism in OVX mice;

[0023] Figure 7 Effects of lyophilized recombinant human fibroblast growth factor-18 drug composition on bone morphology and structure in OVX mice. Detailed Implementation

[0024] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0025] Example 1: MTT assay for detecting the proliferative biological activity of recombinant human fibroblast growth factor-18 native and truncated sequences on osteoblasts.

[0026] Using MC3T3-E1 osteoblasts as the test cell line, the proliferative biological activities of recombinant human fibroblast growth factor-18 native sequence (rhFGF18), N-terminal truncated protein (rhFGF18ΔN), and C-terminal truncated protein (rhFGF18ΔC) on the target cell line were determined by cell proliferation assay / MTT colorimetric method. Specific procedures are as follows:

[0027] 1) Preparation of the test solution: Dilute FGF18 protein with maintenance culture medium to approximately 160 ng / ml. Perform serial 2-fold dilutions in a 96-well cell culture plate, for a total of 9 dilutions, with 2 wells for each dilution. All operations were performed under aseptic conditions.

[0028] 2) Biological activity assay: MC3T3-E1 cell lines were cultured in DMEM complete medium at 37°C and 5% CO2. The culture medium in the culture flasks was discarded, and the cells were digested with 0.25% trypsin-EDTA and collected. The cell concentration was adjusted to 5 × 10⁶ cells / mL using complete culture medium. 4 Cell suspension at 100 μl / ml was seeded into 96-well cell culture plates and cultured at 37°C and 5% CO2. After 24 h, the suspension was replaced with 100 μl of DMEM starvation medium per well, and cultured for another 24 h at 37°C and 5% CO2. The maintenance medium in the 96-well plates was discarded, and the test solution diluted with fresh DMEM starvation medium was added to each well to achieve a protein concentration gradient of 0, 0.06, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 ng / ml, at 100 μl per well. A PBS group was set up as a negative control, and the plates were cultured for another 6 h at 37°C and 5% CO2. 20 μl of MTT (5 mg / ml) was added to each well, and the plates were incubated at 37°C and 5% CO2 for 4 h. After discarding the liquid in the 96-well plates, 25 μl of dimethyl sulfoxide was added to each well, and the plates were shaken to mix for 10 min. Place the sample in an ELISA reader, use 630nm as the reference wavelength, measure the absorbance at a wavelength of 490nm, and record the results.

[0029] The biological activity of recombinant human fibroblast growth factor-18 natural sequence protein (rhFGF18), N-terminal truncated protein (rhFGF18ΔN), and C-terminal truncated protein (rhFGF18ΔC) on the proliferative effects on MC3T3-E1 osteoblasts was detected by biological activity assay. Results are as follows: Figure 1 As shown, the natural sequence of rhFGF18 exhibits better biological activity.

[0030] Example 2: Effect of the combination ratio of recombinant human fibroblast growth factor-18 and type I collagen on the stability of its biological activity and its proliferative biological activity on osteoblasts.

[0031] Recombinant human fibroblast growth factor-18 (rhFGF18) protein and type I collagen were mixed at ratios of 1:0, 1:2.5, 1:5, and 1:10 (by mass), and other formulation excipients were added before lyophilization. The lyophilized product was stored at 4°C, and samples were taken at 0, 1, 3, 6, and 12 months. Using MC3T3-E1 osteoblasts as the detection cell line, the biological activity of recombinant human fibroblast growth factor-18 in the formulation was determined by a cell proliferation assay / MTT colorimetric method, following the same procedure as in Example 1. The results are as follows: Figure 2 As shown, type I collagen has a protective effect against the biological activity of rhFGF18, and also has a certain proliferative effect on osteoblasts, exhibiting a dose-dependent effect within a certain range. Based on the experimental results ( Figure 2 Taking into account factors such as cost, the preferred ratio of active substances added to the freeze-dried composition is FGF18: type I collagen = 1:5 (mass ratio), which can effectively protect the biological activity of rhFGF18 protein and also exert a certain synergistic effect.

[0032] Example 3: Preparation method of lyophilized formulation of recombinant human fibroblast growth factor-18 pharmaceutical composition

[0033] The lyophilized formulation solution of the pharmaceutical composition and the filling method were prepared according to the preferred ratio (FGF18: type I collagen = 1:5 (mass ratio)) in Example 2. The components are shown in Table 1.

[0034] 1) Preparation of protein composition solution: The purified rhFGF18 protein and type I collagen were dialyzed and concentrated by ultrafiltration in 20 mM PBS buffer (pH 7.0). A 1 M stock solution of arginine and lysine hydrochloride was prepared and added to the protein solution to a final concentration of 50 μM, rhFGF18 protein concentration of 4.0 mg / ml, and type I collagen concentration of 20 mg / ml. The solution was then sterilized and filtered through a sterile 0.22 μm microporous membrane before use. All the above operations were performed at 2-8℃.

[0035] 2) Preparation of lyophilized excipient solution: Accurately weigh each component according to the proportion of lyophilized excipients in Table 1, fully dissolve in 20mM PBS buffer (pH 7.0), adjust the pH to 7.0, filter sterile through a sterile 0.22μm microporous membrane, and pre-cool at 2-8℃ before use;

[0036] 3) Mix the protein solution and the excipient solution at a ratio of 1:1 (v / v) to obtain a lyophilized formulation solution with a rhFGF18 protein concentration of 2.0 mg / ml and a type I collagen concentration of 20 mg / ml.

[0037] 4) Accurately measure 2 ml of the lyophilized formulation solution, dispense it into 7 ml dry heat sterilized vials, partially stopper them, and place them in a pre-cooled lyophilizer for lyophilization.

[0038] Freeze-drying process and storage:

[0039] 1) Pre-freezing: Cool at a rate of 1℃ / min, with a pre-freezing temperature of -40℃, and hold for 2 hours;

[0040] 2) Single drying: Vacuum degree controlled at 100 mTorr, temperature -40~-20℃, drying for 32h. Each temperature rise of approximately 5℃ is considered a node (i.e. -35, -30, -25, -20℃), the temperature rise rate of the partition is 0.5℃ / min, and each temperature node is maintained for 8h.

[0041] 3) Secondary drying: Vacuum degree controlled at around 30 mTorr, temperature -20~20℃. The temperature rise rate of the partition is 1℃ / min, and it is maintained at 20℃ for 6 hours.

[0042] 4) After the secondary drying is completed, the vial is stoppered under certain vacuum conditions, and finally sealed with an aluminum cap and stored at 2-8℃.

[0043] Table 1

[0044]

[0045] Example 4: Effects of recombinant human fibroblast growth factor-18 protein on the viability and anti-apoptotic activity of MC3T3-E1 osteoblasts in a H2O2-induced oxidative damage model.

[0046] The MC3T3-E1 osteoblast culture, cell plating (96-well plate), and cell starvation methods were the same as in Example 1. The old starvation culture medium was discarded, and H2O2 was diluted with fresh DMEM starvation culture medium to prepare a final concentration of 25 μM. This medium was used to stimulate MC3T3-E1 osteoblasts for 4 h to create an H2O2 cell oxidative damage model. The culture medium was then discarded, and the cells were washed with 1 ml of PBS. They were then incubated with rhFGF18 (0, 5, 10, 20, 40, 80, 160 ng / ml) for 4 h. Cell viability was assessed using the MTT assay, following the same procedure as in Example 1.

[0047] Western blot analysis was performed to detect the effects of rhFGF18 on apoptotic and anti-apoptotic proteins in H2O2-stimulated osteoblasts. Total cell protein samples were thoroughly mixed with buffer at a ratio of 4:1 (V / V), boiled for 10 min, and centrifuged at 13000 rpm for 10 min to prepare samples for later use. The samples were processed according to the following procedure: electrophoresis, membrane transfer, blocking, washing, incubation with primary antibody, washing, incubation with secondary antibody, washing, and ECL staining. Finally, the images were analyzed using ImageLab software (version 5.2) for optical density scanning; grayscale values ​​represent protein expression levels.

[0048] Apoptosis in MC3T3-E1 osteoblasts was detected by flow cytometry and the FITC Annexin V apoptosis assay kit. Cell culture was performed as described above. MC3T3-E1 osteoblasts were seeded in 6-well plates and stimulated with 25 μM H2O2 for 4 h. The culture medium was then discarded, and the cells were washed with 1 ml PBS and incubated with rhFGF18 (20, 40, and 80 ng / ml) for 4 h. Cell culture medium was collected from each well of the corresponding 6-well plate, and 1 ml PBS was slowly added along the wall for washing and recovery. 500 μl of trypsin digestion solution (EDTA-free) was added, and the cells were observed under a microscope. Once the cell edges brightened, 2 ml PBS was added to dilute the trypsin digestion solution, ensuring all cells were in a floating state. The cells were then collected. The cells were repeatedly washed with PBS, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Add 100 μl of 1×Buffer, gently pipette to resuspend, transfer to a flow cytometry tube, add 5 μl of PI and 5 μl of AV for staining for 15 min, then add 400 μl of 1×Buffer for detection.

[0049] The effect of rhFGF18 protein on the viability of H2O2-stimulated MC3T3-E1 osteoblasts, such as Figure 3 As shown in Figure A, administration of rhFGF18 protein at concentrations ranging from 5 to 160 ng / ml significantly enhanced the viability of osteoblasts damaged by H2O2 oxidation, with the most significant protective effect against oxidative damage observed at a concentration of 80 ng / ml. Western blot analysis confirmed this effect. Figure 3As shown in BD. Administration of 20–80 ng / ml rhFGF18 protein significantly promoted the expression of the anti-apoptotic protein Bcl-2, while significantly inhibiting the expression of apoptotic proteins Bax and caspase-3, thus alleviating H2O2-induced apoptosis in MC3T3-E1 cells. Further, flow cytometry analysis revealed… Figure 4 As shown in A and B, the apoptosis level of MC3T3-E1 osteoblasts significantly increased after H2O2 stimulation. Figure 4 As shown in CF, administration of 20–80 ng / ml rhFGF18 protein significantly improved apoptosis in osteoblasts damaged by oxidative stress in a dose-dependent manner.

[0050] Example 5: Effects of lyophilized recombinant human fibroblast growth factor-18 drug composition on bone metabolism and bone morphology in OVX mice

[0051] Eight-week-old female ICR mice were used as experimental animals. A PMOP model was established by ovariectomy (OVX). The effect of the rhFGF18 drug composition lyophilized formulation on osteoporosis was investigated by comparing the rhFGF18 drug composition administration group with the negative and SHAM groups. The specific procedures are as follows:

[0052] 1) Test drug: A lyophilized formulation of recombinant human fibroblast growth factor-18 prepared according to the method described in Example 3, with a specification of 4 mg / vial. It is prepared with physiological saline to a concentration of 0.5 μg / ml and 5 μg / ml before use, and should be prepared fresh each time.

[0053] Negative control: 0.9% physiological saline.

[0054] 2) Experimental animals: 7-week-old female ICR mice, SPF grade, weighing 20-22g. After purchase, they were acclimatized in an SPF-grade animal facility for 1 week before modeling.

[0055] 3) Experimental grouping and administration: Mice were randomly divided into four groups (n=8 per group): SHAM group (sham surgery, intraperitoneal injection of 0.3 ml of physiological saline per injection); negative control group (ovarian removal surgery, intraperitoneal injection of 0.3 ml of physiological saline per injection); low-dose rhFGF18 group (ovarian removal surgery, intraperitoneal injection of 0.15 μg / 0.3 ml of rhFGF18 protein per injection); and high-dose rhFGF18 group (ovarian removal surgery, intraperitoneal injection of 1.5 μg / 0.3 ml of rhFGF18 protein per injection). All formulations were prepared according to the requirement of equal volumes (0.3 ml) with varying concentrations. Intraperitoneal administration began on the second day after surgery, with all groups receiving the medication every other day.

[0056] 4) Observation indicators: Weighing and recording were performed weekly. After 8 weeks of treatment, mice were anesthetized with chloral hydrate solution, and blood was collected from the eyeballs. The levels of E2, BALP, and TRACP in mouse serum were detected by enzyme-linked immunosorbent assay (ELISA). Bilateral femurs were harvested, and the samples were decalcified with 10% EDTA. After the syringe needle could easily penetrate the tissue, the samples were dehydrated, cleared, and paraffin-impregnated. They were then embedded, sectioned, and stained. The morphology and structure of the distal femoral trabeculae were observed and photographed under a microscope. Micro-CT scanning was used to analyze the morphology and structure of the mouse femur. The voltage was set to 70kV, the current to 200μA, the resolution to 10μm, and the exposure time to 300ms. The distal femoral trabeculae were selected for three-dimensional reconstruction, and the following parameters were obtained: bone mineral density (BMD), bone volume fraction (BV / TV), number of trabeculae (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and structural pattern index (SMI).

[0057] Experimental results are as follows Figure 5 As shown, compared with the SHAM group mice that did not have their ovaries removed, the OVX group mice that had their ovaries removed had a significant increase in body weight, while the mice treated with the rhFGF18 drug combination had a significantly lower body weight compared with the OVX group.

[0058] Experimental results are as follows Figure 6 As shown in Figure A, the E2 content in mice was significantly reduced after ovariectomy, indicating a decrease in estrogen levels. Compared with the OVX group, the E2 content in the rhFGF18 treatment group was significantly increased, suggesting that rhFGF18 can improve estrogen deficiency in OVX mice. Figure 6 In mice in groups B and 6C, serum BALP levels were significantly decreased while TRACP levels were increased, indicating reduced bone formation and correspondingly increased bone resorption. Similarly, after treatment with rhFGF18, BALP levels increased in the treated group, promoting bone formation; TRACP levels decreased, slowing bone resorption.

[0059] Experimental results are as follows Figure 7 As shown in A and 7B, ovariectomy in mice altered the trabecular structure of the distal femur, reducing the number of trabeculae. Treatment with rhFGF18 effectively alleviated this condition, demonstrating a good therapeutic effect. Experimental results are as follows... Figure 7 As shown in the CH diagram, the relevant morphological and structural parameters obtained from Micro-CT scanning analysis showed consistent results with those from pathological sections. In OVX mice, bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), and trabecular bone thickness (Tb.Th) were all decreased, while trabecular bone separation (Tb.Sp) and structural pattern index (SMI) were significantly increased. Similarly, treatment with rhFGF18 significantly altered this trend.

[0060] Experimental results showed that the lyophilized formulation of recombinant human fibroblast growth factor-18 drug composition could significantly improve bone loss caused by estrogen deficiency and oxidative stress.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A lyophilized formulation of a recombinant human fibroblast growth factor-18 (rhFGF18) protein pharmaceutical composition for treating osteoporosis, characterized in that, The active ingredients of the composition are recombinant human fibroblast growth factor-18 (rhFGF18) protein and type I collagen, wherein the mass ratio of rhFGF18 to type I collagen is 1:

5. The drug composition is administered by injection. The components of the drug composition are as follows: rhFGF18 protein 2.0 mg / mL, type I collagen 10 mg / mL, L-arginine 8.71 mg / mL, lysine hydrochloride 9.13 mg / mL, mannitol 30 mg / mL, trehalose 20 mg / mL, Tween-80 0.1 mg / mL, disodium EDTA 2 mM, PBS buffer system 20 mM, pH 7.

0.

2. The use of the lyophilized formulation as described in claim 1 in the preparation of a medicament for treating osteoporosis, characterized in that, The lyophilized formulation is administered by injection.

3. The application as described in claim 2, characterized in that, The osteoporosis mentioned is postmenopausal osteoporosis.

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