A composition comprising a polypeptide and its use for bone growth in children

Abstract

The application discloses a polypeptide-containing composition and its use for bone growth of children. The application also discloses a growth-promoting polypeptide which is screened and separated from a horse foal leg bone and can effectively promote proliferation of osteoblasts. The growth-promoting polypeptide can effectively promote weight increase of a rat femur, can significantly improve bone strength and bone density, can increase ALP activity, promote maturation of osteoblasts and start calcification, can significantly improve calcium ion content in the bone, and has a good effect of promoting bone growth and improving bone strength.

Description

Technical Field

[0001] This application relates to the biological field, specifically to a composition comprising a polypeptide and its use for bone growth in children. Background Technology

[0002] Short stature is defined as a height that is more than two standard deviations below the average height of children of the same age, race, and sex under similar living conditions, or below the third percentile of the normal growth curve. Its causes are complex and can be classified into several types, including growth hormone neurosecretion disorder (GHND), idiopathic short stature, intrauterine growth retardation, idiopathic precocious puberty, constitutional delay of puberty, Turner syndrome, primary hypothyroidism, and achondroplasia. Vitamin D promotes calcium and phosphorus metabolism, osteoblast proliferation, and bone matrix formation, thereby maintaining the body's growth and development.

[0003] Vitamin D affects calcium and phosphorus metabolism. Vitamin D deficiency reduces calcium and phosphorus levels in the body, affecting bone mineralization and thus impacting height. Studies have shown that when 120 healthy children undergoing physical examinations were divided into three groups based on serum 25-(OH)D levels—mild deficiency, severe deficiency, and normal—the bone mineral density, growth rate standard deviation ratio (GVSDS), height, and weight were all lower in the mild and severe deficiency groups than in the normal group. The bone mineral density, GVSDS, height, and weight were all lower in the severe deficiency group than in the mild deficiency group. When comparing the prevalence of short stature, the severe deficiency group > mild deficiency group > normal group. Yan et al.'s investigation of the prevalence of short stature among 12-year-old children in Zhuzhou showed a prevalence of 4.21% among 1756 children, with a vitamin D deficiency rate of 58.11%. Research by Lu Yanfei et al. showed that serum 25-(OH)D levels in children with short stature were significantly lower than in normal children. In children with idiopathic short stature (ISS), 25-(OH)D levels were positively correlated with height and weight. Based on the above results, it can be concluded that vitamin D deficiency or insufficiency is correlated with children's growth, and children with short stature have lower vitamin D levels than normal children. Vitamin D deficiency increases the incidence of short stature, and this deficiency is related to the degree of deficiency; the more severe the deficiency, the higher the incidence of short stature. Growth hormone deficiency and idiopathic short stature are the most common types of short stature. Chen Jin et al. divided 101 children with short stature into three groups based on their growth hormone secretion status: complete growth hormone deficiency (CGHD), partial growth hormone deficiency (PGHD), and idiopathic short stature (ISS). The vitamin D deficiency or insufficiency rates in all three groups were higher than in the normal group, with CGHD showing the highest rate at 73.08%. In patients with growth hormone deficiency (GHD) caused by pituitary stalk interruption syndrome (PSIS), a significant positive correlation was found between peak growth hormone levels and 1,25(OH)2D. GH can increase 25-(OH)D levels through the action of IGF-1, and better vitamin D levels may help GHD patients achieve normal IGF-1 levels. Even in patients with severely impaired GH secretion and low IGF-1 levels, the aforementioned interactions between the GH / IGF1 axis and vitamin D persist. This suggests that vitamin D levels may be a contributing factor to growth hormone deficiency.

[0004] rhGH is an exogenous growth hormone, a protein hormone produced using recombinant DNA technology. This hormone is created through gene recombination of secretory expression in *E. coli*, and it has similar effects to growth hormone (GH). GH treatment can promote linear growth and increase final height (Ht) in children and adolescents with or without growth hormone deficiency, and is therefore often used to treat related diseases of insufficient GH secretion. rhGH mainly promotes height growth by regulating the GH-IGF axis, promoting the production of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3). IGF-1 is a polypeptide protein essential for growth hormone production, promoting cell mitosis and differentiation, and regulating cell growth, proliferation, and apoptosis. When the concentration of IGF-1 synthesis and secretion reaches a certain level, it will in turn stimulate the hypothalamus-pituitary-IGF-1 axis, regulating growth hormone secretion in the body and preventing excessive growth hormone. IGFBP-3 is the main binding protein of IGF-1, which can regulate IGF-1, induce apoptosis, and exert biological effects in conjunction with growth factors. However, current rhGH treatment is expensive and not always successful for patients. Therefore, developing a cost-effective treatment is an important research direction. Summary of the Invention

[0005] The inventors discovered that the lactation period is the fastest growth stage for foals. By the time they are one year old, their weight can reach 64% of their adult weight, and by two years old, it can reach 85%. Their growth rate in the first two years far exceeds that of most mammals. Therefore, isolating growth-promoting peptides from foal bones is an important research direction.

[0006] In one aspect, the present invention provides a growth-promoting polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] The growth-promoting polypeptide of this invention can significantly promote height increase.

[0008] Specifically, the polypeptides of the present invention can be modified or replaced but still retain the corresponding polypeptide activity.

[0009] A "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is a conserved substitution. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art.

[0010] Specifically, the growth-promoting polypeptide can significantly promote the proliferation of bone cells, thereby promoting bone growth and thus increasing height.

[0011] Specifically, the present invention provides a pharmaceutical composition for promoting bone growth in children, characterized in that it contains a growth-promoting polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0012] Furthermore, the present invention also provides the use of the growth-promoting peptide in the preparation of a pharmaceutical composition for promoting bone growth, wherein the amino acid sequence of the growth-promoting peptide is shown in SEQ ID NO: 1.

[0013] Specifically, the drug of the present invention contains a pharmaceutically acceptable carrier or excipient.

[0014] The pharmaceutical compositions of the present invention may include additional excipients (also referred to herein as co-excipients). Suitable examples of additional excipients include pH-stabilizing buffers, preservatives, surfactants, stabilizers, antioxidants, tensioning agents, and ionic and nonionic polymers as defined herein.

[0015] Preservatives are compounds added to pharmaceutical preparations to reduce bacterial activity or undesirable chemical alterations. Examples of preservatives include benzyl alcohol, ethanol, methanol, isopropanol, butylparaben, ethylparaben, methylparaben, propylparaben, cathechol, 2-chlorophenol, m-cresol, phenol, resorcinol, xylitol, 2,6-dimethylcyclohexanol, 2-methyl-2,4-pentanediol, polyvinylpyrrolidone, benzyl chloride, thimerosal, benzoic acid, benzalkonium chloride, chlorobutanol, sodium benzoate, sodium propionate, and cetylpyridinium chloride.

[0016] A buffer is an excipient that stabilizes the pH of a pharmaceutical composition. Suitable buffers are well known in the art and can be found in the literature. Examples of suitable buffers include histidine-buffers, citrate-buffers, succinate-buffers, acetate-buffers, and phosphate-buffers, or mixtures thereof. The most preferred buffer comprises citrate, L-histidine, or a mixture of L-histidine and L-histidine hydrochloride. Another preferred buffer is a citrate buffer. Independent of the buffer used, the pH can be adjusted using acids or bases known in the art, such as hydrochloric acid, acetic acid, phosphoric acid, sulfuric acid, citric acid, sodium hydroxide, and potassium hydroxide.

[0017] Ionic polymers suitable for use as additional excipients include ionic carboxymethyl cellulose (CMC), hyaluronic acid, poly(glutamic acid), poly(aspartic acid), poly(glutamic acid-co-glycine), poly(aspartic acid-co-glycine), poly(glutamic acid-co-alanine), poly(aspartic acid-co-alanine), sodium glycolate starch, polygalacturonic acid, poly(acrylic acid), carrageenan, and alginate.

[0018] Surfactants are surface-active agents or substances that tend to reduce the surface tension of liquids in which they are dissolved. Suitable surfactants include polysorbates, poloxamers, tritons, sodium lauryl sulfate, sodium lauryl sulfate, and betaine. For example, surfactants include polyoxyethylene (20) lauryl sorbitan (20, e.g. from Sigma-Aldrich), polyoxyethylene (20) palmitolean (40), polyoxyethylene (20) dehydrated sorbitol monooleate (80), poloxamer 188, polyoxyethylene-polyoxypropylene block copolymer (F-68), polyethylene glycol 660-12-hydroxystearate (HS15, BASF), cocamidopropyl betaine, linoleyl betaine, myristyl betaine, cetyl betaine, polyethoxylated castor oil (currently Kolliphor BASF), and lecithin.

[0019] The methods and compositions of the present invention can be applied to conditions characterized by or resulting in bone loss, such as osteoporosis (including secondary osteoporosis), short stature, and developmental delays in children.

[0020] In one embodiment, the present invention provides a method for achieving a beneficial effect in a subject suffering from a growth hormone-related disease, disorder, or condition, the method comprising the step of administering a therapeutically or preventively effective amount of a polypeptide to the subject, wherein the administration results in an improvement in one or more biochemical or physiological parameters or clinical endpoints associated with the growth hormone-related disease, disorder, or condition. The effective amount produces a beneficial effect in aiding in the treatment (e.g., cure or reduction of severity) or prevention (e.g., reduction of the likelihood of onset or severity) of the growth hormone-related disease, disorder, or condition. In some cases, methods for achieving beneficial effects include administering a therapeutically effective amount of the peptide drug to treat subjects with growth hormone-related diseases, disorders, or conditions, including but not limited to congenital or acquired GH deficiency in adults and children, Turner syndrome, Predwell syndrome, chronic renal failure, intrauterine growth retardation, idiopathic short stature, AIDS-related wasting, obesity, multiple sclerosis, aging, fibromyalgia, Crohn's disease, ulcerative colitis, muscular dystrophy, low muscle mass (e.g., fitness), low bone mineral density, or any other indication for which GH can be utilized (but not necessarily a deficiency in endogenous growth hormone levels in the subject).

[0021] Osteoporosis can be caused by or associated with a variety of factors. For women, especially postmenopausal women, low body weight and sedentary lifestyles are risk factors for osteoporosis (loss of bone mineral density, leading to increased risk of fractures). Individuals with any of the following characteristics may be candidates for peptide therapy: postmenopausal women not taking estrogen or other hormone replacement therapy; tall (over 5 feet 7 inches) or thin (less than 125 pounds) postmenopausal women; men with clinical conditions associated with bone loss; individuals using medications known to cause bone loss, including glucocorticoids such as prednisone, various anticonvulsants such as phenytoin (Dilantin), certain barbiturates, or high doses of thyroid replacement therapy; individuals with type 1 diabetes, liver disease, kidney disease, or a family history of osteoporosis; individuals with high bone turnover (e.g., excessive collagen in urine); individuals with thyroid conditions such as hyperthyroidism; individuals with fractures following minor trauma; individuals with vertebral fractures or other radiographic evidence of osteoporosis.

[0022] For oral, sublingual, and sublingual administration, acceptable solid dosage forms include: powders, suspensions, granules, tablets, pills, capsules, soft capsules, and microcapsules. Preparation methods include, for example, mixing the polypeptides of the present invention with at least one additive, such as starch or other additives. Suitable additives include: sucrose, lactose, cellulose sugars, mannitol, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, or glycerides. Alternatively, oral dosage forms may contain other components to aid administration, such as inert diluents, or lubricants such as magnesium stearate, or preservatives such as parabens or sorbic acid, or antioxidants such as ascorbic acid, vitamin E, or cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, or aromatizers. Tablets and pills may also be further treated with suitable coating materials known in the art.

[0023] The composition can be encapsulated or injected as a form released to a target tissue site (e.g., bone). In some embodiments, the medicament of the present invention includes a matrix capable of releasing peptides to a target tissue site (e.g., bone), providing a structure for developing tissue and, ideally, being absorbed into the body. For example, the matrix can provide a slow release of the peptide. Such a matrix can be formed from substances already used in other implantable medical applications.

[0024] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms contain inert diluents (such as water or other solvents), solubilizers and emulsifiers commonly used in the art (such as alcohol, isopropanol, ethyl carbonate, ethyl acetate, phenylpropanol, benzyl benzoate, propylene glycol, 1,3-butanediol, oils (particularly cottonseed, peanut, corn, germ, olive, castor, and sesame oils), glycerin, tetrahydrofuran, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions also include adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings, colorings, fragrances, and preservatives.

[0025] This invention also covers the administration of peptides used according to the methods provided herein in combination with other therapeutic and pharmaceutical compositions for the treatment of growth hormone-related diseases, disorders, and conditions, or conditions for which growth hormone is a combination therapy (e.g., insulin resistance and poor glycemic control). Such compositions include, for example, DPP-IV inhibitors, insulin, insulin analogs, PPARγ agonists, dual-effect PPAR agonists, GLP-1 agonists or analogs, PTP1B inhibitors, SGLT inhibitors, insulin secretion promoters, RXR agonists, glycogen synthase kinase-3 inhibitors, insulin sensitizers, immunomodulators, β-3 adrenergic receptor agonists, Pan-PPAR agonists, 11β-HSD1 inhibitors, biguanides, α-glucosidase inhibitors, chloroanisidines, thiazolidinediones, sulfonylureas, and other diabetes medications or antihypertensive medications, calcium channel blockers, and related products known in the art. In some implementations, the administration of the peptide allows for the use of lower doses of the co-administered pharmaceutical composition to achieve similar clinical effects or measurement parameters for a subject’s disease, ailment, or condition.

[0026] On the other hand, the present invention provides a kit that facilitates the use of peptides. The kit includes the pharmaceutical composition provided herein, a label identifying the pharmaceutical composition, and instructions regarding the storage, reconstitution, and / or administration of the pharmaceutical composition to a subject. In some embodiments, the kit preferably includes: (a) an amount of peptide sufficient to treat a disease, condition, or ailment when administered to a subject with appropriate need; and (b) a pharmaceutically acceptable carrier; together forming a formulation for immediate injection or reconstitution with sterile water, buffer, or glucose; a label identifying the peptide drug and storage and handling conditions, and an insert indicating the approved indications for the drug, instructions regarding the reconstitution and / or administration of the peptide drug for the prevention and / or treatment of the approved indications, appropriate dosage and safety information, and information identifying the batch number and expiration date of the drug. In another embodiment described above, the kit may include a second container capable of carrying a suitable diluent for the peptide composition, which will provide the user with an appropriate concentration of the peptide to be delivered to the subject.

[0027] Beneficial effects This invention discloses a composition comprising polypeptides and its use in children's bone growth. The invention also isolates and screens growth-promoting polypeptides from foal leg bones that effectively promote osteoblast proliferation. These growth-promoting polypeptides effectively promote the increase of rat femur weight, significantly improve bone strength and bone density, increase ALP activity to promote osteoblast maturation and initiate calcification, and significantly increase the calcium ion content in bone, exhibiting good effects in promoting bone growth and improving bone strength. Attached Figure Description

[0028] Figure 1 Figure 1 shows the results of ALP enzyme activity detection in cells of each group.

[0029] Figure 2 Images showing the results of femoral strength measurements for each group. Detailed Implementation

[0030] Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0031] Example 1: Screening and preparation of growth-promoting peptides The foal leg bones were crushed, and the surface blood was rinsed off with ultrapure water at a ratio of 400g:800mL. The mixture was then boiled for 2 hours, filtered through double-layered gauze, and allowed to stand at 4°C for 2 days to remove the upper layer of fat. After thawing, the mixture was centrifuged at 5000 rpm for 30 minutes, and the supernatant was collected. Alkaline protease was added at a ratio of 5000 U / g, and the mixture was enzymatically hydrolyzed at pH 10 and 50°C for 4 hours. After hydrolysis, the pH was adjusted to 7.0, and the enzyme was inactivated at 100°C for 10 minutes. After cooling, the mixture was centrifuged at 10000×g for 15 minutes, and the supernatant was collected. The supernatant was then ultrafiltered using a Minimate Tangential Flow Filtration system to obtain foal polypeptide fractions with a molecular weight of less than 15000. These fractions were identified as effectively promoting osteoblast proliferation. The component was further concentrated and freeze-dried. The dried product was separated by G-25 gel electrophoresis, yielding seven elution peaks. Peak 2, exhibiting the highest osteoblast proliferation-promoting activity, was collected. After concentration and freeze-drying, it was further separated by reversed-phase high-performance liquid chromatography (HPLC) gradient elution, yielding four elution peaks. The component with the strongest proliferative activity was selected. HPLC analysis confirmed that the extract was a single component. The component was identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1. This polypeptide sequence was synthesized using solid-phase synthesis technology, and the purity was verified to be 99.2% by HPLC-MS.

[0032] Example 2: Activity Identification of Growth-Promoting Peptides Mouse preosteoblastic cell line MC3T3-E1 cells were used at a rate of 2×10⁻⁶ 4100 μL of each of the cells / mL was seeded into 96-well plates. After incubation at a constant temperature for 24 h, the original culture medium was removed. Subsequently, a series of growth-promoting peptide solutions of the identified in Example 1 (concentrations of 10, 50, 100, and 200 μg / mL) were prepared and added to each well at a concentration gradient of 200 μL. The plates were then incubated continuously at 37°C with 5% CO2 for 48 h. An equal volume of culture medium was added to the blank group, and EGF (10 ng / mL, catalog number HEGFP-05013, Cyagen (Suzhou) Biotechnology Co., Ltd.) was added to the positive control group. The proliferation rate was determined by the CCK-8 assay, and each experiment was performed in triplicate. The relative proliferation rate of pre-osteoblasts was calculated using the formula: relative cell proliferation rate (%) = [A sample / A blank] * 100. Where: A sample is the absorbance of the sample solution; A blank is the absorbance of the blank solution. The results are shown in Table 1.

[0033] Table 1. Effects of growth-promoting peptides on osteoblast proliferation.

[0034] As can be seen from Table 1, the growth-promoting polypeptide prepared in this invention has a dose-dependent effect on promoting the proliferation of osteoblasts, and has a good effect on promoting osteoblast proliferation.

[0035] Mouse preosteoblastic cell line MC3T3-E1 cells were used at a rate of 2×10⁻⁶ 4 Cells were seeded at a concentration of 100 μL / well in 96-well plates. After incubation at a constant temperature for 24 h, the original culture medium was removed. Subsequently, a series of growth-promoting peptide solutions of the identified in Example 1 (concentrations of 10, 50, 100, and 200 μg / mL) were prepared and added to each well at a concentration of 200 μL. The plates were then incubated at 37°C with 5% CO2 for 9 days. An equal volume of culture medium was added to the blank group, and EGF (10 ng / mL, catalog number HEGFP-05013, Cyagen (Suzhou) Biotechnology Co., Ltd.) was added to the positive control group. Equal amounts of cells were collected from each group and processed according to the instructions of the alkaline phosphatase (ALP) assay kit. The absorbance of each well was measured using an ELISA reader to determine the intracellular ALP activity. The results are shown below. Figure 1 As shown.

[0036] from Figure 1 As can be seen, with the increase of the concentration of the growth-promoting peptide, the activity of ALP enzyme gradually increased, and the difference compared with the blank group was statistically significant (P<0.05). This also indicates that the growth-promoting peptide of the present invention can increase ALP activity, promote osteoblast maturation and initiate calcification, and has a good effect on promoting bone growth.

[0037] Example 3 Animal experiments with growth-promoting peptides Four-week-old weaned Wistar rats, clean grade, weighing 62-72g, male, 10 rats per group.

[0038] The low-calcium control group (model group) was fed low-calcium rat food (150mg calcium / 100g feed) daily, and was given deionized water and 5ml (kg.d) of distilled water by gavage.

[0039] Control group: fed normal rodent food, given deionized water, and gavaged with 5 ml (kg.d) of distilled water.

[0040] Positive control group: fed low-calcium rat food (150mg calcium / 100g feed) daily, drinking deionized water, and gavaged with 50mg calcium carbonate (kg.d) distilled water.

[0041] High-dose growth-promoting peptide group: fed low-calcium rat food (150mg calcium / 100g feed) daily, drinking deionized water, and administered 10mg (kg.d) of the growth-promoting peptide of Example 1 by gavage in distilled water.

[0042] Low-dose growth-promoting peptide group: Rats were fed low-calcium rat food (150 mg calcium / 100 g feed) daily, and given deionized water to drink. They were also given 5 mg (kg·d) of the growth-promoting peptide from Example 1 via gavage. The rats in each group were fed for 3 months.

[0043] Rats in each group were sacrificed, and the right femur was dissected, ensuring the entire right femur, including the femoral head, was dissected. The femurs were then baked in a 105°C oven until constant weight, and the dry weight of the bone was measured. The results are shown in Table 2.

[0044] Table 2. Effects of growth-promoting peptides on rat femur weight

[0045] As shown in Table 2, compared with the model group, the positive control group, the high-dose growth-promoting peptide group, and the low-dose growth-promoting peptide group had significant differences in their effects on rat femoral weight (P<0.05). Moreover, both the high-dose and low-dose growth-promoting peptide groups had a better effect on promoting rat bone growth than the positive control group.

[0046] Remove the left femur from each group of rats, locate the midpoint of the femur, and then use a bone strength tester to measure the maximum load on the femur at the midpoint. After removing the rat skeleton, remove any attached muscles and ligaments, keep it clean and dry, place it on a rack, cover it with a protective cover, and run the instrument. The display screen will show the pressure the skeleton experiences when it breaks at the midpoint, i.e., the maximum lateral load on the skeleton. The results are as follows: Figure 2 As shown.

[0047] Depend on Figure 2It was found that, compared with the blank group, the femoral strength of the model group was significantly reduced due to calcium deficiency (P<0.01). Compared with the model group, the positive control group, the high-dose growth peptide group, and the low-dose growth peptide group showed significant differences in their effects on rat femoral weight (P<0.05). In particular, the femoral strength of the high-dose growth peptide group reached (2.71±0.02) kg, which was significantly enhanced compared with (2.03±0.03) kg in the model group. This also indicates that growth peptides can significantly improve bone strength and bone mineral density.

[0048] The calcium content in the bones of rats in each group was determined. The calcium ion content in the bones was determined according to the second method of the National Food Safety Standard for the Determination of Calcium in Food - EDTA titration. Calcium forms a complex with the indicator. When titrated with EDTA, at the equivalence point, within an appropriate pH range, EDTA forms a stable metal complex with calcium, and the indicator is released. At this time, the solution shows the color of the free indicator. Therefore, the calcium content is calculated based on the amount of EDTA used. The calcium content in the sample is calculated according to formula (1): Calcium content = [T×(V1-V0)×V2×1000] / (m×V3). Where: T - EDTA titer (mg / mL); V1 - volume of EDTA solution diluted 10 times consumed when titrating the sample solution (mL); V0 - volume of EDTA solution diluted 10 times consumed when titrating the blank solution (mL); V2 - final volume of the sample digest (mL); 1000 - conversion factor; M - sample mass or transfer volume (g or mL); V3 - volume of the sample solution used for titration (mL). The results are shown in Table 3.

[0049] Table 3. Effects of growth-promoting peptides on calcium ion content in rat bones.

[0050] As shown in Table 3, compared with the model group, the calcium ion content in the bones of rats in the positive control group, the high-dose group of growth-promoting peptide, and the low-dose group of growth-promoting peptide was significantly increased (P<0.05). This also shows that the growth-promoting peptide of the present invention can significantly increase the calcium ion content in bone and has a good effect on promoting bone growth and improving bone strength.

[0051] When implementing or testing embodiments of the present invention, optional methods and materials similar to or equivalent to those described in this specification may be used, although preferred methods, apparatus, and materials are described in this specification. However, before describing the materials and methods of the present invention, it should be understood that the specific sizes, shapes, dimensions, materials, methods, means, etc., described in this specification can be modified according to conventional experimental methods and for optimization purposes; therefore, the present invention is not limited to these. Furthermore, it should be understood that the technical terms used in this specification are only used to describe specific types or embodiments and are not intended to limit the scope of the present invention, which is limited only by the scope of the appended claims.

Claims

1. A growth-promoting polypeptide, characterized in that... The amino acid sequence is shown in SEQ ID NO:

1.

2. A pharmaceutical composition for promoting bone growth, characterized in that... It contains a growth-promoting polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

1.

3. Use of the growth-promoting peptide in the preparation of a pharmaceutical composition for promoting bone growth, wherein the amino acid sequence of the growth-promoting peptide is shown in SEQ ID NO:

1.

4. The use as described in claim 3, characterized in that... The drug contains a pharmaceutically acceptable carrier or excipient.

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