CN1163262C - Osteogenic growth peptide pharmaceutical composition, preparation method and application - Google Patents

Abstract

The invention relates to a pharmaceutical composition containing osteogenic growth peptide (Osteogenic Growth Peptide, OGP). The OGP of the present invention is artificially synthesized and has the same structure as the naturally occurring OGP in serum. The pharmaceutical composition has the functions of promoting the proliferation of osteoblasts and fibroblasts, promoting the osteogenesis of the body and improving the density of trabecular bone, preventing bone mass Lost effect. It has broad application prospects in the prevention and treatment of osteoporosis and the treatment of fractures. The invention provides a preparation method.

Description

Osteogenic growth peptide pharmaceutical composition, preparation method and application

The invention belongs to biological preparations, and specifically relates to a pharmaceutical composition containing osteogenic growth peptide, a preparation method and application.

Osteogenic Growth Peptide (Osteogenic Growth Peptide) is a 14-amino acid polypeptide that can promote the growth of bone cells discovered in humans and animals in recent years. In addition to the osteogenic response, there is also a systemic osteogenic response [Bab I, et al. (1985) Calcif Tissue Int. 37: 551-555; Foldes J, et al. (1989) J Bone Miner Res. 4: 643-646]. In-depth experimental research shows that the healed bone marrow tissue can release several osteogenic factors into the blood circulation, resulting in enhanced osteogenic response throughout the body. It has been confirmed, separated and purified by research, and one of the factors is osteogenic growth peptide (Osteogenic Growth Peptide, OGP) [Bab I, et al. (1988) Endocrinology 123: 345-352; Bab I, et al. (1992) EMBO J 11: 1867-1873]. Further studies have shown that the amino acid sequence of OGP is ALKRQRGTLYGFGG, which is completely consistent in mice and humans, and its functional properties are also the same.

Relevant in vitro experimental studies have shown that artificially synthesized OGP (consistent with the OGP structure naturally present in serum) acts on ROS, MC3T3, E1 and NH3T3 fibroblasts of the osteoblast cell line, and has the ability to promote the proliferation of osteoblasts and fibroblasts , Increased activity of alkaline phosphatase. In addition, it was also found that when the concentration of OGP in serum exceeds a certain critical point, its effect turns to inhibition instead, that is to say, the effect of OGP shows a bidirectional performance.

The relevant in vivo experimental reports and our pre-experimental studies have shown that artificially synthesized OGP injected into rats can promote the proliferation of osteoblasts, promote the osteogenesis of the body, and increase the density of trabecular bone.

The object of the present invention is to provide a pharmaceutical composition of an OGP polypeptide having the following general structural formula or a pharmaceutically acceptable salt thereof:

R ₁ -Ala-Leu-Lys-Arg-Glu-Arg-Gly-Thr-Leu-Tyr-Gly-Phe-Gly-Gly-R ₂

Wherein R ₁ represents a terminal selected from CH ₃ COO- or NH ₂ -; R ₂ represents a terminal of -NH ₂ or -OH.

The pharmaceutical composition mentioned herein is composed of OGP polypeptide as the active ingredient and a pharmaceutical carrier, and can contain OGP in any proportion of 0.01-99.99% and pharmaceutical carrier in an arbitrary ratio of 99.99%-0.01% to form a 100% composition .

Conventional peptide chemical synthesis techniques can be used to synthesize OGP, the main component of the OGP pharmaceutical composition of the present invention, in a solid-phase or liquid-phase method, but it is preferred to use a solid-phase synthesis method (see, for example, Birr, C., Aspect of the Merrifield Peptide Synthesis, Springer-Verlag, Heidelberg, 1978; Stewart et al., Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem.Co., Rockford, IL, 1984; Barany, G. and Merrifield R.B. in The Peptides, Vol. .2; Gross, E. & Meienhoffer J., eds., Academic Press, New York, pp3-284, 1979). It can also be produced by recombinant DNA technology.

Another object of the present invention is to provide a method for preparing the above-mentioned osteogenic growth peptide (OGP polypeptide). According to the designed and given amino acid sequence, an appropriate activator and condensing agent are used to convert The C-terminal amino acid residues of the protected peptide chains are linked to a solid support. The solid phase support described therein is divinylbenzene cross-linked polystyrene, polyacrylamide resin, Kiseseguhr polyamide, glass fiber or beads with controlled pore size, cellulose, polypropylene membrane and acrylic coated polystyrene carrier. Preferred solid supports include divinylbenzene crosslinked polypropylene resins, chloromethyl resins, methylol resins, p-acetylmethyl resins, benzhydrylamine (BHA) resins, 4-methylbenzhydrylamine (MBHA) resin, 4-(2',-4'-dimethoxyphenylaminomethyl)-phenoxymethyl resin, 2,4-dimethoxybenzylaminoamine resin and (2, 4-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHA resin (ie Rink Amide MBHA resin).

A preferred protecting group for the α-amino acid is the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

When using the Fmoc system for solid-phase synthesis, the following protected amino acid residues can be selected: Fmoc-Leu, Fmoc-Thr (But), Fmoc-Gly, Fmoc-Lys (Boc), Fmoc-Gln, Fmoc-Tyr ( But) and Fmoc-Arg (PMC). When using the Boc system for solid-phase synthesis, the following protected amino acid residues can be selected: Boc-Lys (ClZ), Boc-Thr (Bzl), Boc-Leu, Boc-Gly, Boc-Arg (Tos), Boc - Gln, Boc-Tyr (Brz).

Various coupling agents and coupling methods known in the field of peptide chemical synthesis can be used to couple the amino acid residues, for example, diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC) can be used ) for direct coupling, or via active esters such as pentafluorophenyl ester, or using Fmoc-amino acid-carboxylic anhydride. Hydroxybenzotriazole (HOBt) or 7-azahydroxybenzotriazole (HOAt) or 2-(1H-benzotriazol-1-yl), 1,1,3,3-tetra Urea hexafluorophosphate (HBTU) activates amino acids.

The synthesis of the above-mentioned human calcitonin analogue polypeptide can be completed by manual method or synthesizer, but for convenience, it is best to use a commercially available peptide automatic synthesizer, such as the ABI 430A type produced by Applied Biosystems or ABI 431A type peptide. In the Fmoc synthesis system, the first amino acid at the C-terminus (C-terminal Fmoc-Pro) is first installed in a reactor containing a hydroxyl resin, and extended from the C-terminus to the N-terminus one by one according to the given amino acid sequence. Among them, the α-amino groups of various amino acids are protected by Fmoc. The side chain protecting groups of each amino acid are: Thr is protected by tert-butyl (But); Arg is protected by Mtr; Lys is protected by Boc; Tyr is protected by But. HOBT and DCC were used to activate the carboxyl terminus of each amino acid and complete the condensation between amino acid molecules.

After the synthesis is completed, the synthesized OGP peptide is cleaved from the resin with or without a reducing agent (such as mercaptoethanol) in aqueous trifluoroacetic acid solution and the protective groups are removed. The crude peptide can be separated by filtration or ether precipitation. After lyophilization of the resulting product solution, the desired peptide is purified by gel filtration or reverse phase high pressure liquid chromatography.

In the solid phase synthesis of Boc protected systems, PAM resins are typically used. The difference is that various amino acid α-amino groups are protected by Boc. The amino acid side chain protection groups are: Lys uses oxybenzyloxycarbonyl (CL-Z) to protect the ε-amino group; Arg uses tosyl (Tos) to protect the side chain hydroxyl group; Thr uses benzyl (Bzl) to protect the side chain hydroxyl group; Tyr uses BrZ Protect. In a cycle of deprotection, neutralization, coupling, the protecting group (Boc) was removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA)/dichloromethane. After the peptide chain condensation is completed, treat with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0° C. for 1-2 hours to cut the peptide chain from the resin and remove all protective groups at the same time. Peptides are extracted with 10%-50% acetic acid (containing a small amount of mercaptoethanol), and the peptides are oxidized. After lyophilization, it was further separated and purified by molecular sieve Sephadex G10 or Tsk-40f, and then purified by high-pressure liquid chromatography _C8 column to obtain HPLC pure OGP polypeptide.

The OGP peptides prepared by the above method or by recombinant DNA technology can be prepared into pharmaceutically acceptable salts, especially base addition salts, by known methods. For example, base addition salts of acidic amino acids can be prepared by treating these peptides with an appropriate base according to methods well known to those skilled in the art.

The OGP of the present invention can be made into a pharmaceutical composition suitable for a specific clinical administration according to conventional methods known in the field of pharmacy. For example, appropriate carriers or diluents, such as water, physiological saline, and isotonic glucose solution, can be added to the calcitonin analogs of the present invention to prepare solutions, injections, emulsions, drops, etc. that can be administered through routes other than the gastrointestinal tract. Nasal agents, eye drops. Excipients or carriers such as starch, lactose, talcum powder, sucrose, glucose or glycerin, liquid paraffin, liposome or gelatin can also be added, and the OGP of the present invention can be made into suppositories and tablets that can be administered by gastrointestinal route. agent, powder, granule, capsule or liposomal encapsulation. In addition to the active ingredients and appropriate carriers or excipients, these preparations can also add some other auxiliary ingredients as needed, such as one or more diluents, fillers, emulsifiers, preservatives, surfactants, absorption Accelerators, buffers, fragrances and colorants.

Another object of the present invention is to provide the above pharmaceutical composition which can improve the activities of osteoblasts and fibroblasts, increase bone mass and prevent bone loss in humans and mammals. And the application in the preparation of medicines for preventing and treating osteoporosis, promoting bone healing of fractures, shortening healing time and wound healing.

The results of in vivo experiments that we utilize the rat osteoporosis model to do show that compared with salmon calcitonin, which is commonly used clinically and has a strong effect of improving bone mass and preventing bone loss, the OGP pharmaceutical composition of the present invention It can improve the activity of osteoblasts and fibroblasts, and at the same time promote the increase of bone mass and prevent bone loss. In comparison, salmon calcitonin mainly increases bone mass by inhibiting the activity of osteoclasts, while the OGP pharmaceutical composition of the present invention mainly promotes the activity of osteoblasts, improves bone mass through the increase of osteogenesis, and has the effect of improving bone mass. Peak bone mass, slow down the bone loss time and other advantages. It can be used to prevent and treat fracture healing and osteoporosis, promote bone healing after fracture and wound healing. In addition, the main components that exert medicinal effects in the pharmaceutical composition of OGP of the present invention have the same structure as that of naturally occurring human OGP, thereby significantly avoiding possible immunogenicity after long-term use.

As mentioned above, the OGP pharmaceutical composition of the present invention can significantly improve the activity of osteoblasts and fibroblasts, while promoting the increase of bone mass and preventing bone loss. It is superior to salmon calcitonin, which is commonly used clinically at present. Compared with salmon calcitonin, it mainly inhibits the activity of osteoclasts to increase bone mass, while the OGP drug combination of the present invention mainly promotes the activity of osteoblasts to increase bone mass through the increase of osteogenesis. It has the advantages of increasing the peak bone mass and slowing down the time of bone loss.

Osteogenic Growth Peptide Promotes Bone Mass in Animal Models of Osteoporosis

This experiment observes the effect of osteogenic growth peptide (OGP) on the increase of bone mass, and finds a safe and effective drug for the treatment of osteoporosis.

50 SD rats, 12 weeks old, with an average body weight of 240 grams, were raised for 6 months to become osteoporosis models through low-calcium diet (Ca: 0.1%, P: 0.6%), and then randomly divided into 5 groups, Ten rats in each group were fed with common diet (Ca: 1.1%, P: 1.1%). Grouping experiments are as follows: Group A (low dose OGP) 0.2ug/0.5ml; Group B (high dose OGP) 2ug/0.5ml; Group C (salmon calcitonin) 0.1ug/0.5ml; Group D (normal saline) 0.5 ml control, each subcutaneous injection every other day, a total of 12 weeks. Then perform the following detection.

1) Bone density measurement: 6-7 animals were randomly selected from each group, a total of 33 animals were given intraperitoneal anesthesia with 0.25% pentobarbital sodium (50 mg/kg body weight), and the level of lumbar spine 3, right femoral neck and tibia were measured respectively. Bone mineral density (BMD) at the top.

2) 5ml of blood was collected from the heart, and serum alkaline phosphatase (AKP) and osteocalcin (BGP) were measured respectively.

3) Bone histomorphological measurement: 14 days before the rats were sacrificed, they were intraperitoneally injected with 0.25% tetracycline hydrochloride (35ml/kg) for 2 days every day, and then injected with the same dose for 2 days 4 days before the sacrifice. After intraperitoneal anesthesia with excessive pentobarbital sodium, the 3rd lumbar vertebral body was intercepted, soft tissues were removed, fixed with acetone, dehydrated with ethanol with an ascending gradient, embedded in methyl methacrylate, and sliced without decalcification on the pyramidal surface, thickness There are two kinds of 12μ and 5μ. 12μ sections were used for unstained fluorescence observation, and 5μ sections were stained with 1% toluidine blue. Sections were observed with an Olympus Vanox-1 microscope. Structural measurement parameters were analyzed with automatic graphics software Mias-300: ① TBV (%), the volume of trabecular bone in a certain slice; ② Average width of trabecular bone, MTT (μm); ③ Average thickness of bone cortex, MTC (μm) .

4) Scanning electron microscope observation: Take the 3rd lumbocone, remove the soft tissue, saw it cross-sectioned, rinse with normal saline, first double-fix with 2.5% glutaraldehyde and 1% osmic acid, dehydrate with serial alcohol, and replace with n-pentyl acetate , _CO2 critical point drying (HCP-2 type), ion sputtering (IB-3 type) spraying gold, and then observed with a Hitachi S-520 scanning electron microscope.

5) Transmission electron microscope observation: the right femoral head was sectioned along the frontal plane, fixed with 2.5% glutaraldehyde for 2 hours, dehydrated with gradient alcohol and acetone, embedded with epoxy resin, sectioned with LKB-1 ultrathin microtome, JEM-1200EX transmission electron microscope observation.

6) Statistical processing: All data were subjected to analysis of variance and t-test.

The detection indicators are as follows:

1. Body weight measurement results of SD rats at the 12th week:

Table 1 Body weight measurement results of each group (g)

Group A B B C D

Don't n=10 n=10 n=10 n=10

Average 446.5±21.8 445.5±21.2 447.0±24.4 449.0±26.4

It can be seen from Table 1 that there is little difference among groups A, B, C and D. After t test, P>0.05, there is no statistical difference, indicating that there is no significant change in the body weight of rats in each group after the experimental medication.

2. Bone density measurement results:

      Table 2 BMD values of femur, tibia, and lumbar spine in each group (X±SD, g/cm)

Group Femur Tibia Lumbar

A(n＝7) 0.2548±0.0177 0.2623±0.0234 0.2434±0.0270

B(n＝7) 0.2742±0.0264 0.2649±0.0258 0.2816±0.0667

C(n＝6) 0.2635±0.0517 0.2997±0.0416 0.2675±0.0320

D(n＝6) 0.1741±00.0241 0.1686±0.0177 0.1880±00093

It can be seen from Table 2 that the BMD values of femur, tibia, and lumbar spine in group D were significantly lower than those in groups A, B, and C, and the t-test showed a statistically significant difference, P<0.01. However, there is no statistical difference among groups A, B, and C after t test, P>0.05. It shows that after the experimental medication, the bone mass of the control group is obviously loose, while the bone density of the treatment group is increased, suggesting that the treatment is effective.

3. Serum alkaline phosphatase (AKP) and osteocalcin (BGP) measurement results:

Table 3-1 Measurement results of serum alkaline phosphatase (AKP) in each group (X±SD, U/L)

Group A B B C D

Don't n=10 n=10 n=10 n=10

Average 169.7±16.4 165.8±11.7 100.8±9.3 83.4±9.5

It can be seen from 3-1 that groups A and B are significantly higher than group D, P<0.001, and there is a statistically significant difference. At the same time, groups A and B were also higher than group C, P<0.01, and there was a significant difference. But between group C and group E, P>0.05, no difference. It shows that osteogenic peptide is more effective than salmon calcitonin in increasing the content of AKP enzyme, but salmon calcitonin has little effect on increasing the content of AKP enzyme.

Table 3-2 Measurement results of serum osteocalcin (BGP) in each group (X±SD, ng/ml)

Group A B B C D

Don't n=10 n=10 n=10 n=10

Average 2.17±0.20 2.02±0.19 1.43.8±0.15 1.21±0.09

It can be seen from Table 3-2 that there is a statistical difference between groups A and B and group D, P<0.05. There was no difference between group C and group D, P>0.05. It shows that osteogenic peptide can increase the content of osteocalcin (BGP), while salmon calcitonin has little effect.

4. Bone histomorphometry results:

Table 4 Histomorphometric results of lumbar vertebrae in each group (X±S, n=4)

Group TBV MTT MTC

A 37.16±2.56 152.35±8.49 250.93±20.81

B 38.35±2.95 145.10±10.05 249.66±20.72

C 37.62±2.89 147.83±10.60 248.57±21.14

D 15.08±1.38 64.66±4.93 238.69±14.37

It can be seen from Table 4 that there is a highly significant difference between each group A, B, C and D group, P<0.001. However, among groups A, B, and C, P>0.05, there is no difference. It shows that the treatment group is better than the control group in improving bone mass and preventing bone loss.

5. Observation results of scanning electron microscope: the bone trabeculae in the normal saline control group were damaged and absorbed, showing that the bone trabeculae became thinner, sharper, and broken, and the gaps between the bone trabeculae increased. absorption (Figure 1). In the OGP treatment group, the bone trabeculae were thick, complete and continuous (Fig. 2).

6. The results of transmission electron microscope observation: in the normal saline control group, the bone cells were mostly in the degenerative phase, with nuclear pyknosis, a large nucleus/plasma ratio, more vacuoles, decreased cytoplasm, increased floc in the lacuna gap, and There are osmiophilic lamellae (Figure 3). In the OGP treatment group, most of the bone cells were in the osteogenic phase, with abundant cytoplasm, a small nucleus/plasma ratio, and small lacuna gaps (Fig. 4).

7. Observation results by light microscope: the light microscope performance of the trabecular bone section without decalcification in the normal saline control group, the trabecular bone became thinner (Fig. The trabeculae are thick and continuous (Figure 6). In the normal saline control group without decalcification, the fluorescent markers were less and darker (Figure 7), while in the OGP treatment group without decalcification, the fluorescent markers were more and brighter (Figure 8).

Synthetic OGP can promote the proliferation of osteoblasts and fibroblasts, increase the activity of osteoblast AKP, increase the volume of bone formation and trabecular bone in the whole body, prevent bone loss, and induce new bone in the treatment of osteoporosis. Accumulation improves the biomechanical properties of bone. Promote wound healing after body injury, promote healing of damaged bone marrow, and provide a new way for osteoporosis and fracture treatment.

Figure 1 The trabecular bone became thinner and the gap increased in the normal saline control group (SEM, X100)

Figure 2 The trabeculae in the OGP treatment group are thick, with small gaps and complete continuity (SEM, X100)

Figure 3 Osteocytes in the control group showed degenerative phase, nuclear pyknosis, less cytoplasm, and enlarged lacuna (TEM, X6000)

Figure 4 Osteocytes in the OGP treatment group showed osteogenic phase, more cytoplasm and organelles, and narrow lacuna (TEM, X6000)

Figure 5 The light microscope appearance of the trabecular bone section without decalcification in the control group, the trabecular bone became thinner (stained with toluidine blue, X100)

Figure 6 The light microscope appearance of the trabecular bone section without decalcification in the OGP treatment group, the trabecular bone is thick and continuous (stained with toluidine blue, X100)

Figure 7 The section without decalcification in the control group has less and darker fluorescent markers (fluorescence microscope, X100)

Figure 8 The section without decalcification in the OGP treatment group showed more and brighter fluorescent markers (fluorescent microscope, X100)

The dosage of the pharmaceutical composition of the present invention will depend on various factors such as the nature and severity of the disease to be treated, the patient's body weight, general health condition, and administration route. Generally, the dosage of the pharmaceutical composition of the present invention is about 0.5 to 5 micrograms per kilogram of body weight per day, preferably 1-2 micrograms per kilogram of body weight.

The pharmaceutical composition of the present invention can be used through various conventional routes of administration, for example, it can be administered through routes such as gastrointestinal tract, subcutaneous, intradermal, intranasal, intravenous, intramuscular, rectal, and intraocular, but preferred The route of administration is intramuscular injection, subcutaneous injection, nasal spray or oral administration.

Example 1:

(1) Synthesize the following structure (1) OGP peptide with the Boc system:

NH ₂ -Ala-Leu-Lys-Arg-Glu-Arg-Gly-Thr-Leu-Try-Gly-Phe-Gly-Gly-OH(1)

The starting PAM resin (1 g, 0.5 mmol) was placed in the ABI 430 automatic peptide synthesizer reactor, and each protected amino acid was condensed from the C-terminus to the N-terminus as shown in the table below. After the completion of each reaction step, the reagents were removed from the resin by filtration under positive pressure of nitrogen. The condensation reaction between the first protected amino acid Boc-Gly at the C-terminal and the resin is carried out in steps 5 to 14 in the following table, and the condensation of other amino acids is carried out in steps 1 to 14. step Reagent Reagent dosage (ml) repeat times Mixing time (minutes) 1 DCM 30 3 2 2 TFA/DCM 30 1 3 TFA/DCM 30 15 4 DCM 30 3 2 5 DIEA/DMF 30 1 6 DIEA/DMF 30 10 7 DMF 30 3 2 8 DCM 30 3 2 9 Boc-amino acid, 2 mmol HOBT/DCC in DMF 20 120 10 DCM 30 3 2 11 DMF 30 3 2 12 DCM 30 3 2 13 Boc-amino acid, 1 mmol HOBT/DCC in DMF 20 120 14 Repeat steps 10-12 15 Repeat steps 1 to 14 to extend the C-terminal amino acids to the N-terminal one by one

Each amino acid condensation reaction was carried out for an average of 2 hours. Before entering the next amino acid condensation cycle, use the ninhydrin method to measure the free amino group to determine whether the condensation is complete [Kaiser ET, et al. Anal. Biochem. 34: 595-598, 1969]. According to the above reaction steps, the peptide chain is extended from the C-terminal amino acid to the N-terminal one by one to obtain the target peptide chain resin, and the resin is removed from the reactor and then vacuum-dried.

(2) Hydrogen fluoride (HF) cleavage reaction of peptide chain resin

Dry peptide resin (1 g) and p-cresol (ml) were placed in the reactor. Put a dry ice/ethanol cooling bath outside the container to cool, and collect about 20ml of liquid HF, then stir the reaction under the ice bath for 1 hour. After the reaction was complete, the ice bath was removed and HF was evaporated under vacuum. Anhydrous ethyl acetate was added into the reactor to wash the resin three times (30ml each time), and then the reagent was filtered off. The crude peptide was extracted twice with 50% acetic acid (5ml each time). The resulting filtrates were combined, diluted with 100 ml of deionized water, and freeze-dried to remove the solvent.

(3) Purification of OGP crude peptide

Weigh 50 mg of the above crude product, dissolve it in 1 ml of 1N acetic acid, pass the resulting solution through a Sephadex G10 column (2.0×25 cm), and elute with 1N acetic acid for desalting. After collecting all the peaks, freeze-dry to obtain 37 mg of desalted crude product. Further, the obtained desalted product was passed through a high-pressure liquid-phase reverse chromatography column Beckman Octyl C18, 10mm×25cm. The main peaks were collected, combined, acetonitrile removed in vacuo, and lyophilized. The purity of the purified sample is over 95%, and the results of amino acid composition analysis and mass spectrometry analysis are in line with theoretical values.

Embodiment 2: (1) synthesize OGP peptide with Fmoc system:

Starting Rink Amide hydroxyl resin (1 g, 0.45 mmol) was charged in a 30 ml vial and treated with DCM for 30 minutes. Condensate each protected amino acid one by one from the C-terminus to the N-terminus as shown in the table below. After the completion of each step of the reaction, the reagents are removed by negative pressure filtration and separated from the peptide resin. The condensation reaction between the first protected amino acid Fmoc-Gly at the C-terminal and the resin is carried out according to steps 1-15 in the following table, and the condensation of other amino acids is carried out according to steps 1-15. step Reagent Reagent dosage (ml) repeat times Mixing time (minutes) 1 DMF 20 3 2 2 Hexahydropyridine/DCM 20 5 3 Hexahydropyridine/DCM 20 15 4 DCM 20 3 2 5 DMF 20 2 6 Dioxane/H ₂ O 20 2 7 DMF 20 3 2 8 DCM 20 3 2 9 Fmoc-AA, 1.5mmol HOBT/DCC in DMF 15 120 10 DCM 20 3 2 11 ethanol 20 3 2 12 DMF 20 3 2 13 DCM 20 3 2 14 Fmoc-AA, 0.5mmol HOBT/DCC dissolved in DMF 15 120 15 Repeat steps 10 to 13 16 Repeat 1 to 15 to complete the extension of the C-terminal amino acids to the N-terminus one by one

Each amino acid condensation reaction was carried out for an average of 2 hours. Before entering the next amino acid condensation cycle, use the ninhydrin method to measure the free amino group to determine whether the condensation is complete. According to the above reaction steps, the peptide chain is extended from the C-terminal amino acid to the N-terminal one by one to obtain the target peptide chain resin, and the resin is removed from the reactor and then vacuum-dried.

(2) TFA cleavage reaction of peptide chain resin

Dry peptide resin (0.5 g) and m-cresol (0.5 ml) were placed in the reactor and pre-cooled in an ice bath for 30 minutes. Add 0.5ml of water, 9ml of TFA and stir the reaction for 1 hour under ice bath condition. The reaction solution was filtered off, the residual resin was washed three times with a small amount of TFA, and then washed three times with DCM, and then the filtrates were combined. DCM and TFA were removed by evaporation under vacuum. When the liquid is basically drained and becomes viscous, add pre-cooled ethyl acetate, shake quickly and gradually precipitate the crude peptide in solid form and centrifuge to precipitate it.

(3) Purification method is the same as (3) method in the above-mentioned embodiment 1.

Embodiment 3, OGP freeze-dried product needle preparation method

OGP 100μg per ampere bottle (purity of over 98% identified by HPLC)

20% Mannitol 1ml

6% Low Molecular Dextran 9mg Low Molecular Dextran Specifications: Molecular weight 10,000-20,000 meets the quality standard for injection, dissolved in 0.5ml of ultra-pure water, freeze-dried to dilute the lyophilized injection with physiological saline, and the dosage is 1ml each time.

Claims (4)

1. the purposes of an osteogenic growth peptide, it is characterized in that, be used for preparing the medicine that improves osteocalcin content. 2. The use according to claim 1, characterized in that the medicine contains 0.01-99.99% osteogenic growth peptide and 99.99-0.01% pharmaceutical carrier, by weight. 3. The use according to claim 1, characterized in that the drug is also used to increase bone density. 4. The use according to claim 1, characterized in that the dosage of the drug is 0.2ug/240g body weight.

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