CN118791592A - A polyethylene glycol site-specifically modified teriparatide and its preparation method and application - Google Patents

Abstract

The invention discloses a polyethylene glycol site-specific modified teriparatide, a preparation method and application thereof, wherein polyethylene glycol is modified at least one site of 13, 26 or 27 sites of the teriparatide; the modified structure of polyethylene glycol is shown as follows: The half-life of the polyethylene glycol site-specific modified teriparatide in the rat body is obviously prolonged, which is 5 times that of the teriparatide, and the administration frequency of patients can be reduced; the pharmacodynamics effect of treating osteoporosis is superior to teriparatide under the same dosage.

Description

A polyethylene glycol site-specifically modified teriparatide and its preparation method and application

Technical Field

The present invention belongs to the technical field of teriparatide modification, and specifically relates to a polyethylene glycol site-specifically modified teriparatide, and a preparation method and application thereof.

Background Art

The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

With the aging of the population, osteoporosis is gradually becoming one of the common diseases in the world. Osteoporosis can reduce the strength of patients' bones and increase the risk of fractures. Osteoporotic fractures and increased bone fragility seriously reduce the quality of life of patients and bring economic burdens to patients and society. According to literature reports, among patients with hip fractures over the age of 50, about 20% of patients will die each year and 50% of patients will become disabled. This shows that effective diagnosis and treatment of osteoporosis are crucial for preventing fractures. According to their mechanism of action, osteoporosis treatment drugs can be divided into bone resorption inhibitors and bone formation promoters. Currently, bone resorption inhibitors used in clinical practice mainly include selective estrogen receptor modulators, calcitonin, estrogen, bisphosphonates and RANKL inhibitors, and bone formation promoters mainly include parathyroid hormone analogs (teriparatide and abaloparatide). Some studies have shown that teriparatide can improve bone density and bone biomechanical properties and accelerate fracture healing.

Teriparatide is the N-terminal active fragment of human parathyroid hormone, composed of 34 amino acids, generally referred to as PTH (1-34), with a half-life of 1 hour after subcutaneous injection. It has been approved worldwide for the treatment of osteoporosis in men and postmenopausal women. However, PTH (1-34) has the problem of rapid metabolism in the body and a short half-life, and requires daily injection, which brings great inconvenience to patients.

PEG is a linear or branched polymer with a terminal hydroxyl group, and its structural formula is HO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH. Monomethoxy PEG is often used to modify proteins and peptides because it has a single reactive group. Its structural formula is CH ₃ O-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH. PEG is a neutral amphiphilic polymer that can be dissolved in both aqueous and organic phases. Each of its monomers can bind 2-3 molecules of water, so the hydrodynamic radius of PEG is 5-10 times higher than that of globular proteins of the same molecular weight. Monomethoxy PEG is often further functionalized and used for PEG modification of peptides and proteins. PEG modification may occupy the active site or receptor binding site of the protein, or may change the conformation of the protein, ultimately leading to protein inactivation. This prompted researchers to look for site-specific PEGylation technology and produce mono-PEGylated products. Site-specific PEG modification is an effective strategy to introduce PEG into specific amino acid sites of different proteins or peptides. Currently, there are modification strategies such as introducing cysteine at the end of teriparatide to modify it with polyethylene glycol. However, when the peptide reacts with polyethylene glycol, the reaction conditions used will affect the activity of teriparatide.

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) can form 1,4-disubstituted triazoles with a fast reaction rate. It has been widely used in organic synthesis, polymer functionalization, drug discovery and chemical biology. However, the application of CuAAC in bioconjugation still has some limitations.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of the present invention is to provide a polyethylene glycol site-specifically modified teriparatide and a preparation method and application thereof.

In order to achieve the above object, the present invention is implemented through the following technical solutions:

In a first aspect, the present invention provides a polyethylene glycol site-specifically modified teriparatide, wherein the polyethylene glycol is modified at at least one of positions 13, 26, or 27 of teriparatide;

The modified structure of polyethylene glycol is shown below:

In a second aspect, the present invention provides a method for preparing the polyethylene glycol site-specifically modified teriparatide, comprising the following steps:

DIBO-mPEG was prepared by reacting 4-dibenzocyclooctanol DIBO with methoxypolyethylene glycol for nucleophilic substitution.

Teriparatide derivatives were reacted with DIBO-mPEG through bioorthogonal reaction to prepare polyethylene glycol-site-modified teriparatide;

The structures of the amino acid side chains in the teriparatide derivatives that undergo bioorthogonal reactions are:

The side chain is located at at least one of position 13, position 26 or position 27 of teriparatide.

Derivatives of 4-dibenzocyclooctanol (DIBO) have the characteristics of rapid reaction with sugars and amino acids containing azide groups and can be used to visualize metabolic labeling of living cells. Therefore, site-specific PEG modification of PTH (1-34) through SPAAC reaction can effectively solve the problem of short half-life of PTH (1-34).

In some embodiments, the catalyst for the nucleophilic substitution of DIBO and methoxypolyethylene glycol is triethylamine, and the solvent is dichloromethane.

Preferably, the temperature of the nucleophilic substitution is 20-30° C., and the time of the nucleophilic substitution is 10-15 h.

In some embodiments, the temperature of the bioorthogonal reaction is 1-5°C and the reaction time is 2-6 hours.

Preferably, the temperature of the bioorthogonal reaction is 3-5°C and the reaction time is 3-5h.

More preferably, the molar ratio of the teriparatide derivative to DIBO-mPEG is 1:0.8-1.2, preferably 1:1.

In some embodiments, the molecular weight of the polyethylene glycol is 10-20 kDa.

In some embodiments, the method further includes the step of isolating and purifying the prepared polyethylene glycol site-specifically modified teriparatide.

Preferably, the separation and purification method is ion exchange chromatography or gel filtration chromatography.

In a third aspect, the present invention provides the use of the polyethylene glycol site-specifically modified teriparatide in the preparation of a drug for treating osteoporosis.

The beneficial effects achieved by one or more embodiments of the present invention are as follows:

The half-life of the polyethylene glycol site-specifically modified teriparatide in rats is significantly prolonged, which is 5 times that of teriparatide, and can reduce the frequency of drug administration for patients.

The polyethylene glycol site-specifically modified teriparatide of the present invention has a better pharmacodynamic effect in treating osteoporosis than teriparatide at the same dosage.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

FIG1 is a purity analysis chart of PTH(1-34) _-N3 and its PEG-modified products in Example 2 of the present invention;

FIG2 is an SDS-PAGE analysis of PTH(1-34)-N ₃ and its PEG-modified products in Example 3 of the present invention. Lane M: protein markers; Lane 1: PTH(1-34)-N ₃ ; Lane 2: mPEG _10k -PTH(1-34); Lane 3: mPEG _20k -PTH(1-34);

Figure 3 is a mass spectrometry analysis of PTH(1-34)-N ₃ and its PEG-modified products in Example 3 of the present invention, wherein (A) is PTH(1-34)-N ₃ ; (B) is mPEG _10k -PTH(1-34); (C) is mPEG _20k -PTH(1-34);

FIG4 shows the effects of PTH (1-34) and its PEG-modified products on the proliferation of mouse BMSCs in Example 4 of the present invention, wherein (A) is PTH (1-34); (B) is mPEG _10k -PTH (1-34); (C) is mPEG _20k -PTH (1-34) (n=3);

5 is a comparison chart of the effects of PTH (1-34), mPEG _10k -PTH (1-34) and mPEG _20k -PTH (1-34) on osteogenic differentiation of mouse BMSCs evaluated by ALP staining in Example 4 of the present invention (Scale bar: 100 μm);

FIG6 is an ARS staining test for evaluating the effects of PTH (1-34), mPEG _10k -PTH (1-34) and mPEG _20k -PTH (1-34) on osteogenic differentiation of mouse BMSCs in Example 4 of the present invention (Scale bar: 100 μm);

FIG. 7 is a graph showing the blood concentration curves of PTH (1-34) and mPEG _10k -PTH (1-34) in rats in Example 5 of the present invention (n=3);

Figure 8 is an in vivo pharmacodynamics experiment in Example 6 of the present invention, wherein (A) is a 3D reconstructed image of the femur of each group of mice; (B) is a parameter statistical diagram of the bone density (BMD) of each group of mice; (C) is a parameter statistical diagram of the number of trabeculae (Tb.N) of each group of mice; (D) is a parameter statistical diagram of the cortical bone thickness (Ct.Th) of each group of mice; (E) is a parameter statistical diagram of the trabecular separation (Tb.Sp) of each group of mice, statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ^# p<0.05, ^## p<0.01, ^### p<0.001, ^#### p<0.0001;

FIG. 9 is an evaluation of the effect of drug administration on the osteogenic differentiation ability of BMSCs in postmenopausal osteoporotic mice by ARS and ALP staining (Scale bar: 100 μm).

DETAILED DESCRIPTION

It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

The present invention will be further described below in conjunction with the embodiments.

Example 1

A method for preparing polyethylene glycol-site-modified teriparatide is provided, and its synthetic route is as follows:

Preparation of DIBO and DIBO-mPEG:

Preparation of compound I-3: Under argon protection and dry conditions, compound I-1 (1.00 g, 5 mmol, 1 eq) was added to a 100 mL side-mouth bottle, 10 mL of ultra-dry dichloromethane was added, BF ₃ -Et ₂ O (926 μL, 7.5 mmol, 1.5 eq) was added at -10°C, the reaction was continued for 1 h, I-2 (8 mL, 16 mmol, 3.2 eq) was slowly added dropwise, the reaction was continued for 3 h, ice water was added to quench the reaction, the aqueous phase was extracted 3 times with dichloromethane, the organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product was purified by column chromatography using petroleum ether: ethyl acetate (4:1) to obtain a white solid powder compound I-3 (926 mg, yield 84%). ¹ H NMR (400MHz, Chloroform-d) δ8.25(d,J＝8.0Hz,1H),7.50(t,J＝8.0Hz,1H),7.40(dd,J＝24.2,7.7Hz,2H),7.34–7.27(m,2H),7.25–7.19(m,2H),7.05(d,J＝3. 6Hz, 2H), 4.06 (s, 2H). ¹³ C NMR (101MHz, CDCl ₃ )δ196.74,137.00,136.38,135.58,133.94,133.26,132.56,132.54,131.54,130.72,129.39,128.94,128.09,127.41,127.00,48.57. (See Experiment Log 2374-p72)

Preparation of compound 1-4: Compound I-3 (926 mg, 4.21 mmol, 1 eq) was added to a 250 mL round-bottom flask, 42 mL of a mixed solution of ethanol:tetrahydrofuran (1: 1, volume ratio) was added, sodium borohydride (318.5 mg, 8.42 mmol, 2 eq) was added at room temperature, and spot plate detection was performed. After the reaction was completed, glacial acetic acid was slowly added dropwise to quench, and the mixture was concentrated under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product was purified by column chromatography using petroleum ether: ethyl acetate (4: 1, volume ratio) to obtain compound I-4 (818 mg, yield 87%) as a white solid powder. ¹ H NMR (400MHz, Chloroform-d) δ7.49–7.43(m,1H),7.26–7.07(m,7H),6.90–6.80(m,2H),5.29(dd,J＝10.1,6.2Hz,1H),3.46(dd,J＝13.7,6.2Hz,1H),3.33(dd,J ＝13.7,10.1Hz,1H). ¹³ CNMR(101MHz,CDCl ₃ )δ140.88,136.97,136.32,134.60,131.75,131.69,130.31,130.03,129.53,128.78,127.56,127.30,127.13,126.07,74.70,42.66. (See Experiment Log 2374-p75)

Preparation of compound I-5: Compound I-4 (877 mg, 3.95 mmol, 1 eq) was added to a 50 mL eggplant-shaped bottle, 12 mL of a mixed solution of pyridine: dichloromethane (1:4) was added, and the mixture was stirred vigorously. Tert-butyldimethylsilyl chloride (2.973 g, 19.73 mmol, 5 eq) was added, and the mixture was reacted at 40 °C for 12 h. The reaction was detected by spot plate. After the reaction was completed, the mixture was diluted with water, extracted with DCM, and the organic phase was washed with a saturated sodium chloride solution. After drying over anhydrous sodium sulfate, the concentrated product was concentrated under reduced pressure. The concentrated product was purified by column chromatography using petroleum ether: ethyl acetate (10:1) to obtain a colorless oily compound I-5 (1.293 g, yield 97%). ¹ H NMR (400MHz, Chloroform-d) δ7.53 (d, J = 7.7 Hz, 1H), 7.20 (td, J = 7.5, 1.5 Hz, 1H), 7.17–7.01 (m, 6H), 6.91–6.74 (m, 2H), 5.44 (dd, J = 9.6, 6.1 Hz, 1H), 3.47 (dd, J = 15.3, 6.1 Hz, 1H), 3.15 (dd, J = 15.3, 9.7 Hz, 1H), 0.89 (s, 9H), 0.00 (s, 3H), -0.07 (s, 3H). (See experimental record 7526-p14)

Preparation of compound I-6: Compound I-5 (840 mg, 2.5 mmol) was dissolved in CDCl ₃ (15 mL), and Br ₂ (400 mg, 2.5 mmol) diluted with CDCl ₃ was added. The mixture was reacted at room temperature overnight, and single plate detection was performed. When the reaction was complete, the organic phase was washed with Na ₂ S ₂ O ₃ , dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and then directly proceeded to the next step.

Preparation of compound I-7: Under argon protection, compound I-6 (573 mg, 1.5 mmol) and THF (25 mL) were added to a bottle under dry conditions, and then LDA (2.5 mL, 5 mmol) was added dropwise. The reaction was carried out at room temperature for 3 h, and the plate was tested. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product was purified by column chromatography using petroleum ether: ethyl acetate (4:1) to obtain a white solid powder compound (147 mg, 0.67 mmol, 45%).

Preparation of compound I-9: Under argon protection and dry conditions, compound I-7 (110 mg, 0.5 mmol) and dichloromethane (15 mL) were added to a bottle, followed by addition of p-nitrophenyl chloroformate (0.2 g, 1 mmol), and finally pyridine (0.2 mL, 2.5 mmol). The reaction was carried out at room temperature, and the plate was tested. After the reaction was complete, the mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrated product was purified by column chromatography using petroleum ether: ethyl acetate (5:1) to obtain compound I-9 (173 mg, 0.45 mmol, 89%).

Preparation of DIBO-mPEG _10k : Under argon protection, compound I-9 (16 mg, 40 μmol, 4 eq) was added to a 10 mL vial, 2 mL of ultra-dry dichloromethane was added, and then mPEG _10k (100 mg, 10 μmol, 1 eq) and triethylamine (7 μL, 50 μmol, 5 eq) were added. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic phase was washed with 1N hydrochloric acid, then washed with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Then, cold ether was used for precipitation twice to obtain a white solid powder DIBO-mPEG _10k (80 mg, yield 80%).

Preparation of DIBO-mPEG _20k : Under argon protection, compound I-9 (16 mg, 40 μmol, 4 eq) was added to a 10 mL vial, 2 mL of ultra-dry dichloromethane was added, and then mPEG _20k (200 mg, 10 μmol, 1 eq) and triethylamine (7 μL, 50 μmol, 5 eq) were added. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, the organic phase was washed with 1N hydrochloric acid, then washed with water, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Finally, cold ether was used for precipitation twice to obtain a white solid powder DIBO-mPEG _20k (179 mg, yield 90%).

Example 2

Preparation and purification of teriparatide modified with polyethylene glycol:

Preparation of PTH(1-34)-N ₃ : 1) Weigh 5.91 g (2.5 mmol) of Fmoc-Phe-Wang Resin with a degree of substitution of 0.423 mol/g, place it in a reactor, add an appropriate amount of DMF and soak for 2 h;

2) Drain DMF, add appropriate amount of 20% Pip/DMF, deprotect with nitrogen for 0.5 h, wash with DMF 5 times, and detect with ninhydrin to see dark blue;

3) Add raw materials in an equivalent ratio (AA (acrylic acid): DIC (N, N'-diisopropylcarbodiimide): HoBt (1-hydroxybenzotriazole) = 3:3:3), add appropriate amount of DMF, and react with nitrogen until the ninhydrin test is transparent. After drying, wash with DMF three times;

4) repeating steps 2) and 3) to obtain H-Ser(tBu)-Val-Ser(tBu)-Glu(OtBu)-Ile-Gln(Trt)-Leu-Met-His(Trt)-Asn(Trt)-Leu-Gly-Lys(N3)-His(Trt)-Leu-Asn(Trt)-Ser(tBu)-Met-Glu(OtBu)-Arg(Pbf)-Val-Glu(OtBu)-Trp(Boc)-Leu-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Le u-Gln(Trt)-Asp(OtBu)-Val-His(Trt)-Asn(Trt)-Phe-Wang Resin;

5) Drain the resin in the reactor, transfer it to a cutting tube, add E solution (TFA: EDT: H ₂ O: phenol: TIS = 90: 2.5: 2.5: 2.5: 2.5), and shake on a shaker to control the temperature for 2.5 hours;

6) Filter by suction, collect the cut filtrate into a centrifuge tube, add it into 6 volumes of methyl tert-butyl ether, collect the solid by centrifuge precipitation, and wash the precipitated crude product with methyl tert-butyl ether 3 times to obtain the final crude product;

7) The crude product was placed in a drying pot, vacuum dried overnight, and purified by HPLC to finally obtain the fine product of PTH(1-34)-N ₃ .

Preparation of mPEG _10k -PTH(1-34): Compound PTH(1-34)-N ₃ (14 mg, 3.4 μmol, 1 eq) was dissolved in 4 mL phosphate buffer solution (PBS), and compound DIBO-mPEG10k (100 mg, 10 μmol, 3 eq) was added, and the mixture was reacted at 4°C for 4 h. After the reaction was completed, further purification was performed.

Preparation of mPEG _20k -PTH(1-34): Compound PTH(1-34)-N ₃ (12 mg, 2.9 μmol, 1 eq) was dissolved in 4 mL phosphate buffer solution (PBS), and compound DIBO-mPEG20k (174 mg, 8.7 μmol, 3 eq) was added, and the mixture was reacted at 4°C for 4 h. After the reaction was completed, further purification was performed.

The product was purified using an AKTA protein purifier, a cation exchange column and Superdex 200Increase 10/300GL.

mPEG-PTH (1-34) was purified using a cation exchange column. The specific steps were as follows: the reaction mixture was dissolved in a 20 mmol/L, pH 4.5 CH ₃ COOH-CH ₃ COONa buffer solution to a final concentration of 0.5 mg/mL, and filtered using a 0.45 μm filter membrane.

The balancing solution was 20mmol/L CH ₃ COOH-CH ₃ COONa, and the pH value was 4.5; the eluent included 20mmol/L CH ₃ COOH-CH ₃ COONa, 1mol/L NaCl, and the pH value was 4.5.

Method: ① Use pure water to clean the A and B pump inlet pipes. ② Install the chromatography column and use pure water to balance the chromatography column. Note: When installing the chromatography column, input a lower flow rate to prevent exceeding the tolerance pressure of the chromatography column. In addition, the chromatography column should be installed while the instrument is running to prevent bubbles from entering the chromatography column and damaging the chromatography column. ③ Place the A and B pumps in the buffer solution and clean them respectively, use the balance solution to balance the chromatography column, and then use the eluent to clean the B pump. ④ Load the mPEG-PTH (1-34) sample to be purified and run the elution collection program. ⑤ Use the balance solution and pure water to clean the pipes and chromatography columns. Combine the main elution peaks and concentrate them using a 10kDa ultrafiltration tube.

mPEG-PTH (1-34) was further purified using Superdex 200 Increase 10/300GL, and the buffer solution was pH=6.0, 20 mmol/L NaH ₂ PO ₄ —Na ₂ HPO ₄ 100 mmol/L NaCl.

Method: ① Use pure water to clean the pipe. ② Run the elution program, set the flow rate to 0.2mL/min, install the chromatography column, and then use pure water to complete an elution program. ③ Use buffer solution to balance the chromatography column at a flow rate of 0.2mL/min. ④ Load the sample and run the elution program. ⑤ Use the equilibration solution and pure water to clean the chromatography column. Collect the main elution peak, ultrafilter and freeze-dry.

The purity of the sample can be analyzed by HPLC. Sample treatment: PTH(1-34)-N ₃ and its PEG-modified product were dissolved in pure water at a concentration of 2 mg/mL, and filtered using a 0.22 μm filter membrane. Mobile phase A was H ₂ O + 0.1% TFA, and mobile phase B was MeCN + 0.1% TFA.

Method: Use 10% mobile phase B for equilibration, inject 10 μL each time, and then use 10-100% MeCN (0.1% TFA)-H ₂ O (0.1% TFA) system for gradient elution. The results of liquid chromatography analysis showed that the purity of mPEG _20k -PTH (1-34) and mPEG _10k -PTH (1-34) after purification was greater than 95%.

The purity analysis chart of PTH(1-34)-N ₃ and its PEG-modified products is shown in FIG1 , and the contents in Table 1 are the purity analysis results.

Table 1 Peak area and elution time of PTH(1-34)-N ₃ and its PEG-modified products

Example 3

Characterization of teriparatide modified with polyethylene glycol

(a) Coomassie Brilliant Blue staining (SDS-PAGE)

Prepare 15% separation gel (10 mL): Use a pipette to accurately measure 2.3 mL of triple-distilled water, 5 mL of 30% acrylamide solution, 2.5 mL of 1.5 mol/L tri (pH 8.8), 100 μL of 10% SDS, 100 μL of APS, and 4 μL of TEMED into a beaker, use a pipette to blow and mix evenly, then pour the gel, and add isopropanol to the upper layer of the separation gel.

Prepare 4 mL of concentrated gel: Use a pipette to accurately measure 2.7 mL of triple-distilled water, 0.67 mL of 30% acrylamide solution, 0.5 mL of 1.5 mol/L tri (pH 6.8), 40 μL of 10% SDS, 40 μL of APS, and 4 μL of TEMED into a beaker. Use a pipette to blow and mix evenly, then pour the gel and insert a 1.5 mm 12-hole comb. Remove the comb after the gel solidifies.

SDS-PAGE sample processing and electrophoresis: PTH(1-34)-N ₃ , mPEG _10k -PTH(1-34) and mPEG _20k -PTH(1-34) were dissolved in PBS to the appropriate concentration, β-mercaptoethanol and loading buffer were added and mixed, heated at 100℃ for 5min, cooled, loaded, the stacking gel voltage was 80V, the separation gel voltage was 130V, the stacking gel time was set to 30min, and the separation gel time was set to 60min. Then staining and decolorization were performed.

The results are shown in Figure 2. The apparent molecular weight of mPEG _10k -PTH (1-34) is about 20 kDa, and the apparent molecular weight of mPEG _20k -PTH (1-34) is about 37 kDa. Both PEG-modified products are single electrophoretic bands, indicating that the purity of mPEG _20k -PTH (1-34) and mPEG _10k -PTH (1-34) is very high.

(b) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS)

The molecular weights of PTH(1-34)-N ₃ , mPEG _10k -PTH(1-34) and mPEG _20k -PTH(1-34) were determined using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. 700 μL of double distilled water, 300 μL of chromatographic acetonitrile and 1 μL of trifluoroacetic acid were added to prepare 1 mL of solution (solution A). 200 μL of solution A was taken and 3 mg of α-cyano-4-hydroxycinnamic acid was dissolved to prepare the matrix solution. PTH(1-34) and its derivatives were prepared into a 10 μg/μL sample solution using solution A. 5 μL of sample solution and 5 μL of matrix solution were mixed, and 2 μL was applied to the plate. After the sample crystallized on the plate, the plate was inserted into the MALDI target plate and the sample was analyzed using MALDI-TOF-MS. The obtained data were then processed using OriginPro 9.1 software.

The results are shown in Figure 3. The actual molecular weight of PTH(1-34)-N ₃ is 4142.638Da, and the actual molecular weights of mPEG _10k -PTH(1-34) and mPEG _20k -PTH(1-34) are 15514.962Da and 24981.136Da, respectively, which are consistent with their theoretical molecular weights. SDS-PAGE and MALDI-TOF-MS results show that the purified peptides are single PEG chain modified peptides with a single structure.

Example 4

Effects of teriparatide modified with polyethylene glycol on proliferation and differentiation of mouse BMSCs

(a) In vitro evaluation of the effect of mPEG-PTH (1-34) on the proliferation of BMSCs, and calculation of the relative cell proliferation rate. BMSCs in the logarithmic growth phase were evenly seeded in a 96-well plate. The cells were placed in an incubator at 37°C and 5% _CO2 overnight. After the cells adhered to the wall, PTH (1-34), mPEG _10k -PTH (1-34) and mPEG _20k -PTH (1-34) were applied to the cells at a concentration gradient of (0, 0.24, 1.2, 2.4, 12 nmol/mL) for 0, 12, 24, 36, and 48 h, respectively. After adding 10 μL of CCK-8 solution to each well and continuing to incubate, the OD value of each well at 450 nm was measured using an ELISA reader to calculate the relative cell proliferation rate.

The experimental results showed that, as shown in Figure 4A, B and C, compared with the Control group, 2.4 and 12 nmol/mL PTH(1-34) and mPEG _10k -PTH(1-34) could significantly promote the proliferation of mouse BMSCs, while mPEG _20k -PTH(1-34) had no activity in promoting the proliferation of mouse BMSCs at all concentrations.

(b) Effect of mPEG-PTH(1-34) on osteogenic differentiation of mouse BMSCs

Mouse BMSCs were obtained and induced to osteogenic differentiation using osteogenic induction solution and different concentrations of PTH(1-34), mPEG _10k -PTH(1-34) and mPEG _20k -PTH(1-34). ARS and ALP staining were used to observe the osteogenic differentiation of mouse BMSCs.

ALP staining to detect osteogenic differentiation of BMSCs:

(1) Take 40 μL of staining reagent A (provided by the ALP staining kit, the ALP staining kit was purchased from Shanghai ELISA Biotechnology Co., Ltd.) and add it to 1 mL of reaction buffer (provided by the ALP staining kit, the ALP staining kit was purchased from Shanghai ELISA Biotechnology Co., Ltd.), mix well, then add 40 μL of staining reagent B (provided by the ALP staining kit, the ALP staining kit was purchased from Shanghai ELISA Biotechnology Co., Ltd.), mix well, and obtain the reaction working solution.

(2) After inducing BMSCs to differentiate into osteoblasts for 7 days, the culture medium was removed and 2 mL of PBS solution was added to each well. The cells were allowed to stand for 1 min and the operation was repeated once.

(3) Fix the cells with fixative for 30 min at room temperature.

(4) Aspirate the fixative solution and wash twice with washing solution, 500 μL per well.

(5) Add 500 μL of the reaction working solution prepared in the first step to each well and stain at room temperature for 30 min or longer (no more than 24 h) until the color development meets the expected requirements.

(6) Wash twice with washing buffer, 500 μL per well, and take pictures of the stained cells using a microscope.

ARS staining to detect osteogenic differentiation of BMSCs:

(1) After osteogenic differentiation was induced for 14 days, the culture medium was removed and the cells in the 24-well plate were washed three times with PBS solution.

(2) Fix the cells with 4% paraformaldehyde for 10 min at room temperature.

(3) Wash with PBS three times and stain with 40 mmol/L ARS (pH 4.2) at 37°C for 15 min.

(4) Wash with PBS three times and take pictures of the stained cells using a microscope.

As shown in Figures 5 and 6, within a certain drug concentration range, increasing the drug concentration, the staining of PTH (1-34) and mPEG _10k -PTH (1-34) groups deepened, the number of mineralized nodules increased, and the ability to induce osteogenic differentiation of mouse BMSCs was enhanced. However, there was no significant change in the mineralized nodules of the mPEG _20k -PTH (1-34) group. This shows that PTH (1-34) and mPEG _10k -PTH (1-34) can promote the osteogenic differentiation of mouse BMSCs, and mPEG _20k -PTH (1-34) has no effect on inducing osteogenic differentiation of mouse BMSCs.

Example 5

In vivo half-life study of teriparatide modified with polyethylene glycol

Collection of plasma samples: Female SD rats (190-210 g) aged 8 weeks were selected and divided into two groups, PTH (1-34) and mPEG _10k -PTH (1-34), with 3 rats in each group. The subcutaneous injection dose of PTH (1-34) and mPEG _10k -PTH (1-34) was 42 nmol/kg. The rats were fasted for 12 hours before administration and had free access to water. 300 μL of blood was collected from the ocular venous plexus of the rats before administration (0), 5 minutes, 15 minutes, 30 minutes, 1, 2, 4, 8, 12, 24, 48, and 72 hours after administration, and placed in a 1.5 mL centrifuge tube soaked with 10 mg/mL heparin sodium, centrifuged at 3000 rpm for 10 minutes, and the upper layer of light yellow plasma was placed in a 1.5 mL tube and frozen at -80°C for later use. The drug concentration in plasma was subsequently determined using a competitive quantitative enzyme-linked immunosorbent assay (ELISA) kit.

Establishment of standard curve: Use the PTH (1-34) standard provided by the kit to establish a standard curve. Add 1 mL of washing buffer to the PTH (1-34) standard and vortex to mix. The stock solution is recorded as S0, with a concentration of 1000 ng/mL. Let it stand at room temperature for 10 minutes to fully dissolve. Then, take 100 μL of each diluted solution of stock solution S0 and add it to a 1.5 mL centrifuge tube containing 900 μL of washing buffer to obtain 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL, and 0.01 ng/mL PTH (1-34) standard solutions.

The blood drug concentration was measured using the following steps:

Blood drug concentration determination steps:

(1) Take out the 96-well plate and equilibrate it at room temperature for 30 minutes before use.

(2) Add 50 μL of washing buffer to each well except the blank well.

(3) Add 50 μL of pre-diluted PTH (1-34) standard, positive control, and sample to the corresponding wells.

(4) Add 25 μL of primary antibody to each well except the blank well.

(5) Add 25 μL of biotinylated peptide to each well except the blank well.

(6) Seal the 96-well plate with plate sealant and incubate on a microplate shaker at 300-400 rpm for 2 h at room temperature.

(7) Discard the solution in each well and wash the 96-well plate with washing buffer. Repeat this step 4 times. After the last wash, absorb the washing buffer with absorbent paper.

(8) Add 100 μL of horseradish peroxidase-labeled streptavidin to each well.

(9) Seal the 96-well plate with plate sealant and incubate on a microplate shaker at 300-400 rpm for 1 h at room temperature.

(10) Repeat step (6).

(11) Add 100 μL of color development solution to each well.

(12) Seal the 96-well plate with plate sealant and incubate on a microplate shaker at 300-400 rpm for 1 h at room temperature in the dark.

(13) Discard the sealing film and add 100 μL of stop solution to each well to stop the reaction. Gently tap the plate to mix the solution evenly. The color of the solution changes from yellow to blue.

(14) Use a microplate reader to read the OD value at 450 nm.

The results are shown in Figure 7, and the pharmacokinetic parameters are shown in Table 2. The elimination half-life of mPEG _10k -PTH (1-34) was 5.3h, which was 13.3 times that of PTH (1-34). The C _max of mPEG _10k -PTH (1-34) was 1.5 times that of PTH (1-34), and the AUC of mPEG _10k -PTH (1-34) was 5 times that of PTH (1-34). Compared with PTH (1-34), the AUC, T _max , C _max and elimination half-life of mPEG _10k -PTH (1-34) were significantly increased.

Table 2 Pharmacokinetic parameters of PTH (1-34) and mPEG _10k -PTH (1-34) in rats

Example 6

In vivo activity experiment of teriparatide modified with polyethylene glycol

Eight-week-old female C57BL/6 mice weighing 18-22 g were selected. After one week of adaptive feeding, the mice were randomly divided into 6 groups: sham, OVX, PTH (1-34), high-dose mPEG _10k -PTH (1-34), medium-dose mPEG _10k -PTH (1-34), and low-dose mPEG _10k -PTH (1-34), with 5 mice in each group. The sham group underwent bilateral sham surgery, and the rest of the groups underwent ovariectomy to establish a postmenopausal osteoporosis mouse model. One week after recovery, medication was started, including mPEG _10k- PTH (1-34) high-dose group (15nmol/kg), mPEG _10k- PTH (1-34) medium-dose group (10nmol/kg), mPEG _10k -PTH (1-34) low-dose group (5nmol/kg), PTH (1-34) group (10nmol/kg), and OVX group (0.9% saline). Each group was dosed once every two days for 8 consecutive weeks, and the weight of the mice was recorded during medication.

24 hours after the last administration, the mice were eyeballed and blood was collected. The mice were then killed by cervical dislocation, and their livers and kidneys were removed. The mice were weighed and photographed. Finally, the mouse tissues were fixed with tissue fixative, stained with H&E, and observed and photographed under a microscope.

The results of three-dimensional reconstruction images of the femur of each group of mice under Micro-CT scanning, as well as the bone density (BMD), trabecular number (Tb.N), cortical bone thickness (Ct.Th) and trabecular separation (Tb.Sp) of each group of mice showed that compared with the sham operation group, the mice in the postmenopausal osteoporosis model group had severe bone loss. Both PTH (1-34) and mPEG _10k -PTH (1-34) could reduce the bone loss of postmenopausal osteoporosis mice, and the effect of mPEG _10k -PTH (1-34) was better than PTH (1-34) at the same dosage. The effect of dosage on the effect of mPEG _10k -PTH (1-34) was further investigated. It was found that within a certain dosage range, increasing the dosage enhanced the effect of improving bone loss in osteoporotic mice, and high dose (15nmol/kg) of mPEG _10k -PTH (1-34) had the best effect, as shown in Figure 8.

BMSCs of mice in each group were extracted for osteogenic induction, and ARS and ALP staining were performed 7 days and 14 days after osteogenic induction to analyze the effects of PTH (1-34) and mPEG _10k -PTH (1-34) on the osteogenic differentiation ability of BMSCs in postmenopausal osteoporotic mice. The results are shown in Figure 9. Compared with the OVX group, the mineralized nodules in the PTH (1-34) and mPEG _10k -PTH (1-34) groups increased and the staining became deeper. This indicates that both PTH (1-34) and mPEG _10k -PTH (1-34) can improve the osteogenic differentiation ability of BMSCs in postmenopausal osteoporotic mice, and at the same dosage, the ability of mPEG _10k -PTH (1-34) to improve the osteogenic differentiation ability of BMSCs in postmenopausal osteoporotic mice is stronger than that of PTH (1-34). When investigating the effect of dosage on the osteogenic differentiation ability of BMSCs in postmenopausal osteoporotic mice, it was found that high-dose mPEG _10k -PTH (1-34) had a stronger effect on improving the osteogenic differentiation ability of BMSCs than low-dose.

To further prove the safety of mPEG _10k -PTH(1-34), the tissues and organs (liver and kidney) of each group of mice were sliced and analyzed by H&E staining. PTH(1-34) and mPEG _10k -PTH(1-34) had no obvious toxicity to the liver and kidney of mice. The above results show that compared with the positive drug PTH(1-34), mPEG _10k -PTH(1-34) has a better effect on improving bone loss in osteoporotic mice and has good safety.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A polyethylene glycol site-specifically modified teriparatide, characterized in that the polyethylene glycol is modified at at least one of positions 13, 26 or 27 of teriparatide; The modified structure of polyethylene glycol is shown below: 2. The method for preparing teriparatide modified with polyethylene glycol according to claim 1, characterized in that it comprises the following steps: DIBO-mPEG was prepared by reacting 4-dibenzocyclooctanol DIBO with methoxypolyethylene glycol for nucleophilic substitution. Teriparatide derivatives were reacted with DIBO-mPEG through bioorthogonal reaction to prepare polyethylene glycol-site-modified teriparatide; The structures of the amino acid side chains in the teriparatide derivatives that undergo bioorthogonal reactions are: The side chain is located at at least one of position 13, position 26 or position 27 of teriparatide. 3. The method for preparing teriparatide modified with polyethylene glycol according to claim 2, characterized in that the catalyst for nucleophilic substitution of DIBO and methoxypolyethylene glycol is triethylamine, and the solvent is dichloromethane. 4. The method for preparing teriparatide modified with polyethylene glycol according to claim 2, characterized in that the temperature of nucleophilic substitution is 20-30°C, and the time of nucleophilic substitution is 10-15h. 5. The method for preparing teriparatide modified with polyethylene glycol according to claim 2, characterized in that the temperature of the bioorthogonal reaction is 1-5°C and the reaction time is 2-6h; Preferably, the temperature of the bioorthogonal reaction is 3-5°C and the reaction time is 3-5h. 6. The method for preparing teriparatide modified with polyethylene glycol according to claim 2, characterized in that the molar ratio of the teriparatide derivative to DIBO-mPEG is 1:0.8-1.2. 7. The method for preparing teriparatide modified with polyethylene glycol according to claim 2, characterized in that the molecular weight of the polyethylene glycol is 10-20 kDa. 8. The method for preparing teriparatide modified with polyethylene glycol according to claim 2, characterized in that it also comprises the step of separating and purifying the prepared teriparatide modified with polyethylene glycol. 9. The method for preparing teriparatide modified with polyethylene glycol according to claim 8, characterized in that the separation and purification method is ion exchange chromatography or gel filtration chromatography. 10. Use of the polyethylene glycol site-specifically modified teriparatide according to claim 1, or the polyethylene glycol site-specifically modified teriparatide prepared by the preparation method according to any one of claims 2 to 9 in the preparation of a drug for treating osteoporosis.