Arginine glycosylation modified teriparatide and use thereof

Abstract

The application relates to the technical field of medicines, and discloses arginine glycosylation modified teriparatide and application thereof. First, a glycosylation donor is synthesized through a chemical synthesis method, then, according to a template PTH: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH2 amino acid sequence, arginines at positions 20 and 25 are replaced by ornithine with a Dde protecting group, the arginine glycosylation modified teriparatide is obtained by using the glycosylation donor to modify the same through a solid-phase synthesis method. The method is simple and easy to implement, has high purity and high yield. Further experiments prove that the arginine glycosylation modified teriparatide can significantly promote osteoblast differentiation, has high serum stability, and has potential application value in the treatment of diseases such as osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to arginine glycosylation-modified teriparatide and its applications, which promotes osteoblast formation and can be used to treat osteoporosis. Background Technology

[0002] Osteoporosis, a disease characterized by low bone mass and bone structure deterioration, leading to decreased bone strength and increased fracture risk, has become a significant and increasingly serious public health problem, once considered an inevitable consequence of aging. Osteoporosis and its related complications are also common causes of morbidity and mortality in the elderly. With increasing life expectancy worldwide, the incidence of fractures is also rising. Globally, osteoporosis causes more than 8.9 million fractures annually, a fracture occurring every 3 seconds. It is estimated that half of women and a quarter of men over 50 will experience an osteoporotic fracture. Currently, many safe and effective drug therapies are available for treating osteoporosis; for example, bisphosphonates or denoxamethasone can reduce the risk of low-traumatic fractures in patients with bone disease.

[0003] Teriparatide is a truncated parathyroid hormone (PTH) for the treatment of osteoporosis. It offers a different anabolic mechanism of action than oral bisphosphonates, justifying daily injection. Parathyroid hormone (PTH) is a key regulator of calcium metabolism in the body, composed of 84 amino acids. Studies have found that although bone loss is associated with hyperparathyroidism, intermittent PTH use leads to increased bone mass, possibly because the anabolic effects of PTH are more significant than its catabolic effects. Teriparatide is a recombinant polypeptide containing the first 34 amino acids of the N-terminus of PTH, including the biologically active N-terminal domain of PTH. Currently, most osteoporosis treatments focus on inhibiting bone resorption, while teriparatide is the only drug that promotes bone formation. Teriparatide mediates osteogenic metabolism by inhibiting osteoblast apoptosis, activating osteoblast lining cells, and enhancing osteoblast differentiation.

[0004] However, teriparatide is primarily administered subcutaneously and cannot be taken orally, making it more inconvenient to carry and store compared to oral medications, thus reducing its ease of use. Furthermore, the injection method increases the cost of administration and can cause pain and fear in some patients, reducing their comfort. Therefore, this paper proposes arginine glycosylation modification of teriparatide, its preparation method, and its applications to address the aforementioned problems. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides arginine glycosylation-modified teriparatide and its applications, which have advantages such as enhanced water solubility, improved enzyme stability and anti-osteoporosis activity, thus solving the problems of poor membrane permeability and weak resistance to hydrolytic enzymes in existing anti-osteoporosis peptide drugs.

[0007] (II) Technical Solution

[0008] To achieve the aforementioned objectives of enhancing water solubility, improving enzyme stability, and increasing anti-osteoporosis activity, the present invention provides the following technical solution:

[0009] A teriparatide modified with arginine glycosylation, wherein the teriparatide is one of the following:

[0010] 1) Using SVSEIQLMHNLGKHLNSMERX1VEWLRKKLQDVHNF-NH2 as a peptide template, the R at position 20 was replaced by Orn(Dde) and coupled with X1.

[0011] 2) Using SVSEIQLMHNLGKHLNSMERVEWLRX2KKLQDVHNF-NH2 as a peptide template, the R at position 25 is replaced by Orn(Dde) and coupled with X2.

[0012] 3) Using SVSEIQLMHNLGKHLNSMERX1VEWLRX2KKLQDVHNF-NH2 as a peptide template, the R at positions 20 and 25 were replaced with Orn(Dde) and coupled to X1 and X2 respectively.

[0013] Wherein, X1 represents a glucose donor, or a galactose donor, or a mannose donor, or a rhamnose donor, or a ribose donor, or an N-acetylglucosamine donor, or a xylose donor, or a lactose donor, or a maltose donor; X2 represents a glucose donor, or a galactose donor, or a mannose donor, or a rhamnose donor, or a ribose donor, or an N-acetylglucosamine donor, or a xylose donor, or a lactose donor, or a maltose donor.

[0014] The specific structure is shown in equations 1)-3) below:

[0015] 1) ;

[0016] 2) ;

[0017] 3) .

[0018] In this document, "the arginine glycosylation modified teriparatide of the present invention" refers to the polypeptide having the structure shown in formulas 1)-3) of the present invention. In this document, such polypeptide may be referred to as "polypeptide fragment" or "polypeptide of the present invention".

[0019] In this invention, the N-terminal amino group of the polypeptide is acetylated to form -Ac, and the C-terminal carboxyl group is amidated to form -NH2.

[0020] The methods used in this article to represent polypeptides, amino acids, and chemical groups are all recognized in the relevant fields. The abbreviations for amino acids can be found in Table 1. Unless otherwise specified, amino acids in this article generally refer to L-type amino acids.

[0021] Table 1. Amino Acid Abbreviations

[0022]

[0023] The anti-osteoporosis effect of the peptides of this invention can be verified by conventional experimental methods in the field, such as cell experiments. In the specific embodiments of this invention, cell experiments such as ALP experiments, alizarin red staining experiments, and gene-level verification experiments are preferred. Through these experiments, it was found that the teriparatides modified with arginine glycosylation of formulas 1)-3) involved in this invention all have in vitro anti-osteoporosis effects.

[0024] For ease of understanding, the present invention will be described below with reference to specific embodiments and accompanying drawings. It should be noted that these descriptions are merely exemplary and do not constitute a limitation on the scope of the present invention.

[0025] (III) Beneficial Effects

[0026] Compared with the prior art, the present invention provides an anti-osteoporosis active compound modified with arginine glycosylation, its preparation method and application, which has the following beneficial effects:

[0027] This study reports on the arginine glycosylation-modified anti-osteoporosis active compounds, their preparation methods, and applications. It has been shown that chemically modifying the arginine residue in the teriparatide sequence to increase its membrane permeability and enzyme stability is an effective strategy to address the poor drug-likeness of teriparatide. Therefore, we designed and synthesized a series of novel PTH glycopeptide active molecules using an arginine glycosylation modification strategy, aiming to enhance their cell permeability, improve enzyme stability, and enhance their anti-osteoporosis activity, making them more suitable for safer use in osteoporosis patients. Attached Figure Description

[0028] Figure 1 This is the mass spectrum of PTH-1a in Table 2 of this invention;

[0029] Figure 2 This is the mass spectrum of PTH-1b in Table 2 of this invention;

[0030] Figure 3 This is the PTH-1c mass spectrum in Table 2 of this invention;

[0031] Figure 4 The mass spectrum of PTH-1d in Table 2 of this invention;

[0032] Figure 5 The mass spectrum of PTH-1e in Table 2 of this invention;

[0033] Figure 6 The mass spectrum of PTH-1f in Table 2 of this invention;

[0034] Figure 7 This is the mass spectrum of PTH-1g in Table 2 of this invention;

[0035] Figure 8 This is the mass spectrum of PTH-1g in Table 2 of this invention;

[0036] Figure 9 The mass spectrum of PTH-1i in Table 2 of this invention;

[0037] Figure 10 This is the mass spectrum of PTH-2a in Table 2 of this invention;

[0038] Figure 11 This is the mass spectrum of PTH-2b in Table 2 of this invention;

[0039] Figure 12 The mass spectrum of PTH-2c in Table 2 of this invention;

[0040] Figure 13 The mass spectrum of PTH-2d in Table 2 of this invention;

[0041] Figure 14 The mass spectrum of PTH-2e in Table 2 of this invention;

[0042] Figure 15 The mass spectrum of PTH-2f in Table 2 of this invention;

[0043] Figure 16 This is the mass spectrum of PTH-2g in Table 2 of this invention;

[0044] Figure 17 The mass spectrum of PTH-2h is shown in Table 2 of this invention;

[0045] Figure 18 The mass spectrum of PTH-2i in Table 2 of this invention;

[0046] Figure 19 This is the mass spectrum of PTH-3a in Table 2 of this invention;

[0047] Figure 20 This is the mass spectrum of PTH-3b in Table 2 of this invention;

[0048] Figure 21 This is the PTH-3c mass spectrum in Table 2 of this invention;

[0049] Figure 22 The mass spectrum of PTH-3d in Table 2 of this invention;

[0050] Figure 23 The mass spectrum of PTH-3e in Table 2 of this invention;

[0051] Figure 24 The mass spectrum of PTH-3f in Table 2 of this invention;

[0052] Figure 25 This is the mass spectrum of PTH-3g in Table 2 of this invention;

[0053] Figure 26 This is the PTH-3h mass spectrum in Table 2 of this invention;

[0054] Figure 27 The mass spectrum of PTH-3i in Table 2 of this invention;

[0055] Figure 28 Synthetic route diagram for glycosylation donors;

[0056] Figure 29 This is a solid-phase synthetic route for glycosylated teriparatide, showing the boxes and the Glu inside them. 19 -Orn-Phe 34 The symbols represent amino acids at positions 19-34, and the circles (○) in the diagram represent resin.

[0057] Figure 30 The percentage of glycosylated teriparatide ALP-positive staining area;

[0058] Figure 31 The percentage of area positive for Alizarin Red S staining;

[0059] Figure 32 This is a diagram showing changes in the expression of osteogenic-related genes. Detailed Implementation

[0060] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] The preparation method of the arginine glycosylation modified teriparatide of the present invention is as follows: First, a glycosylation donor is synthesized by chemical synthesis (the synthetic route of the glycosylation donor is as follows). Figure 28(As shown), then, following the template PTH: SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-NH2 amino acid sequence, the arginine at positions 20 and 25 were replaced with ornithine with a Dde protecting group, using a solid-phase synthesis method (solid-phase synthesis route as shown). Figure 29 As shown in the figure, the teriparatide was modified using the above-mentioned glycosylation donor to obtain arginine glycosylated teriparatide. The method of the present invention is simple, easy to implement, has high purity, and high yield.

[0062] The names, structural formulas, and mass spectrometry data of the compounds synthesized in this invention are shown in Table 2.

[0063] Table 2. Names, glycosylation groups, mass spectra, and mass spectra data of arginine-glycosylated teriparatide molecules.

[0064]

[0065] Example 1: Preparation method of glycosylation intermediate compound, chemical synthesis 5a, the specific steps are as follows:

[0066] Using glucose as a starting material, acetyl chloride was added as a solvent to the starting material, resulting in a one-step reaction to obtain triacetyl-protected α-chloroglycol. The chloroglycol was unstable and proceeded directly to the next step. In anhydrous acetonitrile, the chloroglycol was treated with potassium thiocyanate and tetrabutylammonium iodide to obtain glucose isothiocyanate 4a. Potassium tert-butoxide and Pbf-NH2 were premixed and immediately alkylated with iodoethane, yielding the key sugar intermediate 5a via a two-step one-pot reaction.

[0067] Example 2: Preparation method of arginine glycosylation modified anti-osteoporosis active compound, solid-phase synthesis of PTH-1a, the specific steps are as follows:

[0068] First, the linear peptide was synthesized using a solid-phase peptide synthesis (SPPS) strategy, with Rink Amide MBHA resin as the solid support, N,N'-diisopropylcarbodiimide / ethyl 2-oxime cyanoacetate as the condensing agent, and Fmoc-Orn(Dde)-OH replacing arginine as the initiating amino acid for glycosylation. The general method used for coupling teriparatide onto this resin was as follows: the Fmoc protecting group was removed by adding 20% ​​piperidine / N,N-dimethylformamide as a deprotecting agent (5 min × 2), followed by filtration of the resin and washing three times sequentially with DCM, DMF, and DCM. During peptide coupling, the Fmoc-protected amino acid and ethyl 2-oxime cyanoacetate were dissolved in NMP, then activated with N,N-diisopropylethylamine, and shaken at 45°C for 20 min. After obtaining a linear peptide by sequentially coupling the amino acid sequence of teriparatide onto the resin, the Dde protecting group of the amino side chain was removed under the action of 2% hydrazine hydrate. Then, the amino and thioester glycosylation reaction was carried out on the peptide-carrying resin using silver nitrate catalysis to obtain the glycopeptide coupled to the resin. Subsequently, the acetyl protecting group on the glycointermediate was removed using 5% hydrazine hydrate. Finally, the glycopeptide was released from the resin using a cleavage reagent (triisopropylsilane:trifluoroacetic acid = 5:95), purified by preparative RP-HPLC, and lyophilized to obtain a white lyophilized powder with a purity ≥97.0%.

[0069] Example 3: Glycosylation modification of teriparatide can enhance its osteogenic effect.

[0070] 1) Cell biology experiments

[0071] Extraction of pre-osteoblasts: Skulls from one-day-old C57BL / 6 (CAVENS.LA, Guangzhou, China) mice were collected and washed with 10×, 5×, and 1× antibiotics and PBS. Cells were incubated overnight in a cell culture incubator (37℃, 5% CO2) with collagenase II (C8150; Solarbio, Beijing, China). After removing impurities through a 70μm sieve and washing with PBS, the cells were cultured in α-MEM medium. The resulting cells were passaged and used for subsequent experiments.

[0072] ALP and Alizarin Red S staining: Extracted pre-osteoblasts were seeded at 50,000 cells / well in 24-well plates. After 24 hours of culture, cells were cultured in media containing / without 50 μg / mL L-ascorbic acid, 5 mM sodium β-glycerophosphate, and 50 nM of each drug, with medium changes every 2 days. After 2 days of culture, cells without L-ascorbic acid and sodium β-glycerophosphate were used for ALP staining. After 14 days of culture, cells containing L-ascorbic acid and sodium β-glycerophosphate were used for Alizarin Red S staining. For ALP staining, cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS for 10 minutes each time, and then stained using an alkaline phosphatase assay kit (P0321; Beyotime, Jiangsu, China) according to the manufacturer's instructions. For Alizarin Red staining, cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS for 10 minutes each time, and washed three times with 70% ethanol for 10 minutes each time. The samples were then stained with Alizarin Red S staining solution (G1452; Solarbio, Beijing, China), washed once with 50% ethanol, and air-dried. Subsequent images were captured using a microplate reader, exported, and analyzed using ImageJ software.

[0073] RNA extraction: Extracted pre-osteoblasts were plated at 200,000 cells / well in 6-well plates. After 24 h of culture, the cells were cultured in a medium containing 50 μg / mL L-ascorbic acid, 5 mM sodium β-glycerophosphate, and 50 nM of each drug. RNA extraction was performed at 0, 3, and 7 days as follows: 1 mL of RNA iso plus (#9109; TaKaRa, Tokyo, Japan) was added to each well of the 6-well plate, placed on ice for 10 min, and the cells were collected into 1.5 mL EP tubes. 0.2 mL of chloroform was added, the plates were vortexed for 15 s, and incubated at room temperature for 10 min. Centrifuge at 12000×g for 15 min at 4°C. Transfer 500 μL of supernatant to a new 1.5 mL EP tube, add 500 μL of isopropanol, let stand for 10 min, centrifuge at 12000g for 15 min at 4°C, remove the supernatant, resuspend the RNA in 75% ethanol, and centrifuge at 7500g at 4°C for 15 min. Discard the ethanol, and when the ethanol has completely evaporated, dissolve the RNA in 30 μL of DEPC water and quantify using a microplate reader.

[0074] RT-qPCR: Total RNA extracted from 1.0 μg was reverse transcribed using PrimeScript™ RT Master Mix (#RR036A; Tokyo Takahara, Japan) and used as a template for subsequent qPCR reactions. A qPCR instrument (Analytik Jena, Jena, Germany) was used. The reaction system contained MonAmp™ SYBR® Green qPCR Mix (#MQ10101S; Wuhan, China), cDNA, and forward and reverse primers. The reaction conditions were 95°C for 3 min; then 40 cycles of 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s were performed; the final step was 72°C for 20 s. The following primer sequences were used: alkaline phosphatase (ALP) (forward: 5ʹ-GCTGATCATCCCACGTTT-3ʹ, reverse: 5ʹ-ACCATAGATGGCCGTGA-3ʹ); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5ʹ-ACCCAGAAGACTGGATGG-3ʹ, reverse: 5ʹ-CACATTGGGTAGAACAC-3ʹ); osteocalcin (OCN) (forward: 5ʹ-CCCTGAGTCGAAAGCT-3ʹ, reverse: 5ʹ-GCGGTCTTCAAGCATAG-3ʹ); Runt-related transcription factor 2 (Runx2) (forward: 5ʹ-GCGCATTCACAGTA-3ʹ, reverse: 5ʹ-TGGAGTGGATGGAT-3ʹ). The expression levels of the target genes were analyzed using the 2-ΔΔCT method.

[0075] Experimental Results: ALP staining analysis of three series of glycopeptides, PTH-1, PTH-2, and PTH-3, revealed that PTH-1g and PTH-2i significantly promoted osteoblast differentiation compared to teriparatide, while the disaccharidated glycopeptides, namely the PTH-3 series, had little effect. Further Alizarin Red staining analysis of the PTH-1 and PTH-2 series showed that PTH-1a, PTH-1g, PTH-2a, and PTH-2i exhibited more pronounced calcium nodules than teriparatide, indicating a better effect in promoting osteoblast formation. Next, gene-level analysis of the osteogenic differentiation-promoting effects of these four well-performing glycopeptides was performed. ALP and Alizarin Red staining results were consistent: the compound PTH-2i, obtained by maltose glycosylation modification of teriparatide at arginine position 25, showed a significantly better osteogenic differentiation-promoting effect than other drugs. The results are as follows: Figure 30-32 As shown.

[0076] Example 4: Glycosylation modification of teriparatide can improve enzyme stability and serum stability.

[0077] 1) Enzyme stability experiment

[0078] Enzyme stability assay: 1 mg of teriparatide glycopeptide was dissolved in a specific amount of PBS buffer to prepare a 1 mM peptide stock solution. A certain amount of chymotrypsin was dissolved in 50 mM phosphate buffer (pH 7.4) containing 2 mM calcium chloride to a chymotrypsin concentration of 0.5 ng / μL. 1950 μL of phosphate buffer containing chymotrypsin and 50 μL of peptide stock solution were added to 2 mL centrifuge tubes for enzyme digestion. At time points of 0 min, 10 min, 20 min, 40 min, 80 min, 120 min, and 480 min, 20 μL of 1 M hydrochloric acid was added to quench the chymotrypsin activity. The residual amount of peptide at different time points was analyzed using HPLC.

[0079] 2) Serum stability test

[0080] Serum stability assay: Teriparatide and three teriparatide glycopeptides, PTH-1g, PTH-2a, and PTH-1h, were selected for serum stability studies. The glycopeptide powder was dissolved in water to prepare a 1 mg / mL solution. Human serum and the glycopeptide solution were mixed at a 4:1 ratio and incubated at 37 ℃. Sampling intervals were set at 0 h / 6 h / 12 ​​h / 24 h / 36 h / 48 h / 54 h / 66 h / 78 h. Each time, 50 μL of the mixture was added to 50 μL of ethanol stop solution, centrifuged at 13400 rpm for 10 min, and the supernatant was collected. The supernatant was analyzed by HPLC at a wavelength of 220 nm.

[0081] Experimental results: Compared with unmodified teriparatide, arginine glycosylation-modified teriparatide showed significantly improved stability, with protease stability increasing by 8 times and serum stability increasing by 3 times. The results are shown in Table 3.

[0082] Table 3. Protease half-life and serum half-life

[0083]

[0084] Example 5: Glycosylation modification of teriparatide can effectively prevent bone loss in ovariectomized mice.

[0085] Animal modeling: 6-7 week old female mice were randomly divided into two groups: a non-glycopeptide group (n=6) and a glycopeptide group (n=6). In the non-glycopeptide group, after bilateral ovariectomy, mice were subcutaneously injected with PTH. In the glycopeptide group, after bilateral ovariectomy, mice were subcutaneously injected with PTH-1a.

[0086] Sample processing: Mice were sacrificed after 2 weeks of feeding, and the femurs of both hind limbs and internal organs (heart, liver, spleen, lung, and kidney) were harvested. The right hind limb femur was fixed with 4% paraformaldehyde solution, and the fixed femur was examined by Micro-CT. The distal end of the femur was scanned using a micro-CT scanner to construct two-dimensional and three-dimensional images of the distal metaphysis of the mouse femur. The ratio of bone surface area to tissue volume (BS / TV) and relative bone volume (BV / TV) were analyzed using the built-in software of the CT scanner to obtain quantitative results.

[0087] Experimental Results: Computer analysis of the distal femur revealed that the ratio of bone surface area to tissue volume (BS / TV) and the relative bone volume or bone volume fraction (BV / TV) were 2 times and 1.6 times that of the non-glycopeptide group, respectively. These differences were statistically significant (p<0.001). The results are shown in Table 4.

[0088] Table 4 Bone Surface Area to Tissue Volume Ratio

[0089]

[0090] The beneficial effects of this invention are as follows: Research reports indicate that chemically modifying the arginine glycosylation-modified anti-osteoporosis active compound, its preparation method, and its application demonstrate that increasing arginine glycosylation in the teriparatide sequence to enhance its membrane permeability and enzyme stability is an effective strategy for addressing the poor drug-likeness of teriparatide. Therefore, we designed and synthesized a series of novel PTH glycopeptide active molecules using an arginine glycosylation modification strategy, aiming to enhance their cell permeability, improve enzyme stability and anti-osteoporosis activity, making them more suitable for safer use in osteoporosis patients.

[0091] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A teriparatide modified with arginine glycosylation, characterized in that, The teriparatide is one of the following: 1) PTH-1a: Using SVSEIQLMHNLGKHLNSMERX1VEWLRKKLQDVHNF-NH2 as a peptide template, the R at position 20 is replaced by Orn(Dde) and coupled with a glucose donor; 2) PTH-1g: Using SVSEIQLMHNLGKHLNSMERX1VEWLRKKLQDVHNF-NH2 as a peptide template, the R at position 20 is replaced by Orn(Dde) and coupled with a xylose donor; 3) PTH-2a: Using SVSEIQLMHNLGKHLNSMERVEWLRX2KKLQDVHNF-NH2 as a peptide template, the R at position 25 is replaced by Orn(Dde) and coupled with a glucose donor; 4) PTH-2i: Using SVSEIQLMHNLGKHLNSMERVEWLRX2KKLQDVHNF-NH2 as a peptide template, the R at position 25 is replaced by Orn(Dde) and coupled with a maltose donor.

2. The use of teriparatide, which is arginine glycosylated modified as described in claim 1, in the preparation of a medicament for treating osteoporosis.

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