阅读说明：本技术 新的特立帕肽糖基化衍生物及其应用 (Novel glycosylated teriparatide derivative and application thereof ) 是由 胡宏岗 丛薇 刘超 何世鹏 宋慧 汪楠 于 2020-05-18 设计创作，主要内容包括：本发明涉及医药技术领域,且公开了新的特立帕肽糖基化衍生物,具体指具有式(I)结构的多肽,具有式(I)结构的多肽及其药学上可接受的盐或酯：X1VX2EIQLMH X3LGKHL X4X5MERVEWLRKKLQ X6VHNF (I),其中,X1,X2,X5表示Ser(GlcNAc)或Ser(Glc)或Ser(Fuc)或Ser(Rib)或Ser(Gal)或Ser(GalNAc)或Ser(Lyx)或Ser(Rha)或Ser(Man)或Ser(ManNAc)或Ser(Cel)或Ser(Mal)或Ser(Suc)或Ser(Lac)或Ser(Tre)或Ser,X3,X4,X6表示Asn(GlcNAc)或Asn(Glc)或Asn(Fuc)或Asn(Rib)或Asn(Gal)或Asn(GalNAc)或Asn(Lyx)或Asn(Rha)或Asn(Man)或Asn(ManNAc)或Asn(Cel)或Asn(Mal)或Asn(Suc)或Asn(Lac)或Asn(Tre)或Asn。该新的特立帕肽糖基化衍生物及其制备方法与应用,该系列衍生物具有促进成骨细胞分化的作用,可用于治疗骨质疏松症,且不会损坏骨晶体结构,不会影响骨强度,使用更加安全可靠。(The invention relates to the technical field of medicines, and discloses a novel teriparatide glycosylation derivative, in particular to a polypeptide with a structure shown in a formula (I), a polypeptide with the structure shown in the formula (I) and pharmaceutically acceptable salts or esters thereof: x1VX2EIQLMH X3LGKHL X4X5MERVEWLRKKLQ X6VHNF (I), wherein X1, X2, X5 represent Ser (GlcNAc) or Ser (Glc) or Ser (Fuc) or Ser (Rib) or Ser (Gal) or Ser (GalNAc) or Ser (Lyx) or Ser (Rha) or Ser (Man) or Ser (ManNAc) or Ser (Cel) or Ser (Mal) or Ser (Suc) or Ser (Lac) or Ser (Trec) or Ser, X3, X4, X6 represent Asn (GlcNAc) or Asn (Glc) or Asn (Fuc) or Asn (Rib) or Asn (Gal) or Asn (NAc) or LyAsn (Lyx) or Asn (Rha) or ManAsn (Asn) or Asn (Asn) or Cel (Asn) or Asn (C) or Asn (Asn) or Asn (C). The series of derivatives have the effect of promoting osteoblast differentiation, can be used for treating osteoporosis, can not damage a bone crystal structure, can not influence bone strength, and is safer and more reliable to use.)

1. Novel glycosylated teriparatide derivatives, in particular polypeptides having the structure of formula (I).

A polypeptide having the structure of formula (I) and pharmaceutically acceptable salts or esters thereof:

X1VX2EIQLMH X3LGKHL X4X5MERVEWLRKKLQ X6VHNF (I)

wherein the content of the first and second substances,

x1, X2, X5 represents Ser (GlcNAc) or Ser (glc) or Ser (Fuc) or Ser (Rib) or Ser (Gal) or Ser (GalNAc) or Ser (Lyx) or Ser (Rha) or Ser (Man) or Ser (ManNAc) or Ser (Cel) or Ser (Mal) or Ser (Suc) or Ser (Lac) or Ser (Tre) or Ser.

X3, X4, X6 represents Asn (GlcNAc) or Asn (glc) or Asn (Fuc) or Asn (Rib) or Asn (Gal) or Asn (GalNAc) or Asn (Lyx) or Asn (Rha) or Asn (Man) or Asn (ManNAc) or Asn (Cel) or Asn (Mal) or Asn (Suc) or Asn (Lac) or Asn (Tre) or Asn.

2. Pharmaceutical composition comprising a polypeptide fragment having the structure of formula (I) as defined above, characterized in that it may comprise a polypeptide according to claim 1, preferably only one glycosylated teriparatide derivative.

3. The pharmaceutical composition comprising the polypeptide fragment having the structure of formula (I) as claimed in claim 2, further comprising a pharmaceutically acceptable diluent, excipient or carrier.

4. The pharmaceutical composition of claim 3, wherein the carrier is one or more of ethanol, glycerol, or water.

5. The application of the novel teriparatide glycosylation derivative and the pharmaceutical composition of the polypeptide fragment with the structure of the formula (I) can be used for treating osteoporosis.

Technical Field

The invention relates to the technical field of medicines, in particular to a novel teriparatide glycosylated derivative and a preparation method and application thereof.

Background

Osteoporosis, which is a common systemic bone metabolic disease, mainly manifests as low bone mass, destruction of bone microstructure and increase of bone fragility, thereby causing the occurrence of secondary fracture, and can occur in different gender and age stages, but is mostly seen in postmenopausal women and elderly men, and the national institutes of health in 2001 proposes that bone mineral density and bone quality are two main aspects affecting bone strength, wherein bone quality is maintained by bone absorption of osteoclasts and matrix deposition and mineralization of osteoblasts, if bone formation is weakened, bone absorption is strengthened, bone homeostasis is changed, osteoporosis is caused, the drug therapy of osteoporosis is divided into anti-catabolic drugs and anabolic drugs, and the anti-catabolic drugs mainly comprise estrogen, selective estrogen receptor regulator, bisphosphonates, calcium, vitamin D and the like, these drugs increase bone mass mainly by reducing bone turnover, and although effective in preventing bone fracture, they bring about a reduction in bone mass which increases the risk of potential bone fracture, and anabolic drugs such as fluoride have not been widely used because of adverse effects on bone strength due to damage to bone crystal structure, and parathyroid hormone is considered to be the most promising anabolic drug as one of important hormones for regulating calcium-phosphorus metabolism and bone metabolism at present.

Teriparatide (Terparatide) is a human parathyroid hormone analogue synthesized by genetic engineering recombination technology, the amino acid sequence of the Teriparatide is completely identical to the N-terminal 34-site amino acid sequence of natural human parathyroid hormone, the analogue has similar affinity to parathyroid hormone receptors and can activate the same signal channel of osteoblasts so as to promote bone formation, the Teriparatide is approved to be on the market by the FDA in 2002 in the United states and is the only currently approved bone anabolic preparation, the biological function of acetylglucosamine glycosylation in parathyroid hormone and Teriparatide of human parathyroid hormone analogue is not reported, but researches show that glycosylation can improve the properties and functions of polypeptide and protein from various aspects, such as increasing hydrophilicity, bioavailability, limiting or maintaining conformation, increasing resistance to hydrolytic enzyme, reducing immunogenicity and the like, the inventor carries out a series of novel glycosylation derivatives of teriparatide based on teriparatide sequence with different sites including O-linked glycosylation and N-linked glycosylation modification, and the preferred compounds have good osteoblast differentiation promoting effect, thereby providing a new choice for treating osteoporosis.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a novel teriparatide glycosylated derivative, a preparation method and application thereof, has the advantages of better osteoblast differentiation promoting effect and the like, and solves the problem that the existing medicament for treating osteoporosis cannot be widely applied because the side effect of the existing medicament on the bone strength is influenced by the damage of the bone crystal structure.

(II) technical scheme

In order to achieve the above-mentioned goal of having better osteoblast differentiation promoting effect, the invention provides the following technical scheme: novel glycosylated teriparatide derivatives, in particular polypeptides having the structure of formula (I).

A polypeptide having the structure of formula (I) and pharmaceutically acceptable salts or esters thereof:

X1VX2EIQLMH X3LGKH LX4X5MERVEWLRKKLQ X6VHNF (I)

wherein the content of the first and second substances,

x1, X2, X5 represents Ser (GlcNAc) or Ser (glc) or Ser (Fuc) or Ser (Rib) or Ser (Gal) or Ser (GalNAc) or Ser (Lyx) or Ser (Rha) or Ser (Man) or Ser (ManNAc) or Ser (Cel) or Ser (Mal) or Ser (Suc) or Ser (Lac) or Ser (Tre) or Ser.

X3, X4, X6 represents Asn (GlcNAc) or Asn (glc) or Asn (Fuc) or Asn (Rib) or Asn (Gal) or Asn (GalNAc) or Asn (Lyx) or Asn (Rha) or Asn (Man) or Asn (ManNAc) or Asn (Cel) or Asn (Mal) or Asn (Suc) or Asn (Lac) or Asn (Tre) or Asn.

The amino acid residue at position 1 of the polypeptide fragment of formula (I) (X1) is preferably Ser (GlcNAc).

The amino acid residue at position 3 of the polypeptide fragment of formula (I) (X2) is preferably Ser (GlcNAc).

The amino acid residue at position 10 of the polypeptide fragment of formula (I) (X3) is preferably Asn (GlcNAc).

The amino acid residue at position 16 (X4) of the polypeptide fragment in formula (I) is preferably Asn (GlcNAc).

The amino acid residue at position 17 (X5) of the polypeptide fragment of formula (I) is preferably Ser (GlcNAc).

The amino acid residue (X6) at position 30 of the polypeptide fragment of formula (I) is preferably Asn (GlcNAc).

Herein, the "glycosylated teriparatide derivative of the present invention" refers to the polypeptide of the present invention having the structure represented by formula (I), and such a polypeptide may be referred to as "glycosylated teriparatide derivative", "polypeptide fragment" or "polypeptide of the present invention" herein.

The amino group at the N-terminus and the carboxyl group at the C-terminus and the amino acid side chain groups of the polypeptide of formula (I) may be unmodified or modified without substantially affecting the activity of the polypeptide of the invention, such as forming a "pharmaceutically acceptable ester", the modification of the N-terminal amino group including but not limited to de-amino, N-lower alkyl, N-di-lower alkyl and N-acyl modifications, the modification of the C-terminal carboxyl group including but not limited to amide, lower alkyl amide, dialkyl amide and lower alkyl ester modifications, the invention prefers that the amino group at the N-terminus of the polypeptide of formula (I) is not acetylated, i.e., -NH2, and the carboxyl group at the C-terminus is amidated, i.e., -CONH 2.

As used herein, the polypeptide and the amino acid and chemical group are commonly known in the art, wherein the abbreviations of the amino acids are as defined in Table 1, the structures of the specific amino acids and sugar amino acids are as defined in Table 2, and the amino acids are generally referred to as L-form amino acids unless otherwise specified.

TABLE 1 amino acid abbreviation table

TABLE 2 Special amino acid abbreviations Table

"pharmaceutically acceptable salts" refers to salts of certain small acidic or basic compounds with polypeptides, which generally increase the solubility of the polypeptide, and which do not substantially alter the activity of the polypeptide, e.g., acids which typically form salts with the polypeptides of the invention are hydrochloric, phosphoric, sulfuric, acetic, succinic, maleic, and citric acids, and the like; bases capable of forming salts with the polypeptide of the present invention include hydroxides, ammonium and carbonates of alkali metals or alkaline earth metals, and the like.

The osteoblast promoting effect of the polypeptide of the present invention can be verified by conventional experimental methods in the art, such as cytological experiments, etc., and in particular embodiments of the present invention, preferably, by cytological experiments, such as alkaline phosphatase (ALP) staining of mouse osteoblast samples, through which it is found that the polypeptide fragments of formula (I) according to the present invention have an in vitro osteoblast promoting effect, and can be used for treating osteoporosis.

In addition, another aspect of the present invention provides a pharmaceutical composition comprising the polypeptide fragment having the structure of formula (I) as described above, which can be used for treating osteoporosis, the composition may comprise one or more of the polypeptide fragments of the present invention, preferably only one glycosylated teriparatide derivative, the composition may comprise one or more pharmaceutically acceptable diluents, excipients or carriers, preferably the composition is in unit dosage form, such as tablets, films, pills, capsules (including sustained or delayed release forms), powders, granules, syrups or emulsions, sterile injectable solutions, suspensions or lyophilized powders, aerosols or liquid sprays, drop-on-demand devices or suppositories, and the active pharmaceutical ingredient may be combined with a non-toxic pharmaceutically acceptable inert carrier, such as ethanol, glycerol, water or a combination thereof, the glycosylated teriparatide derivative of formula (I) of the present invention is preferably used as a sterilized aqueous solution for injection.

The pharmaceutical compositions of the present invention may be administered by any of the means well known to those skilled in the art of administration, such as oral, rectal, sublingual, pulmonary, transdermal, iontophoretic, vaginal and intranasal administration.

The pharmaceutical compositions of the invention are preferably administered parenterally, such as by subcutaneous, intramuscular or intravenous injection

The names, structural formulae and mass spectral data of some of the preferred compounds synthesized in accordance with the present invention are shown in Table 3.

Table 3 partial name, structural formula and Mass Spectrometry data of preferred Compounds

For the purpose of facilitating understanding, the present invention will be described below by way of specific embodiments and accompanying drawings. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention.

(III) advantageous effects

Compared with the prior art, the invention provides a novel teriparatide glycosylated derivative, a preparation method and application thereof, and the novel teriparatide glycosylated derivative has the following beneficial effects:

1. the novel glycosylated teriparatide derivative, the preparation method and the application thereof are provided

2. The novel glycosylated teriparatide derivative, the preparation method and the application thereof are provided

Drawings

FIG. 1 shows ALP staining results of mouse osteoblasts with glycosylated derivatives of teriparatide;

FIG. 2 is a schematic representation of the structural formula of PTHG-2 in Table 3;

FIG. 3 is a schematic representation of the structural formula of PTHG-4 in Table 3;

FIG. 4 is a schematic representation of the structural formula of PTHG-9 in Table 3.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.