PEG-leuprorelin conjugate and preparation method thereof
阅读说明：本技术 Peg-亮丙瑞林缀合物及其制备方法 (PEG-leuprorelin conjugate and preparation method thereof ) 是由 张拥军 付免 关英 于 2019-11-01 设计创作，主要内容包括：本发明提供了一种新的PEG-亮丙瑞林缀合物及其制备方法,所述方法包括,将单甲氧基聚乙二醇琥珀酰亚胺活泼酯直接与未进行保护的亮丙瑞林反应,得到亮丙瑞林2-位组氨酸上咪唑基修饰的PEG-亮丙瑞林缀合物。该缀合物既保留亮丙瑞林活性,同时半衰期延长,血浆清除率降低,药代动力学性能改善。(The invention provides a novel PEG-leuprorelin conjugate and a preparation method thereof, wherein the method comprises the step of directly reacting monomethoxy polyethylene glycol succinimide active ester with unprotected leuprorelin to obtain the PEG-leuprorelin conjugate modified by an imidazolyl group on leuprorelin 2-histidine. The conjugate not only retains the activity of the leuprorelin, but also has prolonged half-life, reduced clearance rate of blood plasma and improved pharmacokinetic performance.)
1. A PEG-leuprolide conjugate having the structure of formula (I) or (II):
wherein x is an integer value of 10 to 1300, and y is an integer value of 0, 0.5, 1 to 5.
2. A method of making a PEG-leuprolide conjugate comprising: the monomethoxy polyethylene glycol succinimide active ester directly reacts with unprotected leuprorelin to obtain the PEG-leuprorelin conjugate modified by the imidazolyl on the leuprorelin 2-histidine, and the chemical structure is shown as formula I or formula II.
3. The method of claim 2, wherein the leuprolide comprises leuprolide and leuprolide acetate.
4. The preparation method of claim 2, wherein the monomethoxypolyethylene glycol succinimide active ester has the structure
Wherein x is an integer value of 10-1300, the structure of polyethylene glycol comprises a linear structure and a branched structure, and the molecular weight is 400-60 KDa; y is an integer of 1 to 5, and includes carbonate, acetate, succinate and the like of monomethoxypolyethylene glycol succinimide.
5. The production method according to claim 2, wherein the solvent for reaction is distilled water.
6. The preparation method according to claim 2, wherein the molar ratio of the leuprorelin to the monomethoxy polyethylene glycol succinimide active ester is 1: 1-1: 8.
7. The method according to claim 2, wherein the reaction temperature is 4 to 50 ℃.
8. The preparation method according to claim 2, wherein the reaction time is 1 to 24 hours.
9. Use of the PEG-leuprolide conjugate of claim 1 in the field of drug delivery.
Technical Field
The invention relates to the field of medicinal chemistry, in particular to a PEG-leuprorelin conjugate and a synthetic method thereof.
Background
Leuprorelin is a polypeptide consisting of nine amino acids, the amino acid sequence of which is: GLP-HIS-TRP-SER-TYR-DLEU-LEU-ARG-PRO-NHET is an artificially synthesized gonadotropin releasing hormone (GnRH, also known as luteinizing hormone releasing hormone, LHRH) analogue, and the activity of the analogue is far higher than that of natural GnRH. When the medicine is used for a short time, the medicine can promote the pituitary to secrete gonadotropin and the testis or the ovary to secrete steroid hormone due to the exciting effect of the medicine on a gonadotropin releasing hormone receptor. When the medicine is used for a long time, the secretion of gonadotropin is inhibited due to the influence of the down-regulation of a receptor, and further, the secretion of steroid hormones from reproductive organs is inhibited. Thus, leuprolide is used clinically to treat or ameliorate a variety of hormone-dependent diseases including: prostate cancer, endometriosis, uterine fibroids, precocious puberty, and the like.
As a polypeptide drug, leuprorelin has the disadvantages of easy degradation by in vivo enzymes and short half-life. Several techniques have been developed to extend the duration of action. For example, sustained-release microsphere injection, which is prepared by coating leuprorelin with polylactic-co-glycolic acid (PLGA) microspheres, has been used clinically. But has the problems of strong pain feeling, burst release, inflammatory reaction and the like. Bayer developed subcutaneous implant devices driven by osmotic pressure changes, achieved sustained release of leuprolide, and performed clinical trials. But the cost is too high to terminate the research of the project. The literature (Journal of Controlled Release,2014,185,62-70) discloses a technology for realizing sustained Release of leuprorelin by using sorbitan monooleate liquid crystal as a drug carrier. The literature (Journal of Controlled Release,2015,205,98-108) discloses a technique for conjugation of leuprolide to polymeric micelles via the hydroxyl residue of serine at position 4, thereby extending the half-life of the drug.
Pegylation is a common method for improving the pharmacokinetic properties of therapeutic drugs, particularly polypeptides/proteins. Polyethylene glycol (PEG) is a water-soluble polymer, chemically stable, non-antigenic, low-toxicity, biocompatible, and has been approved by FDA. Conjugation of polypeptides/proteins to PEG can increase drug molecular weight, reduce its renal clearance, protect it from proteolytic enzyme degradation, thereby increasing its stability and extending its circulating half-life. In addition, pegylation can also reduce its immunogenicity and antigenicity.
The amino terminal and the carboxyl terminal of the leuprorelin are blocked, and the structure does not have common modifying groups such as amino, sulfydryl and the like, so that the PEG modification of the leuprorelin is relatively difficult, and the research is less. At present, only Chinese patent CN105237762B discloses a technology for preparing PEG leuprorelin. According to the technology, leuprorelin is protected, then coupled with PEG active ester, and finally deprotected to obtain PEGylated leuprorelin taking hydroxyl of serine at 4-position as PEGylation site. The loss is large due to the multiple synthesis steps.
Disclosure of Invention
The invention aims to provide a novel PEG-leuprorelin conjugate and a synthesis method thereof, which can keep the activity of leuprorelin, prolong the half-life period, and have simple and convenient operation and easy commercialization.
The present invention provides a novel PEG-leuprorelin conjugate having the structure of formula I or formula II:
wherein x is an integer value of 10 to 1300, and y is an integer value of 0, 0.5, 1 to 5.
In order to achieve the purpose, the invention provides the following technical scheme: mono-methoxy polyethylene glycol succinimide active ester (CH)3O-(CH2CH2O)x-(CH2CH2)y-NHS) and Leuprorelin (LEU) to obtain the PEG-leuprorelin conjugate (I or II) modified by the imidazole group on the leuprorelin 2-histidine at a single site.
Wherein the leuprorelin comprises leuprorelin and leuprorelin acetate.
Wherein the structure of the monomethoxy polyethylene glycol succinimide active ester is
Wherein the solvent used in the reaction is distilled water with the pH value of 5.5-6.0.
Wherein the molar ratio of the leuprorelin to the monomethoxy polyethylene glycol succinimide active ester is 1: 1-1: 8, preferably 1: 1-1: 5.
Wherein the reaction temperature is 4-50 ℃, and preferably 10-40 ℃.
Wherein the reaction time is 1-24 h, preferably 4-10 h.
The invention provides an application of PEG-leuprorelin conjugate in the field of drug release.
The invention has the advantages of
The invention provides a novel PEG-leuprorelin conjugate and a synthetic method thereof. Firstly, the PEG-leuprorelin conjugate which is obtained by the invention and takes the imidazolyl on the 2-bit histidine as the PEGylation site is structurally different from the PEGylated leuprorelin which is obtained by taking the hydroxyl of the serine at the 4-bit position as the PEGylation site in the prior art. Secondly, the synthesis method provided by the invention does not need to protect the leuprorelin, so that the steps of protection and deprotection are not needed, the synthesis route is short, the loss is small, the synthesis method is efficient, simple and convenient, and the commercialization is easy.
The PEG-leuprorelin conjugate prepared by the invention is used for pharmacokinetic study in rats by taking leuprorelin as a control, and the result is shown in table 1. The data in the table below show that the half-life of the PEG-leuprolide conjugate is extended compared to leuprolide, in particular the half-life of PEG5K-LEU is extended 3-fold over the half-life of leuprolide. Compared with leuprorelin, the area under the curve of PEG-leuprorelin conjugate is increased by 5 times, and the drug absorption condition is obviously improved; and meanwhile, the plasma clearance rate is obviously reduced by 80 percent compared with the plasma clearance rate of the leuprorelin, and the pharmacokinetic property of the leuprorelin is obviously improved.
TABLE 1 pharmacokinetic parameters of leuprorelin and PEG-leuprorelin conjugates
Note: t is t1/2For half-life of the drug, CmaxIs the peak concentration, TmaxTime to peak, AUClastAs the area under the curve (reflecting drug absorption) at drug time, Cl _ F is plasma clearance.
Drawings
FIG. 1 is a MALDI-TOF mass spectrum of PEG-leuprorelin conjugates prepared in examples 1 and 2, PEG2K-LEU and PEG5K-LEU, respectively. Molecular weights are 3235.6 and 6144.2, respectively. The molecular weights of both PEG-leuprorelin conjugates were exactly the sum of the molecular weights of leuprorelin and PEG, indicating that only 1 PEG chain was attached to each leuprorelin molecule.
FIG. 2. preparation of leuprolide, PEG-leuprolide conjugates prepared in examples 1 and 2, respectively1H NMR spectrumIndicating that the PEG segment is attached to the imidazolyl group of leuprorelin 2-histidine.
FIG. 3. rat in vivo pharmacokinetic profiles of leuprolide and PEG-leuprolide conjugates.
Figure 4. variation of blood testosterone concentration in male SD rats injected daily with leuprolide or PEG-leuprolide conjugate.
Detailed Description
The invention provides a new PEG-leuprorelin conjugate and a synthesis method thereof, through directly reacting monomethoxy polyethylene glycol succinimide active ester with unprotected leuprorelin, PEG-leuprorelin conjugate (I or II) modified by imidazolyl on leuprorelin 2-histidine is obtained, and the reaction formula is shown as the following figure:
in the invention, the leuprolide comprises leuprolide and leuprolide acetate. The structure of the monomethoxy polyethylene glycol succinimide active ester is CH3O-(CH2CH2O)x-(CH2CH2) y-NHS; x is an integer value of 10-1300, the structure of the polyethylene glycol comprises a linear structure and a branched structure, and the molecular weight is 400-60 KDa; y is an integer of 0, 0.5, 1 to 5, and includes carbonate, acetate, propionate, succinate, valerate, etc. of monomethoxypolyethylene glycol succinimide. In the reaction, the molar ratio of the leuprorelin to the monomethoxy polyethylene glycol succinimide active ester is 1: 1-1: 8, preferably 1: 1-1: 5.
The conditions under which the reaction is carried out include: the solvent is distilled water with the pH value of 5.5-6.0; the reaction temperature is 4-50 ℃, and preferably 10-40 ℃; the reaction time is 1-24 h, preferably 4-15 h.
The invention provides an application of PEG-leuprorelin conjugate in the field of drug release.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.