CN114057863B - Parathyroid hormone related peptide analogue and application thereof - Google Patents

Parathyroid hormone related peptide analogue and application thereof Download PDF

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CN114057863B
CN114057863B CN202010780705.5A CN202010780705A CN114057863B CN 114057863 B CN114057863 B CN 114057863B CN 202010780705 A CN202010780705 A CN 202010780705A CN 114057863 B CN114057863 B CN 114057863B
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leu
compound
pharmaceutical composition
parathyroid hormone
preventing
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CN114057863A (en
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谢鑫
黄亮
曹春来
周翠
何秀仪
邓慧兴
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Zhuhai United Laboratories Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Abstract

The invention provides parathyroid hormone related peptide analogues and application thereof. The parathyroid hormone related peptide analogue is a compound shown in a general formula I or a salt or solvate thereof. The parathyroid hormone related peptide analogue has good stability, and the stability is superior to teriparatide and is not inferior to abamectin; the compound has very high agonistic activity to PTHR1, has remarkable agonistic activity as well as Yu Teli Pa peptide and Abaparib peptide, and has the characteristics of excellent medicinal activity and good osteogenesis effect when being used as a medicament for preventing and/or treating bone diseases and related complications thereof.

Description

Parathyroid hormone related peptide analogue and application thereof
Technical Field
The invention relates to the field of medicines, in particular to a parathyroid hormone related peptide analogue and application thereof.
Background
The number of patients suffering from osteoporosis worldwide is over 2 hundred million, and on average, fracture cases caused by osteoporosis occur every 3 seconds, and the mortality rate of hip fracture patients is up to 20% within 1 year after fracture. While this number is so apparent, it is increasing as the global population ages. There is an increasing need to find safe and effective medicaments for the treatment of these diseases caused by osteopenia.
Through many years of research and study, parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) have been found to share many biological effects, including binding to parathyroid receptor 1 (PTHR 1), and regulation of calcium and phosphorus metabolism and bone turnover following binding, which has a close association with bone mass reduction. The active fragments of the two are PTH (1-34), PTH (1-84), PTHrP (1-34), PTHrP (1-36) and the like, and a series of PTH analogues and PTHrP analogue medicaments are developed on the basis.
hPTH (1-34), the amino acid sequence is:
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH(SEQ ID NO:1)。
hPTHRP (1-34), the amino acid sequence is:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-I1e-Gln-Asp-Leu-Arg-Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-NH 2 (SEQ ID NO:2)。
recombinant human PTH (1-34), teriparatide, trade name developed by Gift corporationThe injection is the first bone morphogenic drug approved by the U.S. FDA for the treatment of osteoporosis in month 12 of 2002, and is subsequently also approved by the european union.
However, teriparatide is poorly chemically stable and its solutions are susceptible to degradation, and the drug degrades mainly in the three forms of oxidation, deamidation and cleavage of peptide bonds, the most prominent of which is oxidation of Met8 and Met18 residues, deamidation at Asn10, asn16 and Asn33 resulting in cleavage of peptide bonds at Asn16, asn33, asp30 (Ruchi Kothari et al Modes of Degradation and Impurity Characterization in rhPTH (1-34) two-stage Studies.2011,65 (4): 348-62.). The liquid injection must be stored in a refrigerator at 2-8 ℃ for the reasons, and the liquid injection must be stored at 2-8 ℃ after unsealing for use, so that the liquid injection has high requirements on storage and transportation conditions and is unfavorable for patients to use.
To overcome the disadvantage of the susceptibility of PTH (1-34) to degradation, the preferred parathyroid hormone analogues are substituted with at least one amino acid, at one or more of the following positions 8-11, 13, 16-19, 21, 22, 29 to 34, in particular 8-11, 16-19, 33 and/or 34 of the parathyroid hormone sequence. For example, sandoz corporation developed a novel analog of PTH (1-34) SDZ PTS 893 of the formula [ Leu8, asp10, lys11, ala16, gln18, thr33, ala34] -hPTH- (1-34), which enhanced chemical stability and oxidation resistance in solution, stimulated adenylate cyclase in the binding affinity of kidney cells, increased intracellular calcium, and the like 2 to 5 fold that of the natural fragment (R.Gamse et al SDZ PTS 893:Pharmacological Profile of A Highly Potent Novel PTH Analog.ASBMR 19th Annual Meeting:F366). The therapeutic effects of PTH (1-34), PTHrP (1-36) and SDZ PTS 893 were compared in the ovariectomized rat model, and the bone synthesis capacity was ranked as SDZ PTS 893> PTH (1-34) > PTHrP (1-36), but the side effects of rats in the SDZ PTS 893 group were also greatest, and these rats exhibited moderate hypercalcemia and significant renal calcium accumulation with mortality of 13% (A.F. Stewart et al six-Month Daily Administration of Parathyroid Hormone and Parathyroid Hormone-Related Protein Peptides to Adult Ovariectomized Rats Markedly Enhances Bone Mass and Biomechanical Properties: A Comparison of Human Parathyroid Hormone 1-34,Parathyroid Hormone-Related Protein 1-36, and SDZ-Parathyroid Hormone 893.Journal of Bone and Mineral Research:the official journal of the American Society for Bone and Mineral Research.2000,15 (8): 1517-25.). Unfortunately, such drugs have no information on marketing to date.
Abaparib ([ Glu ] developed by Radius Health Co 22 ' 25 ,Leu 23 ' 28 ' 31 ,Aib 29 ,Lys 26 ' 30 ]hPTHrP(l-34)NH 2 ) The amino acid sequence is:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-I1e-Gln-Asp-Leu-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu-His-Thr-Ala-NH 2 (SEQ ID NO: 3) thereofThe injection is approved by FDA to be marketed in 2017, 4 and 28, and is PTHrP analogue medicine for treating osteoporosis by promoting bone formation, and has the trade name of
The abapatatin modifies 8 amino acids on the basis of hPTHRP (l-34), enhances the pharmaceutical activity and has excellent solution stability, the liquid injection is transported and stored at 2-8 ℃ before being unused, can be stored at 20-25 ℃ for 30 days after being unsealed for use, greatly reduces the storage requirement, is convenient for patients to use at home or carry out, but has 29-site amino acid which is unnatural amino acid Aib, and is synthesized by SPSS (specific pathogen Health) company, and the production cost is higher than the biosynthesis.
A novel all-natural amino acid PTHrP analog developed by Roche, RS66271, i.e. [ Glu 22 ' 25 ' 29 ,Leu 23 ' 28 ' 31 ,Lys 26 ' 30 ]hPTHrP(l-34)NH 2 Doses of 50 μg and 100 μg increased lumbar BMD in postmenopausal osteoporotic women by 2.2% and 8.8%, respectively, over a 6 month treatment period, but further development was stopped by the appearance of cortical bone loss (John Fox, developments in parathyroid hormone and related peptides as bone-information agents, current opiion in pharmacology 2002, 2:338-344.).
The effect of both teriparatide and abamectin on bone depends on the pattern of systemic exposure, administration in a pulsatile manner, requiring daily subcutaneous injections, and poor patient compliance and compliance. Since the use of both in the cancerogenic studies in rats has been shown to increase the incidence of osteosarcoma, a malignancy, both treatment cycles were up to 24 months, with patients receiving only one treatment for 24 months throughout the life.
Therefore, the PTHrP analogue with excellent solution normal temperature stability, convenient use and carrying for patients, low production cost, suitability for large-scale production and better pharmaceutical activity in a limited treatment time is urgently needed at present.
Disclosure of Invention
The primary object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide a parathyroid hormone-related peptide (PTHrP) analog. The polypeptide sequence of the parathyroid hormone related peptide (PTHrP) analogue is composed of natural amino acids, can be produced through biological fermentation, has low production cost, and has the characteristics of excellent solution stability, plasma stability and physical stability.
A second object of the present invention is to provide an application of the above parathyroid hormone related peptide (PTHrP) analogue in the preparation of a medicament for preventing and/or treating skeletal diseases and related complications thereof, and the parathyroid hormone related peptide (PTHrP) analogue has the characteristics of excellent pharmaceutical activity and good osteogenic effect.
The aim of the invention is achieved by the following technical scheme:
a parathyroid hormone-related peptide (PTHrP) analogue which is a compound having the general formula i:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-X 11 -Gly-Lys-Ser-I1e-Ala-Asp-Ala-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-X 29 -Lys-Leu-His-Thr-Ala-NH 2 (general formula I)
Wherein, the liquid crystal display device comprises a liquid crystal display device,
X 11 selected from Arg, lys or His;
X 29 selected from Leu, ile, val or Phe.
In some embodiments, the provided PTHrP analog is selected from compound 1 having an amino acid sequence shown as SEQ ID NO.4, compound 2 having an amino acid sequence shown as SEQ ID NO.5, compound 3 having an amino acid sequence shown as SEQ ID NO.6, or compound 4 having an amino acid sequence shown as SEQ ID NO. 7:
compound 1:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Arg-Gly-Lys-Ser-I1e-Ala-Asp-Ala-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Leu-Lys-Leu-His-Thr-Ala-NH 2 (SEQ ID NO.4)；
compound 2:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Arg-Gly-Lys-Ser-I1e-Ala-Asp-Ala-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Ile-Lys-Leu-His-Thr-Ala-NH 2 (SEQ ID NO.5)；
compound 3:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Arg-Gly-Lys-Ser-I1e-Ala-Asp-Ala-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Val-Lys-Leu-His-Thr-Ala-NH 2 (SEQ ID NO.6)；
compound 4:
Ala-Va1-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Arg-Gly-Lys-Ser-I1e-Ala-Asp-Ala-Arg-Arg-Arg-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Phe-Lys-Leu-His-Thr-Ala-NH 2 (SEQ ID NO.7)。
the parathyroid hormone related peptide (PTHrP) analogues can be prepared by methods including synthesis and recombinant DNA biotechnology.
The application of the parathyroid hormone related peptide (PTHrP) analogue in preparing medicaments for preventing and/or treating bone diseases and related complications thereof.
The medicine for preventing and/or treating the bone diseases and related complications thereof comprises a medicine composition for preventing and/or treating osteoporosis.
The parathyroid hormone related peptide (PTHrP) analogs have agonistic activity at PTH receptor 1 (PTHR 1), and thus the parathyroid hormone related peptide (PTHrP) analogs may also act as PTHR1 agonists. By "agonistic activity" is meant that the compound agonizes a particular receptor cell to produce cAMP, and the cell used may be a human embryonic kidney cell (HEK 293), COS-7 cell or CHO-K1 cell that is constructed by one of skill in the art to overexpress PTH receptor 1. The receptor agonism activity can be achieved by using compounds that agonize the EC of the receptor cells to produce cAMP 50 The value is taken as a measurement. EC (EC) 50 The values are the drug concentration values required to achieve half the maximum activity (50% activity) of the compound in a particular assay system.
A pharmaceutical composition for preventing and/or treating osteoporosis comprises an effective amount of a compound and pharmaceutically acceptable auxiliary materials; an effective amount of a compound is a parathyroid hormone related peptide (PTHrP) analog as described above, or a combination of a parathyroid hormone related peptide (PTHrP) analog as described above with other agents for treating osteoporosis disorders.
By an effective amount of a compound is meant an amount of the compound that produces both a disease modifying or therapeutic effect and a non-damaging effect. The effective amount can be suitably determined by the attending physician according to the severity of the disease, age, sex, weight, general health, etc., of the patient. The patient is a mammal; more preferably at least one of cow, horse, goat, sheep, dog, chimpanzee, rabbit, mouse, rat, monkey, pig and human; most preferably a human.
The auxiliary materials include, but are not limited to, at least one of diluents, excipients, lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, gelling agents, disintegrants, pH adjusters and solubilizing agents.
The auxiliary materials are used for assisting in preparing the pharmaceutical composition into different dosage forms, including but not limited to injection or freeze-dried powder, tablets, patches, pills, troches, soft capsules, hard capsules, granules, powder, solutions, suspensions and syrups; more preferably an injection or a lyophilized powder.
The pharmaceutical composition may be loaded into a drug carrier material and/or a drug delivery device for use.
The drug carrier material is preferably at least one of a microcapsule, a microsphere, a nanoparticle and a liposome.
The other medicines for treating the osteoporosis diseases comprise at least one of bisphosphate, calcitonin, estrogens, selective estrogen receptor modulators, strontium salts, active vitamin D and analogues thereof and traditional Chinese medicine medicines for protecting and strengthening bones.
The administration modes of the pharmaceutical composition include, but are not limited to, at least one or more of oral administration, inhalation administration and parenteral administration; the parenteral administration is preferably by intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, intradermal injection or transdermal microneedle means.
The frequency of administration of the pharmaceutical composition can be selected according to the actual situation; preferably at least once a day, once a week or once a month.
Compared with the prior art, the invention has the following advantages and effects:
(1) The compounds of the invention have good stability (solution stability, plasma stability and physical stability), which is superior to teriparatide and not inferior to abamectin.
(2) The compound disclosed by the invention has very high agonistic activity on PTHR1, has remarkably high agonistic activity on Yu Teli Pa peptide and Abacalcet peptide, and has the characteristics of excellent medicinal activity and good osteogenesis effect when being used as a medicament for preventing and/or treating bone diseases and related complications thereof.
(3) The polypeptide sequence of the compound consists of natural amino acids, and can be produced by biological fermentation, so that the production cost is low.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
The abbreviations used in the present invention have the following specific meanings:
PTHR 1: parathyroid receptor 1
cAMP: adenosine cyclophosphate
HPLC-MS: high performance liquid chromatography-mass spectrometry
HPLC: high performance liquid chromatography
HEK-293: human embryonic kidney cells
CHO-K1: chinese hamster ovary cells
PBS: phosphate buffer solution
FBS: fetal bovine serum
EC 50 : concentration of half-maximal effect
DMSO: dimethyl sulfoxide
ThT: thioflavin T
The teriparatide and the abaparatide are both consigned to be prepared by synthesis by the Hefei national peptide biotechnology Co., ltd; de-Glutamine was purchased from Norand Norde Inc.
Example 1: synthesis of Compounds
All the compounds of the invention are entrusted to the chemical synthesis method of the compound fertilizer national peptide biotechnology limited company, and the synthesis method provided by the company has the following process. Each of the specific synthetic steps described may be combined in different materials and manners to synthesize a plurality of corresponding compounds of the invention or salts thereof. The reagents and starting materials used are readily available to those of ordinary skill in the art. In particular, the following examples are only illustrative of the invention and should not be construed as limiting the scope of the invention in any way.
Materials:
the materials and reagents used in the invention are purchased from commercial products, and the protective amino acids used in the whole synthesis process are as follows: fmoc-Ser (tBu) -OH, fmoc-Pro-OH, fmoc-Ala-OH, fmoc-Gly-OH, fmoc-Asp (OtBu) -OH, fmoc-Leu-OH, fmoc-Trp (Boc) -OH, fmoc-Gln (Trt) -OH, fmoc-Val-OH, fmoc-Phe-OH, fmoc-Arg (Pbf) -OH, fmoc-Glu (OtBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Thr (tBu) -OH, fmoc-His (Trt) -OH, fmoc-Tyr (tBu) -OH, fmoc-Ile-OH.
The synthetic preparation of the compounds of the present invention will be described below by taking compound 3 (SEQ ID NO: 6) as an example.
The method comprises the following steps:
(1) Pretreatment of Rink amino resin: 1g of dry Rink amino resin was weighed, soaked in DMF and swelled for 10min, and the solvent was removed.
(2) Removing the protecting group Fmoc: 20% (v/v) piperidine/DMF solution was added to the treated Rink amino resin and the reaction was stirred for 20min. The extent of the reaction was monitored during the reaction using ninhydrin chromogenic method, if the resin developed a color change (turning purple), indicating successful Fmoc removal. After the reaction is finished, filtering to remove the solvent, adding DMF into the reaction system, stirring and washing the resin for 1min, and repeating the washing for 3 times to obtain the amino resin with Fmoc protecting groups removed.
(3) Coupling reaction (peptide bond formation): and (3) adding the prepared corresponding Fmoc-protected amino acid solution into an amino resin reactor for removing Fmoc protecting groups in the step (2), then adding DIC/DMF solution, and stirring for reaction for 1.5h. The progress of the reaction was monitored during the reaction using ninhydrin chromogenic method, indicating successful coupling if the resin had no change in color (still colorless). After the reaction was completed, the solvent was removed by filtration, and the resin was washed with DMF under stirring for 1min, followed by repeated washing 3 times. Repeating the above steps, and sequentially adding corresponding amino acid solution until peptide chain synthesis is completed. The last amino acid was coupled with Fmoc-Ala-OH. The sequence of amino acid coupling was added to the compound 3 main peptide sequence in the order Fmoc-Ala-OH, fmoc-Thr (tBu) -OH, fmoc-His (Trt) -OH, fmoc-Leu-OH, fmoc-Lys (Boc) -OH, fmoc-Val-OH, fmoc-Leu-OH (2X), fmoc-Lys (Boc) -OH, fmoc-Glu (OtBu) -OH, fmoc-Leu-OH (2X), fmoc-Glu (OtBu) -OH, fmoc-Arg (Pbf) -OH (3X), fmoc-Ala-OH, fmoc-Asp (OtBu) -OH, fmoc-Ala-OH, fmoc-Ile-OH, fmoc-Ser (tBu) -OH, fmoc-Lys (Boc) -OH, fmoc-Gly-OH, fmoc-Glu (Pbu) -OH, fmoc-Leu-OH (2X), fmoc-Glu (OtBu) -OH, fmoc-OH (Fmoc-OH), fmoc-Glu-OH (OtBu) -OH, fmoc-OH (Fmoc-OH), fmoc-OH (FtBu) -Fhu) -Glu (Fmoc-OH).
(4) Peptide resin post-treatment: the resin was cut by adding the cutting reagent K. Filtering the filtrate, precipitating and centrifuging the filtrate to obtain a white solid which is a crude product of the target compound 3.
Purification of crude peptide compound:
the crude peptide obtained was purified by reversed-phase C18 preparative chromatography (Shimadzu, inertsil ODS 20X 250mm 5 μm) to a purity of more than 95%. Starting with 95% (v/v) buffer A (0.065% (v/v) TFA/water) and 5% (v/v) buffer B (0.05% (v/v) TFA/acetonitrile), the ratio of buffer B was gradually increased to 65% at a rate of 2%/min, and the elution was performed continuously for 30min to collect the target peptide fraction. The purified peptide compounds were confirmed by analytical HPLC/MS analysis.
Based on the above synthesis method, the invention synthesizes and characterizes polypeptide compounds 1 to 4 with the amino acid sequences shown in SEQ ID NO.4 to 7 (see Table 1).
TABLE 1 list of synthetic peptide compounds, molecular weight and purity
Example 2: compound stability test
1. Teriparatide, abamectin, compounds 1-4 were dissolved in sodium acetate buffer (5 mg/mL, formulated with sodium acetate trihydrate) and pH adjusted with appropriate amount of acetic acid to give final concentrations of 2mg/mL, pH 5.1, and filtered with a 0.22 μm sterile filter head. The above compound solutions were separately aspirated and passed through HPLC (Shimadzu LC-20,Waters XBridge C18 column, solution A:0.1% (v/v) TFA-water solution, solution B:0.1% (v/v) TFA-ACN solution, and the solvents in solution A and solution B were water and ACN, respectively). The peak area of 15. Mu.L of injectate was analyzed, and the result of this analysis was the initial point (T 0 )。
The compound sample solution subjected to the stability test is placed in a constant temperature box at 25 ℃, and is stored for 6 days in a sealed and light-proof mode. Samples were taken at regular intervals each day, after the end of the procedure, the sample solution was centrifuged at 4500rpm for 10min, the supernatant solution was gently aspirated and 10. Mu.L of the peak area of the injectate was analyzed by HPLC, the analysis result being the endpoint of the compound stability test (T 6 )。
By comparison of T 0 And measuring the target peak area and the relevant impurity peak area of the compound at each time point, and calculating the residual peptide amount of the measured peptide. The calculation formula is as follows:
residual peptide (%) = (main peak area/T at each time point 0 Main peak area) ×100%;
the chemical stability of the peptide compounds was evaluated by comparing the peptide residual amounts of the peptide compounds, and the measurement results are shown in table 2.
Table 2 results of analysis of the stability of sodium acetate buffer of peptide compound
Analysis of results: from the table it is evident that compound 1, compound 2, compound 3, compound 4 are better stable than teriparatide in sodium acetate buffer than abaparatide.
2. Frozen plasma was thawed in a 37 ℃ water bath prior to the experiment, the plasma was centrifuged at 4000rpm for 5min, and if there was a clot, the clot was removed. The compound solution (12.5. Mu.M) and the positive control drug, bromopropioline solution (100. Mu.M), were prepared with ultrapure water, 2. Mu.L of each of the compound solution and the bromopropioline solution was added to 98. Mu.L of blank plasma, incubated in a 37℃water bath, and 400. Mu.L of stop solution was added to samples taken at 0,1, 2, 3, 4, 5, 6, 8, and 10 hours. Each sample was centrifuged at 4000rpm for 5min, 50. Mu.L of the supernatant was diluted to 100. Mu.L with purified water, and the diluted sample was shaken at 800rpm for 10min and then subjected to HPLC-MS detection (ACQUITY UPLC, XBIdge Protein BEH C4 (Protein analysis) liquid chromatography column, solution A:0.1% (v/v) TFA-aqueous solution, solution B:0.1% (v/v) TFA-ACN solution).
By comparison of T 0 And measuring the target peak area and the relevant impurity peak area of the compound at each time point, and calculating the residual peptide amount of the measured peptide. The calculation formula is as follows:
residual peptide (%) = (main peak area/T at each time point 0 Main peak area) ×100%;
plasma stability of the peptide compounds was evaluated by comparing the peptide residual amounts of the peptide compounds, and the measurement results are shown in table 3.
TABLE 3 results of human plasma stability analysis of peptide compounds
Analysis of results: as is evident from the table, compounds 1 to 4 are all superior to teriparatide and abamectin in human plasma stability tests.
3. Teriparatide, abamectin, compounds 1-4 were dissolved in sodium acetate buffer (5 mg/mL, formulated with sodium acetate trihydrate) and pH adjusted with appropriate amount of acetic acid to give final concentrations of 2mg/mL, pH 5.1, and filtered with a 0.22 μm sterile filter head. 1. Mu.M thioflavin T (ThT) was added to the sample at a volume ratio of ThT: sample = 1:9. additional samples of raw insulin diglucoside (formulated at the same concentration and pH as the abapatatin) were taken and ThT added under the conditions described above. mu.L of sample was added to each well of a 96-well plate, and the wells were sealed by touch using a sealing plate. The plates were incubated with orbital shaking at a temperature of 47℃and an amplitude of 1mm, adjusted to 807cpm, in a Cystation 5 multifunctional microplate reader (Bio-Tek). Fluorescence measurements were performed with excitation through a 440nm filter and measurement of emission from a 485nm filter, spaced 20min apart.
The measurement points were saved in Microsoft Excel format for further processing and curve plotted and fitted using GraphPad Prism. Background emissions from ThT in the absence of fibrils can be ignored.
The physical stability of the peptide compounds was evaluated by comparing the lag time for determining ThT fibril formation of the peptide compounds, see table 4 for the determination results.
TABLE 4 results of peptide compounds ThT fibrillation experimental analysis
Analysis of results: as apparent from the table, the stability of the compound 1, the compound 2, the compound 3 and the compound 4 in the physical stability test is equivalent to that of teriparatide and abaparatide, and the compound is superior to that of the commercial Degu insulin injection, and has good physical stability.
Example 3: determination of the Activity of Compounds on PTH receptor 1
The agonistic activity of the peptide compounds on the corresponding receptor was determined by measuring cAMP signal response of CHO-K1 cells (purchased from Nanj Style Biotechnology Co., ltd.; hereinafter the same) that stably overexpress human PTH receptor 1. Intracellular cAMP content was determined using a kit of Cisbio corp (cAMP Gs dynamic kit) based on HTRF (homogeneous time resolved fluorescence) technology.
CHO-K1 cells (purchased from Nanjing Style Biotechnology Co., ltd.) stably overexpressing human PTH receptor 1 were cultured in the presence of 10% (v/v) FBS and 90% ham's F-12KIn the whole culture medium, when the cell grows to 80% -90% density, digesting with 0.25% (w/v) pancreatin-EDTA, gently blowing off the cell mass into single cells after complete digestion, and regulating the cell density to 5.0X10% 5 And each mL.
A96-well plate was prepared, and 25. Mu.L of the cell suspension (cell density: 5.0X10) was added to each well of the 96-well plate 5 and/mL). The test compound was dissolved in 1 XPBS buffer and diluted 4-fold stepwise from 400nM to prepare a total of 8 concentration point compound solutions. 25. Mu.L of the prepared compound solution is respectively added into the corresponding cell suspension of the 96-well plate by using a pipetting gun, and is uniformly mixed by blowing, and then incubated for 30min at 37 ℃. After the completion of the drug incubation, 25. Mu.L of the detection reagent in the kit was added to each well, and the wells were incubated at room temperature for 60 minutes. The plate was placed in a Bio-Tek multifunctional enzyme-labeled instrument (Cystation 5) to determine fluorescence readings at 665nm/620 nm. Compound concentration-response curves were made using GraphPad prism7.00 mapping software and EC was calculated 50 Values.
TABLE 5 average EC of peptide compounds 50 Value of
Analysis of results: it is apparent from the table that compound 1, compound 2, compound 3, and compound 4 all have high agonistic receptor abilities in the receptor agonistic activity assay, yu Teli peptide and abamectin.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Sequence listing
<110> Pinghai Federal pharmaceutical Co., ltd
<120> parathyroid hormone-related peptide analogue and use thereof
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> amino acid sequence of hPTH (1-34)
<400> 1
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210> 2
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> h PTHrP (1-34) amino acid sequence
<400> 2
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His
20 25 30
Thr Ala
<210> 3
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> Abapatide amino acid sequence
<221> ACT_SITE
<222> (29)..(29)
<223> Xaa=Aib
<400> 3
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15
Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu His
20 25 30
Thr Ala
<210> 4
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> amino acid sequence of Compound 1
<400> 4
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Lys Ser Ile Ala
1 5 10 15
Asp Ala Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Leu Lys Leu His
20 25 30
Thr Ala
<210> 5
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> amino acid sequence of Compound 2
<400> 5
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Lys Ser Ile Ala
1 5 10 15
Asp Ala Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Ile Lys Leu His
20 25 30
Thr Ala
<210> 6
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> Compound 3 amino acid sequence
<400> 6
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Lys Ser Ile Ala
1 5 10 15
Asp Ala Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Val Lys Leu His
20 25 30
Thr Ala
<210> 7
<211> 34
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<223> Compound 4 amino acid sequence
<400> 7
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Lys Ser Ile Ala
1 5 10 15
Asp Ala Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Phe Lys Leu His
20 25 30
Thr Ala

Claims (9)

1. A parathyroid hormone-related peptide analog characterized in that,
the parathyroid hormone related peptide analogue is selected from a compound 1 with an amino acid sequence shown as SEQ ID NO.4, a compound 2 with an amino acid sequence shown as SEQ ID NO.5, a compound 3 with an amino acid sequence shown as SEQ ID NO.6 or a compound 4 with an amino acid sequence shown as SEQ ID NO. 7.
2. Use of the parathyroid hormone-related peptide analogue of claim 1 in the manufacture of a medicament for the prevention and/or treatment of bone diseases and their related complications.
3. The use according to claim 2, wherein the medicament for preventing and/or treating bone diseases and complications thereof comprises a pharmaceutical composition for preventing and/or treating osteoporosis.
4. A pharmaceutical composition for preventing and/or treating osteoporosis, characterized by comprising an effective amount of a compound and pharmaceutically acceptable excipients;
the effective amount of the compound is the parathyroid hormone related peptide analogue as claimed in claim 1 or the parathyroid hormone related peptide analogue as claimed in claim 1 in combination with other medicaments for treating osteoporosis diseases.
5. The pharmaceutical composition for preventing and/or treating osteoporosis according to claim 4,
the auxiliary materials comprise at least one of diluents, excipients, lubricants, binders, fillers, preservatives, surfactants, colorants, flavoring agents, emulsifiers, suspending agents, gelling agents, disintegrants, pH regulators and solubilizers.
6. The pharmaceutical composition for preventing and/or treating osteoporosis according to claim 5,
the auxiliary materials are used for assisting in preparing the pharmaceutical composition into different dosage forms, including but not limited to: injection or lyophilized powder, tablet, patch, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, suspension and syrup.
7. The pharmaceutical composition for preventing and/or treating osteoporosis according to claim 4, wherein said other drugs for treating osteoporosis comprise at least one of bisphosphates, calcitonin, estrogens, selective estrogen receptor modulators, strontium salts, active vitamin D and analogues thereof, and bone-protecting and strengthening traditional Chinese medicine.
8. The pharmaceutical composition for preventing and/or treating osteoporosis according to any one of claims 4 to 7, wherein the administration mode of the pharmaceutical composition comprises at least one or more of oral administration, inhalation administration and parenteral administration;
the frequency of administration of the pharmaceutical composition is at least once a day, once a week or once a month.
9. The pharmaceutical composition for preventing and/or treating osteoporosis according to claim 8, wherein said parenteral administration is by intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous, intradermal injection or transdermal microneedle.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
US5589452A (en) * 1992-07-14 1996-12-31 Syntex (U.S.A.) Inc. Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis
CN1193915A (en) * 1995-08-29 1998-09-23 森德克斯(美国)股份有限公司 Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis

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Publication number Priority date Publication date Assignee Title
US5589452A (en) * 1992-07-14 1996-12-31 Syntex (U.S.A.) Inc. Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis
CN1193915A (en) * 1995-08-29 1998-09-23 森德克斯(美国)股份有限公司 Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis

Non-Patent Citations (2)

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Title
Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism;Liu S, Jean-Alphonse FG, White AD, Wootten D, Sexton PM, Gardella TJ, Vilardaga JP, Gellman SH.;《J Am Chem Soc.》;14486-14490 *
一种新型hPTH(1-34)基因相关肽的制备及其应用的初步研究;施小明;王春晓;庄志华;吴洁;李泰明;曹荣月;刘景晶;;东南大学学报(医学版)(第01期);31-36 *

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