CN109620949A - It is a kind of for treating the pharmaceutical composition of osteocarcinoma - Google Patents

It is a kind of for treating the pharmaceutical composition of osteocarcinoma Download PDF

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Publication number
CN109620949A
CN109620949A CN201910056802.7A CN201910056802A CN109620949A CN 109620949 A CN109620949 A CN 109620949A CN 201910056802 A CN201910056802 A CN 201910056802A CN 109620949 A CN109620949 A CN 109620949A
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China
Prior art keywords
polypeptide
osteocarcinoma
ser
gln
pharmaceutical composition
Prior art date
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Application number
CN201910056802.7A
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Chinese (zh)
Inventor
曹帅
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Individual
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane

Abstract

The invention discloses application of the pharmaceutical composition in the drug of preparation treatment osteocarcinoma, described pharmaceutical composition contains ZLGAT polypeptide, can also be administered in combination with bortezomib.Its effective therapeutic dose is 0.1~20mg/Kg weight, can be oral administration, intravenous injection administration, subcutaneous administrations, administered intramuscular when administration.Experiments have shown that: polypeptide of the invention can inhibit growth of tumour cell, have good inhibiting effect to osteocarcinoma, tool has been widely used.

Description

It is a kind of for treating the pharmaceutical composition of osteocarcinoma
Technical field
The present invention relates to the drug of preparation treatment osteocarcinoma and its applications.
Background technique
Osteocarcinoma (Osteosarcoma) is a kind of malignant tumour grown by skeletal system, and is most normal in bone tumour The one kind seen can invade neighbouring tissue and organ, it is also possible to be transferred to other organs of distant place, be apt to occur in the age between ten With child and teenager between year to 20 years old, and male's occurrence probability is slightly above women.The pathogenesis of osteocarcinoma is so far It is unknown, by its original site and the age for mostly occurring in fast-growth, show that it increases and may have with the activity of osteoblast It closes, and inherent cause seems also to play a role in osteocarcinoma, further it is also possible to having with chronic inflammation, radiation or virus infection It closes.
For osteocarcinoma commonly-occurring disease in long bone end, there are about 50% to be located at nearly knee, can cause pain, swelling, even pathology Property fracture, and be likely to occur dyskinesia, part there is lump or other nonspecific symptoms such as weight loss, appetite not Vibration, feveret are had a pain in the back etc., and its death rate is very high, therefore osteocarcinoma is to make us the cancer of the discoloration of news.60 Age mainly takes amputation (amputation) treatment operation, however in the survival rate in 5 years also phase after patient's amputation When low, with the rapid progress of chemotherapy, cooperate the promotion of radiodiagnostics and surgical technic, the five of osteocarcinoma year deposit at present Motility rate, which has improved, to be arrivedBetween, and be expected reach long-term survival (long-term survival), but still without Method fully controls, and merges the Treated with Chemotherapeutic Drugs object used when treatment, there is very strong myelosuppressive effects, therefore develops effective And the therapeutic agent of Small side effects for clinical application be have its necessity, for can both reduce medicine on clinical treatment human bone cancer Object is using concentration and reduces side effect, and up to preferable therapeutic effect.
R. alashanica is perennial acrophyta, has cold-resistant, drought-enduring plant, and inventor passes through biological library batch Amount screening, discovery R. alashanica have stronger degeneration-resistant ability, wherein there may be the substances for inhibiting cancer.
The drug for the treatment of osteocarcinoma is concentrated mainly on Chinese medicine composition and T-2 toxin at present, but these methods all can not What is avoided generates certain side effect to human body, and therapeutic effect is not unusual ideal.Therefore, it develops a kind of efficient , harmless drug seem extremely urgent.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of new pharmaceutical compositions for treating osteocarcinoma.
A technical solution of the invention be it is a kind of for treating the pharmaceutical composition of osteocarcinoma, by ZLGAT polypeptide and Pharmaceutically acceptable carrier is constituted.Shown in the sequence of the polypeptide such as ZLGAT-1~25 are any, sequence is corresponded respectively to Shown in SEQ ID NO:1-25.
The present invention additionally provides a kind of preparation methods of polypeptide, this method comprises: weighing the whole rent strain of R. alashanica 100g is configured to the PBS aqueous solution that concentration of substrate is 5% after smashing to pieces, adjust pH to 8.0, trypsase 4000U/g blade is added, Hydrolysis temperature is 50 DEG C, and after digesting 90min, enzymolysis liquid is brought rapidly up to 90 DEG C, keeps 20min, carries out destroy the enzyme treatment.Then It is rapidly cooled to room temperature, 15min is being centrifuged with 3000r/min, is taking supernatant.According to 100 grams: 100ml ratio in wet macropore It adsorbs and tryptose enzymolysis liquid is added in resin DA201-C, 25 DEG C keep 180min, revolving speed 150r/min in constant-temperature table, use 75% ethanol solution elutes 120min, collects eluent in different time periods.The band for recycling small-molecular-weight, is obtained 103 The sequence of a small peptide.By verifying, wherein thering are 25 small peptides to have the function of that cancer cell is inhibited to be proliferated and killing cancer cell. According to the peak separation time different in chromatographic column, corresponding small peptide can be obtained in batches.Conventional, the polypeptide passes through people Work synthetic method can batch be used for industrialized application, avoid insufficient raw material, bring limitation.
In other embodiments, synergistic effect allows one or more other therapeutic agents and polypeptide of the invention to criticize Quasi- dosage combinations are given, but have and be higher than expected efficiency.
In a typical implementation, polypeptide is enough to allow with 0.2mg/kg to 20mg/kg intravenous administration with concentration Pharmaceutical composition exists.Include suitable for the peptide concentration in the composition and method in certain embodiments, still It is not limited thereto, at least about 0.2mg/kg, at least about 0.75mg/kg, at least about lmg/kg, at least about 2mg/kg, at least about 2.5mg/kg, at least about 3mg/kg, at least about 4mg/kg, at least about 5mg/kg, at least about 6mg/kg, at least about 7mg/kg, until Few about 8mg/kg, at least about 9mg/kg, at least about 10mg/kg, at least about llmg/kg, at least about 12mg/kg, at least about 13mg/ Kg, at least about 14mg/kg, at least about 15mg/kg, at least about 16mg/kg, at least about 17mg/kg, at least about 18mg/kg, at least About 19mg/kg, and at least about 20mg/kg.
Polypeptide can be given with single dose or multi-dose scheme.In general, polypeptide is in the period from about 1 hour to about 24 hours It inside gives, but was typically given within about 1 to 2 hour period.When dosage can repeat about 1 week to about 6 weeks or longer Between, 6 doses or more in total.Typically, dosage weekly, is repeated once every other week, or monthly, and minimum 6 Agent is to most 50 doses.
It can be oral administration, intravenous injection administration, subcutaneous administrations, administered intramuscular when administration.Benefit of the invention Myelodysplastic syndrome is treated with specific killing action of the polypeptide to cancer cell, acts on marrow and other hematopoiesis groups It knits, inhibits growth of tumour cell.There is good killing to osteocarcinoma and myeloproliferative disorder.
Specific embodiment
The present invention is further explained in the light of specific embodiments, these embodiments are merely to illustrate the present invention, and Do not limit the scope of the invention.
The acquisition of 1 polypeptide of embodiment
The whole strain 100g of R. alashanica is weighed, the PBS aqueous solution that concentration of substrate is 5% is configured to after smashing to pieces, adjusts pH extremely 8.0, trypsase 4000U/g blade is added, hydrolysis temperature is 50 DEG C, and after digesting 90min, enzymolysis liquid is brought rapidly up to 90 DEG C, 20min is kept, destroy the enzyme treatment is carried out.Then it is rapidly cooled to room temperature, 15min is being centrifuged with 3000r/min, is taking supernatant. Tryptose enzymolysis liquid is added in wet macroporous absorbent resin DA201-C according to 100 grams: 100ml ratio, 25 DEG C in constant-temperature table 180min, revolving speed 150r/min are kept, 120min is eluted with 75% ethanol solution, collects eluent in different time periods. The band for recycling small-molecular-weight, is obtained the sequence of 103 small peptides.By verifying, inhibit cancer thin wherein there are 25 small peptides to have Born of the same parents are proliferated and kill the function of cancer cell.The sequence of the polypeptide is as any shown such as ZLGAT-1~25 respectively, sequence point It Dui Yingyu not be shown in SEQ ID NO:1-25.
The preparation of 2 pharmaceutical composition of embodiment
0.03 part of ZLGAT-1 polypeptide, 1 part of starch, honey element or/and sucrose is taken to be used as sweetener, benzoic acid 0.1 in right amount Part, it mixes, crosses 50 meshes, after sterilizing, packaging.
The polypeptide of ZLGAT-2~29 is also all prepared into corresponding combination according to the preparation method of ZLGAT-1 polypeptide respectively Object.
The mixing of embodiment 3 pharmaceutical composition and other preparations
ZLGAT-1 polypeptide and bortezomib are mixed according to the ratio of 1:1, then using mixed drug weight as base Plinth number adds 1 part of starch, honey element or/and sucrose and is used as 0.1 part of sweetener, benzoic acid in right amount, mixes, and crosses 50 meshes, After sterilizing, packaging.
The detection of 4 drug safety of embodiment
According to the verification method of this field routine, it can be deduced that the present invention confirms safe and non-toxic, experiment knot through animal experiment Fruit is as follows with conclusion:
(1) acute toxicity testing shows: according to acute toxicity dose grading standard, belonging to practical nontoxicity grade；
(2) Micronucleus test, sperm malformation test, Salmonella reversion test result are feminine gender；
(3) 30 days feeding trials: experimental animal growing state is good, hematological examination, biochemical analysis, main dirty body ratio And histological indications are compared with the control group, are showed no exception, it was demonstrated that the present invention is safe and non-toxic.
The detection of 5 cytology level of embodiment
Myeloma cell strain XG7 is cultivated in the FCS-1640 complete culture solution of 100mL/L and (is contained HEPES25mol/L), every Day changes liquid, passes in time.Cell growth condition is detected using mtt assay, until cell grows into 3.0*108?.
Take 1ml, 3.0*108Cell, continue to cultivate in the FCS-1640 complete culture solution of 10ml, wherein in culture solution The pharmaceutical composition for being added to embodiment 2 and 3 respectively is subject to content of peptides additive amount as 0.2mg.Cell is carried out after culture 48h It counts, as a result as follows:
As can be seen from the above results, drug of the invention has effects that preferably to inhibit myeloma cell strain XG7.
Pharmaceutical activity experiment
(1) cell culture
Myeloma cell strain XG7 is cultivated in the FCS-1640 complete culture solution of 100mL/L and (is contained HEPES25mol/L), every Day changes liquid, passes in time.Cell growth condition is detected using mtt assay, until cell grows into 3.0*108?.
(2) preparation of Rat Model of Osteosarcoma
The myeloma cell strain XG7 of culture is collected, is cleaned 1 time, cell concentration is adjusted；It is subcutaneously big to SD on the outside of right hind Mouse is inoculated with 100ml cell suspension 0.5mL, 15 days after inoculation, 10 qualified SCID mices is taken to be randomly divided into two groups, in control group tumor 2ml physiological saline is injected, experimental group will be dissolved in the conjunction object of the drug of ethyl alcohol mixed liquor of normal saline according to 1.0mg/kg weight Dosage intratumor injection.It the 1st after administration, measures within 2,3,4,5 weeks tumour major diameter a and minor axis b weekly with vernier caliper, and calculates flat Equal diameter, i.e. r=(a+b)/2.After about 5 weeks, tumour can grow into suitable volume.Take the qualified SCID for having injected cell Mouse, control group intratumor injection 2ml physiological saline, experimental group will be dissolved in the pharmaceutical composition of ethyl alcohol mixed liquor of normal saline by According to the dosage intraperitoneal injection of 2.0mg/kg weight.0th, 10,20 day measurement subcutaneous transplantation tumor longest diameter (a) and vertical diameter after administration (d), tumour relative volume is calculated, the results are shown in Table 1 drug to the inhibiting effect (mm of Huppert's disease volume3)
As it can be seen from table 1 drug of the invention has the function of preferably inhibiting tumour growth, and can be significant Reduce the volume of tumour.The function of the growth for the inhibition tumour that it can be cooperateed with especially is administered in combination with bortezomib. The polypeptide of ZLGAT-2~25 can achieve identical with bortezomib administering drug combinations with ZLGAT-1 with bortezomib administering drug combinations Technical effect, specific value will not repeat them here.
Sequence table
<110>Cao Shuai
<120>a kind of for treating the pharmaceutical composition of osteocarcinoma
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
Ser Met Lys Trp Tyr Val Pro Asn Ile Ile Met Leu Tyr Asn Met Pro
1 5 10 15
Gln Phe
<210> 2
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
Met Ser Ile Gly Val Pro Gly Cys Ser Asn Leu Phe Gly Pro Cys Ser
1 5 10 15
Leu Trp
<210> 3
<211> 16
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
Met Phe Asn Ile His Met Tyr Ala Trp Leu Ser Glu Cys Trp Trp Ser
1 5 10 15
<210> 4
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
Leu Asn Tyr Glu Ser Ser Leu Arg Leu Cys Ser Gln His Leu Asp Asn
1 5 10 15
His Glu
<210> 5
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
Thr Thr Phe Tyr Gly Pro Thr Ser Tyr His Met Gly Thr Pro Ile Gly
1 5 10 15
His
<210> 6
<211> 16
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
Asn Tyr Ser Val Phe Gln Met Tyr His Ser Trp Trp Gln Trp Asn Ser
1 5 10 15
<210> 7
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 7
Glu Glu Arg Ile Leu Trp Ile Tyr Asp Phe Leu Pro Arg Gln Met Asp
1 5 10 15
Cys Gln
<210> 8
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 8
Gly Thr Ala Leu Gln Ser Val Thr Cys Thr Gln His Cys Trp Cys Gly
1 5 10 15
Gln
<210> 9
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 9
Pro Pro Arg Gln Ile Pro Leu Trp Arg Tyr Ile Ile Phe Ser Gln Leu
1 5 10 15
Trp Trp
<210> 10
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
Asn Thr Gly Ala Gln Arg Gly Asp Ile Arg Glu Asp Tyr Phe Arg Pro
1 5 10 15
Val
<210> 11
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
Phe Trp Pro Asn Cys Val Pro Phe Gly Ala Pro Ile Trp Lys Trp Ser
1 5 10 15
Ser Gln
<210> 12
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
Phe Cys Ala Arg Val Ser Ser Gln Lys Gly Pro Arg Pro Ala Tyr Leu
1 5 10 15
Ser Arg
<210> 13
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 13
Trp Lys Arg Cys Glu Glu Gly Asp Ala Ser Tyr Leu Tyr Arg Val Ala
1 5 10 15
Met
<210> 14
<211> 18
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 14
Thr Arg Asp Met His Ala Trp Phe Val Asn Ser Lys Arg Asp Pro His
1 5 10 15
Phe Cys
<210> 15
<211> 16
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 15
Gln Tyr His His Cys Gln Thr Pro Trp Trp Arg Asp Asp Ser Met Ser
1 5 10 15
<210> 16
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 16
Thr Met Asn Ala Asp Ser Glu Pro Asp Met Gly Lys Ser Gly Glu Ser
1 5 10 15
Thr
<210> 17
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 17
Ser Gln Gly Asp Lys His Arg Gln Leu Ala Gly Met Ile Lys Val Ile
1 5 10 15
Glu
<210> 18
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 18
Ser Ser Gln Lys Ser His Pro Gln Cys Cys Glu Ala Gln Glu Asn Thr
1 5 10 15
Trp
<210> 19
<211> 17
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 19
Ala Ala Arg Asp Arg Cys Gln Pro Arg Val Glu Ser Gly Ser Cys Ile
1 5 10 15
Leu
<210> 20
<211> 14
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 20
Gln Val Val Leu Gly Ile Glu Gln Pro Glu Ser Lys Glu Val
1 5 10
<210> 21
<211> 13
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 21
Gly His Asp His Gln Lys Ile Asp Leu Ala Ser Trp His
1 5 10
<210> 22
<211> 13
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 22
Pro Tyr Ile Met Ile Gln Gln Gln Ser Asp Gln Pro Met
1 5 10
<210> 23
<211> 14
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 23
Gln Arg Glu Arg Gln Cys Lys Ser Trp His Tyr His Tyr Tyr
1 5 10
<210> 24
<211> 13
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 24
Gln Arg His Ala Gln Ala Met Gly Met Arg Ile Glu Gln
1 5 10
<210> 25
<211> 14
<212> PRT
<213>artificial sequence (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 25
Gln Ile Lys Lys Pro Trp Pro Asn Leu His Asp Arg Thr Asp
1 5 10

Claims (5)

1. a kind of for treating the pharmaceutical composition of osteocarcinoma, characterized in that take 0.01 part of polypeptide, 1 part of starch, honey element or/and Sucrose is used as 0.1 part of sweetener, benzoic acid in right amount, mixes, and crosses 50 meshes, after sterilizing, packaging.
2. pharmaceutical composition as described in claim 1, it is characterised in that: the polypeptide is any polypeptide in ZLGAT-2~25, Sequence is respectively shown in NO:2~25 SEQ ID.
3. composition according to claim 1 to 2, characterized in that bortezomib can also be added.
4. application of the pharmaceutical composition described in claim 1-3 in preparation for the drug of specific treatment osteocarcinoma.
5. a kind of polypeptide, characterized in that be sequence shown in SEQ ID NO:2-25 is any.
CN201910056802.7A 2016-03-13 2016-03-13 It is a kind of for treating the pharmaceutical composition of osteocarcinoma Withdrawn CN109620949A (en)

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FR2860236A1 (en) * 2003-09-25 2005-04-01 Theraptosis PEPTIDES HAVING PARTICULARLY ANTI-ANGIOGENIC ACTIVITY AND THERAPEUTIC APPLICATIONS THEREOF
TW200616659A (en) * 2004-08-27 2006-06-01 Sanol Arznei Schwarz Gmbh Novel use of peptide compounds for treating bone cancer pain, chemotherapy-and nucleoside-included pain
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