CN108324692B - ATAP polypeptide and taxanes pharmaceutical co-crystals medicine and its preparation method and application - Google Patents
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine and its preparation method and application Download PDFInfo
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- CN108324692B CN108324692B CN201810386547.8A CN201810386547A CN108324692B CN 108324692 B CN108324692 B CN 108324692B CN 201810386547 A CN201810386547 A CN 201810386547A CN 108324692 B CN108324692 B CN 108324692B
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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
- A61K38/16—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
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- 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
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
Abstract
The invention discloses ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, eutectic pack contains ATAP polypeptide and taxanes drug such as SEQ ID NO:1 or 2 shown in, and the mass ratio of ATAP polypeptide and taxanes drug is 1~16:4.The invention discloses the preparation methods of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine; include the following steps: Step 1: taking the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:1 or 2 first; freeze drying protectant is added thereto later; and it mixes; wherein, the mass ratio of ATAP polypeptide and taxanes drug is 1~16:4；Step 2: suspension obtained in step 1 is carried out frozen dried, ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder are obtained.The application of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer.
Description
Technical field
The invention belongs to the technical field of pharmaceuticals of oncotherapy, it is related to a kind of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine
And preparation method thereof.
Background technique
As most effective anti-tumor drug, taxol is widely used in the treatment of Several Kinds of Malignancy, acts on machine
Reason keeps tubulin to stablize, inhibits cell primarily as microtubule stabilizer by promoting tubulin polymerization to inhibit depolymerization
Mitosis eventually leads to Apoptosis.Under clinical treatment dosage, also there is enhancing radiation to control for taxol and Docetaxel
The cytotoxicity for the treatment of and the effect of Agiogenesis inhibition.
It can be with selectively targeted mitochondria from the qualitative peptide of amphiphilic stern anchor (ATAP) derived from Bfl-1 (amino acid 1 47-175)
Trigger apoptosis.Compare the treatment that other take mitochondria as targeting, ATAP has and unique can avoid Bcl-2 family protein and lure
Lead the effect of effective apoptosis.In particular, ATAP can not penetrate the film of mitochondria in the presence of Bax and Bak, [Bax and Bak] is logical
It often will affect carrier medicament energy and influence expression of the carrier medicament in cancer cell (including prostatic cell).In addition,
The apoptosis of ATAP induction is not influenced by Bcl-2 or other anti-apoptotic family member, [Bcl-2 or other anti-apoptotic family member]
Usually it is overexpressed in cancer cell.ATAP, which has been shown, can avoid Bcl-2 mediation as therapeutic agent unique advantage i.e. ATAP
Protection mechanism and effectively kill to chemotherapy generate resistance cancer cell.
So, as two kinds of drugs to cancer cell different role mechanism, applicant proposed following technical problem, ATAP
And its derivative whether can with taxanes Drug combination, with for treating tumour.
Summary of the invention
It is excellent it is an object of the invention to solve at least the above problems and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide ATAP polypeptides and taxanes pharmaceutical co-crystals medicine.
A further object of the present invention is to provide the preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine.
The present invention separately has a purpose to be to provide ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer
With the application in lung cancer.
For this purpose, technical solution provided by the invention are as follows:
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the eutectic pack contain the ATAP as shown in SEQ ID NO:1 or 2
The mass ratio of polypeptide and taxanes drug, the ATAP polypeptide and taxanes drug is 1~16:4.
Preferably, in the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the ATAP polypeptide and taxanes
The mass ratio of drug is 4:1,2:1,1:1,1:2 or 1:4.
Preferably, in the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the taxanes drug is Japanese yew
Alcohol, Docetaxel, Paclitaxel liposome or Albumin binding taxol.
Preferably, in the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the ATAP polypeptide is such as SEQ ID
Amino acid sequence shown in NO:2, the taxanes drug are Docetaxel, amino acid shown in the SEQ ID NO:2
The mass ratio of sequence and the Docetaxel is 1:1.
The preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, includes the following steps:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:1 or 2 is taken first, it
Freeze drying protectant is added thereto afterwards, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 1~16:
4, the concentration of the ATAP polypeptide is 0.05~0.15molL-1；
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes are obtained
Pharmaceutical co-crystals medicine freeze-dried powder.
Preferably, in the preparation method of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, in the step 1,
The ATAP polypeptide is the amino acid sequence as shown in SEQ ID NO:2, and the taxanes drug is Docetaxel, described
The mass ratio of amino acid sequence shown in SEQ ID NO:2 and the Docetaxel is 4:1,2:1,1:1,1:2 or 1:4；
The taxanes drug is taxol, Docetaxel, Paclitaxel liposome or Albumin binding taxol；
The freeze drying protectant is glucose, lactose, sucrose, mannitol, the sweet dew alcohol and glucose of mass ratio 1:1, matter
Measure the mannitol and lactose of the mannitol and sucrose or mass ratio 1:1 than 1:1；
The suspension of the ATAP polypeptide and taxanes drug is nanocrystal suspension.
Preferably, in the preparation method of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the frozen-dried protective
Agent is the mannitol and sucrose of mass ratio 1:1, and the concentration of the mannitol and sucrose is 5g/mL.
Preferably, in the preparation method of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, in the step 2,
The specific steps of the frozen dried include:
Suspension obtained in step 1 is divided into 10mL/ parts first, later in temperature -80~60 DEG C 2~4h of pre-freeze, so
It keeps pressure to be dried in vacuo in 30Pa or less, Yu Wendu -50~-40 DEG C of 35~45h of freeze-drying afterwards, is finally gradually warmed up
To+15 DEG C, the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder are obtained.
Preferably, in the preparation method of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, in the step 2,
The specific steps of the frozen dried include:
Suspension obtained in step 1 is divided into 10mL/ parts first, later in temperature -80~60 DEG C 2~4h of pre-freeze, so
Afterwards keep pressure 10h is freeze-dried at 30Pa or less: Yu Wendu -50 DEG C, later at -45 DEG C of temperature freeze-drying 20~
Then 25h is freeze-dried 8~12h at -40 DEG C of temperature, is finally gradually heated to+15 DEG C, obtains the ATAP polypeptide and purple
China fir alcohols pharmaceutical co-crystals medicine freeze-dried powder, wherein the rate of temperature fall of the freeze-drying is 2~3 DEG C/min, is finally gradually warmed up
When heating rate be 0.5~1 DEG C/min.
Application of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer.
The present invention is include at least the following beneficial effects:
Taxanes are aided with acceptable pharmaceutical carrier and various pharmaceutically acceptable systems are made as active constituent
Agent.Know that ATAP-M8 target spot is clear by many experiments, in conjunction with taxanes reasonable employment, can effectively inhibit lung cancer, liver
Cancer, gastric cancer, breast cancer, cervical carcinoma, oophoroma, intestinal cancer, non-small cell lung cancer, to head-neck carcinoma, Small Cell Lung Cancer, cancer of pancreas,
Tumour including melanoma etc. increases.Drug combination of the present invention is used as drug can effectively treat tumour, and joint is used
Medicine is scientific, rational, feasible and effective, is greatly enlarged the treatment spectrum of oncotherapy, reduces toxic side effect, has significant social valence
Value and market value.
Further advantage, target and feature of the invention will be partially reflected by the following instructions, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Detailed description of the invention
Fig. 1 is the chemical structure schematic diagram of the taxol of the present invention in one embodiment；
Fig. 2 is the chemical structure schematic diagram of the Docetaxel of the present invention in one embodiment.
Specific embodiment
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text
Word can be implemented accordingly.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
The present invention provides ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, and the eutectic pack contains such as SEQ ID NO:1 or 2
Shown in ATAP polypeptide and taxanes drug, the mass ratio of the ATAP polypeptide and taxanes drug is 1~16:4.
In the above scheme, preferably, the mass ratio of the ATAP polypeptide and taxanes drug is 4:1,2:1,1:
1,1:2 or 1:4.
It is of the invention in one embodiment, as depicted in figs. 1 and 2, preferably, the taxanes drug is
Taxol, Docetaxel, Paclitaxel liposome or Albumin binding taxol.
It is of the invention in one embodiment, preferably, the ATAP polypeptide be as shown in SEQ ID NO:2
Amino acid sequence, the taxanes drug are Docetaxel, amino acid sequence shown in the SEQ ID NO:2 with it is described
The mass ratio of Docetaxel is 1:1.
The present invention provides the preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, includes the following steps:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:1 or 2 is taken first, it
Freeze drying protectant is added thereto afterwards, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 1~16:
4, the concentration of the ATAP polypeptide is 0.05~0.15molL-1；
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes are obtained
Pharmaceutical co-crystals medicine freeze-dried powder.
In the above scheme, preferably, in the step 1, the ATAP polypeptide is as shown in SEQ ID NO:2
Amino acid sequence, the taxanes drug are Docetaxel, amino acid sequence shown in the SEQ ID NO:2 with it is described
The mass ratio of Docetaxel is 4:1,2:1,1:1,1:2 or 1:4；
The taxanes drug is taxol, Docetaxel, Paclitaxel liposome or Albumin binding taxol；
The freeze drying protectant is glucose, lactose, sucrose, mannitol, the sweet dew alcohol and glucose of mass ratio 1:1, matter
Measure the mannitol and lactose of the mannitol and sucrose or mass ratio 1:1 than 1:1；
The suspension of the ATAP polypeptide and taxanes drug is nanocrystal suspension.
In the above scheme, described sweet preferably, the freeze drying protectant is the mannitol and sucrose of mass ratio 1:1
The concentration for revealing pure and mild sucrose is 5g/mL.
It is of the invention in one embodiment, preferably, in the step 2, the specific step of the frozen dried
Suddenly include:
Suspension obtained in step 1 is divided into 10mL/ parts first, later in temperature -80~60 DEG C 2~4h of pre-freeze, so
It keeps pressure to be dried in vacuo in 30Pa or less, Yu Wendu -50~-40 DEG C of 35~45h of freeze-drying afterwards, is finally gradually warmed up
To+15 DEG C, the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder are obtained.
In the above scheme, preferably, in the step 2, the condition of the pre-freeze is -70 DEG C of pre-freeze 3h, described true
The dry condition of sky is pressure 30Pa hereinafter, -45 DEG C of freeze-drying 40h.
It is of the invention in one embodiment, preferably, in the step 2, the specific step of the frozen dried
Suddenly include:
Suspension obtained in step 1 is divided into 10mL/ parts first, later in temperature -80~60 DEG C 2~4h of pre-freeze, so
Afterwards keep pressure 10h is freeze-dried at 30Pa or less: Yu Wendu -50 DEG C, later at -45 DEG C of temperature freeze-drying 20~
Then 25h is freeze-dried 8~12h at -40 DEG C of temperature, is finally gradually heated to+15 DEG C, obtains the ATAP polypeptide and purple
China fir alcohols pharmaceutical co-crystals medicine freeze-dried powder, wherein the rate of temperature fall of the freeze-drying is 2~3 DEG C/min, is finally gradually warmed up
When heating rate be 0.5~1 DEG C/min.
The present invention also provides the ATAP polypeptides and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer
In application.
In order to enable those skilled in the art to better understand the present invention, following embodiment is now provided to be illustrated:
Embodiment 1
The present invention provides ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, and the eutectic pack contains such as SEQ ID NO:2 institute
The mass ratio of the ATAP polypeptide and taxanes drug shown, the ATAP polypeptide and taxanes drug is 4:4.
ATAP-M8, SEQ ID NO:2:Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe
Leu Glu Val Thr Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys Arg
Gly Asp Lys Gly Pro Asp Cys
The preparation of eutectic medicine freeze-dried powder
The suspension 4mL of ATAP-M8 and taxol is taken, following different types of freeze drying protectant, respectively grape are selected
Sugar, lactose, sucrose, mannitol, mannitol/glucose (1:1), mannitol/sucrose (1:1), mannitol, lactose (1:1), concentration
It is 5g/mL.Last process is set to: mannitol/sucrose (1:1).The concentration of ATAP polypeptide is 0.01molL-1
Above-mentioned freeze drying protectant is mixed in proportion and is dissolved in nanocrystal suspension, is sub-packed in l0mL cillin bottle,
Carry out frozen dried.Refrigerating process: -70 DEG C of pre-freeze 3h are dried in vacuo in 30Pa or less later, in -45 DEG C of freeze-drying 40h,
Then+15 DEG C of eutectic medicine freeze-dried powders to get ATAP-M8 and taxol are gradually heated to.
Embodiment 2
ATAP-M8 peptide molecule, 5-25 milligrams/m of people's dosage range2, it is administered using intravenous injection or intravenous drip
Mode, daily intravenous drip is primary, and dosage period 15-30 days, it is proposed that 3-6 period.Docetaxel, intravenous injection or vein
It instils, single 75-100 milligrams/m of medicine injection amount2.The two drug combination: pass through intravenous injection or intravenous drip, dosage 50-75
Milligram/m2, the intravenous drip time 1 hour, it is repeated once within every 4 weeks.
Embodiment 3
The foundation of animal tumor model: the human liver cancer SMMC-7721 that will be saved in liquid nitrogen, human gastric cancer MGC-803, human lung cancer
H460 carries out tumor cell line recovery, inoculates in 3~5 18-22g kunming mices or C57 mouse armpit, tumour growth
When reaching diameter 2cm or so, mouse cervical dislocation is put to death.Tumor mass is taken out, by the ratio of every gram of tumor tissues 3mL physiological saline
Example, is made tumor cell homogenate, is inoculated with homogenate to 18-22g kunming mice armpit, every mouse is inoculated with tumor liquid 0.2mL.It will move
Object is grouped at random, weighs, if positive and negative control group.Drug is fed with various concentration to animal, 2 times a day, continuous 15-20
It.Cervical dislocation puts to death animal, weighs respectively, knurl weight；Tumor control rate is calculated, and each group result is carried out at statistics
Reason.Formula is as follows: tumor control rate (%)=(control group average knurl weight-treatment group's average knurl weight)/control group average knurl weight ×
100%, each group of data is with mean ± standard deviationIt indicates, is examined with Student t.
Embodiment 4
ATAP-M8 combines Docetaxel to the inhibiting effect of human liver cancer SMMC-7721 transplanted tumor in nude mice
With Jin's formula q=E, (a+b/ ((Ea+Eb)-Ea × Eb) evaluates drug combination to cell in vitro to statistical procedures
Whether poison has synergistic effect: E (a+b) is to share inhibiting rate, and Ea and Eb are respectively the inhibiting rate of A medicine and B medicine.Molecule is that actual measurement is closed
And effect, denominator are that expectation merges effect, q=0.85~1.15 are individually to be added (+), and q=1.15~2.0 are enhancing (++), q
> 2.0 is to be remarkably reinforced (+++), and q=0.55~0.85 is antagonism (-), and q < 0.55 is obvious antagonism (--).Data withIt indicates, carries out variance analysis using SPAA17.0 statistical software.
Human liver cancer SMMC-7721 transplanted tumor in nude mice is treated according to such as the following table 1 experimental setup
The administration setting of table 1
Measurement obtains each group tumor weight (g) and tumour inhibiting rate %, as shown in table 2.
2 each group tumor weight (g) of table and tumour inhibiting rate %
| Group | Knurl weight (g) | Tumour inhibiting rate % |
| G1 | 0.897±0.113 | - |
| G2 | 0.579±0.121* | 35.40 |
| G3 | 0.613±0.115* | 31.66 |
| G4 | 0.514±0.121* | 42.69 |
| G5 | 0.582±0.095** | 35.11 |
| G6 | 0.201±0.089** | 77.59 |
| G7 | 0.275±0.118* | 69.34 |
| G8 | 0.082±0.085** | 90.85 |
| G9 | 0.183±0.092** | 79.59 |
Note: Data Analysis Software SPSS indicates each group average knurl weight with mean value ± SD.Tumour inhibiting rate=(feminine gender group knurl weight-
Treatment group's knurl weight)/negative group knurl weight × 100%, according to T inspection result, P < 0.05 * be it is significant, P < 0.01 * * is extremely significant property
Difference.
Experimental result evaluation
Common drug combination
Q high=E (A+B)/(EA+EB-EA × EB)=77.59%/(35.40%+42.69%-35.40% ×
42.69%)=1.231
Q is low=and E (A+B)/(EA+EB-EA × EB)=69.34%/(31.66%+35.11%-31.66% ×
35.11%)=1.245
According to calculating, ATAP-M8 and Docetaxel form eutectic drug combination and two drug independent medications evaluations
Result is enhancing (++) effect, that is, is acted synergistically.It is observed that animal joint medication state is better than independent medication, weight subtracts
The independent medication that compares less slowly will illustrate toxic side effect reduction.
Eutectic drug combination
Q high=E (A+B)/(EA+EB-EA × EB)=90.85%/(35.40%+42.69%-35.40% ×
42.69%)=1.438
Q is low=and E (A+B)/(EA+EB-EA × EB)=79.59%/(31.66%+35.11%-31.66% ×
35.11%)=1.429
According to calculating, ATAP-M8 and Docetaxel form eutectic drug combination and two drug independent medications evaluations
Result is enhancing (++) effect, that is, is acted synergistically.It is observed that animal joint medication state is better than independent medication, weight subtracts
The independent medication that compares less slowly will illustrate toxic side effect reduction.
Embodiment 5
Human gastric cancer MGC-803 Nude Mouse Model is established according to the method for embodiment 3
Human gastric cancer MGC-803 Nude Mouse Model is tested according to the method for table 1.
Measurement obtains each group tumor weight (g) and tumour inhibiting rate %, as shown in table 3.
Note: Data Analysis Software SPSS indicates each group average knurl weight with mean value ± SD.Tumour inhibiting rate=(feminine gender group knurl weight-
Treatment group's knurl weight)/negative group knurl weight × 100%, according to T inspection result, P < 0.05 * be it is significant, P < 0.01 * * is extremely significant property
Difference.
Experimental result evaluation
Common drug combination
Q high=E (A+B)/(EA+EB-EA × EB)=74.94%/(51.40%+53.86%-51.40% ×
53.86%)=0.966
Q is low=and E (A+B)/(EA+EB-EA × EB)=66.74%/(41.10%+46.37%-41.10% ×
46.37%)=0.975
According to calculating, ATAP-M8 is to be added (+) merely with Docetaxel drug combination with independent medication evaluation result
Effect,.It is observed that animal joint medication state is better than independent medication, weight loss compares independent medication will slowly, explanation
Toxic side effect reduces.
Eutectic drug combination
Q high=E (A+B)/(EA+EB-EA × EB)=88.87%/(51.40%+53.86%-51.40% ×
53.86%)=1.176
Q is low=and E (A+B)/(EA+EB-EA × EB)=80.79%/(41.10%+46.37%-41.10% ×
46.37%)=1.183
According to calculating, ATAP-M8 and Docetaxel form eutectic drug combination and two drug independent medications evaluations
Result is enhancing (++) effect, that is, is acted synergistically.It is observed that animal joint medication state is better than independent medication, weight subtracts
The independent medication that compares less slowly will illustrate toxic side effect reduction.
Embodiment 6ATAP-M8 combines Docetaxel to the inhibiting effect of human lung cancer H460 transplanted tumor in nude mice
Human lung cancer H460 Nude Mouse Model is established according to the method for embodiment 3
Human lung cancer H460 Nude Mouse Model is tested according to the method for table 1.
Measurement obtains each group tumor weight (g) and tumour inhibiting rate %, as shown in table 4.
Note: Data Analysis Software SPSS indicates each group average knurl weight with mean value ± SD.Tumour inhibiting rate=(feminine gender group knurl weight-
Treatment group's knurl weight)/negative group knurl weight × 100%, according to T inspection result, P < 0.05 * be it is significant, P < 0.01 * * is extremely significant property
Difference.
Experimental result evaluation
Common drug combination
Q high=E (A+B)/(EA+EB-EA × EB)=78.64%/(35.93%+43.27%-35.93% ×
43.27%)=1.235
Q is low=and E (A+B)/(EA+EB-EA × EB)=70.28%/(32.09%+36.72%-32.09% ×
36.72%)=1.252
According to calculating, ATAP-M8 and Docetaxel form eutectic drug combination and two drug independent medications evaluations
Result is enhancing (++) effect, that is, is acted synergistically.It is observed that animal joint medication state is better than independent medication, weight subtracts
The independent medication that compares less slowly will illustrate toxic side effect reduction.
Eutectic drug combination
Q high=E (A+B)/(EA+EB-EA × EB)=91.52%/(35.93%+43.27%-35.93% ×
43.27%)=1.437
Q is low=and E (A+B)/(EA+EB-EA × EB)=81.80%/(32.09%+36.72%-32.09% ×
36.72%)=1.434
According to calculating, ATAP-M8 and Docetaxel form eutectic drug combination and two drug independent medications evaluations
Result is enhancing (++) effect, that is, is acted synergistically.It is observed that animal joint medication state is better than independent medication, weight subtracts
The independent medication that compares less slowly will illustrate toxic side effect reduction.
Embodiment 7
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the eutectic pack contain as ATAP shown in SEQ ID NO:1 is more
The mass ratio of peptide and taxanes drug, the ATAP polypeptide and taxanes drug is 1:4.The taxanes drug is purple
China fir alcohol.
ATAP, SEQ ID NO:1:Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe Leu
Glu Val Thr Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys
The preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, includes the following steps:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:1 is taken first, it is backward
Freeze drying protectant is wherein added, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 1:4, the purple
China fir alcohols drug is taxol.The concentration of the ATAP polypeptide is 0.05molL-1；
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes are obtained
Pharmaceutical co-crystals medicine freeze-dried powder.The freeze drying protectant is glucose；The suspension of the ATAP polypeptide and taxanes drug
For nanocrystal suspension.The concentration of the glucose is 5g/mL.The specific steps of the frozen dried include:
Suspension obtained in step 1 is divided into 10mL/ parts first, later in -80 DEG C of pre-freeze 2h of temperature, is then kept
Pressure is dried in vacuo in 30Pa or less, Yu Wendu -50 DEG C of freeze-drying 35h, is finally gradually heated to+15 DEG C, is obtained institute
State ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder.
Application of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer.
Embodiment 8
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the eutectic pack contain as ATAP shown in SEQ ID NO:2 is more
The mass ratio of peptide and taxanes drug, the ATAP polypeptide and taxanes drug is 16:4.The taxanes drug is
Paclitaxel liposome.
ATAP-M8, SEQ ID NO:2:Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe
Leu Glu Val Thr Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys Arg
Gly Asp Lys Gly Pro Asp Cys
The preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, includes the following steps:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:2 is taken first, it is backward
Freeze drying protectant is wherein added, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 16:4, described
The concentration of ATAP polypeptide is 0.15molL-1；The taxanes drug is Paclitaxel liposome.
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes are obtained
Pharmaceutical co-crystals medicine freeze-dried powder.The freeze drying protectant is the mannitol and lactose of mass ratio 1:1, the mannitol and lactose
Concentration is 5g/mL；The suspension of the ATAP polypeptide and taxanes drug is nanocrystal suspension.The frozen dried
Specific steps include: that suspension obtained in step 1 is divided into 10mL/ parts first, later in -60 DEG C of pre-freeze 4h of temperature, so
It keeps pressure to be dried in vacuo in 30Pa or less, Yu Wendu -40 DEG C of freeze-drying 45h afterwards, is finally gradually heated to+15 DEG C,
Obtain the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder.
Application of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer.
Embodiment 9
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the eutectic pack contain as ATAP shown in SEQ ID NO:2 is more
The mass ratio of peptide and taxanes drug, the ATAP polypeptide and taxanes drug is 8:4.The taxanes drug is white
Protein binding taxol.
ATAP-M8, SEQ ID NO:2:Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe
Leu Glu Val Thr Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys Arg
Gly Asp Lys Gly Pro Asp Cys
The preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, includes the following steps:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:2 is taken first, it is backward
Freeze drying protectant is wherein added, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 8:4, the purple
China fir alcohols drug is Docetaxel.The concentration of the ATAP polypeptide is 0.08molL-1, the ATAP polypeptide and taxanes
The suspension of drug is nanocrystal suspension.
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes are obtained
Pharmaceutical co-crystals medicine freeze-dried powder.The freeze drying protectant is the sweet dew alcohol and glucose of mass ratio 1:1, the mannitol and grape
The concentration of sugar is 5g/mL.The specific steps of the frozen dried include: first to be divided into suspension obtained in step 1
10mL/ parts, later in temperature 60 C pre-freeze 4h, then pressure is kept to be freeze-dried 10h at 30Pa or less: Yu Wendu -50 DEG C,
25h is freeze-dried at -45 DEG C of temperature later, 12h is then freeze-dried at -40 DEG C of temperature, is finally gradually heated to+15
DEG C, obtain the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder, wherein the rate of temperature fall of the freeze-drying is
3 DEG C/min, heating rate when being finally gradually warmed up is 1 DEG C/min.
Application of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer.
Embodiment 10
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, the eutectic pack contain as ATAP shown in SEQ ID NO:2 is more
The mass ratio of peptide and taxanes drug, the ATAP polypeptide and taxanes drug is 2:4.The taxanes drug is white
Protein binding taxol.
ATAP-M8, SEQ ID NO:2:Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe
Leu Glu Val Thr Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys Arg
Gly Asp Lys Gly Pro Asp Cys
The preparation method of ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, includes the following steps:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO:2 is taken first, it is backward
Freeze drying protectant is wherein added, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 2:4, the purple
China fir alcohols drug is Docetaxel.The concentration of the ATAP polypeptide is 0.12molL-1, the ATAP polypeptide and taxanes
The suspension of drug is nanocrystal suspension.
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes are obtained
Pharmaceutical co-crystals medicine freeze-dried powder.The freeze drying protectant is the sweet dew alcohol and glucose of mass ratio 1:1, the mannitol and grape
The concentration of sugar is 5g/mL.The specific steps of the frozen dried include: first to be divided into suspension obtained in step 1
10mL/ parts, later in -80 DEG C of pre-freeze 2h of temperature, then pressure is kept to be freeze-dried at 30Pa or less: Yu Wendu -50 DEG C
10h is freeze-dried 20h at -45 DEG C of temperature later, and 8h is then freeze-dried at -40 DEG C of temperature, be finally gradually heated to+
15 DEG C, obtain the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder, wherein the rate of temperature fall of the freeze-drying
For 2 DEG C/min, heating rate when being finally gradually warmed up is 0.5 DEG C/min.
Application of the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine in treatment human liver cancer, gastric cancer and lung cancer.
ATAP polypeptide and taxanes pharmaceutical co-crystals medicine described herein can be treated effectively characterized by abnormal cell increment
Following mammalian diseases: such as tumour, polyp, endometriosis, leukaemia, autoimmune disease, cancer.It is suitable
It shares the disease specific that the compounds of this invention is treated and preferably treats various malignant tumours, the tumprigenicity disease that drug is acted on
Disease is the malignant tumour and cancer that wide spectrum mammal (including people) body causes, wherein preferably acting on Human Lung Cancer, people
Class non-small cell lung cancer, breast cancer, oophoroma and melanoma, especially solid tumor are (for example, lung, oophoroma, mammary gland, stomach and intestine, knot
Intestines, pancreas, bladder, kidney, prostate, brain) and various hemopoietic system cancers (for example, HodgkinShi disease, non-HodgkinShi drench
Bar cancer, white mass formed by blood stasis).Wherein the tumor disease is selected from leukaemia (including acute leukemia, acute lymphatic leukaemia, urgency
Property myelocytic leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocyte
Property leukaemia, erythroleukemia, chronic leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia), genuine erythrocyte increase
More, lymthoma, Hodgkin's disease, non-hodgkin's disease, Huppert's disease, idiopathic macroglobulinemia disease, solid tumor, sarcoma and
Cancer, fibrosarcoma, myxosarcoma, sarcolipoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymph
Pipe sarcoma, lymphangioendothelial sarcoma, synovialoma, celiothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreas
Cancer, breast cancer, oophoroma, prostate cancer, squamous cell carcinoma, basal-cell carcinoma, gland cancer, syringocarcinoma, carcinoma of sebaceous glands, mamillary
Cancer, adenocarcinoma of nipple, cystadenocarcinoma, cephaloma, bronchiolar carcinoma, clear-cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma,
Embryonal carcinoma, wilms' tumor, cervix cancer, uterine cancer, orchioncus, lung cancer, Small Cell Lung Cancer, bladder cancer, epithelioma, mind
Through glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, listen
Neuroma, oligodendroglioma, durosarcoma, melanoma, neuroblastoma and retinal neuronal cell tumor etc..
Module number and treatment scale described herein are for simplifying explanation of the invention.It is more to ATAP of the invention
The application of peptide and taxanes pharmaceutical co-crystals medicine and its preparation method and application, modifications and variations carry out those skilled in the art
Say it is obvious.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
SEQUENCE LISTING
<110>Beijing Ai Taipu biological medicine science and technology limited Company
<120>ATAP polypeptide and taxanes pharmaceutical co-crystals medicine and its preparation method and application
<130> 2017
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> PRT
<213>artificial sequence
<400> 1
Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe Leu Glu Val Thr
1 5 10 15
Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys
20 25 30
<210> 2
<211> 38
<212> PRT
<213>artificial sequence
<400> 2
Lys Lys Phe Glu Pro Lys Ser Gly Trp Met Thr Phe Leu Glu Val Thr
1 5 10 15
Gly Lys Ile Ala Glu Met Leu Ser Leu Leu Lys Gln Tyr Cys Arg Gly
20 25 30
Asp Lys Gly Pro Asp Cys
35
Claims (9)
1.ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, which is characterized in that the eutectic pack contains such as SEQ ID NO: 1 or 2
Shown in ATAP polypeptide and taxanes drug, the mass ratio of the ATAP polypeptide and taxanes drug is 1 ~ 16:4.
2. ATAP polypeptide as described in claim 1 and taxanes pharmaceutical co-crystals medicine, which is characterized in that the ATAP polypeptide and
The mass ratio of taxanes drug is 4:1,2:1,1:1,1:2 or 1:4.
3. ATAP polypeptide as described in claim 1 and taxanes pharmaceutical co-crystals medicine, which is characterized in that the taxanes medicine
Object is taxol, Docetaxel, Paclitaxel liposome or Albumin binding taxol.
4. ATAP polypeptide as described in claim 1 and taxanes pharmaceutical co-crystals medicine, which is characterized in that the ATAP polypeptide is
The amino acid sequence as shown in SEQ ID NO: 2, the taxanes drug are Docetaxel, SEQ ID NO: 2
Shown in the mass ratio of amino acid sequence and the Docetaxel be 1:1.
The preparation method of 5.ATAP polypeptide and taxanes pharmaceutical co-crystals medicine, which comprises the steps of:
Step 1: the suspension of ATAP polypeptide and taxanes drug as shown in SEQ ID NO: 1 or 2 is taken first, it is backward
Freeze drying protectant is wherein added, and mixes, wherein the mass ratio of the ATAP polypeptide and taxanes drug is 1 ~ 16:4, institute
The concentration for stating ATAP polypeptide is 0.05 ~ 0.15 molL-1；
Step 2: suspension obtained in step 1 is carried out frozen dried, the ATAP polypeptide and taxanes drug are obtained
Eutectic medicine freeze-dried powder.
6. the preparation method of ATAP polypeptide as claimed in claim 5 and taxanes pharmaceutical co-crystals medicine, which is characterized in that described
In step 1, the ATAP polypeptide is the amino acid sequence as shown in SEQ ID NO: 2, and the taxanes drug is polyenoid
Taxol, the mass ratio of amino acid sequence and the Docetaxel shown in SEQ ID NO: 2 are 4:1,2:1,1:1,
1:2 or 1:4；
The freeze drying protectant is sweet dew alcohol and glucose, the mass ratio of glucose, lactose, sucrose, mannitol, mass ratio 1:1
The mannitol and sucrose of 1:1 or the mannitol and lactose of mass ratio 1:1；
The suspension of the ATAP polypeptide and taxanes drug is nanocrystal suspension.
7. the preparation method of ATAP polypeptide as claimed in claim 6 and taxanes pharmaceutical co-crystals medicine, which is characterized in that described
Freeze drying protectant is the mannitol and sucrose of mass ratio 1:1, and the concentration of the mannitol and sucrose is 5 g/mL.
8. the preparation method of ATAP polypeptide as claimed in claim 5 and taxanes pharmaceutical co-crystals medicine, which is characterized in that described
In step 2, the specific steps of the frozen dried include:
Suspension obtained in step 1 is divided into 10 mL/ parts first, later in 2 ~ 4 h of temperature -80 ~ -60 DEG C pre-freeze, then
It keeps pressure to be dried in vacuo in 30 Pa or less, Yu Wendu -50 ~ -40 DEG C of 35 ~ 45 h of freeze-drying, is finally gradually heated to
+ 15 DEG C, obtain the ATAP polypeptide and taxanes pharmaceutical co-crystals medicine freeze-dried powder.
9. the preparation method of ATAP polypeptide as claimed in claim 8 and taxanes pharmaceutical co-crystals medicine, which is characterized in that described
In step 2, the specific steps of the frozen dried include:
Suspension obtained in step 1 is divided into 10 mL/ parts first, later in 2 ~ 4 h of temperature -80 ~ -60 DEG C pre-freeze, then
Keep pressure in 30 Pa hereinafter, be freeze-dried 10 h at -50 DEG C of temperature, later at -45 DEG C of temperature freeze-drying 20 ~
Then 25h is freeze-dried 8 ~ 12 h at -40 DEG C of temperature, is finally gradually heated to+15 DEG C, obtains the ATAP polypeptide and purple
China fir alcohols pharmaceutical co-crystals medicine freeze-dried powder, wherein the rate of temperature fall of the freeze-drying is 2 ~ 3 DEG C/min, is finally gradually warmed up
When heating rate be 0.5 ~ 1 DEG C/min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526024A (en) * | 2010-12-27 | 2012-07-04 | 鲁南制药集团股份有限公司 | Anticancer pharmaceutical composition |
| CN103656615A (en) * | 2013-12-11 | 2014-03-26 | 南京安吉生物科技有限公司 | Application HM-3 and platinum, paclitaxel or capecitabine medicine for preparing solid tumor medicine |
| US9102759B2 (en) * | 2010-06-25 | 2015-08-11 | Adamed Sp. Z O.O. | Anticancer fusion protein comprising trail |
-
2018
- 2018-04-26 CN CN201810386547.8A patent/CN108324692B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9102759B2 (en) * | 2010-06-25 | 2015-08-11 | Adamed Sp. Z O.O. | Anticancer fusion protein comprising trail |
| CN102526024A (en) * | 2010-12-27 | 2012-07-04 | 鲁南制药集团股份有限公司 | Anticancer pharmaceutical composition |
| CN103656615A (en) * | 2013-12-11 | 2014-03-26 | 南京安吉生物科技有限公司 | Application HM-3 and platinum, paclitaxel or capecitabine medicine for preparing solid tumor medicine |
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Inventor after: Liu He Inventor after: Guo Xiaochun Inventor after: Yang Huiren Inventor after: Ma Jianjie Inventor after: Guo Erming Inventor after: Wang Xinghui Inventor before: Liu He Inventor before: Shi Weiguo Inventor before: Guo Xiaochun Inventor before: Yang Huiren Inventor before: Ma Jianjie Inventor before: Guo Erming Inventor before: Wang Xinghui |