CN105148256B - A kind of mobilization agent composition and its application, preparation - Google Patents
A kind of mobilization agent composition and its application, preparation Download PDFInfo
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Abstract
The present invention relates to technological field of biochemistry, in particular to a kind of mobilization agent composition and its application, preparation.The mobilization agent composition includes AMD3100, CTX and G-SCF.Mobilization agent composition of the invention mobilizes the time short, needs several days mobilization time compared with prior art, preferably the stabilization of the physical function of protection patient；Mononuclearcell vigor after mobilization is high compared to the control group, and the cell for obtaining expression CD34 is more；Mobilization agent composition each component concentration dosage of the invention is much smaller than what is clinically used, to itself have significant burden organ, system reduce damage；Peripheral blood hematopoietic stem cells after mobilization are able to satisfy the Treatment need of donor, and to the Small side effects of patient, improve patients ' life quality, the physical qualification of good prognosis is kept to patient.
Description
Technical field
The present invention relates to technological field of biochemistry, in particular to a kind of mobilization agent composition and its application, preparation.
Background technique
As the effect of the discovery of the new chemotherapeutics of tumour, the improvement of chemicotherapy technology, killing tumor cell is also continuous
It improving, theoretically sees, the dosage of chemicotherapy and killing tumor cell quantity, therapeutic effect etc. should be linear, but
The side effect of chemicotherapy limits the increased effect of dosage, to influence the prognosis of patient, hematopoietic stem cell transplantation can be certain
This is solved the problems, such as in degree, improves the therapeutic effect for being directed to blood disease and Malignancy, improves patient's prognosis.
Candidate stem cell is the adult stem cell in hematological system, is the origin of all hematopoietic cells and immunocyte, it
Can not only be divided into red blood cell, leucocyte and blood platelet, can also cross-system be divided into the cells of various histoorgans, have from
I update, Multidirectional Differentiation and (i.e. directional migration to hematopoietic tissue organ) potential of going back to the nest.There are two important features for candidate stem cell:
First, self-renewing or the of self-replication capacity of height；Second, all types of haemocytes can be divided into.Candidate stem cell is adopted
With asymmetric divisional mode: being two cells by a cell division.Under normal circumstances, most of multipotential stem cells are in
Stationary state, when body occurs certain changes and needs, a part of multipotential stem cell will division growth.One of cell
The all living things characteristic of stem cell is still maintained, to keep stem cell population in body relatively stable, here it is stem cells certainly
I updates.And another then further Proliferation, Differentiation be all kinds of haemocytes, precursor and mature blood cell, be discharged into peripheral blood
In, it participates in adjusting body items circulation.
Candidate stem cell is divided into peripheral blood hematopoietic stem cells, marrow hemopoietic stem cells, umbilical hemopoietic according to its source difference
Stem cell.Umbilical cord blood transplantation often cannot successfully apply to adult, compared with bone-marrow transplantation, Transplantation of Peripheral Haemopoietic Stem Cells
It is convenient with acquisition, to patient damage it is small, can short time multi collect the advantages of.As blood cell separator is applied to peripheral blood
The acquisition of candidate stem cell, peripheral blood hematopoietic stem cells mobilize, separation and acquisition technique improvement, peripheral blood hematopoietic stem cells move
It is implanted with the trend of gradually substitution marrow hemopoietic stem cells transplanting.
Candidate stem cell (HSC) implantation technique mainly includes the identification of stem cell, determination of activity, the acquisition of stem cell, point
From purifying, to mobilize, amplification, stem cell saves, the purification of tumour cell, stem cell transformation and stem cell manipulation special instrument,
Material and the development and production of reagent etc..Peripheral hematopoietic stem cells (PBSC) are the good substitutes of bone-marrow transplantation.The side that PBSC is mobilized
There are three types of formulas, i.e., high-dose chemotherapy, hemopoieticgrowth factor (HGF) and Papillary is applied alone.Allosome donor is applied alone HGF to mobilize, and swells
Tumor patient then uses high-dose chemotherapy and (or) HGF.HGF it is more common be G-CSF (granulocyte colony stimulating factor) or GM-
CSF (human granulocyte-macrophage colony stimulating factor), G-CSF effect is better than GM-CSF, before the latter's side effect is significantly more than
Person.High-dose chemotherapy mostly uses cyclophosphamide (CTX) 4~7g/m2Intravenous infusion can make peripheral blood CFU-GM improve 50 times or more, change
Treating to share with HGF can make CFU-GM improve hundred times or more, reach as high as 1000 times or so.It is clinical and experimental studies have found that, IL-1,
Though IL-3, IL-6, IL-11, Epo, SCF, PIXY321, MIP-1 α also there are mobilized effects, it is applied alone ineffective.
AMD3100, trade name Plerixafor (Plerixafor), be a series of novel small molecule CXCR4 chemotactic because
Sub- receptor antagonist, multinomial early stage, researches show that the stem cell numbers that can fast and effeciently increase in NHL and MM blood samples of patients circulation.
Plerixafor promotes erythropoietin(EPO) stem cell to enter Fei Huoqi with granulocyte colony stimulating factor (G-CSF) drug combination
Golden lymthoma (NHL) and Huppert's disease (MM) blood stream of patients to collect, subsequent autotransplantation.
Hematopoietic stem cell transplantation Technology application has a extensive future, but when G-CSF, G-CSF/CTX, AMD3100 is used alone, and needs
The stem cell of sufficient amount could be obtained by carrying out multiple cell collection, for the patient of bone marrow suppression, since dosage is big,
In adverse reaction also increase accordingly, hematopoietic function restore slowly or can not restore, cause patient's body adjust disorder or even
It is dead.Therefore it needs to find a kind of quick mobilization and does not damage the mobilization agent of other organs of hematopoietic function and human body.
Summary of the invention
In view of this, the present invention provides a kind of mobilization agent composition and its applications, preparation.Mobilization agent composition joint
It can produce synergistic function after use, can not only greatly shorten the time of mobilization agent mobilization, but also can be improved single after mobilization
The vigor of nucleus greatly reduces the quantity of each component.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of mobilization agent compositions, including AMD3100, CTX (cyclophosphamide) and G-SCF (granulocyte
Colony stimulating factor).
In the present invention, after AMD3100, CTX and G-SCF being used in combination, it can produce synergistic function, it both can be big
The big time for shortening mobilization agent and mobilizing, and the vigor of mononuclearcell after mobilization can be improved, greatly reduce facing for each component
Bed dosage.
Preferably, the mass ratio of AMD3100, CTX and G-SCF are (1000~2000): (1000~3000): (2~
10)。
Preferably, the mass ratio of AMD3100, CTX and G-SCF are 2000:2000:2.
Preferably, the mass ratio of AMD3100, CTX and G-SCF are 1000:3000:5.
Preferably, the mass ratio of AMD3100, CTX and G-SCF are 1500:1000:10.
The present invention also provides the mobilization agent compositions to prepare the application in hematopoietic stem cell mobilization agent；The mobilization agent group
Closing object includes AMD3100, CTX (cyclophosphamide) and G-SCF (granulocyte colony stimulating factor)；Preferably, AMD3100, CTX
Mass ratio with G-SCF is (1000~2000): (1000~3000): (2~10)；Preferably, AMD3100, CTX and G-SCF
Mass ratio be 2000:2000:2；Preferably, the mass ratio of AMD3100, CTX and G-SCF are 1000:3000:5；Preferably,
The mass ratio of AMD3100, CTX and G-SCF are 1500:1000:10.
In some embodiments provided by the invention, candidate stem cell is peripheral blood hematopoietic stem cells.But the present invention is not
It is defined in this, those skilled in the art think that the type of feasible candidate stem cell is within the scope of the present invention.
The present invention also provides a kind of hematopoietic stem cell mobilization agent, including mobilization agent composition of the present invention；The mobilization agent group
Closing object includes AMD3100, CTX (cyclophosphamide) and G-SCF (granulocyte colony stimulating factor)；Preferably, AMD3100, CTX
Mass ratio with G-SCF is (1000~2000): (1000~3000): (2~10)；Preferably, AMD3100, CTX and G-SCF
Mass ratio be 2000:2000:2；Preferably, the mass ratio of AMD3100, CTX and G-SCF are 1000:3000:5；Preferably,
The mass ratio of AMD3100, CTX and G-SCF are 1500:1000:10.
In some embodiments provided by the invention, the dosage form of hematopoietic stem cell mobilization agent is injection.
The present invention also provides a kind of mobilizing methods of the candidate stem cell of non-diagnostic purpose, using mobilization agent group of the present invention
Object is closed, or candidate stem cell is mobilized in hematopoietic stem cell mobilization agent of the present invention.
Preferably, mobilizing method are as follows:
It is moved in three times with mobilization agent composition of the present invention, or hematopoietic stem cell mobilization agent of the present invention as mobilization agent
Member；In mobilization agent, the dosage that the dosage of AMD3100 is 1000~2000 μ g/kg/d, CTX is 1000~3000 μ g/kg/d, G-
The dosage of SCF is 2~10 μ g/kg/d；
It mobilizes for the first time: the mobilization agent of 1/2 dosage of application, time 3h；
Second of mobilization: the mobilization agent of 1/4 dosage of application, time 4h；
Third time is mobilized: the mobilization agent of 1/4 dosage of application, 2~5h of time.
Preferably, the time that third time is mobilized is 3h.
The present invention provides a kind of mobilization agent composition and its applications, preparation.The mobilization agent composition include AMD3100,
CTX and G-SCF.The present invention at least has one of following advantage:
1, mobilization agent composition of the invention mobilizes the time short, needs several days mobilization time compared with prior art, more preferably
Protect the stabilization of the physical function of patient in ground；
2, the mononuclearcell vigor after mobilizing is high compared to the control group, and the cell for obtaining expression CD34 is more；
3, mobilization agent composition each component concentration dosage of the invention is much smaller than what is clinically used, to itself have weight
The organ born greatly, system reduce damage；
4, the peripheral blood hematopoietic stem cells after mobilizing are able to satisfy the Treatment need of donor, and to the Small side effects of patient, mention
High patients ' life quality keeps the physical qualification of good prognosis to patient.
Detailed description of the invention
Fig. 1 shows blood sampling time and PBMC Figure of the quantitative relationship.
Specific embodiment
The invention discloses a kind of mobilization agent composition and its application, preparation, those skilled in the art can be used for reference herein
Content is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
It is it will be apparent that they are considered as being included in the present invention for member.Method and application of the invention has passed through preferably real
Example is applied to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to method described herein
It is modified or appropriate changes and combinations with application, carrys out implementation and application the technology of the present invention.
Agents useful for same, instrument etc. are available on the market in mobilization agent composition provided by the invention and its application, preparation.
Below with reference to embodiment, the present invention is further explained:
The preparation of 1 mobilization agent composition of embodiment
AMD3100 (being purchased from Sigma company) is dissolved with DMSO, and with normal saline at concentration are as follows: 100 μ g/mL's
Solution；
With normal saline dilution CTX (being purchased from Hengrui Medicine Co., Ltd., Jiangsu Prov.) at concentration are as follows: 100 μ g/mL；
With normal saline dilution G-CSF (being purchased from North China Pharmacuetical Jintan Biotechnology Co., Ltd) at concentration are as follows: 1 μ
g/mL；
The AMD3100 of 100 μ g/mL, concentration are the CTX of 100 μ g/mL, and concentration is the G-SCF of 1 μ g/mL, as mobilization agent
Composition.
The test of 2 hematopoietic stem cell mobilization of embodiment
With each 40 rats of male and female (purchase is in Guangdong Medical Lab Animal Center), weight is experiment in 200g or so
Object is randomly divided into eight groups, and every group 10, four groups are control group, and four groups are experimental group.It raises one week, injects under normal condition
Before mobilization agent, rat peripheral blood is acquired, carries out peripheral blood mononuclear cells counting and viability examination, it is ensured that the grouping of each group rat is equal
It is even, as shown in table 1.
Peripheral blood mononuclear cells of rats counts before table 1 is mobilized
As shown in Table 1, control group and experimental group the rat peripheral blood before mobilization are separated to mononuclearcell number and thin
Born of the same parents' vigor does not have difference, it was demonstrated that the grouping of each group rat is uniform.
Administration mode:
Experimental group rat with: with embodiment 1 prepare mobilization agent composition be administered, AMD3100 is 2mg/ with total amount
The dosage of kg/d, CTX are the dosage of 2mg/kg/d with total amount, and G-SCF is mixed with the dosage that total amount is 2 μ g/kg/d, quiet in three times
Arteries and veins injection, injection is injected for the first time with the half of total amount, is spaced and is carried out within three hours injecting for second, the second injection is with residue
The half of amount is injected, and interval carries out third time injection in four hours again, has injected the mobilization agent of all surpluses for the third time；
Control group is then injected with equivalent mL normal saline.
Interval is acquired rat peripheral blood in three hours after having injected mobilization agent, is carried out with percoll cell separating liquid
Mononuclearcell is separated, draws tunica albuginea layer, PBS cleaning counts afterwards three times and its vigor.
Extract 2 × 106A cell, with CD34+Flow cytometry is carried out for surface antibody mark, obtains CD34+Hundred
Divide ratio, peripheral blood PBMC × CD34+Percentage, be candidate stem cell number in peripheral blood, test result is shown in Table 2.
Peripheral blood mononuclear cells of rats after table 2 is mobilized
Note:★Indicate experimental group compared with the control group, P < 0.05.
It is obtained by table 2, after injecting mobilization agent composition, experimental group is compared with control group, P < 0.05, has statistics meaning
Justice, be mobilization agent composition of the present invention can effectively mobilizing hematopoietic stem cells into peripheral blood.
The test of 3 hematopoietic stem cell mobilization of embodiment
(AMD3100+G-CSF+CTX) combination combines comparative experiments with (G-CSF+CTX):
70 rats (purchase is in Guangdong Medical Lab Animal Center) is obscured with every group of 10 male and female, and weight is in 200g
Left and right is experimental subjects, is randomly divided into seven groups, and one group is control group, and six groups are experimental group.Wherein, three groups of experimental groups are used
AMD3100+G-CSF+CTX mobilized peripheral blood candidate stem cell, in addition three groups of experimental groups G-CSF+CTX mobilized peripheral blood hematopoiesis
Stem cell:
(AMD3100+G-CSF+CTX) group: AMD3100 is the dosage of 2mg/kg/d with total amount, and CTX is 2mg/ with total amount
The dosage of kg/d, G-SCF are mixed with the dosage that total amount is 2 μ g/kg/d, are injected intravenously in three times；
(G-CSF+CTX) group: CTX is the dosage of 4mg/kg/d with total amount, and G-SCF is mixed for the dosage of 4g/kg/d with total amount
It closes, is injected intravenously in three times.
The mode being injected intravenously three times are as follows: injection is injected for the first time with the half of total amount, is spaced and is carried out for three hours
Biphasic injection, the second injection are injected with the half of surplus, and interval carries out third time injection for four hours again, and third time is injected
The mobilization agent of complete all surpluses.
Control group is then injected with equivalent mL normal saline.
Injected to be spaced three hours after mobilization agent and be acquired rat peripheral blood 5mL, with percoll cell separating liquid into
Row separation mononuclearcell, draws tunica albuginea layer, and PBS cleaning counts afterwards three times；With CD34+It is thin that streaming is carried out for surface antibody mark
The detection of born of the same parents' art, obtains CD34+Percentage, be peripheral blood PBMC × CD34+Percentage.Test result is as follows:
Peripheral blood mononuclear cells of rats after table 3 is mobilized
Note:▲Indicate experimental group compared with the control group, P < 0.05.
It is obtained by table 3, two groups of experimental groups are compared, and (AMD3100+G-CSF+CTX) group is obtained compared with (G-CSF+CTX) group
PBMC is more, CD34+Expression rate is higher, is that (AMD3100+G-CSF+CTX) group more effectively mobilizes out peripheral blood Hematopoietic Stem
Cell.
The test of 4 hematopoietic stem cell mobilization of embodiment
(AMD3100+G-CSF+CTX) combination organizes comparative experiments with (AMD3100):
70 rats (purchase is in Guangdong Medical Lab Animal Center) is obscured with every group of 10 male and female, and weight is in 200g
Left and right is experimental subjects, is randomly divided into seven groups, and one group is control group, and six groups are experimental group.Wherein, three groups of experimental groups are used
AMD3100+G-CSF+CTX mobilized peripheral blood candidate stem cell, in addition three groups of experimental groups are made with the independent mobilized peripheral blood of AMD3100
Hemocytoblast:
(AMD3100+G-CSF+CTX) group: AMD3100 is the dosage of 2mg/kg/d with total amount, and CTX is 2mg/ with total amount
The dosage of kg/d, G-SCF are mixed with the dosage that total amount is 2 μ g/kg/d, are injected intravenously in three times；
(AMD3100) group: AMD3100 is the dosage of 8mg/kg/d with total amount, is injected intravenously in three times；
It is injected intravenously mode three times are as follows: injection is injected for the first time with the half of total amount, is spaced three hours and is carried out second
Secondary injection, the second injection are injected with the half of surplus, and interval carries out third time injection in four hours again, has injected for the third time
The mobilization agent of all surpluses；
Control group is then injected with equivalent mL normal saline.
Injected to be spaced three hours after mobilization agent and be acquired rat peripheral blood 5mL, with percoll cell separating liquid into
Row separation mononuclearcell, draws tunica albuginea layer, and PBS cleaning counts afterwards three times；With CD34+It is thin that streaming is carried out for surface antibody mark
The detection of born of the same parents' art, obtains CD34+Percentage, be peripheral blood PBMC × CD34+Percentage.Test result is as follows:
Peripheral blood mononuclear cells of rats after table 4 is mobilized
Note: * indicates experimental group compared with the control group, P < 0.05.
It is obtained by table 4, two groups of experimental groups are compared, and (AMD3100+G-CSF+CTX) group is compared with the PBMC that (AMD3100) group obtains
More, CD34+Expression rate is higher, is that (AMD3100+G-CSF+CTX) group more effectively mobilizes out peripheral blood hematopoietic stem cells.
The test of 5 hematopoietic stem cell mobilization of embodiment
Blood sampling time determines scheme:
With each 20 rats of male and female (with purchase in Guangdong Medical Lab Animal Center), weight is real in 200g or so
Object is tested, is randomly divided into four groups, every group 10, one group is control group, and three groups are experimental group, is raised one week under normal condition.
Experimental group rat with: AMD3100 is the dosage of 2mg/kg/d with total amount, and CTX is the dosage of 2mg/kg/d with total amount,
G-SCF is mixed with the dosage that total amount is 2 μ g/kg/d, is injected intravenously in three times, injection is infused for the first time with the half of total amount
It penetrates, is spaced and carries out within three hours second and inject, the second injection is injected with the half of surplus, and interval carries out for four hours again
It injects three times, has injected the mobilization agent of all surpluses for the third time；Control group is then injected with equivalent mL normal saline.Injection
After complete mobilization agent, rat peripheral blood 5mL is acquired with interval 0.5h, 1.0h, 2h, 3h, 4h, 5h, 6h, 7h, 8h respectively, is used
Percoll cell separating liquid carries out separation mononuclearcell, draws tunica albuginea layer, PBS cleaning counts afterwards three times, with CD34+For table
Face antibody labels carry out Flow cytometry, obtain CD34+Percentage, be peripheral blood PBMC × CD34+Percentage.
Test result see the table below:
The optimization experiment of 5 blood sampling time of table
The chart drawn by upper table, as shown in Figure 1.
It is obtained by table 5, after mobilization, first three hour PBMC quantity is ascendant trend, is most peak value to three hours, is more than
Start after three hours in downward trend, is to take a blood sample within three hours after mobilization that more PBMC can be obtained, to obtain more
More candidate stem cells.
The test of 6 hematopoietic stem cell mobilization of embodiment
With each 60 rats of male and female (purchase is in Guangdong Medical Lab Animal Center), weight is experiment in 200g or so
Object is randomly divided into eight groups, and every group 10, four groups are control group, and four groups are experimental group 1, and four groups are experimental group 2.Normal condition
Lower raising one week.
Administration mode:
1 rat of experimental group with: with embodiment 1 prepare mobilization agent composition be administered, AMD3100 is 1mg/ with total amount
The dosage of kg/d, CTX are the dosage of 3mg/kg/d with total amount, and G-SCF is mixed with the dosage that total amount is 5 μ g/kg/d, quiet in three times
Arteries and veins injection, injection is injected for the first time with the half of total amount, is spaced and is carried out within three hours injecting for second, the second injection is with residue
The half of amount is injected, and interval carries out third time injection in four hours again, has injected the mobilization agent of all surpluses for the third time；
2 rat of experimental group with: with embodiment 1 prepare mobilization agent composition be administered, AMD3100 is with total amount
The dosage of 1.5mg/kg/d, CTX are the dosage of 1mg/kg/d with total amount, and G-SCF is mixed with the dosage that total amount is 10 μ g/kg/d,
It being injected intravenously in three times, injection is injected for the first time with the half of total amount, and it is spaced and carries out within three hours injecting for second, the second note
The half injection with surplus is penetrated, interval carries out third time injection in four hours again, injected all surpluses for the third time
Mobilization agent；
Control group is then injected with equivalent mL normal saline.
Interval is acquired rat peripheral blood in three hours after having injected mobilization agent, is carried out with percoll cell separating liquid
Mononuclearcell is separated, draws tunica albuginea layer, PBS cleaning counts afterwards three times and its vigor.
Extract 2 × 106A cell, with CD34+Flow cytometry is carried out for surface antibody mark, obtains CD34+Hundred
Divide ratio, peripheral blood PBMC × CD34+Percentage, be candidate stem cell number in peripheral blood, test result is shown in Table 6.
Peripheral blood mononuclear cells of rats after table 6 is mobilized
Note:★Indicate experimental group compared with the control group, P < 0.05.
It is obtained by table 6, after injecting mobilization agent composition, experimental group 1,2 is compared with control group, P < 0.05, has statistics
Meaning, be mobilization agent composition of the present invention can effectively mobilizing hematopoietic stem cells into peripheral blood.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (5)
1. a kind of mobilization agent composition, which is characterized in that be made of AMD3100, CTX and G-SCF；
The mass ratio of the AMD3100, the CTX and the G-SCF are 2000:2000:2.
2. mobilization agent composition as described in claim 1 is preparing the application in hematopoietic stem cell mobilization agent.
3. application according to claim 2, which is characterized in that the candidate stem cell is peripheral blood hematopoietic stem cells.
4. a kind of hematopoietic stem cell mobilization agent, which is characterized in that including mobilization agent composition as described in claim 1.
5. hematopoietic stem cell mobilization agent according to claim 4, which is characterized in that its dosage form is injection.
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Non-Patent Citations (3)
| Title |
|---|
| Majority of patients receiving initial therapy with lenalidomide-based regimens can be successfully mobilized with appropriate mobilization strategies;S Sinha et al.;《Leukemia》;20111028;第26卷;第1106-1152页 * |
| Plerixafor Added to Chemotherapy Plus G-CSF Is Safe and Allows Adequate PBSC Collection in Predicted Poor Mobilizer Patients with Multiple Myeloma or Lymphoma;I. Attolico et al.;《Biol Blood Marrow Transplant》;20121231;第18卷;第241-249页 * |
| Plerixafor on-demand combined with chemotherapy and granulocyte colony-stimulating factor: significant improvement in peripheral blood stem cells mobilization and harvest with no increase in costs;Giuseppe Milone et al.;《British Journal of Haematology》;20131021(第164期);第113-123页 * |
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