CN112569343A - A composition for treating alopecia and preparation method thereof - Google Patents
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
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Abstract
The invention provides a composition for treating alopecia, which is prepared from botulinum toxin and mesenchymal stem cells, wherein the dosage ratio of the botulinum toxin to the mesenchymal stem cells is 10U:2-50 x 105(ii) a The composition for treating alopecia provided by the invention can effectively treat alopecia and has a good treatment effect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a compound for treating alopecia and a preparation method thereof.
Background
Alopecia is the phenomenon of hair loss, and has physiological and pathological components, physiological alopecia refers to normal hair loss, pathological alopecia refers to abnormal or excessive hair loss, and with the increasing social pressure, faster and faster pace of life, change of dietary habits and other reasons, there are more and more alopecia patients at home and abroad, and although various agents for treating alopecia are available in the market, most of the alopecia patients have a slight effect, and part of the patients choose to transplant hair, but the hair transplantation cost is high.
Disclosure of Invention
In order to solve the above technical problems, the present invention provides a complex for treating alopecia, the active ingredient of the complex comprising a titer/number ratio of 10:2 x 105-5*106The botulinum toxin and the mesenchymal stem cells of (1).
Wherein the titer/number ratio is 10:2 x 105-5*106By botulinum toxin and mesenchymal stem cells is meant 10:2 x 10 per 10U of botulinum toxin5-5*106The mesenchymal stem cell of (4).
In the compound, the botulinum toxin and the mesenchymal stem cells can be newly prepared temporarily, and can also be stored for a period of time for reuse after being prepared, which is within the protection range of the application, but the storage time is not suitable to be overlong.
Further, the complex comprises a first preparation unit and a second preparation unit, wherein the first preparation unit takes the mesenchymal stem cells as an active ingredient, and the second preparation unit takes the botulinum toxin as an active ingredient.
Further, the first formulation unit and the second formulation unit are both liquid formulations.
Further, the second formulation unit is botulinum toxin type a for injection.
Further, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
Wherein, the injection method comprises the following steps: the medicine is administrated by intradermal injection, wherein the injection layer is at the junction of deep dermis and subcutaneous, a 30G or 32G needle is used for injection, and 0.1ml of medicine liquid is injected by 1 needle. Injecting 1 needle per square centimeter, wherein the liquid medicine contains 10U/ml of dry meat toxin and 2 x 105-5*106cell/ml, each injection should not exceed 20 needles, two injections should be 1-3 months apart.
Further, the preparation method of the compound comprises the following steps:
s1 preparing a first formulation unit: the concentration of 2 x 10 is prepared by using physiological saline containing 1% human albumin5-5*106cell/ml stem cell suspension;
s2 preparing a second formulation unit: preparing a botulinum toxin solution with the concentration of 10U/ml by using normal saline;
s3, mixing the first preparation unit and the second preparation unit uniformly to obtain the compound for treating alopecia.
Further, the preparation method of the compound comprises the following steps:
s1 preparing a first formulation unit: the concentration of 2 x 10 is prepared by using physiological saline containing 1% human albumin5-5*106cell/ml stem cell suspension;
s2 preparing a second formulation unit: preparing a botulinum toxin solution with the concentration of 10U/ml by using normal saline;
s3, uniformly mixing the first preparation unit and the second preparation unit according to the ratio of 1:1 to obtain the compound for treating alopecia.
Further, the preparation method of the compound comprises the following steps:
s1 preparing a first formulation unit: the concentration of 5 x 10 was prepared by using physiological saline containing 1% human serum albumin6cell/ml stem cell suspension;
s2 preparing a second formulation unit: preparing a botulinum toxin solution with the concentration of 10U/ml by using normal saline;
s3, uniformly mixing the first preparation unit and the second preparation unit according to the ratio of 1:1 to obtain the compound for treating alopecia.
Specifically, the compound can be prepared by the following method:
observing the growth state of the cells, and starting collection when the fusion degree of the P5 generation cells reaches 80-90%;
taking the old culture medium, transferring the old culture medium into a container, and checking mycoplasma and sterility;
digesting;
centrifugal washing is carried out for 2 times: centrifuging for 5min at 300 g;
sampling, counting and counting the survival rate;
the centrifuged cells were suspended in 1% human serum albumin in physiological saline to 2X 105-5×106cell/ml stem cell suspension;
the cells were split into 0.8 ml/tube and stored temporarily at 4 ℃ for future use.
Adjusting the concentration of the botulinum toxin to 10U/ml by using normal saline;
mixing a solution of botulinum toxin at a concentration of 10U/ml with a solution at a concentration of 2X 10 by syringe5-5×106Uniformly mixing the cell/ml stem cell suspension according to the ratio of 1: 1;
and mixing uniformly for subsequent use.
The invention also provides the application of the compound in preparing a medicine for treating alopecia.
The compound provided by the invention can effectively treat alopecia and has a good treatment effect.
Drawings
FIG. 1 is a diagram showing the state of cells of three control groups 2 days after inoculation in test example 1;
FIG. 2 is a diagram showing the state of cells of five experimental groups 2 days after inoculation in Experimental example 1;
FIG. 3 is a diagram showing the state of cells of three control groups 3 days after inoculation in test example 1;
FIG. 4 is a diagram showing the state of cells of five experimental groups 3 days after inoculation in Experimental example 1;
FIG. 5 is a diagram showing the state of cells of three control groups 4 days after inoculation in test example 1;
FIG. 6 is a diagram showing the state of cells of five experimental groups 4 days after inoculation in Experimental example 1;
FIG. 7 is a diagram showing the state of cells of three control groups after 4 days of inoculation in Experimental example 1 and staining by the cell MTT assay;
FIG. 8 is a diagram showing the state of cells of five experimental groups after 4 days of inoculation and staining by MTT cell assay in Experimental example 1;
FIG. 9 is a diagram showing the state of the cells of three control groups after 4 days of DMSO elution in test example 1;
FIG. 10 is a diagram showing the state of cells of five experimental groups 4 days after inoculation in Experimental example 1 and elution with DMSO;
FIG. 11 is a statistical chart of the effect of treatment on hair loss in mice after administration of each group of drugs.
Detailed Description
Example 1
A compound for treating alopecia is prepared from botulinum toxin and umbilical cord mesenchymal stem cells by the following steps:
s1 preparing a first formulation unit: the concentration of the preparation is 5X 10 by using physiological saline containing 1% human serum albumin61L of cell/ml umbilical cord mesenchymal stem cell suspension;
s2 preparing a second formulation unit: 1L of a 10U/ml botulinum toxin solution is prepared by using normal saline;
s3: uniformly mixing the first preparation unit and the second preparation unit according to the proportion of 1:1 to obtain a compound;
the botulinum toxins used in the invention are type A botulinum toxins for injection, which are purchased from Allergan, with the product number of 92949CH and the batch number of S20171005, and the umbilical cord mesenchymal stem cells in all the examples of the application are commercially available.
Example 2
A compound for treating alopecia is prepared from botulinum toxin and umbilical cord mesenchymal stem cells by the following steps:
s1 preparing a first formulation unit: the concentration of the product is 1 × 10 by using physiological saline containing 1% human albumin61L of cell/ml umbilical cord mesenchymal stem cell suspension;
s2 preparing a second formulation unit: 1L of a 10U/ml botulinum toxin solution is prepared by using normal saline;
s3: and uniformly mixing the first preparation unit and the second preparation unit according to the ratio of 1:1 to obtain the compound.
Example 3
A compound for treating alopecia is prepared from botulinum toxin and umbilical cord mesenchymal stem cells by the following steps:
s1 preparing a first formulation unit: the concentration of the product is 2 × 10 using physiological saline containing 1% human albumin51L of cell/ml umbilical cord mesenchymal stem cell suspension;
s2 preparing a second formulation unit: 1L of a 10U/ml botulinum toxin solution is prepared by using normal saline;
s3: and uniformly mixing the first preparation unit and the second preparation unit according to the ratio of 1:1 to obtain the compound.
Test example 1 Effect of botulinum toxin on umbilical cord mesenchymal Stem cells
This test will perform a gradient concentration test: taking the botulinum toxin with different concentrations as experimental variables to culture the umbilical cord mesenchymal stem cells, wherein the concentration of the botulinum toxin is divided into 100U/ml, 80U/ml, 40U/ml, 20U/ml and 10U/ml; the specific experimental method is as follows:
s1 cell recovery: culturing the recovered umbilical cord mesenchymal stem cells into existing experimental cells in a cell bank, and performing quality part warehousing examination and verification before warehousing, wherein the cells are in good state and the conventional sterile detection results of the cells are qualified;
and (4) preparing the concentration of S2: the concentration of the botulinum toxin is divided into 100U/ml, 80U/ml, 40U/ml, 20U/ml and 10U/ml, the original botulinum toxin (purchased from Allergan, with the product number of 92949CH and the batch number of S20171005) is invisible to the naked eye in an ampere bottle, the specification is 100U/count, and the botulinum toxin contains a small amount of human albumin; the preparation method comprises the following steps of taking complete culture medium (purchased from LONZA) as a diluent, preparing a botulinum toxin mixed culture medium with the concentration of 100U/ml by using the complete culture medium, and then taking a part of samples to dilute the samples by using the complete culture medium, wherein the specific dilution concentration is as follows:
TABLE 1 formulation of botulinum toxin cocktail media at each concentration in examples 1-5.
S3, digesting the recovered umbilical cord mesenchymal stem cells on the first day, sampling and counting, spreading about 20000 cells in each hole of a 96-well plate according to the counting result, adding 200 mu L of complete culture medium into each hole for culture, and marking;
s4, observing the cell adherence condition on the next day, taking pictures and recording the growth condition; sucking out the culture medium in the pore plate, adding the prepared botulinum toxin mixed culture medium with various concentrations (each group is made into two parallel samples) and the culture medium of three control groups, wherein the culture medium added in the control groups is a complete culture medium, and marking each pore;
s3, observing and recording the cell growth condition on the third day, observing the cell state and quantity of the experimental group and the control group, and checking the residual condition of the culture medium in the pore plate;
s4 day four: observing and recording the growth condition of the cells, observing the state and the shape of the cells, observing the number of the cells, and checking the residual condition of the culture medium in the pore plate;
fifth day S5: and observing and recording the growth condition and the growth state of the cells, when the cell fusion degree is more than or equal to 80%, preparing for cell growth state detection, performing cell treatment by using an MTT method, detecting the cell growth activity, and performing statistical analysis.
As shown in fig. 1, the state of the cells was good the first day after the experimental cell inoculation, the number of inoculated cells was large, and the cell debris was small; as shown in FIG. 1, the cell state diagrams of the three control groups for the second day of inoculation, as shown in FIG. 2, the cell state diagrams of the five experimental groups for the second day of inoculation, as shown in FIG. 3, the cell state diagrams of the three control groups for the third day of inoculation, as shown in FIG. 4, the cell state diagrams of the five experimental groups for the third day of inoculation, as shown in FIG. 5, the cell state diagrams of the three control groups for the fourth day of inoculation, as shown in FIG. 6, the cell state diagrams of the five experimental groups for the fourth day of inoculation, the fourth day of cell inoculation, and the third day of drug culture, MTT assay of cells was performed to determine the number of cells and the activity of cells, and the number of cells inoculated with botulinum cell debris was observed to be large experimentally, the cell debris of the experimental groups at a concentration of 10U/ml was not much different from that of the control groups according to the observation under the mirror, whereas the number of cells observed under the mirror was gradually reduced and the cell debris was large since the experimental group had a, the cell fragmentation is severe, after the cell MTT experiment staining, the cell state diagram of three control groups is shown in figure 7, the cell state diagram of five experimental groups is shown in figure 8, the MTT staining can see that the cell volume is gradually increased along with the increase of the concentration and then the cell state diagram is observed after the elution by DMSO, the cell state diagram of three control groups is shown in figure 9, and the cell state diagram of five experimental groups is shown in figure 10;
the fifth day after inoculation, the cells of each group were counted, and the results are shown in table 2;
table 2. statistical table of cell numbers for each group.
According to the cell number statistics, the following results are shown: the cell growth and proliferation capacity is reduced along with the increase of the concentration of the botulinum toxin; according to the above experimental results, the cell proliferation ability decreased with the increase of the concentration of botulinum toxin, and it was shown from the statistical chart that the effect of decrease may be more and more affected according to time; in terms of cell morphology, with the increase of the concentration of the botulinum toxin, the cell volume is larger, the cells with irregular characters are gradually increased, and the cell fragmentation is increased.
Test example 2 Effect of the Compound and method of the present invention for treating alopecia
Laboratory animal
Taking 35 SPF male Wistar rats, randomly dividing the rats into a normal control group, a model control group, a botulinum toxin group, an MSC cell high dose group, and a combination of the botulinum toxin and the MSC cell high, medium and low dose groups according to the weight block, wherein each group comprises 5 rats.
Molding method
Before the experiment, the rats in the groups except the normal control group selected a 4cm × 5cm area on the back to remove hairs, and the depilated area was colored yellow with 3% picric acid solution to determine the observation area of the depilation experiment. Except for the normal control group, rats were injected with testosterone propionate injection (injection dose 5 mg/(kg. d)) subcutaneously in the back of their neck 1 time per day for 60 days, resulting in the SA model. After 4 weeks of continuous subcutaneous injection of testosterone propionate, the rats gradually lose hair, and the residual hair becomes fine and crisp, which proves that the male hormonal alopecia model is successfully established.
Method of treatment
The medicine is administrated by intradermal injection, wherein the injection level is at the junction of deep dermis and subcutaneous, 1 needle is injected every square centimeter every day, and 0.1ml of medicine liquid is injected by a 30 or 32G needle head with 1 needle.
The model control group and the normal control group are injected with normal saline only, the concentration of the botulinum toxin in the botulinum toxin group is 10U/ml, and the MSC cell high dose group is injected with 5 multiplied by 106The concentrations of the high, medium and low dose groups of injected liquid medicines of cell/ml umbilical cord mesenchymal stem cells and the combination of the botulinum toxin and the umbilical cord mesenchymal stem cells are respectively 10U/ml +5 multiplied by 10 of botulinum toxin concentration6cell/ml umbilical cord mesenchymal stem cell, 10U/ml + 1X 106cell/ml umbilical cord mesenchymal stem cell, 10U/ml + 2X 105cell/ml umbilical cord mesenchymal stem cells (i.e., the complexes of examples 1-3). Every 15 days, 10 hairs were plucked from the observation area of the back of each rat, and the length of the hairs was measured with a vernier caliper. After administration for 60 days, the skin of the observation area is taken, conventional tissue dehydration, paraffin embedding, HE staining and optical microscopy are carried out, the histopathological changes of the hair follicle and the sebaceous gland of the skin of the rat are observed, and the experimental result is shown in figure 11.
Results
As can be seen from FIG. 11, the effect on the hair growth of rats, the hair lengths of rats in the high, medium and low dose groups of the botulinum toxin combined umbilical cord mesenchymal stem cells on the administration days 15, 30, 45 and 60 are all longer than those of the model control group, the hair growth length of the MSC cell high dose group is longer than that of the low dose group and is smaller than those of the medium dose group and the high dose group, the hair growth length of the botulinum toxin group is lower than that of the low dose group, the hair growth length of the high dose group is close to that of the normal control group, and the treatment effect is significantly higher than that of the medium dose group and the low dose group.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (9)
1. A composition for treating alopecia, wherein the active ingredients of the composition comprise a potency/number ratio of 10:2 x 105-5*106The botulinum toxin and the mesenchymal stem cells of (1).
2. The complex of claim 1, comprising a first formulation unit having the mesenchymal stem cells as an active ingredient and a second formulation unit having the botulinum toxin as an active ingredient.
3. The complex of claim 2, wherein said first formulation unit and said second formulation unit are both liquid formulations.
4. The complex of claim 3, wherein the second formulation unit is botulinum toxin type A for injection.
5. The complex of claim 1, wherein the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.
6. The compound of claim 2, wherein the compound is prepared by a method comprising the steps of:
s1 preparing a first formulation unit: using a raw material containing 1% human serum albuminThe concentration of the prepared normal saline water is 2 x 105-5*106cell/ml stem cell suspension;
s2 preparing a second formulation unit: preparing a botulinum toxin solution with the concentration of 10U/ml by using normal saline;
s3, mixing the first preparation unit and the second preparation unit uniformly to obtain the compound for treating alopecia.
7. The compound of claim 6, wherein the compound is prepared by a method comprising the steps of:
s1 preparing a first formulation unit: the concentration of 2 x 10 is prepared by using physiological saline containing 1% human albumin5-5*106cell/ml stem cell suspension;
s2 preparing a second formulation unit: preparing a botulinum toxin solution with the concentration of 10U/ml by using normal saline;
s3, uniformly mixing the first preparation unit and the second preparation unit according to the ratio of 1:1 to obtain the compound for treating alopecia.
8. The compound of claim 7, wherein the compound is prepared by a method comprising the steps of:
s1 preparing a first formulation unit: the concentration of 5 x 10 was prepared by using physiological saline containing 1% human serum albumin6cell/ml stem cell suspension;
s2 preparing a second formulation unit: preparing a botulinum toxin solution with the concentration of 10U/ml by using normal saline;
s3, uniformly mixing the first preparation unit and the second preparation unit according to the ratio of 1:1 to obtain the compound for treating alopecia.
9. Use of a complex according to claims 1-8 for the preparation of a medicament for the treatment of alopecia.
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Citations (4)
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|---|---|---|---|---|
| WO2007069813A1 (en) * | 2005-12-16 | 2007-06-21 | Modern Cell & Tissue Technologies Inc. | Methods for culturing mesenchymal stem cell and compositions comprising mesenchymal stem cell for reparing skin defects |
| US20130058906A1 (en) * | 2010-03-11 | 2013-03-07 | Antoine Turzi | Process, tube and device for the preparation of wound healant composition |
| CN107823632A (en) * | 2017-11-10 | 2018-03-23 | 南京九圣生物科技股份有限公司 | A kind of mesenchymal stem cells MSCs parenteral solution and preparation method |
| CN111840330A (en) * | 2020-08-03 | 2020-10-30 | 北京诺赛启研再生医学研究院有限公司 | Anti-hair loss repairing composition and application |
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2020
- 2020-12-23 CN CN202011540343.9A patent/CN112569343A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007069813A1 (en) * | 2005-12-16 | 2007-06-21 | Modern Cell & Tissue Technologies Inc. | Methods for culturing mesenchymal stem cell and compositions comprising mesenchymal stem cell for reparing skin defects |
| US20130058906A1 (en) * | 2010-03-11 | 2013-03-07 | Antoine Turzi | Process, tube and device for the preparation of wound healant composition |
| CN107823632A (en) * | 2017-11-10 | 2018-03-23 | 南京九圣生物科技股份有限公司 | A kind of mesenchymal stem cells MSCs parenteral solution and preparation method |
| CN111840330A (en) * | 2020-08-03 | 2020-10-30 | 北京诺赛启研再生医学研究院有限公司 | Anti-hair loss repairing composition and application |
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