CN104667257A - Method and pharmaceutical composition for applying hair growth peptide - Google Patents

Abstract

The invention provides a method for using peptide and a pharmaceutical composition. Specifically, the pharmaceutical composition comprises a germinal peptide (HGP), wherein the germinal peptide (HGP) is a whole sequence or a partial sequence of an amino acid sequence SEQ ID No: 1. The method comprises administering a germinal peptide (HGP) to the subject, wherein the germinal peptide (HGP) is the full sequence or a partial sequence of amino acid sequence SEQ ID No: 1.

Description

Method and pharmaceutical composition for applying hair growth peptide

technical field

The invention relates to a method and a pharmaceutical composition for applying a hair growth peptide.

Background technique

Alopecia (alopcia) is an abnormality caused by a decrease in the number of hairs in the anagen phase (anagen) of the hair cycle and an increase in the number of hairs in the catagen or telogen phase of the hair cycle Hair reduction. Although the mechanism of baldness has not been clearly revealed, baldness can generally be caused by endocrine disorders (such as hormone imbalance), autonomic nerves disorder, or circulatory disorders ( Excessive sebum production due to circular disorders (such as abnormal blood circulation), deterioration of scalp function due to fungi, allergies, genetic causes, or aging.

Alopecia is also one of the most serious side effects of cancer treatment and is also caused by the administration of various chemotherapeutic agents. Because chemotherapeutics affect cell division (cytokinesis), chemotherapeutics can thrive in areas such as bone marrow, hair, fingernails, toenails, skin, or the gastrointestinal tract cause side effects in the tissues of the body and thus trigger baldness. Although baldness is considered the greatest affliction by 80% or more of patients receiving chemotherapy, no effective method of preventing or treating chemotherapy-induced baldness has been developed.

Baldness will appear about 2 to 4 weeks after chemotherapy. Hair doesn't grow back until 3 to 6 months after chemotherapy ends. The degree of alopecia varies with the drug used, the amount of the drug, and the timing of the administration. Drugs that cause high-grade alopecia include cyclophosphamide, doxorubicin, cisplatin, cytosine arabinoside, and etoposide. The aforementioned drugs can induce alopecia even when administered topically. For example, chemotherapeutic agents can affect cell division of the hair follicle, thereby triggering hair follicle cell death, or can promote the transition from anagen to catagen.

The current clinical and practical methods for treating alopecia are: external application of drugs, oral drugs and autologous hair transplantation (hair implantation), but each has its limitations and shortcomings. Minoxidil or Finasteride are currently two hair growth drugs approved by the US FDA. Patients who use topical minoxidine and oral finasteride must be used continuously to be effective, and the drugs cannot be discontinued. In addition, after using minoxidil or finasteride, the hair cannot be thickened, only the phenomenon of hair loss can be reduced, or only thinning hair can grow, and there will be sexual dysfunction, hypertrichosis (hypertrichosis), fetal defects and other side effects. Therefore, it is not suitable for women of childbearing age. Furthermore, autologous hair follicle transplantation (hair implantation) will leave scars on the transplanted area, and the postoperative recovery time is long, requiring multiple operations, long operation time, and high cost.

Contents of the invention

The present invention provides a method for applying hair growth peptides without facing various problems of autologous hair follicle transplantation (such as scars, long recovery time after surgery, multiple surgeries, etc.).

The present invention provides a method of using peptides. The peptide is selected from the hair growth peptide (HGP), and the hair growth peptide (HGP) is the whole or partial sequence of the amino acid sequence SEQ ID No:1.

The present invention also provides a method of using the peptide. The peptide is selected from hair growth peptide (HGP), and the amino acid sequence of the hair growth peptide (HGP) is about 90% to 99% similar to the amino acid sequence of SEQ ID No: 1. The similarity (similarity) of the present invention is a statistical result obtained by analyzing with Shimadzu LCMS2010 software.

The present invention also provides a use of a peptide for hair growth. The peptide is selected from the hair growth peptide (HGP), and the hair growth peptide (HGP) is the whole or partial sequence of the amino acid sequence SEQ ID No:1.

The present invention also provides a use of the peptide for hair growth. The peptide is selected from hair growth peptide (HGP), and the amino acid sequence of the hair growth peptide (HGP) is about 99% to 90% similar to the amino acid sequence of SEQ ID No: 1.

The invention also provides a pharmaceutical composition for treating alopecia. The pharmaceutical composition comprises hair growth peptide (HGP) and phosphate. The hair growth peptide (HGP) is the whole or partial sequence of the amino acid sequence SEQ ID No:1.

The invention further provides a pharmaceutical composition for treating alopecia. The pharmaceutical composition comprises hair growth peptide (HGP) and phosphate. The amino acid sequence of the hair growth peptide (HGP) is about 99% to 90% similar to the amino acid sequence of SEQ ID No: 1.

Other purposes of the present invention will be partly stated in the subsequent description, and partly can be easily known from the content description, or can be obtained from the implementation of the present invention. Aspects of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the claims. It should be understood that the foregoing general description and the following detailed description are for the purpose of illustration only, and are not intended to limit the present invention.

The technical features and advantages of the present invention have been summarized quite broadly above, so that the following detailed description of the present invention can be better understood. Additional technical features and advantages forming the subject of the claims of the invention will be described hereinafter. Those skilled in the technical field of the present invention should understand that the concepts and specific embodiments disclosed below can be used to modify or design other structures or processes to achieve the same purpose as the present invention. Those with ordinary knowledge in the technical field of the present invention should also understand that such equivalent constructions cannot depart from the spirit and scope of the present invention defined by the claims.

Description of drawings

The foregoing Summary and foregoing Detailed Description of the present invention are better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, presently preferred specific embodiments are shown in each drawing. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

Figure 1 shows a schematic diagram of the experimental steps according to an embodiment of the present invention; and

Fig. 2 shows a schematic diagram of six sections of mouse back skin according to an embodiment of the present invention.

3 shows a schematic diagram of a TCF reporter assay (reporter assay) of a human skin keratinocyte (HaCaT) strain according to an embodiment of the present invention. TOP means that the reporter gene has a binding site for β-catenin binding. FOP means that the binding site for β-catenin in the reporter gene has been mutated and destroyed, so β-catenin cannot be bonded, and it can be used as a negative control. CTL represents HaCaT cell line without any stimulus or peptide treatment. iPept-2 represents the HaCaT cell line stimulated by the amino acid sequence SEQ ID No: 1 peptide. Referring to relative luciferase activity, the activity of β-catenin in HaCaT cell lines stimulated or treated with the peptide of SEQ ID No: 1 was also increased.

4 shows a schematic diagram of a TCF reporter assay of a human hair follicle keratinocyte (HHFK) strain according to an embodiment of the present invention. TOP means that the reporter gene has a binding site for β-catenin binding. FOP means that the binding site for β-catenin in the reporter gene has been mutated and destroyed, so β-catenin cannot be bonded, and it can be used as a negative control. CTL represents HHFK cell line without any stimulus or peptide treatment. iPept-2 represents the HHFK cell line stimulated by the amino acid sequence SEQ ID No: 1 peptide. Referring to relative luciferase activity, HHFK cell lines stimulated or treated with the peptide of SEQ ID No: 1 can be observed to have increased β-catenin activity.

Detailed ways

Hereinafter, the embodiments of the present invention are described in detail with reference to the accompanying drawings. However, the embodiments of the present invention do not necessarily require all technical features to be implemented. In other embodiments, some well-known methods, structures and techniques are not shown so as not to be misleading. "This embodiment" and "other embodiments" and so on mentioned in the specification mean that the relevant special characteristics, sequences, or characteristics included in this embodiment of the present invention are included. The appearances of the phrase "in this embodiment" in various places in the specification do not necessarily all refer to the same embodiment.

The direction of the present invention discussed here is a use of a peptide for hair growth and a pharmaceutical composition for treating alopecia. In order to provide a thorough understanding of the present invention, detailed steps and sequences will be set forth in the following description. It is evident that the practice of the invention is not limited to specific details familiar to those skilled in the relevant art. In other instances, well-known sequences or steps have not been described in detail in order not to unnecessarily limit the invention. The preferred embodiments of the present invention will be described in detail as follows, but in addition to these detailed descriptions, the present invention can also be widely implemented in other embodiments, and the scope of the present invention is not limited, it is based on the appended claims .

The peptide disclosed in the present invention, the hair growth peptide (HGP) or the hair growth peptide in the pharmaceutical composition is, but not limited to, using a mass spectrometer to confirm its specific molecular weight. Specifically, in this case, the mass spectrometer (Time-of-Flight massspectrometry, Sciex QSTAR PULSAR Quadrupole) of Applied Biosystems was used to confirm the molecular weight. Take an appropriate amount of sample and dissolve it in formic acid solution to form a sample solution. In special cases, serine proteases, threonine proteases, and cysteine proteases can also be used to form a sample solution. ), aspartate proteases, glutamic proteases, and metalloproteases to treat the sample, and then carry out the above molecular weight confirmation steps. This sample solution (about 5 μl) was introduced into the above-mentioned mass spectrometer. After the mass spectrometer measures the sample solution, the data is determined by using computer software (such as Shimadzu LCMS2010) to analyze the specific peptide in the measured mass spectrum, for example, when the mass-to-charge ratio values of at least two peaks in the measured mass spectrum can correspond to The specific peptide may be confirmed to have the specific peptide in the sample by using the mass-to-charge ratio in the measured mass spectrum close to the mass-to-charge ratio of the specific peptide. For example, the molecular weight of the amino acid sequence SEQ ID No: 1 is 1258.42. When the peptide has two charges, its mass-to-charge ratio is about 603.21. The mass-to-charge ratio (m/z) formula is (m/z)=(mass M+charge n)/charge n. Therefore, when a peak with a mass-to-charge ratio of about 712.70 appears in the mass spectrum, it can be confirmed that the sample has the amino acid sequence SEQ ID No: 1. In addition, the molecular weights of other amino acid sequences can refer to Table 1.

Similarly, the sample of the pharmaceutical composition can also be confirmed by the above-mentioned measuring steps of the hair growth peptide. However, in some cases, the pharmaceutical composition can also be diluted before performing the above-mentioned measurement steps.

The phosphate contained in the pharmaceutical composition of the present invention is selected from potassium dihydrogen phosphate (KH ₂ PO ₄ ), disodium hydrogen phosphate (Na ₂ HPO ₄ ·2H ₂ O) and mixed salts thereof.

Excipients of the pharmaceutical composition of the present invention are substances that increase the uniformity and stability of the pharmaceutical composition and reduce the irritation and bad smell of the pharmaceutical composition. The excipient of the present invention is non-toxic, non-irritating, non-antigenic, non-sensitizing, non-mutagenic and non-pharmacologically active, and does not hinder the exertion of the drug effect.

Based on the above principles, the excipient of the present invention is selected from lactose (Lactose), dry starch (Starch), starch paste (Starch paste), dextrin (Dextrin), cyclodextrin (Cyclodexrtin), pregelatinized starch (Pregelatinized starch) ), Carboxymethyl Starch Sodium (Carboxymethyl Starch Sodium), HydroXypropy Starch (HPS for short), Microcrystalline Cellulose (Microcrystalline Celiulose), Carboxy Methyl Cellulose (Carboxy Methyl Cellutose for short CMC), cross A mixture of Cross-linked Carboxymethy Celtuiose Sodium, Low substituted Hydroxypropyl Cellulose and at least two of the above excipients.

The confirmation method of the above excipients is selected from Chromatography Methods, Spectrophotometry Methods, Spectroscopy Methods and titration methods.

The peptide of the present invention, the hair growth peptide (HGP) or the hair growth peptide in the pharmaceutical composition is, but not limited to, the solid phase peptide synthesis (solid phase peptide synthesis), also known as the Merifell method ( Merrifield method). In other embodiments, the peptide, the hair growth peptide (HGP) or the hair growth peptide in the pharmaceutical composition can also be produced by protein expression. Since the solid-phase peptide synthesis method or the protein expression method are consistent with the currently known methods, they will not be repeated here. In addition, the hair growth peptide of the present invention can also be synthesized or expressed by Kelowna International Technology Co., Ltd. (Kelowna), so those skilled in this field can easily prepare the hair growth peptide of the present invention with the above assistance. Therefore, the present invention does not need to clearly describe the actual preparation steps and preparation conditions of the hair growth peptide of the present invention.

The pharmaceutical composition of the present invention is but not limited to a solution dosage form. The hair growth peptide is dissolved in 4 ml of phosphate buffered saline (PBS solution) so that the concentration of the hair growth peptide is 2 mM, and then diluted with 2 mM to 8 ml to 0.2 mM . Therefore, the concentration of hair growth peptides ranges from 0.2 to 2 mM to prepare the pharmaceutical composition of the present invention.

The experimental model of the present invention is to apply the hair regeneration mouse animal experimental model (mouse model) developed by Academician Zhong Zhengming of University of Southern California.

Mice used in this mouse animal experiment model: C57BL/6 eight-week-old female mice.

According to literature (Muller-Rover et al., 2001; Plikus et al., 2009), after all the hairs on the back skin of female rats are plucked or waxed, the hair follicles will enter the anagen phase (anagen) after the seventh day. At this time, the hair on the back skin of the female mouse will re-grow. The growth period is about 14 days. The above steps synchronize the anagen phase of all hair follicles.

The hair follicles of the back skin of the female mouse will enter the absolute non-responsive resting period (refractory telogen) after the anagen period. The absolute non-response telogen (refractory telogen) is about 28 days. The present invention uses drugs to treat hair follicles in the absolutely unresponsive telogen phase to observe whether the drugs help hair growth.

As shown in Figure 1, after all the hair follicles on the back skin of the female mouse were removed, the hair on the back skin of the female mouse was removed on the 21st day. As shown in Figure 2, the back skin from which the hair was removed was divided into six blocks, and the distance between each block was at least 3 cm to avoid mutual interference. Drugs will be administered continuously for 10 days per block. Methods of application include but are not limited to injection (50 microliters) and smearing (10 microliters). In addition, the daily dose of the drug includes but is not limited to two doses, namely a high dose (2,000 μM) and a low dose (200 μM).

In order to ensure the credibility of the experiment and avoid errors in human operation, as shown in Figure 2, the first block 1 continuously applies cyclosporine (cyclosporine) 10 times (1 time per day), wherein the present invention uses cyclosporine ( cyclosporine) as a positive control group (positive control), the concentration of the positive control group is 0.5% by weight (prepared with 100% alcohol), and 10 microliters are applied each time; the second block 2 is continuously applied with a high dose of hair growth peptide or Medicinal composition 10 times (1 time per day); third block 3 continuously smears low-dose hair growth peptide or pharmaceutical composition 10 times (1 time per day); fourth block 4 continuously smears 100% alcohol 10 times (every day 1 time, smearing 10 microliters each time), this group was used as the negative control group (negative control); the fifth block 5 was continuously injected with high-dose hair growth peptide or pharmaceutical composition 10 times (once a day); and the sixth Block 6 continuously injects low doses of hair growth peptide or pharmaceutical composition 10 times (once a day). The above-mentioned hair growth peptides all have the same peptide sequence.

Based on the literature (Maurer et al., 1997) report, if cyclosporin is continuously applied to the skin of the back of the mouse for 10 days, the skin hair will be induced to regenerate. Therefore, all the hair growth peptides or pharmaceutical compositions to be screened will be tested using the above-mentioned experimental mode and the hair regeneration situation will be recorded every day. Since the absolute non-response telogen (refractory telogen) of mice is about 28 days, if no hair regeneration is observed after 30 days of starting to apply the drug, the hair growth peptide or pharmaceutical composition is considered as having no promoting effect. The ability to grow hair. In other words, if any hair regrowth is observed within 30 days after the application of the drug, the hair growth peptide or pharmaceutical composition of this experimental group is considered to have the ability to promote hair growth.

The Wnt gene family plays important roles in many developmental stages, including hair follicle growth and differentiation. General Wnt signaling will stabilize β-catenin protein and aggregate β-catenin, which in turn will cause nuclear translocation and activation of LEF/TCF transcription factors to regulate gene expression. Academic journals have deduced that Wnt/β-catenin has important functions in hair morphology and differentiation (Kishimoto et al. 2000; Fuchs et al. 2001; Millar 2002).

In addition, hair growth is related to Wnt/β-catenin (β-catenin) and bone morphogenetic protein 2/4 (BMP2/4) signaling pathways, so the hair growth peptide or pharmaceutical composition of the present invention seems to be possible through This signaling pathway regulates hair growth. In other words, the hair growth peptide or the pharmaceutical composition of the present invention may affect the Wnt/β-catenin (β-catenin) and bone morphogenetic protein 2/4 (BMP2/4) signal transmission pathways by acting on hair follicles.

In this case, the relevant amino acid sequences were further applied to human skin keratinocytes (HaCaT) and hair follicle keratinocytes (HHFK) to verify the hair growth induced by these amino acid sequence peptides.

In the present invention, HaCaT and HHFK are used as cell models for studying related mechanisms. The reporter assay (reporter assay) is used as a biological activity test to analyze how the amino acid sequence SEQ ID No: 1 peptide can enhance hair growth.

In the reporter gene experiment shown in Figures 3 and 4, the dose of Wnt3a was 50ng/ml as the positive control group. According to the results, 200 μM of SEQ ID No: 1 peptide can enhance the activity of β-catenin in HaCaT cells and HHFK cells.

Table 1 shows the correspondence between the numbers of the sequence listing and the amino acid sequences of the present invention.

Table 1

Table 2 shows the comparative relationship between the experimental group number and the sequence listing number of the hair growth peptide and its pharmaceutical composition of the present invention. The similarity of the sequences of the present invention is a statistical result obtained by analyzing with Shimadzu LCMS2010 software.

Table 2

Experimental group number Sequence listing number P1 SEQ ID No:1 P2 SEQ ID No:2 P3 SEQ ID No: 3 P4 SEQ ID No:4 P5 SEQ ID No:5 P6 SEQ ID No:6 P7 SEQ ID No:7 P8 SEQ ID No:8 P9 SEQ ID No:9 P10 SEQ ID No:10 P11 SEQ ID No: 11 P12 SEQ ID No:12 P13 SEQ ID No:13 P14 SEQ ID No:14 P15 SEQ ID No:15 P16 SEQ ID No:16 P17 SEQ ID No:17 P18 SEQ ID No:18 P19 SEQ ID No:19 P20 SEQ ID No: 20 P21 SEQ ID No: 21 P22 SEQ ID No:22 P23 SEQ ID No:23 P24 SEQ ID No:24 P25 SEQ ID No:25

P26 SEQ ID No:26 P27 SEQ ID No:27 P28 SEQ ID No:28 P29 SEQ ID No:29 P30 SEQ ID No: 30 P31 SEQ ID No: 31 P32 SEQ ID No: 32 P33 SEQ ID No: 33 P34 SEQ ID No: 34 P35 SEQ ID No: 35 P36 SEQ ID No: 36 P37 SEQ ID No: 37

Each group of the above-mentioned experimental groups P1 to P37 was tested using the steps of the aforementioned Figure 1 and Figure 2, and the results were observed in the following table 3. The number X in table 3 represents the phenomenon of hair regeneration observed on the X day (for example, X=5 means hair regrowth was observed on day 5). And NA represents not yet analyzed, pending further experiments. In addition, in the experimental results in Table 3, the phenomenon of hair regeneration was observed in both the positive and control groups within 30 days, so the experimental results should be accepted. In addition, "None" means no hair growth within 30 days after peptide treatment.

table 3

Experimental group number Injection or application (high dose) P1 twenty two P2 none P3 twenty two P4 twenty two P5 twenty two P6 none P7 twenty two P8 none P9 19 P10 twenty two P11 twenty two P12 twenty two P13 twenty two P14 twenty two P15 none P16 none P17 none P18 none P19 none P20 none P21 none P22 none

P23 none P24 none P25 none P26 none P27 twenty two P28 none P29 twenty two P30 none P31 twenty two P32 none P33 none P34 none P35 none P36 none P37 twenty two

The technical content and technical features of the present invention have been disclosed above, but those with ordinary knowledge in the technical field of the present invention should understand that the teachings and disclosures of the present invention can be made in various ways without departing from the spirit and scope of the present invention defined by the claims. replacement and modification. For example, many of the elements disclosed above can be implemented differently or replaced by other sequences with the same function, or a combination of the above two ways can be used.

Furthermore, the scope of rights in this case is not limited to the sequence of specific embodiments disclosed above. Those with ordinary knowledge in the technical field of the present invention should understand that, based on the teachings and disclosed sequences of the present invention, no matter what exists now or will be developed in the future, it performs substantially the same function in substantially the same way as the person disclosed in the embodiment of this case, and Achieving substantially the same result can also be used in the present invention. Accordingly, the appended claims are intended to cover such sequences.

Symbol Description

1 first block

2 Second block

3 The third block

4 The fourth block

5 fifth block

6 The sixth block

Claims (24)

1. A method of using a peptide, comprising administering a hair growth peptide (HGP) to a recipient, wherein the hair growth peptide is the entire or partial sequence of the amino acid sequence SEQ ID No:1. 2. The method of claim 1, wherein the amino acid sequence of the hair growth peptide (HGP) is about 90% to 99% similar to the amino acid sequence of SEQ ID No: 1. 3. The method according to claim 1, wherein the carboxy-terminal of the amino acid sequence SEQ ID No: 1 deletes 1 or 7 any amino acids. 4. The method according to claim 1, wherein the carboxy-terminal of the amino acid sequence SEQ ID No: 1 replaces 1 or 7 any amino acids. 5. The method according to claim 1, wherein any amino acid from 1 to 3 is added to the carboxy-terminal of the amino acid sequence SEQ ID No:1. 6. The method according to claim 1, wherein the amino terminal of the amino acid sequence SEQ ID No: 1 deletes any 6 or 7 amino acids. 7. The method according to claim 1, wherein the amino-terminal of said amino acid sequence SEQ ID No: 1 replaces 6 or 7 any amino acids. 8. The method according to claim 3, wherein the hair growth peptide (HGP) is selected from SEQ ID No:2 and SEQ ID No:3. 9. The method of claim 5, wherein the hair growth peptide (HGP) is SEQ ID No:4. 10. The method according to claim 6, wherein said hair growth peptide (HGP) is selected from SEQ ID No:2 and SEQ ID No:5. 11. The method according to claim 1, wherein the dosage of the hair germinal peptide (HGP) is between 200 μM and 2000 μM. 12. The method of claim 1, wherein the hair growth peptide (HGP) acts on hair follicles. 13. A pharmaceutical composition for hair growth comprising: Hair growth peptide (HGP), wherein the hair growth peptide (HGP) is the whole or partial sequence of the amino acid sequence SEQ ID No: 1; and phosphate. 14. The pharmaceutical composition for hair growth according to claim 13, wherein the amino acid sequence of the hair growth peptide is about 90% to 99% similar to the amino acid sequence of SEQ ID No: 1. 15. The pharmaceutical composition for hair growth according to claim 13, wherein the carboxy-terminal of the amino acid sequence SEQ ID No: 1 deletes 1 or 7 any amino acids. 16. The pharmaceutical composition for hair growth according to claim 13, wherein the carboxy-terminal of said amino acid sequence SEQ ID No: 1 replaces 1 or 7 any amino acids. 17. The pharmaceutical composition for hair growth according to claim 13, wherein 1 to 3 any amino acids are added to the carboxy-terminal of the amino acid sequence SEQ ID No: 1. 18. The pharmaceutical composition for hair growth according to claim 13, wherein the amino terminal of said amino acid sequence SEQ ID No: 1 deletes any 6 or 7 amino acids. 19. The pharmaceutical composition for hair growth according to claim 13, wherein the amino terminal of said amino acid sequence SEQ ID No: 1 replaces 6 or 7 of any amino acids. 20. The pharmaceutical composition for hair growth according to claim 15, wherein the hair growth peptide (HGP) is selected from SEQ ID No:2 and SEQ ID No:3. 21. The pharmaceutical composition for hair growth according to claim 17, wherein the hair growth peptide (HGP) is SEQ ID No:4. 22. The pharmaceutical composition for hair growth according to claim 18, wherein the hair growth peptide (HGP) is selected from SEQ ID No:2 and SEQ ID No:5. 23. The pharmaceutical composition for hair growth according to claim 13, wherein the dosage of the hair growth peptide (HGP) is between 200 μM to 2000 μM. 24. The pharmaceutical composition for hair growth according to claim 13, wherein the hair growth peptide (HGP) acts on hair follicles.