WO2023210402A1 - Method for evaluating hair regeneration ability of dermal sheath cup (dsc) cells, composition for hair regeneration and manufacturing method thereof - Google Patents
Method for evaluating hair regeneration ability of dermal sheath cup (dsc) cells, composition for hair regeneration and manufacturing method thereof Download PDFInfo
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- WO2023210402A1 WO2023210402A1 PCT/JP2023/015123 JP2023015123W WO2023210402A1 WO 2023210402 A1 WO2023210402 A1 WO 2023210402A1 JP 2023015123 W JP2023015123 W JP 2023015123W WO 2023210402 A1 WO2023210402 A1 WO 2023210402A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
Definitions
- the present invention relates to a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells, a composition for regenerating hair, and a method for producing the same.
- DSC hair bulb root sheath
- hair is produced by hair follicles present in the skin.
- a hair follicle is a tissue layer that surrounds hair, and is composed of the ectoderm-derived hair matrix (hair matrix cells), inner root sheath, and outer root sheath, as well as the mesoderm-derived dermal root sheath, dermal papilla, etc. .
- Hair matrix cells surrounding the dermal papilla are induced to divide repeatedly by nutrients and proteins supplied from the dermal papilla, and form hair by keratinization.
- Thinning hair and alopecia occur due to problems with hair growth. Thinning hair and alopecia include alopecia in the middle years, alopecia areata, and telogen effluvium, with the most common being alopecia in the middle years. Middle age alopecia is mainly caused in men by the influence of male hormones, and is also called androgenetic alopecia. Hair is regrown while repeating a hair cycle consisting of a growth phase, a regression phase, and a resting phase. Male pattern baldness is a symptom caused by a shortening of the growth phase in this hair cycle and an increase in the proportion of thin and short hair.
- the topical drug minoxidil and the oral drug finasteride are mainly used to treat middle-aged hair loss. All of these treatments have been confirmed to be effective and safe, but they are not necessarily effective for all cases of middle age alopecia.
- autologous hair transplantation is being carried out as a treatment for middle age alopecia.
- Autologous hair transplantation is a surgical procedure in which hair including hair roots collected from the temporal region or the back of the head is transplanted to the hair loss area of one's own body.
- this surgical method involves transplanting the patient's own hair to another location, it does not increase the total number of hair.
- DSC dermal sheath cup cell
- the purpose of the present invention is to provide a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells, a composition for regenerating hair, and a method for producing the same.
- DSC hair bulb root sheath
- the present invention includes the following inventions.
- a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells using the level of mitochondrial respiration as an index [2] When the level of mitochondrial respiration in the DSC cell is lower than a predetermined threshold, the hair regeneration ability of the DSC cell is evaluated to be high, The method according to item 1, wherein the hair regeneration ability of the DSC cells is evaluated to be low when the level of mitochondrial respiration in the DSC cells is higher than a predetermined threshold. [3] The method according to item 1 or 2, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells. [4] The method according to item 2, wherein the level of mitochondrial respiration is a preliminary respiratory capacity. [5] The method according to any one of items 1 to 4, wherein the hair regeneration ability is the ability to increase hair diameter and/or hair density.
- OCR oxygen consumption rate
- a composition for regenerating hair comprising hair bulb root sheath (DSC) cells with a low level of mitochondrial respiration.
- DSC hair bulb root sheath
- OCR oxygen consumption rate
- the step of reducing the level of mitochondrial respiration includes performing hypoxic culture and/or adding one or more agents selected from the group consisting of mitochondrial complex inhibitors and pseudohypoxia inducers.
- the method according to item 10 or 11 wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
- OCR oxygen consumption rate
- the method according to any one of items 10 to 12, wherein the step of reducing the level of mitochondrial respiration is a step of reducing the preliminary respiratory capacity of the DSC cells to a predetermined threshold value or less.
- composition containing DSC cells for regenerating hair obtained by the method according to any one of claims 10 to 13.
- composition according to item 14 which increases hair diameter and/or hair density.
- the present inventors have discovered for the first time that there is an inverse correlation with the level of mitochondrial respiration, a factor in DSC cells that is linked to actual therapeutic outcomes.
- DSC cells By evaluating DSC cells using the level of mitochondrial respiration as an index, it becomes possible to predict the therapeutic effect of DSC cells even before they are used for treatment.
- the level of mitochondrial respiration As an index, it is also possible to improve the process (eg, culture conditions, etc.) for producing DSC cells with high therapeutic effects.
- by reducing the level of mitochondrial respiration of DSC cells it is also possible to produce a composition with high hair regeneration ability.
- the results of hair regeneration treatment are determined by the rate of change in total hair density and cumulative hair diameter (relative to 0 months [0M] or placebo ratio [P] (site where DSC cells were not transplanted)) after transplantation. It was calculated and used every month, 6 months, 9 months, and 12 months. The correlation coefficient between the activity of mitochondrial respiratory parameters and the results of hair regeneration treatment is calculated and shown. Comparison of evaluation items for DSC cells derived from each subject when divided into two groups, high activity or low activity, based on preliminary respiratory capacity. The subjects whose DSC cell reserve respiratory capacity value was >90 pmol/min were divided into the "high activity group"; the subjects whose DSC cell reserve respiratory capacity value was ⁇ 90 pmol/min were divided into the "low activity group”. .
- DSC cells were divided into groups (low efficacy group), and the activities of mitochondrial respiratory parameters (maximum respiration/respiratory reserve capacity) of DSC cells were calculated in each group and compared between the groups. Mitochondrial respiratory activity was shown to be significantly lower in DSC cells of the subject group with high therapeutic effects. **p ⁇ 0.01, *p ⁇ 0.05 (Student's t-test).
- the deepest swollen part of the hair inside the skin is called the hair bulb, and the part made of mesenchymal cells in the center of the hair bulb is called the dermal papilla.
- the dermal papilla is filled with capillaries and nerves that take in nutrients and oxygen from food and are responsible for hair development and growth.
- HF Heair Follicle
- hair matrix hair matrix cell
- inner root sheath and outer root sheath, which are epithelial cells
- dermal hair root which is mesenchymal cells.
- the tissues or cells used in the present invention may be derived from any animal, but are preferably derived from vertebrates, more preferably from mammals, and most preferably from humans.
- DS Dermathelial Sheath
- ⁇ -SMA actin positive cells
- the dermal hair root sheath is continuous with the dermal papilla at the lowest end of the hair bulb. As described below, in this specification, the dermal root sheath includes the hair bulb root sheath and the upper dermal hair root layer.
- DSC skin sheath cup
- hair bulb root sheath cells are cells that constitute the hair bulb root sheath.
- Hair bulb root sheath cells are known to be precursor cells of dermal papilla cells, and it is known that transplanting hair bulb root sheath cells into the skin induces hair follicles at the transplant site. ing.
- a hair follicle-derived cell group containing a high proportion of hair bulb root sheath cells and/or a high activity thereof is used. If a cell group or composition can be selected, it is possible to provide a hair follicle-derived cell group with higher hair follicle-inducing ability or a composition for regenerating hair follicles containing a hair follicle-derived cell group. becomes.
- UDS cells refer to cells of the dermal hair root sheath excluding the above-mentioned hair bulb root sheath portion.
- cell group derived from hair follicles refers to a cell group containing the cells that constitute the hair follicles described above.
- a composition for regenerating hair that contains hair bulb sheath (DSC) cells includes, for example, a biocompatible substance in addition to DSC cells.
- biocompatible substances include water, physiological saline, phosphate buffer, cell culture medium, biocompatible hydrogels (chitosan gel, collagen gel, gelatin, peptide gel, laminin gel, fibrin gel, etc.), etc. It may be possible, but is not limited to these.
- the composition of the present invention can also be a composition in which DSC cells are combined with one or more other components, such as excipients, carriers, and/or diluents.
- the composition and form of the composition are arbitrary and may be appropriately selected depending on the conditions such as the active ingredient and the intended use.
- the composition can be manufactured by a conventional method in a formulation in which the composition is appropriately combined with excipients, carriers and/or diluents, and other components depending on the dosage form.
- DSC cells with a low level of mitochondrial respiration have a high hair regeneration effect, and have completed the present invention.
- hair regeneration ability refers to the ability to regenerate hair, and includes, for example, hair diameter (e.g., integrated value of hair diameters in any region (integrated hair diameter)) and/or hair density (e.g., It can be evaluated by the number of hairs present in any area (total hair density).
- the level of mitochondrial respiration refers to the intracellular level of respiration in which cells use energy oxygen, that is, among the three processes of aerobic respiration (glycolysis, citric acid cycle, and electron transport chain). It refers to the activity level of the citric acid cycle and electron transport chain mediated by the organelle mitochondria.
- the level of mitochondrial respiration can be evaluated by measuring intracellular activity. For example, and without limitation, it may be evaluated by measuring the cellular oxygen consumption rate (OCR value), the extracellular acidification rate (ECAR value), or the OCR and ECAR values (e.g., Non-Patent Document 8). checking).
- OCR value cellular oxygen consumption rate
- ECAR value extracellular acidification rate
- Non-Patent Document 8 Non-Patent Document 8
- Intracellular activities can be measured, for example, by using the extracellular flux analyzer
- the state of cells can be measured non-invasively and with high sensitivity over time.
- OCR measurement methods using fluorescent or phosphorescent oxygen probes for example, Oxygen Consumption Rate Assay Kit (Cayman Chemical), Extracellular Oxygen Consumption Assay (Abcam)) use, etc.
- various metabolite analysis methods using enzymatic methods e.g.
- Intracellular activity can also be measured by using methods such as Glycolysis/OXPHOS Assay Kit (Dojindo Laboratories, etc.), or metabolome analysis by mass spectrometry (e.g., use of LC/TOF-MS, etc.).
- the above devices, kits, or methods are merely examples, and any device, kit, or method can be used to evaluate intracellular activity. It is not limited to the use of
- Parameters to indicate mitochondrial respiration include, for example, basal respiration and maximum respiration calculated from the cellular oxygen consumption rate (OCR value) measured when a predetermined treatment is performed using the extracellular flux analyzer XFe24. , respiratory reserve capacity, or ATP production can be used (see Table 1, Figure 1).
- glycolysis is a biochemical reaction pathway that exists within living organisms (cells), which decomposes (catabolizes) glucose into organic acids such as pyruvic acid, and uses the high binding energy contained in glucose.
- a metabolic process that converts substances into forms that are easy for living organisms to use. While glycolysis is a typical metabolic system in anaerobic respiration, it also functions as a part of aerobic respiration by transferring the resulting reducing power and pyruvate to the electron transport system and citric acid cycle. do.
- the activity level of glycolysis may be evaluated, for example, by measuring the extracellular acidification rate (ECAR value) using the above-mentioned extracellular flux analyzer XFe24.
- the present invention provides a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells using the level of mitochondrial respiration as an index. According to the present invention, by measuring the level of mitochondrial respiration in DSC cells or a cell group containing DSC cells, it is possible to predict hair regeneration ability in advance even before administration to a subject.
- DSC hair bulb root sheath
- the method of evaluating the hair regeneration ability of a hair bulb root sheath (DSC) cell of the present invention comprises: when the level of mitochondrial respiration in the DSC cell is lower than a predetermined threshold, the hair regeneration ability of the DSC cell is determined. When the level of mitochondrial respiration in the DSC cell is higher than a predetermined threshold value, the hair regeneration ability of the DSC cell may be evaluated as low.
- the predetermined threshold value serving as an evaluation index is not a specific value that is uniquely determined, but for example, the DSC transplanted to each subject in a subject group with increased hair diameter and/or hair density. determined by comparing the level of mitochondrial respiration of the cell with the level of mitochondrial respiration of DSC cells transplanted into each subject in a group of subjects in which hair diameter and/or hair density did not increase; Good too.
- the level of mitochondrial respiration may be a reserve respiratory capacity.
- the predetermined threshold values are, for example, 60 pmol/min, 65 pmol/min, 70 pmol/min, 75 pmol/min, 80 pmol/min, 85 pmol/min, 90 pmol/min, 95 pmol/min, 100 pmol/min, 105 pmol/min, Or it may be 110 pmol/min.
- the present inventors have found that subjects transplanted with DSC cells whose preliminary respiratory capacity used for transplantation is below a predetermined threshold have high hair regeneration ability.
- the hair regeneration capacity of DSC cells is evaluated to be low, and when the preliminary respiratory capacity of DSC cells is lower than a predetermined threshold value, the hair regeneration capacity of DSC cells It may be evaluated that the regeneration ability is high.
- the hair regeneration ability of each DSC cell may be evaluated, or the hair regeneration ability of the entire cell group including a plurality of DSC cells may be evaluated.
- the hair regeneration ability of the entire cell group containing multiple DSC cells is determined by the DSC cells included in the cell group that have a low level of mitochondrial respiration (for example, DSC cells whose level of mitochondrial respiration is below a predetermined threshold). may be evaluated by measuring the proportion of
- the present invention provides a composition for regenerating hair that contains hair bulb root sheath (DSC) cells with reduced levels of mitochondrial respiration.
- DSC hair bulb root sheath
- the present invention provides a composition for regenerating hair that contains hair bulb root sheath (DSC) cells with reduced levels of mitochondrial respiration.
- DSC cells with a low level of mitochondrial respiration have a high ability to regenerate hair, so hair regeneration can be expected by transplanting the composition of the present invention to a site where hair regeneration is desired.
- the DSC cells with low levels of mitochondrial respiration applied to the present invention are DSC cells with mitochondrial respiration lower than a predetermined threshold.
- the predetermined threshold value is not uniquely determined, for example, the mitochondrial respiration of DSC cells transplanted to each subject in a subject group with increased hair diameter and/or hair density may be considered as the predetermined threshold value. and the level of mitochondrial respiration of DSC cells transplanted into each subject in a group of subjects in which hair diameter and/or hair density did not increase.
- the level of mitochondrial respiration of DSC cells included in the compositions of the invention is lower than the average level of mitochondrial respiration of DSC cells in a group of subjects in which hair diameter and/or hair density did not increase (e.g., 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0. 1 times or less, or 0.05 times or less), and in the cell group included in the composition of the present invention, such DSC cells may be, for example, 10% or more (for example, 10%, 20%, 30% or less). %, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more).
- the invention provides a method for producing a composition for regenerating hair comprising hair bulb root sheath (DSC) cells, the method comprising: reducing the level of mitochondrial respiration of the DSC cells. , provides a method.
- DSC hair bulb root sheath
- the method for reducing the level of mitochondrial respiration may be according to a known method, for example, a step of performing hypoxic culture, and/or one or more selected from the group consisting of mitochondrial complex inhibitors and pseudohypoxia inducers. It is possible to reduce the level of mitochondrial respiration by carrying out the step of adding an agent that is The hypoxic culture may be carried out, for example, by referring to the method of Papandreou et al. Culture may be performed for any length of time (eg, at least 0.5 hours, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours) in an atmosphere containing 1% or less oxygen.
- mitochondrial complex inhibitors examples include mitochondrial complex I inhibitors (e.g., rotenone (D J Horgan et al., J Biol Chem, 1968 Feb 25;243(4):834-43.), Complex III inhibitors such as antimycin A (E. C. SLATER, Biochim Biophys Acta., 1973 Dec 7;301(2):129-54.) may also be used.
- pseudohypoxia inducing agents include cobalt (II) chloride (CoCl 2 ), deferoxamine, and dimethyloxaloylglycine (P. Jaakkola et al., Science. 2001 Apr 20;292(5516): 468-72.), L-mimosine (C.
- DSC cells that can be used in the present invention can be obtained using known methods (for example, Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R , Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.). A composition with high hair regeneration ability can be produced by reducing the level of mitochondrial respiration using the cell group containing the DSC cells obtained in this way.
- the method of the invention determines the respiratory reserve capacity of DSC cells at a predetermined threshold (e.g., 60 pmol/min, 65 pmol/min, 70 pmol/min, 75 pmol/min, 80 pmol/min, 85 pmol/min, 90 pmol/min). /min, 95 pmol/min, 100 pmol/min, 105 pmol/min, or 110 pmol/min).
- a predetermined threshold e.g. 60 pmol/min, 65 pmol/min, 70 pmol/min, 75 pmol/min, 80 pmol/min, 85 pmol/min, 90 pmol/min.
- a predetermined threshold e.g. 60 pmol/min, 65 pmol/min, 70 pmol/min, 75 pmol/min, 80 pmol/min, 85 pmol/min, 90 pmol/min.
- the present inventors have found that subjects transplanted with DSC cells whose preliminary respiratory capacity
- reducing the level of mitochondrial respiration of a DSC cell may be adjusted such that the respiratory reserve capacity of the DSC cell is below a predetermined threshold.
- the DSC cells with a reduced level of mitochondrial respiration contained in the composition produced by the production method of the present invention are the DSC cells in a subject group in which hair diameter and/or hair density did not increase.
- DSC cells may have a , 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more).
- Method 1-1 Preparation of DSC cells Tsuboi et al. (Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, DSC cells were prepared according to the method described in Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116. ( Figure 1).
- FCCP Seahorse Nido-p-trifluoromethoxyphenylhydrazone
- rotenone mitochondrial complex I inhibitor, final concentration 0.5 ⁇ M
- antimycin A mitochondrial complex III inhibitor, final concentration 0.5 ⁇ M
- the correlation coefficient between the total hair density or cumulative hair diameter score of each subject and the mitochondrial respiration parameter values (maximum respiration and preliminary respiratory capacity) of DSC cells was calculated.
- the absolute value of the correlation coefficient was greater than 0.3, it was determined that there was a weak correlation or a weak inverse correlation, and when the absolute value of the correlation coefficient was greater than 0.5, it was determined that there was a correlation or an inverse correlation.
- DSC cells were divided into two groups, and mitochondrial respiratory parameter (maximum respiration/respiratory capacity) activity of DSC cells was examined in each group, and a significant difference test was performed to determine whether a difference was observed between the groups.
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Abstract
The present invention provides a method for evaluating the hair regeneration ability of dermal sheath cup (DSC) cells using a mitochondrial respiration level as an indicator. The present invention also provides a composition for hair regeneration that contains DSC cells with a low mitochondrial respiration level, and a method for manufacturing the same.
Description
本発明は、毛球部毛根鞘（ＤＳＣ）細胞の毛髪再生能を評価する方法、毛髪を再生するための組成物及びその製造方法に関する。
The present invention relates to a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells, a composition for regenerating hair, and a method for producing the same.
毛髪は皮膚に存在する毛包によって産生される。毛包とは、毛を取り囲む組織層であり、外胚葉由来の毛母（毛母細胞）、内毛根鞘、外毛根鞘や、中胚葉由来の真皮毛根鞘、毛乳頭などから構成されている。毛乳頭を取り囲む毛母細胞が、毛乳頭から供給される栄養やタンパク質によって誘導されて分裂を繰り返し、角質化することによって毛を形成する。
Hair is produced by hair follicles present in the skin. A hair follicle is a tissue layer that surrounds hair, and is composed of the ectoderm-derived hair matrix (hair matrix cells), inner root sheath, and outer root sheath, as well as the mesoderm-derived dermal root sheath, dermal papilla, etc. . Hair matrix cells surrounding the dermal papilla are induced to divide repeatedly by nutrients and proteins supplied from the dermal papilla, and form hair by keratinization.
毛髪の発育に問題が生じることで薄毛・脱毛症が生じる。薄毛・脱毛症には、壮年性脱毛症、円形脱毛症、休止期脱毛症などがあり、最も頻度が多いのが壮年性脱毛症である。壮年性脱毛症は、男性において、主に男性ホルモンの影響によって引き起こされるものであり、男性型脱毛症とも呼ばれる。毛は、成長期、退行期及び休止期からなるヘアサイクルを繰り返しながら生え替わる。男性型脱毛症は、このヘアサイクルの中でも成長期が短くなり、細く短い毛髪の割合が増えることで引き起こされる症状である。
Thinning hair and alopecia occur due to problems with hair growth. Thinning hair and alopecia include alopecia in the middle years, alopecia areata, and telogen effluvium, with the most common being alopecia in the middle years. Middle age alopecia is mainly caused in men by the influence of male hormones, and is also called androgenetic alopecia. Hair is regrown while repeating a hair cycle consisting of a growth phase, a regression phase, and a resting phase. Male pattern baldness is a symptom caused by a shortening of the growth phase in this hair cycle and an increase in the proportion of thin and short hair.
現在、壮年性脱毛症の治療として、外用剤のミノキシジルや、経口薬のフィナステリドが主に用いられている。これらの治療法は、いずれも有効性や安全性が確認されているが、必ずしも全ての壮年性脱毛症に有効というわけではない。
Currently, the topical drug minoxidil and the oral drug finasteride are mainly used to treat middle-aged hair loss. All of these treatments have been confirmed to be effective and safe, but they are not necessarily effective for all cases of middle age alopecia.
その他、壮年性脱毛症の治療として、自家植毛術が実施されている。自家植毛術とは、側頭部や後頭部から採取した毛根を含む毛髪を、自己の脱毛部に移植する術式である。しかしながら、当該術式は、自己の毛髪を別の場所へ植え替える術式であるため、毛髪の総数を増やすものではない。
In addition, autologous hair transplantation is being carried out as a treatment for middle age alopecia. Autologous hair transplantation is a surgical procedure in which hair including hair roots collected from the temporal region or the back of the head is transplanted to the hair loss area of one's own body. However, since this surgical method involves transplanting the patient's own hair to another location, it does not increase the total number of hair.
近年、様々な疾患に対する再生医療技術が開発されており、毛髪再生分野においても新たな技術が研究開発されている。例えば、毛包の中でも、毛包の再外層に位置する真皮毛根鞘、特に毛球部の底部に位置する毛球部毛根鞘（ｄｅｒｍａｌ ｓｈｅａｔｈ ｃｕｐ ｃｅｌｌ：ＤＳＣ）細胞には高い毛包誘導能力を有することが示唆されており、マウスを用いた試験でＤＳＣ細胞の移植により発毛が認められること、ヒトにおいても脱毛症の治療に有効であることが報告されている（非特許文献１、２）。また、ＤＳＣ細胞が、発毛において重要な役割を有する毛乳頭（Ｄｅｒｍａｌ ｐａｐｉｌｌａ：ＤＰ）細胞の前駆細胞であることも示されており、ＤＳＣ細胞に注目が集まっている（非特許文献３）。
In recent years, regenerative medical technology for various diseases has been developed, and new technologies are also being researched and developed in the field of hair regeneration. For example, within the hair follicle, the dermal root sheath located in the outer layer of the hair follicle, especially the dermal sheath cup cell (DSC) cells located at the bottom of the hair bulb, have a high ability to induce hair follicles. It has been reported that transplantation of DSC cells results in hair growth in mice, and that it is effective in treating alopecia in humans (Non-patent Documents 1, 2). ). It has also been shown that DSC cells are precursor cells of dermal papilla (DP) cells that play an important role in hair growth, and DSC cells are attracting attention (Non-Patent Document 3).
毛髪再生の分野において毛包由来の細胞群を用いた治療法が開発されつつあるが、ＤＳＣ細胞を移植したとしても、必ずしも毛髪を再生する効果を発揮するとは限らない。従って、ＤＳＣ細胞の毛髪再生効果の有効性に関する指標が同定されれば、それを用いた治療の担保（品質保証）及び、細胞の改良、組成物の改良などへの活用により治療改善が期待される。
In the field of hair regeneration, treatments using cell groups derived from hair follicles are being developed, but even if DSC cells are transplanted, they do not necessarily have the effect of regenerating hair. Therefore, if an index regarding the effectiveness of the hair regeneration effect of DSC cells is identified, it is expected that treatment will be improved by using it to guarantee treatment (quality assurance), improve cells, improve compositions, etc. Ru.
これまでに、ＤＳＣ細胞の毛髪再生能に影響を与える因子として、Ｗｎｔシグナルや、ＰＤＧＦＲシグナル等が報告されているが、その他の因子についてはほとんど報告されていない（非特許文献４～６）。
To date, Wnt signals, PDGFR signals, and the like have been reported as factors that affect the hair regeneration ability of DSC cells, but almost no other factors have been reported (Non-Patent Documents 4 to 6).
これまでにｉｎｔａｃｔな毛包中の毛球部毛根鞘に特異的に発現する遺伝子を探索したところ、ＧＲＥＭ２遺伝子がＤＳＣ細胞のマーカーとなり得ることが報告されている（非特許文献７、特許文献１）。
As a result of searching for genes that are specifically expressed in the hair bulb root sheath in intact hair follicles, it has been reported that the GREM2 gene can be a marker for DSC cells (Non-patent Document 7, Patent Document 1). ).
本発明は、毛球部毛根鞘（ＤＳＣ）細胞の毛髪再生能を評価する方法、毛髪を再生するための組成物及びその製造方法を提供することを目的とする。
The purpose of the present invention is to provide a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells, a composition for regenerating hair, and a method for producing the same.
これまで、ＤＳＣ細胞を同定するためのマーカー候補について探索されてきたが、実際の治療成績とリンクするようなバイオマーカーやパラメーターについては、ほとんど知られていない。
To date, marker candidates for identifying DSC cells have been searched for, but little is known about biomarkers and parameters that are linked to actual treatment outcomes.
本発明者らは、既に実施済みの臨床研究を詳細に解析することにより、毛髪再生の治療成績能と相関性があるＤＳＣ細胞における因子を見出し、本発明を完成するに至った。すなわち、本発明は、以下の発明を包含する。
Through detailed analysis of clinical studies that have already been conducted, the present inventors have discovered factors in DSC cells that are correlated with hair regeneration treatment performance, and have completed the present invention. That is, the present invention includes the following inventions.
［１］ ミトコンドリア呼吸のレベルを指標とする、毛球部毛根鞘（ＤＳＣ）細胞の毛髪再生能を評価する方法。
［２］ 前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より低い場合に、前記ＤＳＣ細胞の毛髪再生能が高いと評価し、
前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より高い場合に、前記ＤＳＣ細胞の毛髪再生能が低いと評価する、項目１に記載の方法。
［３］ 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、項目１又は２に記載の方法。
［４］ 前記ミトコンドリア呼吸のレベルが予備呼吸能である、項目２に記載の方法。
［５］ 前記毛髪再生能が、毛髪径及び／又は毛髪密度を増加させる能力である、項目１～４のいずれか１項に記載の方法。 [1] A method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells using the level of mitochondrial respiration as an index.
[2] When the level of mitochondrial respiration in the DSC cell is lower than a predetermined threshold, the hair regeneration ability of the DSC cell is evaluated to be high,
The method according to item 1, wherein the hair regeneration ability of the DSC cells is evaluated to be low when the level of mitochondrial respiration in the DSC cells is higher than a predetermined threshold.
[3] The method according to item 1 or 2, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
[4] The method according to item 2, wherein the level of mitochondrial respiration is a preliminary respiratory capacity.
[5] The method according to any one of items 1 to 4, wherein the hair regeneration ability is the ability to increase hair diameter and/or hair density.
［２］ 前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より低い場合に、前記ＤＳＣ細胞の毛髪再生能が高いと評価し、
前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より高い場合に、前記ＤＳＣ細胞の毛髪再生能が低いと評価する、項目１に記載の方法。
［３］ 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、項目１又は２に記載の方法。
［４］ 前記ミトコンドリア呼吸のレベルが予備呼吸能である、項目２に記載の方法。
［５］ 前記毛髪再生能が、毛髪径及び／又は毛髪密度を増加させる能力である、項目１～４のいずれか１項に記載の方法。 [1] A method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells using the level of mitochondrial respiration as an index.
[2] When the level of mitochondrial respiration in the DSC cell is lower than a predetermined threshold, the hair regeneration ability of the DSC cell is evaluated to be high,
The method according to item 1, wherein the hair regeneration ability of the DSC cells is evaluated to be low when the level of mitochondrial respiration in the DSC cells is higher than a predetermined threshold.
[3] The method according to item 1 or 2, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
[4] The method according to item 2, wherein the level of mitochondrial respiration is a preliminary respiratory capacity.
[5] The method according to any one of items 1 to 4, wherein the hair regeneration ability is the ability to increase hair diameter and/or hair density.
［６］ ミトコンドリア呼吸のレベルが低い毛球部毛根鞘（ＤＳＣ）細胞を含有する、毛髪を再生するための組成物。
［７］ 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、項目６に記載の組成物。
［８］ 前記ミトコンドリア呼吸のレベルが予備呼吸能であり、前記予備呼吸能が所定の閾値以下のＤＳＣ細胞を含有する、項目６に記載の組成物。
［９］ 毛髪径及び／又は毛髪密度が増加することを特徴とする、項目６～８のいずれか１項に記載の組成物。 [6] A composition for regenerating hair, comprising hair bulb root sheath (DSC) cells with a low level of mitochondrial respiration.
[7] The composition according to item 6, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
[8] The composition according to item 6, which contains DSC cells in which the level of mitochondrial respiration is a reserve respiratory capacity, and the reserve respiratory capacity is below a predetermined threshold.
[9] The composition according to any one of items 6 to 8, which increases hair diameter and/or hair density.
［７］ 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、項目６に記載の組成物。
［８］ 前記ミトコンドリア呼吸のレベルが予備呼吸能であり、前記予備呼吸能が所定の閾値以下のＤＳＣ細胞を含有する、項目６に記載の組成物。
［９］ 毛髪径及び／又は毛髪密度が増加することを特徴とする、項目６～８のいずれか１項に記載の組成物。 [6] A composition for regenerating hair, comprising hair bulb root sheath (DSC) cells with a low level of mitochondrial respiration.
[7] The composition according to item 6, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
[8] The composition according to item 6, which contains DSC cells in which the level of mitochondrial respiration is a reserve respiratory capacity, and the reserve respiratory capacity is below a predetermined threshold.
[9] The composition according to any one of items 6 to 8, which increases hair diameter and/or hair density.
［１０］ 毛球部毛根鞘（ＤＳＣ）細胞を含む、毛髪を再生するための組成物の製造方法であって、
ＤＳＣ細胞のミトコンドリア呼吸のレベルを低下させる工程、
を含む、方法。
［１１］ 前記ミトコンドリア呼吸のレベルを低下させる工程が、低酸素培養を行う工程、並びに／又は、ミトコンドリア複合体阻害剤及び疑似低酸素誘導剤からなる群から１又は複数選択される剤を添加する工程である、項目１０に記載の方法。
［１２］ 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、項目１０又は１１に記載の方法。
［１３］ 前記ミトコンドリア呼吸のレベルを低下させる工程が、前記ＤＳＣ細胞の予備呼吸能を所定の閾値以下に低下させる工程である、項目１０～１２のいずれか１項に記載の方法。 [10] A method for producing a composition for regenerating hair, comprising hair bulb root sheath (DSC) cells, comprising:
reducing the level of mitochondrial respiration in DSC cells;
including methods.
[11] The step of reducing the level of mitochondrial respiration includes performing hypoxic culture and/or adding one or more agents selected from the group consisting of mitochondrial complex inhibitors and pseudohypoxia inducers. The method according to item 10, which is a step.
[12] The method according to item 10 or 11, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
[13] The method according to any one of items 10 to 12, wherein the step of reducing the level of mitochondrial respiration is a step of reducing the preliminary respiratory capacity of the DSC cells to a predetermined threshold value or less.
ＤＳＣ細胞のミトコンドリア呼吸のレベルを低下させる工程、
を含む、方法。
［１１］ 前記ミトコンドリア呼吸のレベルを低下させる工程が、低酸素培養を行う工程、並びに／又は、ミトコンドリア複合体阻害剤及び疑似低酸素誘導剤からなる群から１又は複数選択される剤を添加する工程である、項目１０に記載の方法。
［１２］ 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、項目１０又は１１に記載の方法。
［１３］ 前記ミトコンドリア呼吸のレベルを低下させる工程が、前記ＤＳＣ細胞の予備呼吸能を所定の閾値以下に低下させる工程である、項目１０～１２のいずれか１項に記載の方法。 [10] A method for producing a composition for regenerating hair, comprising hair bulb root sheath (DSC) cells, comprising:
reducing the level of mitochondrial respiration in DSC cells;
including methods.
[11] The step of reducing the level of mitochondrial respiration includes performing hypoxic culture and/or adding one or more agents selected from the group consisting of mitochondrial complex inhibitors and pseudohypoxia inducers. The method according to item 10, which is a step.
[12] The method according to item 10 or 11, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
[13] The method according to any one of items 10 to 12, wherein the step of reducing the level of mitochondrial respiration is a step of reducing the preliminary respiratory capacity of the DSC cells to a predetermined threshold value or less.
［１４］ 請求項１０～１３のいずれか１項に記載の方法により得られる、毛髪を再生するためのＤＳＣ細胞を含む組成物。
［１５］ 毛髪径及び／又は毛髪密度が増加することを特徴とする、項目１４に記載の組成物。 [14] A composition containing DSC cells for regenerating hair, obtained by the method according to any one of claims 10 to 13.
[15] The composition according to item 14, which increases hair diameter and/or hair density.
［１５］ 毛髪径及び／又は毛髪密度が増加することを特徴とする、項目１４に記載の組成物。 [14] A composition containing DSC cells for regenerating hair, obtained by the method according to any one of claims 10 to 13.
[15] The composition according to item 14, which increases hair diameter and/or hair density.
本発明者らは、実際の治療成績とリンクしたＤＳＣ細胞の因子、すなわちミトコンドリア呼吸のレベルと逆相関することを初めて見出した。ミトコンドリア呼吸のレベルを指標とし、ＤＳＣ細胞を評価することにより、治療に用いられる前であってもＤＳＣ細胞の治療効果を予測可能となる。また、ミトコンドリア呼吸のレベルを指標とすることにより、治療効果の高いＤＳＣ細胞を製造するプロセス（例えば、培養条件など）を改善することも可能となる。また、ＤＳＣ細胞のミトコンドリア呼吸のレベルを低下させることにより、毛髪再生能が高い組成物を製造することも可能となる。
The present inventors have discovered for the first time that there is an inverse correlation with the level of mitochondrial respiration, a factor in DSC cells that is linked to actual therapeutic outcomes. By evaluating DSC cells using the level of mitochondrial respiration as an index, it becomes possible to predict the therapeutic effect of DSC cells even before they are used for treatment. Moreover, by using the level of mitochondrial respiration as an index, it is also possible to improve the process (eg, culture conditions, etc.) for producing DSC cells with high therapeutic effects. Furthermore, by reducing the level of mitochondrial respiration of DSC cells, it is also possible to produce a composition with high hair regeneration ability.
以下、本発明を実施するための形態について説明するが、本発明の技術的範囲は下記の形態のみに限定されない。なお、本明細書において引用されている先行技術文献は、本明細書においても援用され、その全体が本明細書において取り込まれる。
Hereinafter, embodiments for carrying out the present invention will be described, but the technical scope of the present invention is not limited only to the following embodiments. Note that the prior art documents cited in this specification are also cited in this specification, and are incorporated herein in their entirety.
毛の皮膚内部最深部の膨らんだ部分を毛球部（ｈａｉｒ ｂｕｌｂ）といい、毛球部の中央部にある間葉系細胞からなる部分を毛乳頭（ｄｅｒｍａｌ ｐａｐｉｌｌａ）という。毛乳頭には毛細血管や神経が入り込んでいて、食物からの栄養や酸素を取り入れ、毛の発生や成長をつかさどっている。毛乳頭に接したところに毛母細胞（ｈａｉｒ ｍａｔｒｉｘ ｃｅｌｌ）があり、毛はこの部位で産生される。すなわち毛母細胞は、毛乳頭に入り込んでいる毛細血管から栄養や酸素を取り込み、分裂を繰り返すことにより毛が形成される。
The deepest swollen part of the hair inside the skin is called the hair bulb, and the part made of mesenchymal cells in the center of the hair bulb is called the dermal papilla. The dermal papilla is filled with capillaries and nerves that take in nutrients and oxygen from food and are responsible for hair development and growth. There are hair matrix cells in contact with the dermal papilla, and hair is produced at this site. That is, hair matrix cells take in nutrients and oxygen from the capillaries that enter the dermal papilla, and hair is formed by repeating division.
本明細書において、「毛包（Ｈａｉｒ Ｆｏｌｌｉｃｌｅ：ＨＦ）」とは、上皮系細胞であるの毛母（毛母細胞）、内毛根鞘、外毛根鞘や、間葉系細胞であるの真皮毛根鞘、毛乳頭、及びメラノサイトなどを含む、毛を取り囲む組織層をいう。本発明において使用される組織又は細胞は、いずれの動物由来であってもよいが、脊椎動物由来が好ましく、哺乳動物由来がより好ましく、ヒト由来であることが最も好ましい。
As used herein, "Hair Follicle (HF)" refers to the hair matrix (hair matrix cell), inner root sheath, and outer root sheath, which are epithelial cells, and the dermal hair root, which is mesenchymal cells. The tissue layer that surrounds hair, including the sheath, dermal papilla, and melanocytes. The tissues or cells used in the present invention may be derived from any animal, but are preferably derived from vertebrates, more preferably from mammals, and most preferably from humans.
本明細書において、「真皮毛根鞘（Ｄｅｒｍａｌ Ｓｈｅａｔｈ：ＤＳ）」とは、毛包の最外層を包む一層又は数層の真皮性細胞層（ビメンチン陽性）よりなる組織であって、平滑筋型αアクチン（α－ＳＭＡ）陽性細胞を含む。真皮毛根鞘は、毛球部の最下端において毛乳頭と連続している。後述するように、本明細書において、真皮毛根鞘には、毛球部毛根鞘と上部真皮毛根層が含まれる。
As used herein, "Dermal Sheath (DS)" is a tissue consisting of one or several dermal cell layers (vimentin positive) surrounding the outermost layer of the hair follicle, and is composed of smooth muscle type α Contains actin (α-SMA) positive cells. The dermal hair root sheath is continuous with the dermal papilla at the lowest end of the hair bulb. As described below, in this specification, the dermal root sheath includes the hair bulb root sheath and the upper dermal hair root layer.
本明細書において、「毛球部毛根鞘（ｄｅｒｍａｌ ｓｈｅａｔｈ ｃｕｐ：ＤＳＣ）」とは、真皮毛根鞘の一部であり、毛球部の底部に位置する組織をいう（例えば、国際公開第２０１９／１７６８８１号の図１を参照）。「毛球部毛根鞘（ｄｅｒｍａｌ ｓｈｅａｔｈ ｃｕｐ：ＤＳＣ）細胞」（以下、「ＤＳＣ細胞」という。）とは、毛球部毛根鞘を構成する細胞である。毛球部毛根鞘細胞は、毛乳頭細胞の前駆細胞であることが知られており、毛球部毛根鞘細胞を皮膚に移植することにより、移植部位において毛包が誘導されることが知られている。すなわち、毛包由来の細胞群又は毛包由来の細胞群を含有する毛包を再生するための組成物において、毛球部毛根鞘細胞の割合が高い及び／又はその活性が高い毛包由来の細胞群又は組成物を選択することができれば、より毛包誘導能が高い毛包由来の細胞群又は毛包由来の細胞群を含有する毛包を再生するための組成物を提供することが可能となる。
As used herein, "dermal sheath cup (DSC)" refers to a tissue that is part of the dermal hair root sheath and is located at the bottom of the hair bulb (for example, International Publication No. 2019/ 176881). "Dermal sheath cup (DSC) cells" (hereinafter referred to as "DSC cells") are cells that constitute the hair bulb root sheath. Hair bulb root sheath cells are known to be precursor cells of dermal papilla cells, and it is known that transplanting hair bulb root sheath cells into the skin induces hair follicles at the transplant site. ing. That is, in a composition for regenerating a hair follicle-derived cell group or a hair follicle containing a hair follicle-derived cell group, a hair follicle-derived cell group containing a high proportion of hair bulb root sheath cells and/or a high activity thereof is used. If a cell group or composition can be selected, it is possible to provide a hair follicle-derived cell group with higher hair follicle-inducing ability or a composition for regenerating hair follicles containing a hair follicle-derived cell group. becomes.
本明細書において、「上部毛根鞘（Ｕｐｐｅｒ Ｄｅｒｍａｌ Ｓｈｅａｔｈ：ＵＤＳ）細胞」とは、真皮毛根鞘のうち、上記の毛球部毛根鞘の部分を除いた組織の細胞をいう。
As used herein, "Upper Dermal Sheath (UDS) cells" refer to cells of the dermal hair root sheath excluding the above-mentioned hair bulb root sheath portion.
本明細書において、「毛包由来の細胞群」とは、上記の毛包を構成する細胞を含む細胞群をいう。
As used herein, the term "cell group derived from hair follicles" refers to a cell group containing the cells that constitute the hair follicles described above.
本明細書において、「毛球部毛根鞘（ＤＳＣ）細胞を含有する、毛髪を再生するための組成物」とは、ＤＳＣ細胞の他、例えば、生体適合可能な物質を含むものである。生体適合可能な物質とは、例えば、水、生理食塩水、リン酸緩衝液、細胞培養培地、生体適合性ハイドロゲル（キトサンゲル、コラーゲンゲル、ゼラチン、ペプチドゲル、ラミニンゲル及びフィブリンゲルなど）等であってもよく、これらに限定されない。本発明の組成物は、ＤＳＣ細胞の他、１種又は２種以上の他の成分、例えば賦形剤、担体及び／又は希釈剤等と組み合わせた組成物とすることもできる。組成物の組成や形態は任意であり、有効成分や用途等の条件に応じて適切に選択すればよい。当該組成物は、その剤形に応じ、賦形剤、担体及び／又は希釈剤等及び他の成分と適宜組み合わせた処方で、常法を用いて製造することができる。
As used herein, "a composition for regenerating hair that contains hair bulb sheath (DSC) cells" includes, for example, a biocompatible substance in addition to DSC cells. Examples of biocompatible substances include water, physiological saline, phosphate buffer, cell culture medium, biocompatible hydrogels (chitosan gel, collagen gel, gelatin, peptide gel, laminin gel, fibrin gel, etc.), etc. It may be possible, but is not limited to these. The composition of the present invention can also be a composition in which DSC cells are combined with one or more other components, such as excipients, carriers, and/or diluents. The composition and form of the composition are arbitrary and may be appropriately selected depending on the conditions such as the active ingredient and the intended use. The composition can be manufactured by a conventional method in a formulation in which the composition is appropriately combined with excipients, carriers and/or diluents, and other components depending on the dosage form.
本発明者らは、鋭意研究を行った結果、ミトコンドリア呼吸のレベルが低いＤＳＣ細胞は毛髪再生効果が高いことを見出し、本発明を完成させるに至った。
As a result of intensive research, the present inventors discovered that DSC cells with a low level of mitochondrial respiration have a high hair regeneration effect, and have completed the present invention.
本明細書において、「毛髪再生能」は、毛髪を再生させる能力をいい、例えば、毛髪径（例えば、任意の領域における毛髪径の積算値（積算毛髪径））及び／又は毛髪密度（例えば、任意の領域に存在する毛髪の数（総毛髪密度））によって評価され得る。
As used herein, "hair regeneration ability" refers to the ability to regenerate hair, and includes, for example, hair diameter (e.g., integrated value of hair diameters in any region (integrated hair diameter)) and/or hair density (e.g., It can be evaluated by the number of hairs present in any area (total hair density).
本明細書において、「ミトコンドリア呼吸のレベル」とは、細胞がエネルギー酸素を使う呼吸、すなわち好気呼吸の３つの過程（解糖系、クエン酸回路、および電子伝達系）のうち、細胞内小器官であるミトコンドリアが介在するクエン酸回路及び電子伝達系の活性レベルを指す。
In this specification, "the level of mitochondrial respiration" refers to the intracellular level of respiration in which cells use energy oxygen, that is, among the three processes of aerobic respiration (glycolysis, citric acid cycle, and electron transport chain). It refers to the activity level of the citric acid cycle and electron transport chain mediated by the organelle mitochondria.
ミトコンドリア呼吸のレベルは、細胞内活性を測定することで評価することができる。限定されるわけではないが、例えば、細胞の酸素消費速度（ＯＣＲ値）、細胞外酸性化速度（ＥＣＡＲ値）、又はＯＣＲ及びＥＣＡＲ値を測定することによって評価し得る（例えば、非特許文献８を参照のこと）。
The level of mitochondrial respiration can be evaluated by measuring intracellular activity. For example, and without limitation, it may be evaluated by measuring the cellular oxygen consumption rate (OCR value), the extracellular acidification rate (ECAR value), or the OCR and ECAR values (e.g., Non-Patent Document 8). checking).
細胞内活性は、例えば、Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ（旧Ｓｅａｈｏｒｓｅ Ｂｉｏｓｃｉｅｎｃｅ）社製の細胞外フラックスアナライザーＸＦｅ２４（２４ｗｅｌｌ用）を用いることによって細胞の主要なエネルギー代謝経路である解糖系、及びミトコンドリアによる好気呼吸の状態を、細胞に対して無侵襲・高感度に経時的計測を行うことができる。また、蛍光又はりん光性酸素プローブを用いたＯＣＲ測定方法（例えば、Ｏｘｙｇｅｎ Ｃｏｎｓｕｍｐｔｉｏｎ Ｒａｔｅ Ａｓｓａｙ Ｋｉｔ（ケイマン ケミカル）、Ｅｘｔｒａｃｅｌｌｕｌａｒ Ｏｘｙｇｅｎ Ｃｏｎｓｕｍｐｔｉｏｎ Ａｓｓａｙ（アブカム）の使用等）、酵素法による各種代謝物解析法（例えば、Ｇｌｙｃｏｌｙｓｉｓ／ＯＸＰＨＯＳ Ａｓｓａｙ Ｋｉｔ（同仁化学研究所）の使用等）、又は質量分析によるメタボローム解析法（例えば、ＬＣ／ＴＯＦ－ＭＳの使用等）などの方法を用いることによっても細胞内活性を測定することができる。しかしながら、上記装置、キット又は方法は例示であり、細胞内活性の測定には任意の装置、キット又は方法を用いて評価することができるため、細胞内活性の測定は、上記装置、キット又は方法の利用に限定されない。
Intracellular activities can be measured, for example, by using the extracellular flux analyzer The state of cells can be measured non-invasively and with high sensitivity over time. In addition, OCR measurement methods using fluorescent or phosphorescent oxygen probes (for example, Oxygen Consumption Rate Assay Kit (Cayman Chemical), Extracellular Oxygen Consumption Assay (Abcam)) use, etc.), various metabolite analysis methods using enzymatic methods (e.g. Intracellular activity can also be measured by using methods such as Glycolysis/OXPHOS Assay Kit (Dojindo Laboratories, etc.), or metabolome analysis by mass spectrometry (e.g., use of LC/TOF-MS, etc.). be able to. However, the above devices, kits, or methods are merely examples, and any device, kit, or method can be used to evaluate intracellular activity. It is not limited to the use of
ミトコンドリア呼吸を示すためのパラメーターとしては、例えば、細胞外フラックスアナライザーＸＦｅ２４を用いて、所定の処置を行った場合に測定される細胞の酸素消費速度（ＯＣＲ値）から算出される基礎呼吸、最大呼吸、予備呼吸能、又はＡＴＰ産生を使用することができる（表１、図１を参照）。
Parameters to indicate mitochondrial respiration include, for example, basal respiration and maximum respiration calculated from the cellular oxygen consumption rate (OCR value) measured when a predetermined treatment is performed using the extracellular flux analyzer XFe24. , respiratory reserve capacity, or ATP production can be used (see Table 1, Figure 1).
本明細書において「解糖系」とは、生体（細胞）内に存在する生化学反応経路であり、グルコースをピルビン酸などの有機酸に分解（異化）し、グルコースに含まれる高い結合エネルギーを生物が使いやすい形に変換していくための代謝過程をいう。解糖系は、嫌気呼吸における代謝系の代表的なものである一方で、得られる還元力やピルビン酸が電子伝達系やクエン酸回路に受け渡されることで好気呼吸の一部としても機能する。解糖系の活性レベルは、例えば、上述の細胞外フラックスアナライザーＸＦｅ２４を用いて、細胞外酸性化速度（ＥＣＡＲ値）を測定することによって評価してもよい。
In this specification, "glycolysis" is a biochemical reaction pathway that exists within living organisms (cells), which decomposes (catabolizes) glucose into organic acids such as pyruvic acid, and uses the high binding energy contained in glucose. A metabolic process that converts substances into forms that are easy for living organisms to use. While glycolysis is a typical metabolic system in anaerobic respiration, it also functions as a part of aerobic respiration by transferring the resulting reducing power and pyruvate to the electron transport system and citric acid cycle. do. The activity level of glycolysis may be evaluated, for example, by measuring the extracellular acidification rate (ECAR value) using the above-mentioned extracellular flux analyzer XFe24.
一実施態様において、本発明は、ミトコンドリア呼吸のレベルを指標とする、毛球部毛根鞘（ＤＳＣ）細胞の毛髪再生能を評価する方法を提供する。本発明により、ＤＳＣ細胞又はＤＳＣ細胞を含む細胞群において、ミトコンドリア呼吸のレベルを測定することによって、対象に投与する前であっても、毛髪再生能を予め予測することが可能となる。
In one embodiment, the present invention provides a method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells using the level of mitochondrial respiration as an index. According to the present invention, by measuring the level of mitochondrial respiration in DSC cells or a cell group containing DSC cells, it is possible to predict hair regeneration ability in advance even before administration to a subject.
一実施態様において、本発明の毛球部毛根鞘（ＤＳＣ）細胞の毛髪再生能を評価する方法は、前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より低い場合に、前記ＤＳＣ細胞の毛髪再生能が高いと評価し、前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より高い場合に、前記ＤＳＣ細胞の毛髪再生能が低いと評価するものであってもよい。
In one embodiment, the method of evaluating the hair regeneration ability of a hair bulb root sheath (DSC) cell of the present invention comprises: when the level of mitochondrial respiration in the DSC cell is lower than a predetermined threshold, the hair regeneration ability of the DSC cell is determined. When the level of mitochondrial respiration in the DSC cell is higher than a predetermined threshold value, the hair regeneration ability of the DSC cell may be evaluated as low.
評価の指標となる所定の閾値は、特定の値が一意的に決定されるものではないが、例えば、毛髪径及び／又は毛髪密度が増加した被検体群におけるそれぞれの被検体に移植されたＤＳＣ細胞のミトコンドリア呼吸のレベルと、毛髪径及び／又は毛髪密度が増加しなかった被検体群におけるそれぞれの被検体に移植されたＤＳＣ細胞のミトコンドリア呼吸のレベルとの比較によって決定されるものであってもよい。
The predetermined threshold value serving as an evaluation index is not a specific value that is uniquely determined, but for example, the DSC transplanted to each subject in a subject group with increased hair diameter and/or hair density. determined by comparing the level of mitochondrial respiration of the cell with the level of mitochondrial respiration of DSC cells transplanted into each subject in a group of subjects in which hair diameter and/or hair density did not increase; Good too.
また、一実施態様において、ミトコンドリア呼吸のレベルは予備呼吸能であってもよい。この場合、所定の閾値は、例えば、６０ｐｍｏｌ／分、６５ｐｍｏｌ／分、７０ｐｍｏｌ／分、７５ｐｍｏｌ／分、８０ｐｍｏｌ／分、８５ｐｍｏｌ／分、９０ｐｍｏｌ／分、９５ｐｍｏｌ／分、１００ｐｍｏｌ／分、１０５ｐｍｏｌ／分、又は１１０ｐｍｏｌ／分としてもよい。本発明者らは、移植に用いられるＤＳＣ細胞の予備呼吸能が所定の閾値以下のＤＳＣ細胞が移植された被検体では、毛髪再生能が高いことを見出した。従って、例えば、ＤＳＣ細胞の予備呼吸能が所定の閾値より高い場合は、ＤＳＣ細胞の毛髪再生能が低いと評価し、ＤＳＣ細胞の予備呼吸能が所定の閾値より低い場合は、ＤＳＣ細胞の毛髪再生能が高いと評価してもよい。
Additionally, in one embodiment, the level of mitochondrial respiration may be a reserve respiratory capacity. In this case, the predetermined threshold values are, for example, 60 pmol/min, 65 pmol/min, 70 pmol/min, 75 pmol/min, 80 pmol/min, 85 pmol/min, 90 pmol/min, 95 pmol/min, 100 pmol/min, 105 pmol/min, Or it may be 110 pmol/min. The present inventors have found that subjects transplanted with DSC cells whose preliminary respiratory capacity used for transplantation is below a predetermined threshold have high hair regeneration ability. Therefore, for example, when the preliminary respiratory capacity of DSC cells is higher than a predetermined threshold value, the hair regeneration capacity of DSC cells is evaluated to be low, and when the preliminary respiratory capacity of DSC cells is lower than a predetermined threshold value, the hair regeneration capacity of DSC cells It may be evaluated that the regeneration ability is high.
ＤＳＣ細胞の毛髪再生能は、１細胞ごとのＤＳＣ細胞における毛髪再生能が評価されてもよく、複数のＤＳＣ細胞が含まれる細胞群全体としての毛髪再生能が評価されてもよい。また、複数のＤＳＣ細胞が含まれる細胞群全体としての毛髪再生能は、その細胞群に含まれる、ミトコンドリア呼吸のレベルが低いＤＳＣ細胞（例えば、ミトコンドリア呼吸のレベルが所定の閾値以下のＤＳＣ細胞）の割合を測定することによって評価されてもよい。
As for the hair regeneration ability of DSC cells, the hair regeneration ability of each DSC cell may be evaluated, or the hair regeneration ability of the entire cell group including a plurality of DSC cells may be evaluated. In addition, the hair regeneration ability of the entire cell group containing multiple DSC cells is determined by the DSC cells included in the cell group that have a low level of mitochondrial respiration (for example, DSC cells whose level of mitochondrial respiration is below a predetermined threshold). may be evaluated by measuring the proportion of
一実施態様において、本発明は、ミトコンドリア呼吸のレベルが低い毛球部毛根鞘（ＤＳＣ）細胞を含有する、毛髪を再生するための組成物を提供する。上述のようにミトコンドリア呼吸のレベルが低いＤＳＣ細胞は毛髪再生能が高いことから、毛髪再生を所望する部位に本発明の組成物を移植することにより毛髪の再生が期待できる。
In one embodiment, the present invention provides a composition for regenerating hair that contains hair bulb root sheath (DSC) cells with reduced levels of mitochondrial respiration. As mentioned above, DSC cells with a low level of mitochondrial respiration have a high ability to regenerate hair, so hair regeneration can be expected by transplanting the composition of the present invention to a site where hair regeneration is desired.
一実施態様において、本発明に適用されるミトコンドリア呼吸のレベルが低いＤＳＣ細胞は、ミトコンドリア呼吸が所定の閾値より低いＤＳＣ細胞である。所定の閾値は、特定の値が一意的に決定されるものではないが、例えば、毛髪径及び／又は毛髪密度が増加した被検体群におけるそれぞれの被検体に移植されたＤＳＣ細胞のミトコンドリア呼吸のレベルと；毛髪径及び／又は毛髪密度が増加しなかった被検体群におけるそれぞれの被検体に移植されたＤＳＣ細胞のミトコンドリア呼吸のレベルとの比較によって決定されるものであってもよい。例えば、本発明の組成物に含まれるＤＳＣ細胞のミトコンドリア呼吸のレベルは、毛髪径及び／又は毛髪密度が増加しなかった被検体群におけるＤＳＣ細胞のミトコンドリア呼吸のレベルの平均よりも低い（例えば、０．９倍以下、０．８倍以下、０．７倍以下、０．６倍以下、０．５倍以下、０．４倍以下、０．３倍以下、０．２倍以下、０．１倍以下、又は０．０５倍以下）ものであってよく、本発明の組成物に含まれる細胞群において、そのようなＤＳＣ細胞が、例えば１０％以上（例えば、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、９５％又は９９％以上）含まれるように選択又は濃縮してもよい。
In one embodiment, the DSC cells with low levels of mitochondrial respiration applied to the present invention are DSC cells with mitochondrial respiration lower than a predetermined threshold. Although the predetermined threshold value is not uniquely determined, for example, the mitochondrial respiration of DSC cells transplanted to each subject in a subject group with increased hair diameter and/or hair density may be considered as the predetermined threshold value. and the level of mitochondrial respiration of DSC cells transplanted into each subject in a group of subjects in which hair diameter and/or hair density did not increase. For example, the level of mitochondrial respiration of DSC cells included in the compositions of the invention is lower than the average level of mitochondrial respiration of DSC cells in a group of subjects in which hair diameter and/or hair density did not increase (e.g., 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0. 1 times or less, or 0.05 times or less), and in the cell group included in the composition of the present invention, such DSC cells may be, for example, 10% or more (for example, 10%, 20%, 30% or less). %, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more).
一実施態様において、本発明は、毛球部毛根鞘（ＤＳＣ）細胞を含む、毛髪を再生するための組成物の製造方法であって、 ＤＳＣ細胞のミトコンドリア呼吸のレベルを低下させる工程、を含む、方法を提供する。
In one embodiment, the invention provides a method for producing a composition for regenerating hair comprising hair bulb root sheath (DSC) cells, the method comprising: reducing the level of mitochondrial respiration of the DSC cells. , provides a method.
ミトコンドリア呼吸のレベルを低下させる方法は、公知の方法に従えばよく、例えば、低酸素培養を行う工程、並びに／又は、ミトコンドリア複合体阻害剤及び疑似低酸素誘導剤からなる群から１又は複数選択される剤を添加する工程を実施することによってミトコンドリア呼吸のレベルを低下させることが可能である。低酸素培養は、例えば、Ｐａｐａｎｄｒｅｏｕらの方法（Ｃｅｌｌ Ｍｅｔａｂ． ２００６ Ｍａｒ；３（３）：１８７－９７）を参考に実施してもよく、例えば、１０％以下、５％以下、２％以下、１％以下の酸素を含む雰囲気下で、任意の時間（例えば、少なくとも０．５時間、１時間、２時間、６時間、１２時間、１８時間、又は２４時間）培養してもよい。ミトコンドリア複合体阻害剤としては、例えば、ミトコンドリア複合体Ｉ阻害剤（例えば、ロテノン（D J Horgan et al., J Biol Chem, 1968 Feb 25;243(4):834-43.）など）や、ミトコンドリア複合体III阻害剤（例えば、アンチマイシンA（E. C. SLATER, Biochim Biophys Acta., 1973 Dec 7;301(2):129-54.)など）を用いてもよい。また、疑似低酸素誘導剤としては、例えば、塩化コバルト（ＩＩ）（ＣｏＣｌ２）、デフェロキサミン、ジメチルオキサロイルグリシン（Dimethyloxaloylglycine）（P. Jaakkola et al., Science. 2001 Apr 20;292(5516):468-72.)、L-ミモシン（C. Warnecke et al., FASEB J. 2003 Jun;17(9):1186-8.)、メトホルミン（A. Luengo, et al., BMC Biol. 2014 Oct 24;12:82.)、又はコハク酸（H. Mao et al., Stem Cells Int. 2020 Mar 10;2020:2016809.）などを用いてもよい。
The method for reducing the level of mitochondrial respiration may be according to a known method, for example, a step of performing hypoxic culture, and/or one or more selected from the group consisting of mitochondrial complex inhibitors and pseudohypoxia inducers. It is possible to reduce the level of mitochondrial respiration by carrying out the step of adding an agent that is The hypoxic culture may be carried out, for example, by referring to the method of Papandreou et al. Culture may be performed for any length of time (eg, at least 0.5 hours, 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, or 24 hours) in an atmosphere containing 1% or less oxygen. Examples of mitochondrial complex inhibitors include mitochondrial complex I inhibitors (e.g., rotenone (D J Horgan et al., J Biol Chem, 1968 Feb 25;243(4):834-43.), Complex III inhibitors such as antimycin A (E. C. SLATER, Biochim Biophys Acta., 1973 Dec 7;301(2):129-54.) may also be used. In addition, examples of pseudohypoxia inducing agents include cobalt (II) chloride (CoCl 2 ), deferoxamine, and dimethyloxaloylglycine (P. Jaakkola et al., Science. 2001 Apr 20;292(5516): 468-72.), L-mimosine (C. Warnecke et al., FASEB J. 2003 Jun;17(9):1186-8.), metformin (A. Luengo, et al., BMC Biol. 2014 Oct 24 ;12:82.) or succinic acid (H. Mao et al., Stem Cells Int. 2020 Mar 10;2020:2016809.).
本発明に用いられ得るＤＳＣ細胞は、公知の方法（例えば、Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.）を参考とすることにより調製することが可能である。そのようにして得られたＤＳＣ細胞を含む細胞群を用いて、ミトコンドリア呼吸のレベルを低下させることにより、毛髪再生能の高い組成物を製造することができる。
DSC cells that can be used in the present invention can be obtained using known methods (for example, Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R , Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.). A composition with high hair regeneration ability can be produced by reducing the level of mitochondrial respiration using the cell group containing the DSC cells obtained in this way.
一実施態様において、本発明の方法は、ＤＳＣ細胞の予備呼吸能を、所定の閾値（例えば、６０ｐｍｏｌ／分、６５ｐｍｏｌ／分、７０ｐｍｏｌ／分、７５ｐｍｏｌ／分、８０ｐｍｏｌ／分、８５ｐｍｏｌ／分、９０ｐｍｏｌ／分、９５ｐｍｏｌ／分、１００ｐｍｏｌ／分、１０５ｐｍｏｌ／分、又は１１０ｐｍｏｌ／分）以下に低下させるものであってもよい。本発明者らは、移植に用いられるＤＳＣ細胞の予備呼吸能が所定の閾値以下のＤＳＣ細胞が移植された被検体では、毛髪再生能が高いことを見出した。従って、例えば、ＤＳＣ細胞のミトコンドリア呼吸のレベルを低下させる工程は、ＤＳＣ細胞の予備呼吸能が所定の閾値より低くなるよう調整されてもよい。また、他の態様において、本発明の製造方法によって作製される組成物に含まれる、ミトコンドリア呼吸のレベルが低下したＤＳＣ細胞は、毛髪径及び／又は毛髪密度が増加しなかった被検体群におけるＤＳＣ細胞のミトコンドリア呼吸のレベルの平均よりも低い（例えば、０．９倍以下、０．８倍以下、０．７倍以下、０．６倍以下、０．５倍以下、０．４倍以下、０．３倍以下、０．２倍以下、０．１倍以下、又は０．０５倍以下）ものであってよく、そのようなＤＳＣ細胞が、例えば１０％以上（例えば、１０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、９５％又は９９％以上）含まれるよう調整されるものであってもよい。
In one embodiment, the method of the invention determines the respiratory reserve capacity of DSC cells at a predetermined threshold (e.g., 60 pmol/min, 65 pmol/min, 70 pmol/min, 75 pmol/min, 80 pmol/min, 85 pmol/min, 90 pmol/min). /min, 95 pmol/min, 100 pmol/min, 105 pmol/min, or 110 pmol/min). The present inventors have found that subjects transplanted with DSC cells whose preliminary respiratory capacity used for transplantation is below a predetermined threshold have high hair regeneration ability. Thus, for example, reducing the level of mitochondrial respiration of a DSC cell may be adjusted such that the respiratory reserve capacity of the DSC cell is below a predetermined threshold. In another embodiment, the DSC cells with a reduced level of mitochondrial respiration contained in the composition produced by the production method of the present invention are the DSC cells in a subject group in which hair diameter and/or hair density did not increase. lower than the average level of cellular mitochondrial respiration (e.g., 0.9 times or less, 0.8 times or less, 0.7 times or less, 0.6 times or less, 0.5 times or less, 0.4 times or less, 0.3 times or less, 0.2 times or less, 0.1 times or less, or 0.05 times or less), and such DSC cells may have a , 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more).
以下に、本発明を実施例に基づいて更に詳しく説明するが、これらは本発明を何ら限定するものではない。
Hereinafter, the present invention will be explained in more detail based on Examples, but these are not intended to limit the present invention in any way.
１．方法
１－１．ＤＳＣ細胞の調製
Tsuboiらの文献（Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.）に記載の方法に従って、ＤＳＣ細胞を調製した(図１）。 1. Method 1-1. Preparation of DSC cells Tsuboi et al. (Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, DSC cells were prepared according to the method described in Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116. (Figure 1).
１－１．ＤＳＣ細胞の調製
Tsuboiらの文献（Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.）に記載の方法に従って、ＤＳＣ細胞を調製した(図１）。 1. Method 1-1. Preparation of DSC cells Tsuboi et al. (Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, DSC cells were prepared according to the method described in Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116. (Figure 1).
１－２．臨床試験の概要
本実施例では、Tsuboiらの文献（Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.）で評価した被検体から採取されたＤＳＣ細胞のうち、移植に用いられなかったものを拡大培養して得られたＤＳＣ細胞について細胞内活性を測定し、治療成績（積算毛髪径（３か月、６か月、９か月、１２か月）、総毛髪密度（３か月、６か月、９か月、１２か月）、対プラセボ（ＤＳＣ細胞移植無し）、対０か月）のスコアとの相関性を解析した。 1-2. Overview of the clinical trial In this example, we will use the literature by Tsuboi et al. (Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116. We measured the intracellular activity of the DSC cells obtained by expanding the DSC cells that were not present, and measured the treatment results (cumulative hair diameter (3 months, 6 months, 9 months, 12 months), total hair density (3 The correlation with the scores of 2 months, 6 months, 9 months, 12 months), vs. placebo (no DSC cell transplantation), vs. 0 months) was analyzed.
本実施例では、Tsuboiらの文献（Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116.）で評価した被検体から採取されたＤＳＣ細胞のうち、移植に用いられなかったものを拡大培養して得られたＤＳＣ細胞について細胞内活性を測定し、治療成績（積算毛髪径（３か月、６か月、９か月、１２か月）、総毛髪密度（３か月、６か月、９か月、１２か月）、対プラセボ（ＤＳＣ細胞移植無し）、対０か月）のスコアとの相関性を解析した。 1-2. Overview of the clinical trial In this example, we will use the literature by Tsuboi et al. (Autologous cell-based therapy for male and female pattern hair loss using dermal sheath cup cells: A randomized placebo-controlled double-blinded dose-finding clinical study. Tsuboi R, Niiyama S, Irisawa R, Harada K, Nakazawa Y, Kishimoto J. J Am Acad Dermatol. 2020 Jul;83(1):109-116. We measured the intracellular activity of the DSC cells obtained by expanding the DSC cells that were not present, and measured the treatment results (cumulative hair diameter (3 months, 6 months, 9 months, 12 months), total hair density (3 The correlation with the scores of 2 months, 6 months, 9 months, 12 months), vs. placebo (no DSC cell transplantation), vs. 0 months) was analyzed.
具体的には、それぞれのＤＳＣ細胞５×１０５個を１０％ＦＢＳ含有ＤＭＥＭ培地（ＧＩＢＣＯ）でサブコンフルエントになるまで培養した。その後、細胞培養用マイクロプレート（ＸＦｅ２４用）に、２×１０４個／ウェルのＤＳＣ細胞を、推奨濃度のグルコース、ピルビン酸、グルタミン酸を含むＸＦ用ＤＭＥＭ培地で播種し一晩培養した。細胞外フラックスアナライザーＸＦｅ２４による細胞のエネルギー代謝活性の評価には、Ｓｅａｈｏｒｓｅ ＸＦ ミトストレスキット（Ａｇｉｌｅｎｔ Ｔｅｃｈｎｏｌｏｇｉｅｓ社）を用い、製品のマニュアルに従って、オリゴマイシン（ＡＴＰ合成酵素阻害剤、終濃度１μＭ）、カルボニルシアニド-ｐ-トリフルオロメトキシフェニルヒドラゾン（ＦＣＣＰ、脱共役剤、終濃度３μＭ）、ロテノン（ミトコンドリア複合体Ｉ阻害剤、終濃度０．５μＭ）およびアンチマイシンＡ（ミトコンドリア複合体ＩＩＩ阻害剤、終濃度０．５μＭ）を順次添加し、ＯＣＲを測定した（図２）。
Specifically, 5 x 10 5 DSC cells of each were cultured in DMEM medium containing 10% FBS (GIBCO) until subconfluent. Thereafter, 2×10 4 DSC cells/well were seeded in a DMEM medium for XF containing recommended concentrations of glucose, pyruvate, and glutamic acid in a cell culture microplate (for XFe24), and cultured overnight. For evaluation of cellular energy metabolic activity using the extracellular flux analyzer XFe24, we used the Seahorse Nido-p-trifluoromethoxyphenylhydrazone (FCCP, uncoupler, final concentration 3 μM), rotenone (mitochondrial complex I inhibitor, final concentration 0.5 μM) and antimycin A (mitochondrial complex III inhibitor, final concentration 0.5 μM) was added sequentially, and OCR was measured (FIG. 2).
２．結果
３４名の被検体由来のＤＳＣ細胞の細胞内活性（ＯＣＲ値、ＥＣＡＲ値）を測定した。それらの値に基づき、最大呼吸及び予備呼吸能を算出した結果、各被検体由来ＤＳＣ細胞の最大呼吸及び予備呼吸能には、差異が認められた（図３）。 2. Results Intracellular activity (OCR value, ECAR value) of DSC cells derived from 34 subjects was measured. As a result of calculating the maximum respiration and preliminary respiratory capacity based on these values, differences were observed in the maximum respiration and preliminary respiratory capacity of the DSC cells derived from each subject (FIG. 3).
３４名の被検体由来のＤＳＣ細胞の細胞内活性（ＯＣＲ値、ＥＣＡＲ値）を測定した。それらの値に基づき、最大呼吸及び予備呼吸能を算出した結果、各被検体由来ＤＳＣ細胞の最大呼吸及び予備呼吸能には、差異が認められた（図３）。 2. Results Intracellular activity (OCR value, ECAR value) of DSC cells derived from 34 subjects was measured. As a result of calculating the maximum respiration and preliminary respiratory capacity based on these values, differences were observed in the maximum respiration and preliminary respiratory capacity of the DSC cells derived from each subject (FIG. 3).
そこで、各被検体の総毛髪密度又は積算毛髪径のスコアと、ＤＳＣ細胞のミトコンドリア呼吸パラメーターの値（最大呼吸及び予備呼吸能）との相関係数を算出した。相関係数の絶対値が０．３より大きい場合を弱い相関、弱い逆相関があると判定し、相関係数の絶対値が０．５より大きい場合を相関、逆相関があると判定した。
Therefore, the correlation coefficient between the total hair density or cumulative hair diameter score of each subject and the mitochondrial respiration parameter values (maximum respiration and preliminary respiratory capacity) of DSC cells was calculated. When the absolute value of the correlation coefficient was greater than 0.3, it was determined that there was a weak correlation or a weak inverse correlation, and when the absolute value of the correlation coefficient was greater than 0.5, it was determined that there was a correlation or an inverse correlation.
その結果、特に、総毛髪密度において、最大呼吸と予備呼吸能で負の相関が認められた（図４）。また、それは３か月又は６か月の移植後早期の段階で特に相関が高い傾向が見られた。
As a result, a negative correlation was observed between maximum respiration and preliminary respiratory capacity, especially in total hair density (Figure 4). Furthermore, there was a tendency for the correlation to be particularly high in the early stages after transplantation, 3 months or 6 months.
そこで、ＤＳＣ細胞の予備呼吸能により二群に分けた場合の治療成績を解析した。ＤＳＣ細胞の予備呼吸能の値が＞９０ｐｍｏｌ／分となる被検体群を「高活性群」；ＤＳＣ細胞の予備呼吸能の値が≦９０ｐｍｏｌ／分となる被検体群を「低活性群」に分けた。それぞれの群において、ＤＳＣ細胞移植６か月後の対プラセボ比のスコア化された総毛髪密度又は積算毛髪径を比較した。
Therefore, we analyzed the treatment results when DSC cells were divided into two groups based on their preliminary respiratory capacity. A group of subjects whose DSC cell reserve respiratory capacity value is >90 pmol/min is defined as a "high activity group"; a subject group whose DSC cell reserve respiratory capacity value is ≤90 pmol/min is defined as a "low activity group". divided. In each group, the scored total hair density or integrated hair diameter in comparison with the placebo ratio was compared 6 months after DSC cell transplantation.
その結果、ミトコンドリア呼吸の高活性群／低活性群において、総毛髪密度に有意な差が認められた（図５）。
As a result, a significant difference in total hair density was observed between the high mitochondrial respiration activity group and the low activity group (Figure 5).
さらに、投与後３か月・６か月における治療成績（総毛髪密度、対プラセボ比）を基に、治療成績が高い被験者群（高有効性群）と、低い被験者群（低有効性群）の２群に分け、各群でそれぞれＤＳＣ細胞のミトコンドリア呼吸パラメーター（最大呼吸・予備呼吸能）活性を調べ、群間で差が認められるか、有意差検定を実施した。
Furthermore, based on the treatment results (total hair density, vs. placebo) at 3 and 6 months after administration, we determined a group of subjects with high treatment results (high efficacy group) and a group of subjects with low treatment results (low efficacy group). DSC cells were divided into two groups, and mitochondrial respiratory parameter (maximum respiration/respiratory capacity) activity of DSC cells was examined in each group, and a significant difference test was performed to determine whether a difference was observed between the groups.
その結果、高い有効性を示す被検体由来のＤＳＣ細胞は、ミトコンドリア呼吸活性が有意に低いことが示された（図６）。
The results showed that DSC cells derived from subjects showing high efficacy had significantly low mitochondrial respiratory activity (Figure 6).
Claims (15)
- ミトコンドリア呼吸のレベルを指標とする、毛球部毛根鞘（ＤＳＣ）細胞の毛髪再生能を評価する方法。 A method for evaluating the hair regeneration ability of hair bulb root sheath (DSC) cells using the level of mitochondrial respiration as an index.
- 前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より低い場合に、前記ＤＳＣ細胞の毛髪再生能が高いと評価し、
前記ＤＳＣ細胞におけるミトコンドリア呼吸のレベルが所定の閾値より高い場合に、前記ＤＳＣ細胞の毛髪再生能が低いと評価する、請求項１に記載の方法。 When the level of mitochondrial respiration in the DSC cell is lower than a predetermined threshold, the hair regeneration ability of the DSC cell is evaluated to be high;
The method according to claim 1, wherein the hair regeneration ability of the DSC cells is evaluated to be low when the level of mitochondrial respiration in the DSC cells is higher than a predetermined threshold. - 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
- 前記ミトコンドリア呼吸のレベルが予備呼吸能である、請求項２に記載の方法。 3. The method of claim 2, wherein the level of mitochondrial respiration is respiratory reserve capacity.
- 前記毛髪再生能が、毛髪径及び／又は毛髪密度を増加させる能力である、請求項１又は２項に記載の方法。 The method according to claim 1 or 2, wherein the hair regeneration ability is the ability to increase hair diameter and/or hair density.
- ミトコンドリア呼吸のレベルが低い毛球部毛根鞘（ＤＳＣ）細胞を含有する、毛髪を再生するための組成物。 A composition for regenerating hair, containing hair bulb root sheath (DSC) cells with a low level of mitochondrial respiration.
- 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、請求項６に記載の組成物。 7. The composition of claim 6, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
- 前記ミトコンドリア呼吸のレベルが予備呼吸能であり、前記予備呼吸能が所定の閾値以下のＤＳＣ細胞を含有する、請求項６に記載の組成物。 The composition according to claim 6, wherein the level of mitochondrial respiration is a respiratory reserve capacity, and the composition contains DSC cells whose reserve respiratory capacity is below a predetermined threshold.
- 毛髪径及び／又は毛髪密度が増加することを特徴とする、請求項６又は７に記載の組成物。 The composition according to claim 6 or 7, characterized in that hair diameter and/or hair density are increased.
- 毛球部毛根鞘（ＤＳＣ）細胞を含む、毛髪を再生するための組成物の製造方法であって、
ＤＳＣ細胞のミトコンドリア呼吸のレベルを低下させる工程、
を含む、方法。 1. A method for producing a composition for regenerating hair, comprising hair bulb sheath (DSC) cells, the method comprising:
reducing the level of mitochondrial respiration in DSC cells;
including methods. - 前記ミトコンドリア呼吸のレベルを低下させる工程が、低酸素培養を行う工程、並びに／又は、ミトコンドリア複合体阻害剤及び疑似低酸素誘導剤からなる群から１又は複数選択される剤を添加する工程である、請求項１０に記載の方法。 The step of reducing the level of mitochondrial respiration is a step of performing hypoxic culture and/or a step of adding one or more agents selected from the group consisting of mitochondrial complex inhibitors and pseudohypoxia inducing agents. , the method according to claim 10.
- 前記ミトコンドリア呼吸のレベルが、前記ＤＳＣ細胞の酸素消費速度（ＯＣＲ）である、請求項１０又は１１に記載の方法。 The method according to claim 10 or 11, wherein the level of mitochondrial respiration is the oxygen consumption rate (OCR) of the DSC cells.
- 前記ミトコンドリア呼吸のレベルを低下させる工程が、前記ＤＳＣ細胞の予備呼吸能を所定の閾値以下に低下させる工程である、請求項１０又は１１に記載の方法。 The method according to claim 10 or 11, wherein the step of reducing the level of mitochondrial respiration is a step of reducing the preliminary respiratory capacity of the DSC cells to a predetermined threshold value or less.
- 請求項１０又は１１に記載の方法により得られる、毛髪を再生するためのＤＳＣ細胞を含む組成物。 A composition containing DSC cells for regenerating hair, obtained by the method according to claim 10 or 11.
- 毛髪径及び／又は毛髪密度が増加することを特徴とする、請求項１４に記載の組成物。 15. The composition according to claim 14, characterized in that the hair diameter and/or hair density is increased.
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