WO2019050480A1 - Méthodes de criblage d'agents de régulation de la pousse des poils/cheveux ou du cycle des poils/cheveux - Google Patents

Méthodes de criblage d'agents de régulation de la pousse des poils/cheveux ou du cycle des poils/cheveux Download PDF

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WO2019050480A1
WO2019050480A1 PCT/SG2018/050454 SG2018050454W WO2019050480A1 WO 2019050480 A1 WO2019050480 A1 WO 2019050480A1 SG 2018050454 W SG2018050454 W SG 2018050454W WO 2019050480 A1 WO2019050480 A1 WO 2019050480A1
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hair
agent
hair growth
cells
hair follicle
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PCT/SG2018/050454
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English (en)
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Andrew Chwee Aun Wan
Tze Chiun Lim
Axel Hillmer
Guo Yan Elaine CHEW
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Agency For Science, Technology And Research
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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/549—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention generally relates to methods of drug screening, in particular methods of screening candidate agents (such as drugs) for hair growth or hair cycle regulating activity (such as hair growth or hair cycle promoting or inhibiting activity), and more particularly methods of screening candidate agents for hair growth or hair cycle regulating activity based on clustering of gene expression profiles of hair follicle cells that have been exposed to the candidate agent.
  • candidate agents such as drugs
  • hair growth or hair cycle regulating activity such as hair growth or hair cycle promoting or inhibiting activity
  • Typical methods to screen novel drugs with hair promotion or inhibition activity involve use of in vitro human hair follicle cultures.
  • a method of screening a candidate agent for hair growth or hair cycle promoting or inhibiting activity comprising:
  • step (c) comparing the profile of genes that are differentially expressed in step (b) against the profile in step (a);
  • step (d) determining the candidate agent as having hair growth or hair cycle promoting activity when the profile of genes that are differentially expressed in step (b) substantially matches the profile of genes that are differentially expressed in hair follicle cells when treated with a reference agent which promotes hair growth or hair cycle obtained in step (a), or as having hair growth or hair cycle inhibiting activity when the profile of genes that are differentially expressed in step (b) substantially matches the profile of genes that are differentially expressed in hair follicle cells when treated with a reference agent which inhibits hair growth or hair cycle obtained in step (a).
  • the screening assay as described herein for agents having hair growth or hair cycle promoting or inhibiting activity is performed on hair follicle cells which are loaded into a scaffold, such as into a whole multi-interfacial polyelectrolyte complex (MIPC) construct, a compartmentalized MIPC fiber section, or combinations thereof.
  • the scaffold is engineered to mimic the cellular structure of the human hair follicle, which is constructed in a manner such that it allows for the epithelial-mesenchymal interactions in the hair follicle to take place, encourages proliferation and prevents apoptosis of hair follicle cells loaded into it.
  • the scaffold is a more suitable model for the hair follicle cells to grow in and be assayed on, as it better mimics in vivo hair growth conditions.
  • the screening assay as disclosed herein circumvents the usage of natural human hair follicles, the isolation and scaling up of which is technically challenging.
  • a plurality of scaffolds can be combined together to form a construct in order to provide a large population of scaffolds for screening multiple agents for their hair growth or hair cycle promoting or inhibiting activity and for data collection in a high throughput fashion.
  • the screening assay as disclosed herein for candidate agents having hair growth or hair cycle promoting or inhibiting activity is based on clustering of gene expression profiles of hair follicle cells that have been exposed to the candidate agent.
  • the quantitative analysis of differential expression of certain genes in hair follicle cells that have been exposed to the candidate agent by techniques such as RNA microarray generates a signature gene expression profile.
  • the activity of the candidate agent on hair growth or hair cycle can be determined more accurately and more efficiently, in comparison to the techniques used in the state of the art to observe the changes to the hair follicle cells due to the presence of a drug, for example, by the naked eye or by a microscopy technique.
  • a method of treating or preventing a condition associated with hair loss in a subject in need thereof comprising administering a therapeutically effective amount of an agent to hair follicle cells or skin of the subject, wherein the agent has been determined as an agent having hair growth or hair cycle promoting activity according to the method as described herein.
  • a cosmetic method of inhibiting hair growth or hair cycle in a subject in need thereof comprising administering a therapeutically effective amount of an agent to hair follicle cells or skin of the subject, wherein the agent has been determined as an agent having hair growth or hair cycle inhibiting activity according to the method as described herein.
  • a method of treating or preventing a condition associated with excessive hair growth in a subject in need thereof comprising administering a therapeutically effective amount of an agent to hair follicle cells or skin of the subject, wherein the agent has been determined as an agent having hair growth or hair cycle inhibiting activity according to the method as described herein.
  • kits for screening an agent as a hair growth or hair cycle promoting agent or a hair growth or hair cycle inhibiting agent according to the method as described herein, comprising one or more of the following:
  • buffer is optionally selected from the group consisting of an extraction buffer, a buffer for dissolving the polycationic polymer in (d) to form a solution of the polycationic polymer, and a buffer for dissolving the poly anionic polymer in (e) to form a solution of the polyanionic polymer;
  • the term "expression” may refer to the expression of an RNA molecule from a gene.
  • expression means allowing or causing the information in a gene or DNA sequence to become manifest, for example by activating the cellular functions involved in transcription of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product” such as mRNA.
  • the expression product itself e.g. the resulting mRNA, may also be said to be “expressed” by the cell.
  • An expression product can be characterized as intracellular, extracellular or secreted.
  • intracellular means something that is inside a cell.
  • extracellular means something that is outside a cell.
  • a substance is "secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • the term "expression profile" refers to the collective pattern of gene expression by a particular cell type or tissue (for example, hair follicle cells, such as dermal papilla cells) under given conditions at a given time.
  • the collective pattern of gene expression may be the differential expression of the genes in the particular cell type or tissue (for example, gene X and gene Y in the hair follicle cell), when treated with a reference agent which promotes hair growth or hair cycle, or a reference agent which inhibits hair growth or hair cycle, or a candidate agent, relative to (or when compared with) the expression of gene X and gene Y in a control hair follicle cell, for example a hair follicle cell which is not treated with any agent (i.e. untreated).
  • the genes of interest may be gene X and gene Y in a hair follicle cell, and thus “differential expression” of these genes refers to the upregulation or downregulation of the expression of gene X and gene Y in the hair follicle cell, when treated with a reference agent which promotes hair growth or hair cycle, or a reference agent which inhibits hair growth or hair cycle, or a candidate agent, relative to (or when compared with) the expression of gene X and gene Y in an untreated hair follicle cell.
  • the expression of gene X when treating a hair follicle cell with a reference agent which promotes hair growth or hair cycle, the expression of gene X may be upregulated and the expression of gene Y may be upregulated, the expression of gene X may be upregulated and the expression of gene Y may be downregulated, the expression of gene X may be downregulated and the expression of gene Y may be upregulated, and the expression of gene X may be downregulated and the expression of gene Y may be downregulated, relative to (or when compared with) the expression of gene X and gene Y in an untreated hair follicle cell.
  • the expression of gene X when treating a hair follicle cell with a reference agent which inhibits hair growth or hair cycle, the expression of gene X may be upregulated and the expression of gene Y may be upregulated, the expression of gene X may be upregulated and the expression of gene Y may be downregulated, the expression of gene X may be downregulated and the expression of gene Y may be upregulated, and the expression of gene X may be downregulated and the expression of gene Y may be downregulated, relative to (or when compared with) the expression of gene X and gene Y in an untreated hair follicle cell.
  • the expression of gene X when treating a hair follicle cell with a candidate agent, the expression of gene X may be upregulated and the expression of gene Y may be upregulated, the expression of gene X may be upregulated and the expression of gene Y may be downregulated, the expression of gene X may be downregulated and the expression of gene Y may be upregulated, and the expression of gene X may be downregulated and the expression of gene Y may be downregulated, relative to (or when compared with) the expression of gene X and gene Y in an untreated hair follicle cell.
  • the term "reference agent” refers to an agent, whose function in regulating hair growth or hair cycle is known in the art.
  • the reference agent may include an agent which promotes hair growth or cycle, and an agent which inhibits hair growth or cycle.
  • promoting and grammatical variations of that term, when used in relation to hair growth or hair cycle, refers to accelerating, enhancing or increasing the growth or development of hair, such as accelerating, enhancing or increasing the growth or development of hair follicle cells, to result in hair which is longer, or to result in hair follicle cells in a higher quantity, relative to the hair length or the quantity of hair follicle cells before the growth or development is accelerated, enhanced or increased.
  • the "promoting” may be determined qualitatively or quantitatively by any method known in the art.
  • the Philpott model may be used, where the length of the hair shaft in an organotypic-cultured hair follicle may be determined visually to have increased or may be measured over time, so that the rate of increase in the length of the hair shaft may be determined as enhanced compared to that of hair the growth or cycle of which is not promoted.
  • the inhibition may be total inhibition, which stops hair growth or development completely, or partial inhibition, which for example reduces the speed of hair growth, or reduces the length of hair grown.
  • the "inhibiting" may be determined qualitatively or quantitatively by any method known in the art.
  • the Philpott model may be used, where the length of the hair shaft in an organotypic-cultured hair follicle may be determined visually to have not increased or may be measured over time, so that the rate of increase in the length of the hair shaft may be determined as reduced compared to that of hair the growth or cycle of which is not inhibited.
  • hair cycle refers to a process from hair starting to grow to hair falling out.
  • a hair cycle typically includes four stages: anagen, catagen, telogen and exogen.
  • anagen growing phase
  • catagen progression phase
  • telogen resting phase
  • exogen shedding phase
  • treatment refers to incubating or otherwise exposing the cell or the sample of the cell (such as a hair follicle cell) to an agent (such as a reference agent or a candidate agent screened by the methods as disclosed herein), by any means, such as by submerging the cell or the sample of the cell in a solution containing the agent, so that the agent is brought into close proximity with the cell or the sample of the cell to exert an effect on the cell or the sample of the cell.
  • the treating may occur in vitro, such as in a test tube, a culture vessel or a plate, or elsewhere outside the living organism, in vivo or ex vivo.
  • treating may also refer to administration of an agent of the disclosure to an organism (such as a subject as described herein) by any appropriate means as described herein.
  • Such treatment includes any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
  • the disease state of symptoms may be those associated with hair loss, or excessive hair growth.
  • treatment includes prophylactic and therapeutic treatment.
  • an agent of the disclosure is exposed to isolated cells outside a subject, and then returned to the subject.
  • hair follicle cells may be extracted from a subject, treated with an agent of the disclosure (for example, in a test tube, a culture vessel or a plate), and then returned to the subject to exert its hair growth or hair cycle promoting or inhibiting effect on the subject.
  • an agent of the disclosure for example, in a test tube, a culture vessel or a plate
  • isolated and grammatical variations of that term refers to removing a substance from, and separating the substance from other components in a mixture, to thereby obtain a pure (or substantially pure) "isolate" of the substance. Accordingly, an “isolated” material is one that is substantially or essentially free from components that accompany it in its original state.
  • isolated aggregate refers to a cell aggregate, which has been purified from other cells surrounding it in an originally occurring state in the scaffold, e.g., a dermal papilla cell (DPC) aggregate which has been removed from normal human epidermal keratinocytes (NHEKs) which are normally adjacent to the DPC aggregate in the scaffold.
  • DPC dermal papilla cell
  • NHEKs normal human epidermal keratinocytes
  • preventing refers to inhibiting completely or in part the development or progression of a disease (such as a disease associated with hair loss).
  • subject refers to patients of human or other mammals, and includes any individual it is desired to be treated using agents and methods of the disclosure. However, it will be understood that “subject” does not imply that symptoms are present. Suitable mammals other than humans that fall within the scope of the disclosure include, but are not restricted to, primates, livestock animals (e.g. sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g. cats, dogs) and captive wild animals (e.g. foxes, deer, dingoes).
  • livestock animals e.g. sheep, cows, horses, donkeys, pigs
  • laboratory test animals e.g. rabbits, mice, rats, guinea pigs, hamsters
  • companion animals e.g. cats, dogs
  • captive wild animals e.g. foxes, deer, dingoes.
  • administering and grammatical variations of that term including “administer” and “administration”, includes contacting, applying, delivering or providing an agent of the disclosure to an organism (such as the subject as described herein), or a surface by any appropriate means, for example, topical application, oral administration, subcutaneous injection, infusion or implantation, intradermal injection, infusion or implantation.
  • terapéuticaally effective amount includes a reference to an amount of a therapeutic agent (for example, an agent which promotes or inhibits hair growth or hair cycle) to treat, ameliorate, or prevent a desired disease or condition (for example, a hair loss condition, or a condition associated with excessive hair growth such as hirsutism (excess hair growth in women) and hypertrichosis), or to exhibit a detectable therapeutic or preventative (prophylactic) effect (for example, an effect of promoting hair growth, or preventing hair loss, or an effect of inhibiting hair growth).
  • a therapeutic agent for example, an agent which promotes or inhibits hair growth or hair cycle
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors: the type and degree of the cellular response to be achieved; activity of the specific agent or composition employed; the specific agents or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent; the duration of the treatment; drugs used in combination or coincidental with the specific agent; and like factors well known in the medical arts.
  • the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician. For example, it is well within the skill of the art to start doses of the agents at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosages until the desired effect is achieved.
  • substantially match when used in relation to gene expression profile in a cell, for example, in a hair follicle cell when treated with a reference agent or a candidate agent, refers to the level of identity between the differential expression profile of a specific group of genes in the hair follicle cell when treated with the reference agent and the differential expression profile of the specific group of genes in the hair follicle cell when treated with the candidate agent.
  • the differential expression profile of the specific group of genes in the hair follicle cell when treated with the reference agent may be considered to "substantially match" the differential expression profile of the specific group of genes in the hair follicle cell when treated with the candidate agent when there is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity between them.
  • the differential expression profile of the specific group of genes in the hair follicle cell when treated with the reference agent may also be considered to "substantially match" the differential expression profile of the specific group of genes in the hair follicle cell when treated with the candidate agent when there is a 100% identity between them, i.e. a complete match.
  • the differential expression profile of the specific group of genes in the hair follicle cell when treated with a reference agent which promotes hair growth or hair cycle is considered to "substantially match” the differential expression profile of the specific group of genes in the hair follicle cell when treated with a candidate agent, i.e.
  • the candidate agent when there is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity between them, then the candidate agent will be determined as having hair growth or hair cycle promoting activity. Conversely, if the differential expression profile of the specific group of genes in the hair follicle cell when treated with a reference agent which inhibits hair growth or hair cycle is considered to "substantially match" the differential expression profile of the specific group of genes in the hair follicle cell when treated with a candidate agent, i.e.
  • the candidate agent when there is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity between them, then the candidate agent will be determined as having hair growth or hair cycle inhibiting activity.
  • range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a method of screening a candidate agent for hair growth or hair cycle promoting or inhibiting activity comprising:
  • step (c) comparing the profile of genes that are differentially expressed in step (b) against the profile in step (a);
  • step (d) determining the candidate agent as having hair growth or hair cycle promoting activity when the profile of genes that are differentially expressed in step (b) substantially matches the profile of genes that are differentially expressed in hair follicle cells when treated with a reference agent which promotes hair growth or hair cycle obtained in step (a), or as having hair growth or hair cycle inhibiting activity when the profile of genes that are differentially expressed in step (b) substantially matches the profile of genes that are differentially expressed in hair follicle cells when treated with a reference agent which inhibits hair growth or hair cycle obtained in step (a).
  • the hair follicle cells used or treated in the methods of the disclosure may be selected from the group consisting of dermal papilla cells, normal epidermal keratinocytes, dermal fibroblasts, dermal papilla fibroblasts, dermal microvascular endothelial cells, keratinocytes, melanocytes, hair follicle dermal papilla cells, hair follicle outer root sheath cells, outer root sheath keratinocytes, hair germinal matrix cells, and combinations thereof.
  • the hair follicle cells used or treated in the methods of the disclosure may be selected from the group consisting of human dermal papilla cells, normal human epidermal keratinocytes, human dermal fibroblasts, human dermal papilla fibroblasts, human dermal microvascular endothelial cells, human keratinocytes, human melanocytes, hair follicle dermal papilla cells, hair follicle outer root sheath cells, outer root sheath keratinocytes, human hair germinal matrix cells, and combinations thereof.
  • the hair follicle cells used or treated in the methods of the disclosure may be obtained from a commercial supplier such as American Type Culture Collection (ATCC), Invitrogen, EMD Millipore, Agilent Technologies, Lonza, Takara Bio Clontech, New England Biolabs, German Collection of Microorganisms and Cell Cultures, Health Protection Agency Culture Collections, Japanese Collection of Research Bioresources, Cambrex, GE Healthcare Life Biosciences, Promocell, Sigma-Aldrich, Promega, AUCells, Mirus, National Institutes of Health AIDS Research and Reference Reagent Program, Riken Cell Bank, Cedarlanelabs, Asterand, Qiagen, Bio-Rad, Stemcell Technologies, Miltenyi Biotec, Cell Applications, Korean Cell Line Bank, Cell Biolabs, Brainbits, BD Biosciences, Dako, InvivoGen, Microbix, Protein Sciences, Provitro, and so on.
  • ATCC American Type Culture Collection
  • Invitrogen EMD Millipore
  • the hair follicle cells used or treated in the methods of the disclosure may be primary hair follicle cells, which are isolated from the skin of a subject.
  • the hair follicle cells used or treated in the methods of the disclosure may be primary hair follicle cells, which are isolated from the scalp of a subject.
  • the human dermal papilla cells are isolated via micro-dissection from the skin biopsy of a human subject.
  • One exemplary method for isolating the cells is the one as described in Helena Topouzi et al. (Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles. Experimental Dermatology. 2017; 26:491-496.)
  • the hair follicle cells are loaded into a scaffold, which encapsulates and supports the assembly of hair follicle cells.
  • the scaffold is a fibrous structure.
  • the fibrous structure may comprise an assembly of hair follicle cells within a fibrous matrix.
  • the fibrous structure may be in the form of a thread-like structure or a hair follicle like structure.
  • the hair follicle like structure may be a three-dimensional hair follicle like structure.
  • the fibrous structure may be used to form a fibrous mat.
  • the fibrous structure may be incorporated into a skin patch, where the hair follicle-like structures form the germ for the development of new hair follicles in vivo.
  • the fibrous structure may have a dimension in the range of about 100 um to about 200 ⁇ , about 120 ⁇ im to about 200 ⁇ , about 140 ⁇ to about 200 ⁇ , ⁇ , about 160 ⁇ xm to about 200 ⁇ , about 180 ⁇ to about 200 ⁇ , about 100 ⁇ to about 120 ⁇ , about 100 ⁇ to about 140 ⁇ , about 100 ⁇ to about 160 ⁇ or about 100 ⁇ to about 180 ⁇ .
  • the above dimension may refer to the diameter of the fibrous structure, which may have a cylindrical shape.
  • the length of the fibrous structure is not limited and depends on the requirements of the user.
  • the length of the fibrous structure may be determined by the size of the support (such as a well or plate) used to culture the construct.
  • the fibrous structure may be placed in a culture medium to promote the formation of the hair follicle like structure.
  • the culture medium to be used depends on the hair follicle cells to be cultured and the person skilled in the art would know what culture medium to use.
  • human follicle dermal papilla cells can be cultured in follicle dermal papilla cell growth medium (Promocell GmbH; Cat. No.: C-26501) supplemented with 1% penicillin- streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA; Cat. No.: 15140122), or in Hair Follicle Dermal Papilla Cell Growth Medium (611-500 Sigma).
  • Human hair germinal matrix cells (HGMC) (ScienCell Research Laboratories, San Diego, CA, Cat. No.: #2410) can be cultured in mesenchymal stem cell medium (ScienCell Research Laboratories, Cat. No.: #7501) supplemented with 1% penicillin-streptomycin (Gibco; Cat. No.: 15140122).
  • Normal human epidermal keratinocytes NHEK (Promocell GnbH, Germany; Cat No.: C-12001) can be cultured in keratinocyte growth medium (Promocell GmbH; Cat. No.: C-20011) supplemented with 1% penicillin- streptomycin (Gibco; Cat. No.: 15140122), or in Keratinocyte Serum-Free Growth Medium for fetal and neonatal cells (131-500 Sigma).
  • the types of the hair follicle cells that can be used in the fibrous structure are those that are able to form a hair follicle architecture when present in the fibrous matrix.
  • the fibrous structure may comprise at least two types of hair follicle cells making up the assembly.
  • the at least two types of hair follicle cells may be able to self- assemble and interact with each other in order to mimic the architecture of an actual hair follicle.
  • one type of hair follicle cells is used. In other embodiments, combinations of two or three or four or more types of hair follicle cells are used. In one embodiment, two types of hair follicle cells are used.
  • the two types of hair follicle cells may be DPC and NHEK.
  • a suitable culture medium such as a mixture of dermal papilla medium with keratinocyte medium in a ratio of 1: 1, at certain cell concentrations, due to the close proximity between the DPC and NHEK, the DPC can aggregate to form spheroids with the NHEK at least partially surrounding the DPC aggregates (or spheroids) to form the fibrous structure.
  • the minimum cell concentration of the DPC in order to form the spheroids may be at least about 100 million cells/ml while that for the NHEK to at least partially surround the DPC spheroids may be at least about 50 million cells/ml.
  • the proper orientation of the DPC and NHEK cells in which the NHEK at least partially surround the DPC aggregates) may result in a fibrous structure having a hair follicle architecture such that the fibrous structure can be viewed as being a hair follicle-like structure.
  • the DPC and NHEK may migrate and self-assemble within the fibrous matrix to form the fibrous structure (or hair follicle like structure).
  • the two types of hair follicle cells may be DPC with outer root sheath cells.
  • the diameter of the spheroids may be in the range of about 80 ⁇ to about 120 ⁇ , about 90 ⁇ to about 120 ⁇ , about 100 ⁇ to about 120 ⁇ , about 110 ⁇ to about 120 ⁇ , about 80 ⁇ to about 90 ⁇ , about 80 ⁇ to about 100 ⁇ or about 80 ⁇ to about 110 ⁇ .
  • the diameter of the spheroids may be about 100 ⁇ .
  • the above diameter may refer to an equivalent diameter of the irregularly- shaped spheroid.
  • the fibrous structure may comprise a plurality of individual fibers making up the fibrous matrix.
  • the individual fiber may be a polyionic fiber.
  • the polyionic fiber may comprise at least one polycationic polymer and at least one polyanionic polymer.
  • the at least one polycationic polymer and/or at least one polyanionic polymer may be biocompatible or biodegradable.
  • polycationic polymer and/or polyanionic polymer are not limited as long as the polycationic polymer and polyanionic polymer can come together to form a fiber.
  • An exemplary polycationic polymer may be chitin, chitosan or methylated collagen.
  • An exemplary polyanionic polymer may be selected from the group consisting of alginate, gellan, heparan sulfate, heparin, acidic keratin, hyaluronic acid, chondroitin sulfate and combinations thereof.
  • the polycationic polymer is water soluble chitin and the polyanionic polymer is alginic acid.
  • the fibrous structure may be implanted into a living organism to form a hair follicle in vivo.
  • a plurality of fibrous structures can be combined together to form a construct in order to provide a larger population for data collection.
  • the number of fibrous structures that can be combined to form a construct is not particularly limited and depends on the needs of the user. In one embodiment, about 10, about 20, about 30, about 40, about 50, about 60, or about 70, fibrous structures can be combined to form a construct.
  • the cell constructs are stable and the cells may be viable for a long period of time. In one embodiment, the cells may be viable for up to two months.
  • the fibrous structure may be formed in a method which comprises the step of providing an assembly of hair follicle cells within a fibrous matrix.
  • the hair follicle cells are loaded into whole multi-interfacial polyelectrolyte complex (MIPC) scaffolds, which encapsulate and support the assembly of hair follicle cells.
  • MIPC multi-interfacial polyelectrolyte complex
  • the hair follicle cells are loaded into compartmentalized MIPC fiber section, which encapsulate and support the assembly of hair follicle cells.
  • Compartmentalized MIPC fiber sections are whole MIPC scaffolds that are sectioned to obtain a larger number of "sliced constructs".
  • a blade can be used to create slices from the whole MIPC scaffolds, for example, by sectioning in a direction perpendicular to the long axis of the fibers of the scaffolds.
  • the large number of compartmentalized MIPC fiber sections allows independent parallel screening of multiple candidate agents.
  • the fibrous structure may be formed using the interfacial polyelectrolyte complexation (IPC) fiber assembly technique.
  • the IPC fiber assembly technique may comprise the step of drawing a fiber from the interface between a polyanionic polymer solution and a polycationic polymer solution to form the fibrous matrix, wherein the hair follicle cells are present in at least one of the polycationic polymer solution and polyanionic polymer solution.
  • Two or more types of hair follicle cells may be loaded in the assembly.
  • the two or more types of hair follicle cells may be present in at least one of the polycationic polymer solution and the polyanionic polymer solution.
  • one type of hair follicle cells may be present in the polycationic solution (or polyanionic solution) while another type of hair follicle cells may be present in the other of the polyanionic solution (or polycationic solution, as the case may be).
  • the additional types of hair follicle cells may be in an admixture with the above types of hair follicle cells in the respective solution or may be present in a second polycationic solution (or polyanionic solution) as desired.
  • the two or more types of hair follicle cells may be present in two or more polycationic polymer solutions such that one type of hair follicle cells is present in one polycationic solution.
  • DPC may be present in one polycationic polymer solution and NHEK may be present in the other polycationic polymer solution.
  • the two or more types of hair follicle cells may be present in two or more polyanionic polymer solutions such that one type of hair follicle cells is present in one polyanionic solution.
  • DPC may be present in one polyanionic polymer solution and NHEK may be present in the other polyanionic polymer solution.
  • the two or more types of hair follicle cells may be present in the same solution, which can either be the polycationic solution or polyanionic solution.
  • the concentration of each type of hair follicle cells present in at least one of the polycationic polymer solution and the polyanionic polymer solution may be in the range of about 70 to about 110 million cells/ml, about 70 to about 80 million cells/ml, about 70 to about 90 million cells/ml, about 70 to about 100 million cells/ml, about 80 to about 110 million cells/ml, about 90 to about 110 million cells/ml or about 100 to about 110 million cells/ml.
  • the at least one polycationic polymer and/or at least one polyanionic polymer may be biocompatible or biodegradable.
  • polycationic polymer and/or polyanionic polymer are not limited as long as the polycationic polymer and polyanionic polymer can come together to form a fiber.
  • An exemplary polycationic polymer may be chitin or chitosan.
  • An exemplary polyanionic polymer may be selected from the group consisting of alginate, gellan, heparan sulfate, heparin, acidic keratin, hyaluronic acid, chondroitin sulfate, methylated collagen combinations thereof.
  • the polycationic polymer is water soluble chitin and the polyanionic polymer is alginic acid.
  • one polyanionic solution and one polycationic solution are required in order to form the fiber.
  • the number of polyanionic solutions and polycationic solutions that can be used is not limited and depends on the number of interfaces required in order to form the fiber as well as the number of types of hair follicle cells that are to be present in the fibrous structure.
  • the formed fibers may be collected on a collector (such as a 2-pronged fork).
  • the fiber may be drawn into a humidified chamber to protect the hair follicle cells from drying.
  • the collector or base plate may be rotated to allow the fibers to fuse together in order to form the fibrous matrix.
  • the rotation speed and number of rotational rounds of the base plate is not particularly limited and can be chosen by a skilled person based on the extent of fusing of the fibers required.
  • An exemplary rotation speed of the base plate may be selected from about 3 to about 7 rpm and an exemplary number of rotational rounds may be selected from about 3 to 7 rounds.
  • the fusing process may be completed by treating the fibrous matrix to form the final construct by dipping the fibrous matrix in a polycationic polymer solution followed by a polyanionic polymer solution.
  • the polycationic polymer solution may be water soluble chitin and the polyanionic polymer solution may be alginate solution.
  • the resultant fibrous matrix with the hair follicle cells therein may be placed in a suitable culture medium to allow the hair follicle cells to assemble in the fibrous matrix to thereby form the fibrous structure (or hair follicle like structure).
  • the hair follicle cells may self-assemble within the fibrous matrix to form the hair follicle structure.
  • DPC and NHEK are used, the DPC aggregate to form spheroids while the NHEK surround the DPC aggregates to form a structure that resembles the hair follicle.
  • the hair follicle cell aggregates formed in the scaffold are then treated with the reference agents or the candidate agents as described herein.
  • the agent is added to the culture medium in which the hair follicle cell aggregates formed in the scaffold are immersed.
  • the treatment may last for a duration of 24-48 h. In some embodiments, the treatment lasts for a duration of 24-45 h, 24-40 h, 24-35 h, 24-30 h, 30-48 h, 35-48 h, 40-48 h, 45-48 h, 30-45 h, 35-40 h, or 37-39 h.
  • the treatment lasts for a duration of about 24 h, about 25 h, about 26 h, about 27 h, about 28 h, about 29 h, about 30 h, about 31 h, about 32 h, about 33 h, about 34 h, about 35 h, about 36 h, about 37 h, about 38 h, about 39 h, about 40 h, about 41 h, about 42 h, about 43 h, about 44 h, about 45 h, about 46 h, about 47 h, or about 48 h.
  • the concentrations of the reference agents or the candidate agents as described herein used for treating the hair follicle cell aggregates formed in the scaffold are at least 0.1 nM, at least 0.2 nM, at least 1 nM, at least 8 nM, at least 10 nM, at least 100 nM, at least 1000 nM, at least 3 ⁇ , at least 25 ⁇ , or at least 300 ⁇ . In some embodiments, the concentrations of the reference agents or the candidate agents as described herein used for treating the hair follicle cell aggregates formed in the scaffold are 1 nM, 100 nM, or 25 ⁇ .
  • the concentrations of the reference agents or the candidate agents as described herein used in treating the hair follicle cell aggregates formed in the scaffold are in the range of 0.2-8 nM, 10-1000 nM, or 3-300 ⁇ . In some embodiments, the concentrations of the reference agents or the candidate agents as described herein used in treating the hair follicle cell aggregates formed in the scaffold are in the range of 0.2-7 nM, 0.2-6 nM, 0.2-5 nM, 0.2-4 nM, 0.2-3 nM, 0.2-2 nM, 0.2-1 nM, 1-8 nM, 2-8 nM, 3-8 nM, 4-8 nM, 5-8 nM, 6-8 nM, 1-7 nM, 2-6 nM, 3-5 nM, 10-900 nM, 10-800 nM, 10-700 nM, 10-600 nM, 10-500 nM, 10-400 nM, 10-300 nM, 10-200 nM, 10-100 nM,
  • cyclosporin A when dexamethasone is used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which inhibits hair growth or hair cycle, its concentration is about 100 nM. In another embodiment, when cyclosporin A is used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which promotes hair growth or hair cycle, its concentration is about 0.2-8 nM. In another embodiment, when cyclosporin A is used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which promotes hair growth or hair cycle, its concentration is about 1 nM.
  • minoxidil when used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which promotes hair growth or hair cycle, its concentration is about 3-300 ⁇ . In another embodiment, when minoxidil is used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which promotes hair growth or hair cycle, its concentration is about 25 ⁇ . In another embodiment, when bimatoprost is used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which promotes hair growth or hair cycle, its concentration is about 10-1000 nM. In another embodiment, when bimatoprost is used for treating the hair follicle cell aggregates formed in the scaffold as a reference agent which promotes hair growth or hair cycle, its concentration is about 100 nM.
  • a reference agent may be an agent which is already known in the art to have hair growth or hair cycle promoting activity.
  • Examples of reference agents which promote hair growth or hair cycle include cyclosporin A, minoxidil and bimatoprost.
  • the reference agents which promote hair growth or hair cycle may exert their activity through pathways involving the exemplary genes listed in Table 1. Thus, any agent (known or unknown in the art) which exerts its effect on such exemplary genes are included within the scope of the disclosure.
  • the reference agent may also be an agent which is already known in the art to have hair growth or hair cycle inhibiting activity. Examples of reference agents which inhibit hair growth or hair cycle include dexamethasone and allopurinol.
  • a candidate agent may refer to any agent that is known or unknown, synthetic or non-synthetic, biological or non-biological.
  • Exemplary biological candidate agents may include nucleic acids, peptides, polypeptides, proteins, cells, bacteria, viruses, fungi, protozoans, and so on.
  • Non-biological candidate agents may include anything that is not derived from living materials, for example, chemicals, metals, drugs, synthetic polymers and so on.
  • the drugs may include a new drug which has been developed but whose function has not been characterized, or a drug which has been developed for applications other than conditions associated with hair growth or loss.
  • isolation of the hair follicle cell aggregates from the scaffold is necessary in order to obtain a pure (or substantially pure) population of the hair follicle cell aggregates for subsequent analysis.
  • isolating means separating the DPC aggregates or spheroids from the NHEK that at least partially surrounds the spheroids, and removing the separated DPC aggregates from the NHEK and the fibrous structure to thereby obtain a pure (or substantially pure) population of the DPC aggregates for further experiments.
  • Common techniques for isolation of cells include enzymatic digestion of the scaffold, and centrifugation, or size-selection of a particular cell population using a filtering means, such as a cell strainer, to exclude cell populations above or below certain size limits.
  • a cell strainer is used, which has a size cut-off that is sufficient to separate single cells from aggregates of cells of different sizes.
  • the size cut-off is 40-120 ⁇ , 40-110 ⁇ , 40-100 ⁇ , 40-90 ⁇ , 40-80 ⁇ , 40-70 ⁇ , 40-60 ⁇ , 40-50 ⁇ , 50-120 ⁇ , 60-120 ⁇ , 70-120 ⁇ , 80-120 ⁇ , 90-120 ⁇ , 100-120 ⁇ , 110-120 ⁇ , 50-110 ⁇ , 60-100 ⁇ , or 70-90 ⁇ .
  • the size cut-off is about 40 ⁇ .
  • the size cut-off is about 80 ⁇ .
  • the size cut-off is about 90 ⁇ .
  • the size cut-off is about 100 ⁇ .
  • the size cut-off is about 110 ⁇ .
  • Total RNA includes all the RNA molecules in a cell, for example, mRNA, polyA RNA, polysomal RNA, tRNA, ribosomal RNA, lincRNA, miRNA, piRNA, siRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, aRNA, crRNA, tasiRNA, rasiRNA, 7SK RNA.
  • Extraction may be done by a method commonly used in the art, for example, by using Qiagen RNeasy kit and on-column DNase treatment according to manufacturer's recommendation, by using TRIzol ® Reagent, by using Guanidinium-Acid-Phenol extraction techniques, and by using SDS and Potassium Acetate reagents.
  • RNA extracted from the cells is then analyzed to ensure that it is of high integrity.
  • Techniques which can be used to analyze the RNA integrity include agarose gel electrophoresis and bioanalyzer (for example, Agilent 2100 Bioanalyzer, Agilent Technologies).
  • an RNA sample with high integrity has RIN value > 8, as measured by a bioanalyzer.
  • RNA samples of high integrity is then subjected to gene expression analysis.
  • the gene expression analysis is done using RNA microarrays.
  • RNA microarrays allow the simultaneous measurement of tens of thousands of RNA transcripts for gene expression for copy number variation analysis.
  • the process of RNA microarray uses microarray chips, prepared commercially, which comprise numerous wells, each of which contains an isolated gene.
  • One or more RNA samples extracted from cells of different states for example, hair follicle cells treated with the hair growth or hair cycle promoting agent, and those treated with the hair growth or hair cycle inhibiting agent representing all of the genes expressed in the cells are labelled using different colored fluorescent labels.
  • the one or more different colored fluorescent probe labelled RNA samples would then be simultaneously applied to a single microarray chip, where they competitively react with the arrayed molecules (for example, cDNA molecules) in the wells of the chip.
  • Each well of the microarray is scanned for the fluorescence intensity of each probe, the intensity of which is proportional to the expression level of that gene in the sample.
  • the ratio of the fluorescent intensities of different probes provides a highly accurate and quantitative measurement of the relative gene expression level in the one or more RNA samples.
  • the gene expression analysis is done using RNA sequencing.
  • Sequencing is a direct measurement of which nucleic acids are present in a sample. Counting sequences is linear with concentration, making sequencing a relatively unbiased approach to measuring which nucleic acids are present in solution. Additionally, sequencing is not dependent on prior knowledge of which nucleic acids may be present, and it is able to independently detect closely related gene sequences, novel splice forms or RNA editing that may be missed due to cross hybridization on microarrays.
  • the RNA sequencing technique is whole transcriptome shotgun sequencing.
  • whole transcriptome shotgun sequencing is done by generating cDNA from RNA with SMARTer Ultra Low Input RNA for Illumina Sequencing (Clontech), followed by RNA-seq library construction with NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).
  • the RNA sequencing technique is single molecule Direct RNA Sequencing (DRSTM). DRSTM sequences RNA molecules directly in a massively-parallel manner without RNA conversion to cDNA or other biasing sample manipulations such as ligation and amplification.
  • DRSTM Direct RNA Sequencing
  • the expression levels of marker nucleic acids may be used to generate expression profiles.
  • An expression profile of a particular sample of hair follicle cells treated with an agent disclosed herein is essentially a "fingerprint" of the state of the sample - while two states may have any particular gene similarly expressed, the evaluation of a number of such genes simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell. This allows hair follicle cells whose growth or cycle is promoted to be distinguished from, for example, hair follicle cells whose growth or cycle is inhibited.
  • Comparing expression profiles in different hair growth or hair cycle states identifies genes that are important in each of these states (for example, being differentially expressed in each of these states).
  • Molecular profiling may distinguish subtypes of a currently collective disease designation, e.g., different forms or stages of a hair loss condition.
  • genes included in the gene expression profile may be genes which have known functions in hair growth or hair cycle. Such exemplary genes are summarized in Table 1.
  • FGF Fibroblast growth factor
  • FGF2 Fibroblast growth factor
  • FGF5 shor form
  • FGF5 long form
  • FGF7 Sonic hedgehog
  • TGF- ⁇ Transforming growth factor- ⁇ [TGF-P-RI, TGF-PRII, TGF- ⁇ , TGF-P2, TGF-P3, BMP2, BMP4, BMP6, Noggin]
  • WNT-3 WNT-3, ⁇ -Catenin, Lef-1, Dishevelled-2
  • IGF Insulin-like growth factor
  • EGF Epidermal growth factors
  • HGF Hepatocyte growth factor
  • the gene expression profile is generated by Hierarchical Clustering.
  • Hierarchical clustering is an algorithm that groups similar objects into groups called clusters.
  • the endpoint is a set of clusters, where each cluster is distinct from each other cluster, and the objects within each cluster are substantially similar to each other.
  • the hierarchical clustering does not require any a priori assumptions about the relative sample relationship or a pre-determined clustering model. By first defining each sample as a cluster then combining the two closest clusters into a new cluster, and by merging two existing clusters into a single cluster in each subsequent step, eventually an unbiased clustering of samples based on their expression profiles can be generated.
  • non-hierarchical clustering requires inputs of postulated sample clustering relationships.
  • non-hierarchical clustering requires a user to partition samples into predetermined number of clusters, which is arbitrary and will likely lead to user-biased clustering output.
  • model based clustering is dependent upon fitting samples to a specific mixture model, which necessitates a pre-existing specific mixture model, and hence is not suitable for use in the present disclosure, as there is no prior knowledge of how the candidate drugs would cluster.
  • the hierarchical clustering may be Divisive and Agglomerative.
  • divisive or top- down clustering method all of the observations are assigned to a single cluster and then the cluster is partitioned to two least similar clusters. Finally, each cluster is partitioned recursively until there is one cluster for each observation.
  • agglomerative or bottom-up clustering method each observation is assigned to its own cluster. The similarity (e.g., distance) between each of the clusters is then computed and the two most similar clusters are joined. Finally, the above steps are repeated until there is only a single cluster left.
  • the gene expression profile is generated by Divisive Hierarchical Clustering.
  • the starting single cluster is split using a flat clustering algorithm, and this procedure is applied recursively until each document is in its own singleton cluster.
  • a second, flat clustering algorithm is also used as a "subroutine".
  • using a flat algorithm like K-means can result in top-down algorithms that are linear in the number of documents and clusters.
  • divisive algorithms can produce accurate hierarchies, as divisive algorithms benefit from complete information about the global distribution when making top-level partitioning decisions.
  • the gene expression profile is generated by Agglomerative Hierarchical Clustering.
  • each data point is defined as a cluster and existing clusters are combined at each step.
  • the following four methods may be used for this approach:
  • Single Linkage In single linkage, the distance between two clusters is defined as the minimum distance between any single data point in the first cluster and any single data point in the second cluster. On the basis of this definition of distance between clusters, at each stage of the process the two clusters with the smallest single linkage distance are combined.
  • Complete Linkage In complete linkage, the distance between two clusters are defined to be the maximum distance between any single data point in the first cluster and any single data point in the second cluster. On the basis of this definition of distance between clusters, at each stage of the process the two clusters that have the smallest complete linkage distance are combined.
  • Average Linkage In average linkage, the distance between two clusters are defined to be the average distance between data points in the first cluster and data points in the second cluster. On the basis of this definition of distance between clusters, at each stage of the process the two clusters that have the smallest average linkage distance are combined.
  • Centroid Method In centroid method, the distance between two clusters is the distance between the two mean vectors of the clusters. At each stage of the process the two clusters that have the smallest centroid distance are combined.
  • a method of treating or preventing a condition associated with hair loss in a subject in need thereof comprising administering a therapeutically effective amount of an agent to hair follicle cells or skin of the subject, wherein the agent has been determined as an agent having hair growth or hair cycle promoting activity according to the method as described herein.
  • the skin is the scalp of the subject.
  • the skin is the facial skin.
  • the skin is the skin of the limbs.
  • the skin is the skin of the torso.
  • the condition associated with hair loss is selected from the group consisting of androgenetic alopecia and telogen effluvium, post-partum hair loss, and hair loss due to anticancer therapy.
  • a cosmetic method of inhibiting hair growth or hair cycle in a subject in need thereof comprising administering a therapeutically effective amount of an agent to hair follicle cells or skin of the subject, wherein the agent has been determined as an agent having hair growth or hair cycle inhibiting activity according to the method as described herein.
  • the skin is the scalp of the subject.
  • the skin is the facial skin.
  • the skin is the skin of the limbs.
  • the skin is the skin of the torso.
  • a method of treating or preventing a condition associated with excessive hair growth in a subject in need thereof comprising administering a therapeutically effective amount of an agent to hair follicle cells or skin of the subject, wherein the agent has been determined as an agent having hair growth or hair cycle inhibiting activity according to the method as described herein.
  • the skin is the scalp of the subject.
  • the skin is the facial skin.
  • the skin is the skin of the limbs.
  • the skin is the skin of the torso.
  • the condition is selected from the group consisting of hirsutism and hypertrichosis.
  • the condition is selected from the group consisting of hirsutism and hypertrichosis.
  • kits for screening an agent as a hair growth or hair cycle promoting agent or a hair growth or hair cycle inhibiting agent according to the method as described herein, comprising one or more of the following:
  • buffer is optionally selected from the group consisting of an extraction buffer, a buffer for dissolving the polycationic polymer in (d) to form a solution of the polycationic polymer, and a buffer for dissolving the polyanionic polymer in (e) to form a solution of the polyanionic polymer;
  • the polycationic polymer may be one that is known in the art, such as chitin, chitosan, and combinations thereof.
  • the polyanionic polymer may be one that is known in the art, such as alginate, gellan, heparan sulfate, heparin, acidic keratin, hyaluronic acid, chondroitin sulfate, methylated collagen, and combinations thereof.
  • the extraction buffer may be any buffer which is known in the art which can be used to extract RNA from the other cellular lysate such as DNA and protein.
  • the extraction buffer is TRIzol ® Reagent (40%w/w Phenol (saturated at pH 4.3), 1 M guanidine thiocyanate, 1 M Ammonium thiocyanate, 0.1 M sodium acetate buffer (pH 5.0), 5%w/w glycerol).
  • the extraction buffer is a Guanidinium buffer such as guanidinium thiocyanate or guanidinium hydrochloride.
  • the extraction buffer is sodium dodecyl sulfate.
  • the extraction buffer is N -Laurylsarcosine. In another embodiment, the extraction buffer is PhenokChloroform: isoamyl alcohol. In another embodiment, the extraction buffer is 8-Hydroxyquinoline. In another embodiment, the extraction buffer is Cesium chloride. In another embodiment, the extraction buffer is Cesium trifluoroacetate. In another embodiment, the extraction buffer is Proteinase K. In another embodiment, the extraction buffer is RNAlater ® (Ambion).
  • the microarray chip may be an RNA microarray chip that is commercially available for RNA hybridization for microarray assay.
  • the microarray chip is HumanHT-12 v4 BeadChip (Illumina, St. Diego, CA).
  • the buffer for dissolving the polycationic polymer to form a polycationic solution should be one that is compatible with the hair follicle cells which are present in the polycationic solution, and has a pKa in the physiological range (6-8).
  • the buffer is phosphate buffered saline (PBS).
  • the buffer is selected from a group consisting of MES (2-(N-morpholino)ethanesulfonic acid), PIPES (Piperazine-N,N'- bis(2-ethanesulfonic acid)), HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), TAPSO (3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid), ACES (N-(2-Acetamido)-aminoethanesulfonic acid), ADA (N-(2-Acetamido)-iminodiacetic acid), BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid), BIS-Tris ([Bis-(2- hydroxyethyl)-imino]-tris-(hydroxymethylmethane)), BIS-Tris-Propane (1,3-
  • the buffer for dissolving the polyanionic polymer to form a polyanionic solution should be one that is compatible with the hair follicle cells which are present in the polyanionic solution, and has a pKa in the physiological range (6-8).
  • the buffer is deionized water.
  • the buffer is selected from a group consisting of MES (2-(N-morpholino)ethanesulfonic acid), PIPES (Piperazine-N,N'-bis(2- ethanesulfonic acid)), HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), TAPSO (3- [N-Tris(hydroxymethyl)methylamino] -2-hydroxypropanesulfonic acid), ACES (N-(2- Acetamido)-aminoethanesulfonic acid), ADA (N-(2-Acetamido)-iminodiacetic acid), BES (N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid), BIS-Tris ([Bis-(2-hydroxyethyl)- imino]-tris-(hydroxymethylmethane)), BIS-Tris-Propane (l,3-Bis
  • the reverse transcriptase converts the extracted RNA as described herein to cDNA, which is then used for the construction of RNA-seq library. Any commercially available reverse transcriptase may be included in the kit.
  • the reverse transcriptase is Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV RT) (Clontech).
  • the RNA-seq library construction kit allows RNA-seq library construction using cDNA prepared from RNA, followed by repair of 3' and 5' ends to form blunt-ended, phosphorylated molecules, and the addition of a non-templated dA-tail before ligation to an adaptor. If necessary to achieve sufficient yields, a final step of PCR amplification of the library is performed. Small RNA libraries are constructed using a different workflow, in which adaptors are ligated directly to the small RNA molecules, followed by reverse transcription and PCR amplification.
  • the RNA-seq library construction kit is NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).
  • RNA-seq library construction kit examples include TruSeq RNA Library Prep Kit (illumina), ScriptSeqTM v2 RNA-Seq Library Preparation Kit (epicentre), NEXTflex ® Rapid Illumina RNA-Seq Library Prep Kit (PerkinElmer), and Ambion ® RNA-Seq Library Construction Kit.
  • FIG. 1 is a heat map representing the top 10% of genes which are most differentially expressed in DPC aggregates treated with reference drugs.
  • each row represents the expression level of one gene in the gene expression profiles of DPC aggregates treated with reference drugs.
  • Heatmap visualization was performed by gplots R package. Shades on the right side of the color key with a positive value represent high gene expression value. Shades on the left side of the color key with a negative value represent low gene expression value.
  • the heatmap includes the same dendrogram as FIG. 2.
  • FIG. 2 is a dendrogram depicting clustering of gene expression in DPC aggregates treated with reference drugs. Clear segregation of gene expression profiles of hair follicle cells treated with reference drugs which promote hair growth or hair cycle (right arm of dendrogram; Cs - cyclosporin A, M - minoxidil, B - bimatoprost) and those of hair follicle cells treated with reference drugs which inhibit hair growth or hair cycle (left arm of dendrogram; D - dexamethasone) is observed.
  • FIG. 3 is a dendrogram depicting the prediction of hair growth promoting or inhibiting properties of candidate drugs.
  • Clustering of gene expression profiles of hair follicle cells treated with candidate drugs (Dia - diazoxide, Alio - allopurinol) with those of hair follicle cells treated with reference drugs which promote hair growth or hair cycle (Cs - cyclosporin A, M - minoxidil, B - bimatoprost), or with those of hair follicle cells treated with reference drugs which inhibit hair growth or hair cycle (D - dexamethasone) is shown.
  • DPC and NHEK were encapsulated in fibrous hydrogel scaffolds and cultured for two days to allow assembly of DP aggregates. Subsequently, the scaffolds were exposed to treatment with reference drugs that promote hair growth or hair cycle at the following concentrations: cyclosporin A (1 nM), minoxidil (25 ⁇ ) and bimatoprost (100 nM) and a reference drug that inhibits hair growth or hair cycle (dexamethasone, 100 nM) for 24 hours 1" . The same treatment was extended to two candidate drugs, diazoxide and allopurinol, that have been suggested in the literature to promote and inhibit hair growth or cycle, respectively 4"6 . Each drug treatment is replicated 3 -5 times. [00115] Isolation of DPC aggregates from MIPC scaffolds
  • All cells in the scaffold were released by dissolving the fibers in a mixture of 0.1 U/ml chitinase (Sigma-Aldrich, USA) and 14.5 U/ml alginate lyase (Sigma-Aldrich, USA) at 37 °C for 15 mins.
  • DPC aggregates were then isolated based on size- selection by filtration of the dissolved fiber and cell mixture obtained through a 40 ⁇ cell strainer (Corning, Durham, NC, USA), as the formed DPC aggregates have diameter of more than 40 ⁇ while NHEK aggregates have diameter of less than 40 ⁇ .
  • RNA samples of high integrity were labelled using TargetAmp Nano-g Biotin-aRNA labelling kit (Epicentre, Madison, WI) before hybridization on HumanHT-12 v4 BeadChip (Illumina, St. Diego, CA).
  • Arrays were scanned using the BeadArray Reader (Illumina, St. Diego, CA) and probe intensities were extracted using GenomeStudio (Illumina, St. Diego, CA).
  • Similar gene expression analysis will be conducted from expression data generated using RNA- sequencing.
  • cDNA was generated from 30 ng of extracted RNA with SMARTer Ultra Low Input RNA for Illumina Sequencing (Clontech), followed by RNA-seq library construction with NEBNext DNA Library Prep Master Mix Set for Illumina (NEB). Constructed libraries were multiplexed in batches of 18 and 151 bp paired-end sequenced with Illumina HiSeq High Output 4000.
  • FIG. 1 The microarray data are shown in the heatmap (FIG. 1), which represents the top 10% of the genes which are most differentially expressed in DPC aggregates treated with reference drugs which promote or inhibit hair growth or hair cycle. Agglomerative hierarchical clustering of the microarray data was used to generate the dendrogram in FIG. 1 and FIG. 2. It was found that expression profiles of DPC aggregates after treatments with reference drugs clustered according to their hair growth or hair cycle promoting or inhibitory properties (FIG. 2).
  • a novel assayed drug will be predicted as a hair growth inhibitor if the gene expression profile of the DPC aggregates treated with it substantially matches that of the DPC aggregates treated with the reference inhibitor drugs instead of the reference promoter drugs, and it could potentially be used to stem hair growth for cosmetic purposes.
  • the assay can also be used to identify drugs which may have the unintended side effect of promoting hair loss.
  • FIG. 3 illustrates the limitation of the screening assay in predicting the hair growth inducing potential of screened drugs if the screened drugs regulates hair growth/cycle through profoundly different pathways and mechanisms as compared to the reference promoters.
  • the addition of more reference drugs will allow predicting and identifying candidate hair growth promoters with greater accuracy.
  • the addition of more reference drugs which exert hair growth inhibitory effects in different pathways and mechanisms as compared to the reference inhibitors used herein will allow predicting and identifying candidate hair growth inhibitors with greater accuracy.
  • compartmentalized MIPC fiber sections can be obtained by sectioning the whole MIPC scaffolds with a blade in a direction perpendicular to the long axis of the fibers of the scaffolds, to create slices from the whole MIPC scaffolds, this assay can be potentially modified to utilize compartmentalized MIPC fiber sections instead of whole MIPC constructs to allow parallel screening of multiple novel drugs/molecules, thus increasing the numbers of novel drugs/molecules screened per assay and improving cost effectiveness.

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Abstract

L'invention concerne une méthode de criblage d'un agent candidat pour favoriser ou inhiber la pousse des poils/cheveux ou le cycle des poils/cheveux, comprenant : (a) l'obtention d'un profil de gènes qui sont exprimés de manière différentielle dans des cellules de follicules pileux lorsqu'ils sont traités avec un(des) agent(s) de référence(s) qui favorise ou inhibe la pousse des poils/cheveux ou le cycle des poils/cheveux, à l'aide d'un microréseau et d'un groupement hiérarchique ; (b) l'obtention d'un profil de gènes qui sont exprimés de manière différentielle dans des cellules de follicules pileux lorsqu'ils sont traités avec l'agent candidat à l'aide d'un groupement hiérarchique ; (c) la comparaison du profil de gènes qui sont exprimés de manière différentielle à l'étape (b) avec le profil de l'étape (a) ; et (d) la détermination de l'agent candidat comme ayant une activité favorisant ou inhibant la pousse des poils/cheveux ou le cycle des poils/cheveux lorsque le profil de gènes qui sont exprimés de manière différentielle à l'étape (b) correspond sensiblement au profil de gènes qui sont exprimés de manière différentielle à l'étape (a). L'invention concerne en outre une méthode de traitement de la perte de poils/cheveux et de la croissance excessive des poils/cheveux à l'aide d'agents identifiés par les méthodes, et un kit associé.
PCT/SG2018/050454 2017-09-07 2018-09-07 Méthodes de criblage d'agents de régulation de la pousse des poils/cheveux ou du cycle des poils/cheveux WO2019050480A1 (fr)

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CN112102914A (zh) * 2020-09-25 2020-12-18 复旦大学附属肿瘤医院 曲妥珠单抗生物类似药的评价方法及系统

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